ISOLATION OF ENDOPHYTIC BACTERIA FROM
NATIVE WESTERN AUSTRALIAN WOODY PLANTS
FOR BIOLOGICAL CONTROL OF PHYTOPHTHORA
CINNAMOMI IN NATURAL ECOSYSTEMS
By
Sian Contarino
Submitted for Fulfillment of
Honours in Molecular Biology
School of Veterinary and Life Sciences
Murdoch University
Perth, Western Australia
November 2013
1
Declaration
This thesis is submitted for completion of Honours in Molecular Biology. I declare
that this thesis has not previously been submitted for the award of any other degree at
another tertiary institution. Unless otherwise stated, the content of this thesis is my
own work, completed between February and November, of 2013.
Word count: 21,522
Sian Contarino
4th November 2013
……………………………………
2
Acknowledgements
I would like to thank my two supervisors Dr Phil O’Brien and Professor Giles Hardy,
who provided me with their knowledge, advice, criticisms, and most importantly their
encouragement during this Honours year. Their assistance during the writing of this
thesis, and in answering my never-ending questions is appreciated. The isolations and
plant sample collections would also not have been possible without their help both in
the lab and in the field.
Thank you to my friend Sophie Monaco, whose assistance in constructing the
rollmops will forever be indebted. I’d still be working on constructing the three
hundred and twenty eight rollmops if it wasn’t for all her hard work and assistance in
cutting and putting them together. Thanks also to Dr Patricia Stasikowski who took
the time to show me the best methods of seed germination and rollmop construction.
Also identification of my six significant endophytes would not have been possible
without the help of CPSM diagnostics, who kindly sequenced my isolates.
To my partner Matthew Tondut, who provided much needed support and put up with
all my stress, thank you. Thanks also to family and friends, for all your love and
encouragements throughout this year.
I’d also like to thank CRC Plant Biosecurity for the generous financial support
provided, and thank you to the Australasian Plant Pathology Society for their financial
support and opportunity to present at the APPS New Zealand conference, 2013.
3
Abstract
The oomycete pathogen Phytophthora cinnamomi is causing massive devastation of
natural forests in the southwestern corner of Western Australia. It is estimated that
2284 of the 5710 described native plant species in the southwest corner of Western
Australia are susceptible to P. cinnamomi, of which 800 are regarded as being highly
susceptible. One option for management of the disease is biological control using a
bacterium or fungus or both to antagonize the pathogen. Bacterial and or fungal
endophytes are a good source of biocontrol agents that can be used to protect plants
against disease. Many endophytes increase the resistance of the host plant to biotic
and abiotic stresses. They increase resistance to disease in a variety of ways: by
inducing the defence systems of the host, by the production of antibiotics to inhibit
pathogenic microorganisms, by production of enzymes to degrade the cell wall of the
pathogen, by competing for space and nutrients. For a biocontrol agent to be
successful, it must be culturable, have the ability to colonise a variety of plant hosts in
different environmental conditions and within different tissue types, and inhibit
different plant pathogens. Most importantly however, it must not be recognized in the
host tissues as a pathogen itself. The aim of this project was to look for a potential
biological control agent that will protect plants against P. cinnamomi. This study
focused on culturable endophytic bacteria within different Australian native plant
species, located in P. cinnamomi infested and non-infested areas. A total of two
hundred and fifty two isolates were recovered from root, stem and leaf samples from a
range of plant species at three sites from healthy and adjacent infested areas. These
isolates were grouped into forty eight morphotypes based on colony morphology.
The morphotype distributions differed significantly across sites and sampling areas
that were infested or not-infested with P. cinnamomi. To determine the potential of
iii
4
these endophytes as biocontrol agents, isolates were tested in vitro using a plate
confrontation assay and in planta with Lupinus angustifolius seedlings against P.
cinnamomi. A number of isolates inhibited the growth of P. cinnamomi in vitro,
suggesting they produced secondary metabolites such as antibiotics. Also, six
endophyte morphotypes significantly decreased lesion lengths caused by P.
cinnamomi in L. angustifolius seedlings, suggesting their ability to reduce infection in
plants against the pathogen. This shows their potential effectiveness as biocontrol
agents; however, further research needs to be undertaken on their viability and
persistence over time within different host plants, and their ability to confer resistance
under different environmental conditions.
5iv
Table of Contents
Abstract ..................................................................................................... 4
1.0 Introduction .............................................................................................................................. 1
1.1 The impact of Phytophthora as a pathogen ................................................................... 1
1.2 Phytophthora cinnamomi ....................................................................................................... 3
1.3 Management of Phytophthora diseases............................................................................... 4
1.4 Biological Control ................................................................................................................... 6
1.5 Mechanism of Action of Endophytes .................................................................................. 7
1.6 The Aims of this Study ........................................................................................................... 9
1.7 Experimental Strategy ........................................................................................................... 9
2.0 Diversity of Endophytic Bacteria in Native Australian Plant
Species ..................................................................................................... 11
2.1 Introduction ............................................................................................................................11
2.2 Materials and Methods ........................................................................................................13
2.2.1 Study Sites .................................................................................................................................... 13
2.2.1.1 Murdoch Woodland Site .................................................................................................................... 14
2.2.1.2 Jarrah Graveyard Sites ....................................................................................................................... 15
2.2.3 Sample Collection ...................................................................................................................... 18
2.2.3.1 Plant Species Used .............................................................................................................................. 19
2.2.3.2 Plant Tissues Sampled .......................................................................................................... 20
2.2.4 Comparison of Endophyte Isolation Methods................................................................... 21
2.2.5 Bacterial Endophyte Isolation from Plant Tissues........................................................... 21
2.2.6 Test of Optimum Bacterial Endophyte Growth Conditions ......................................... 22
2.2.7 Morphotype Identification....................................................................................................... 23
2.2.8 Data Analysis .............................................................................................................................. 23
2.3 Results ......................................................................................................................................24
2.3.1 Test of Isolation Procedures ................................................................................................... 24
2.3.2 Comparison of Bacterial Endophyte Isolation Methods ................................................ 25
2.3.3 Test of Optimum Bacterial Endophyte Growth Conditions ......................................... 25
2.3.4 Test for Morphotype Patch Plate Consistency .................................................................. 25
2.3.5 Bacterial Endophyte Morphotypes Isolated from Each Study Site ............................ 26
2.3.6 Association Between Bacterial Endophyte Morphotypes and Study Sites .............. 29
2.3.6.1 Analyses of Bacterial Endophyte Morphotype Distribution at Different Sites .............. 31
2.3.7 Association Between Bacterial Endophyte Morphotypes and Plant Host Species 32
2.4 Discussion ................................................................................................................................36
2.4.1 Extraction of Bacterial Endophytes ...................................................................................... 36
2.4.2 Differences of Bacterial Endophytes Between Study Sites .......................................... 42
3.0 Screening of Endophytes for Antibiotic Production .................... 45
3.1 Introduction ............................................................................................................................45
3.2 Materials and Methods ........................................................................................................48
3.2.1 Isolate of P. cinnamomi Used ................................................................................................ 48
3.2.2 Preparation of Endophyte Isolates Used ............................................................................. 48
3.2.2 Comparison of Media for Optimal Pathogen and Bacterial Isolate Growth............ 49
3.2.3 In Vitro Confrontation Assays of P. cinnamomi and Bacterial Endophyte Isolates
..................................................................................................................................................................... 50
3.2.4 Data Analysis .............................................................................................................................. 51
3.3 Results ......................................................................................................................................52
3.3.1 Media Comparisons for Optimum Growth of Endophyte Isolates and P.
cinnamomi ............................................................................................................................................... 52
3.3.2 In Vitro Confrontation Plate Assays of Endophytic Bacterial Isolates Against P.
cinnamomi ............................................................................................................................................... 54
3.3.3 Association Between Mean Inhibition Zones and Sampling Area ............................. 58
v6
3.3.4 Analysis of Mean Inhibition Zone, Sampling Area, and Host Plant Species .......... 62
3.4 Discussion ................................................................................................................................63
3.4.1 Diversity of Endophytic Isolates within Morphotypes................................................... 63
3.4.2 Ability of In Vitro Antagonistic Isolates as Potential Biocontrol Agents ................ 64
4.0 Screening Endophytes for in planta Inhibition of Phytophthora
cinnamomi ............................................................................................... 68
4.1 Introduction ............................................................................................................................68
4.2 Materials and Methods ........................................................................................................70
4.2.1 Preparation of Morphotype Representatives for in planta Assays ............................. 70
4.2.2 P. cinnamomi 793 Growth Conditions ................................................................................ 70
4.2.3 Underbark Stem Inoculation Method................................................................................... 71
4.2.4 The Lupin Rollmop Assay. ..................................................................................................... 71
4.2.4.3 Endophyte Effects on Seedling Growth ....................................................................................... 72
4.2.4.4 Endophyte Effects on Lesion Lengths Caused by P. cinnamomi Infection ..................... 73
4.2.5 Data Analysis .............................................................................................................................. 74
4.2.6 Identification of Significant Morphotypes ...................................................................75
4.3 Results ......................................................................................................................................76
4.3.1 Preliminary trial of the Underbark Stem Inoculation Assay for in planta inhibition
of P. cinnamomi. ................................................................................................................................... 76
4.3.2 The Lupin Rollmop Assay for Effect on Dry Weight by Morphotypes ................... 77
4.3.3 Relationship Between Mean Dry Weight of L. angustifolius Seedlings, Sampling
Area, and Number of Plant Species Each Morphotype is Present Within .......................... 80
4.3.4 The Lupin Rollmop Assay for Morphotype Effect on Lesion Lengths by P.
cinnamomi ............................................................................................................................................... 82
4.3.5 Relationship Between Mean Lesion Length of L. angustifolius Seedlings Caused
by P. cinnamomi, Sampling Area, and Number of Plant Species Each Morphotype is
Present Within ........................................................................................................................................ 86
4.3.6 Relationship Between Mean Dry Weight and Mean Lesion Length of L.
angustifolius Seedlings........................................................................................................................ 87
4.3.7 Identification of the Six Endophyte Morphotypes Shown to Significantly Reduce
Lesion Lengths in L. angustifolius Seedlings Caused by P. cinnamomi ............................. 88
4.4 Discussion ................................................................................................................................90
4.4.1 Ability of Endophyte Morphotypes to Enhance Growth and Reduce Lesion
Lengths in L. angustifolius Seedlings ............................................................................................. 91
4.4.2 Morphotypes Tested in In Vivo Assays ............................................................................... 93
5.0 Endophytes and their Future Prospects as Biocontrol Agents.... 96
5.1 Discussion ................................................................................................................................96
6.0 References ....................................................................................... 101
vi7
List of Tables
Table 2.1: Number of bacterial isolates and morphotypes for each study
site, sampling area, and plant tissue type…………………………………..28
Table 2.2: The frequency of occurrence of each morphotype within each
site, sampling area, and plant tissue. (NI= non infested, I= infested;
L, R, S= leaf, root, and stem respectively)………………………………...30
Table 2.3: Frequency of isolation of morphotypes from different host
plant species. (GS1 and GS2= jarrah graveyard site 1 and 2
respectively)……………………………………………………………….32
Table 3.1: Mean inhibition zones (cm ± standard error for each
morphotype against P. cinnamomi within each sampling area
(0 indicates no growth, blank indicates morphotype was not present
within that area; NI = non-infested, I = infested with P. cinnamomi)…….61
Table 4.1: Mean lesion length (cm ± standard error) of under bark stem
inoculation assays between selected endophyte isolates and
P. cinnamomi 793…………………………………………………………77
Table 4.2: DNA sequencing results of morphtoypes found to
significantly reduce lesion length caused by P. cinnamomi………………89
8vii
List of Figures
Figure 2.1: Murdoch and jarrah graveyard sites….…………..……….…………...13
Figure 2.2: Healthy study site within Murdoch University woodlands…………....14
Figure 2.3: Infested (red) and non-infested (yellow) sampling areas within the
Murdoch woodland site (Courtesy Google Earth, 2013)…………………………...15
Figure 2.4: Infested (red) and non-infested (yellow) sampling areas within
Jarrah graveyard sites 1 (A) and 2 (B) located off Albany highway……………….16
Figure 2.5: Infested sampling area within the jarrah forest graveyard site 1……....17
Figure 2.6: Non-infested sampling area within the jarrah forest graveyard
site 1………………………………………………………………………………...18
Figure 2.7: Example of an unhealthy B. attenuata specimen sampled within
the infested sampling area of the Murdoch woodlands site………………………...19
Figure 2.8: Growth of leaf isolated bacterial colonies on LB agar medium
supplemented with 50μg/ml cyclohexamide. No growth occurred on the
sterile rinse water control plate (A), and growth of leaf isolate colonies (B)………24
Figure 2.9: Morphotype patch plates for extracted plant tissue bacterial
endophytes. Arrows indicate the same morphotype (morphotype 9) plated
twice to ensure consistency between plating……………………………………….26
Figure 3.1: LB media plate containing Phytophthora cinnamomi 793……....…….48
Figure 3.2: Confrontation plate assay showing four different bacterial
endophyte isolates on the edges of a Potato Dextrose Agar plate, with a
Phytophthora cinnamomi agar plug placed in the centre…………………………...50
Figure 3.3: In vitro confrontation plate assays between different bacterial
endophyte isolates and P. cinnamomi. Different isolates show different levels
of inhibition (A, B, C, D)………………………………………………….……..…54
Figure 3.4: Mean inhibition zones (cm) and standard error bars of endophyte
isolates against P. cinnamomi on in vitro confrontation assays. Isolates are
within their morphotype groups (shaded green and pink)…………………………..55
Figure 4.1: Germinated L. angustifolius seeds after 2 days incubation…………….71
Figure 4.2: Placement of L. angustifolius seeds on constructed rollmop………..…72
Figure 4.3: Inoculation of L. angustifolius seedling roots contained in rollmops
with agar plugs of P. cinnamomi 793………………………………………………73
viii
9
Figure 4.4: Under bark stem inoculation assays conducted on excised stems
of E. marginata inoculated with P. cinnamomi only as the control (A),
P. cinnamomi challenged with isolate 140BS-1 (B), and P. cinnamomi
challenged with isolate 140BL-2 (C)………………………………………………76
Figure 4.5: Mean dry weight and standard error of L. angustifolius seedlings
after inoculation with endophyte morphotypes…………………………….………78
Figure 4.6: Mean dry weight and standard error of L. angustifolius seedlings
inoculated with morphotypes present across infested, non-infested, and
both sampling areas..………………………………………………………….........81
Figure 4.7: Lesions produced by P. cinnamomi in L. angustifolius seedlings.
Lesions were larger for morphotype 2 (A) compared with morphotype 40 (B).......82
Figure 4.8: Mean lesion length of L. angustifolius rollmop seedlings caused
by P. cinnamomi 793 after inoculation with endophyte morphotypes......................83
Figure 4.9: Mean lesion length and standard error of L. angustifolius seedlings
inoculated with P. cinnamomi 793 and morphotypes present across infested,
non-infested, and both sampling areas......................................................................86
Figure 4.10: Differences in endophyte morphology of the six significant
morphotypes found to reduce lesion length caused by P. cinnamomi in L.
angustifolius seedlings..............................................................................................88
ix
10
Chapter 1
1.0 Introduction
1.1 The impact of Phytophthora as a pathogen
Plant disease epidemics caused by fungal and oomycete pathogens are increasingly
becoming recognized as their impacts on large numbers of susceptible plants species
are reported. The most recent report on the number of native Australian plants at risk
of disease by Phytophthora cinnamomi is from the work of Shearer et al. (2004b),
who showed 2284 out of a total of 5710 native plant taxa in the southwest corner of
Western Australia, a recognized biodiversity hotspot, are susceptible to this
pathogenic oomycete. Also, more than 100 plant species within 30 different families,
all inhabiting different ecosystem types, have also been found to be susceptible to P.
ramorum (Hardham 2005). Even more alarming, approximately 100 million elm trees
and 3.5 billion chestnut trees in the in the United States and United Kingdom were
eradicated from the effects of chestnut blight, caused by Cryphonectria parasitica
(Loo 2009, Giraud et al. 2010).
Effects of Phytophthora caused plant diseases in natural ecosystems have been seen
worldwide (McDougall et al. 2005). As a genus, Phytophthora has been viewed as
one of the most important for causing disease and mortality in a variety of different
tree species (Barber et al. 2013). For example, plant families Ericaceae, Fabaceae,
Proteaceae, and Xanthorrhoeaceae include tree and shrub species considered
especially susceptible to P. cinnamomi (McDougall et al. 2005, Cahill et al. 2008).
Reports have also shown once Phytophthora diseases infect an area, drastic changes
1
result in the plant species composition due to the death of many plants and trees, and
these susceptible plant species are usually replaced with sedges and introduced weed
species (Hardham 2005), which ultimately affects the ecosystem as a whole.
Additionally, as new Phytophthora species are being discovered worldwide from
DNA sequencing (Burgess et al. 2009, Scott et al. 2009, Jung et al. 2011, Rea et al.
2011), new and existing hybridizations can cause devastation to new plant hosts and
different ecosystems (Barber et al. 2013).
The death of native plants and trees caused by P. cinnamomi has been seen
extensively throughout the south-west jarrah forest of Western Australia (Shearer et
al. 2004b). These affected disease fronts (recently killed vegetation that leads into
healthy vegetation beyond) (Shearer et al. 2007) have been shown to extend into
healthy forest areas at a rate of 0.7-2 metres per year (Grant 2003). The rate of disease
movement would also depend on environmental factors such as climate, site, and host
susceptibility (Sturrock et al. 2011). However, although these environmental factors
are important in disease movement, the rate of spread is also closely attributed with
anthropogenic activities such as road works, mining exploration, logging of forests,
(Hansen 2008), contamination of nursery stock (Brasier 2008a), and recreational
activities (Shearer et al. 2004c).
Huge costs and losses are also reported as consequences of P. cinnamomi infection
within both natural and managed environments. For example, control and
management of Phytophthora diseases can be costly, and economic losses due to
affected crops and changed agricultural practices have been reported (Fisher et al.
2012). For example, it has been estimated 10% of avocado trees in California are
infected with P. cinnamomi, which has been shown to result in an annual loss of $36
2
million (McDonald et al. 2007). Additionally, infection with Phytophthora species
have been reported as the primary yield limiting factor in melon crops of India, where
disease has resulted in a yield loss of up to 90% (Guharoy et al. 2006).
1.2 Phytophthora cinnamomi
Species of Phytophthora grow vegetatively as a hyphal mycelium rather like a fungus
(Hardham 2005). For this reason Phytophthora has always been considered to be a
fungus. However biochemical and genetic studies have shown that Phytophthora is
not a fungus but is located within a group called the Stramenophiles (Beakes et al.
2012). The genus contains some of the most destructive plant pathogens. Many
horticultural crops and a wide variety of trees shrubs and ornamental species are
susceptible to Phytophthora (Hardham 2005). Currently there are 121 species of
Phytophthora and more are being discovered with the increased use of DNA
sequencing techniques for phylogenetic analysis (Scott et al. 2013a).
Also, P. cinnamomi is a root infecting pathogen that has caused massive devastation
in the eucalypt forests of WA (Shearer et al. 2004a). Within the root it produces both
sexual spores, oospores and asexual chlamydospores which contribute to prolonged
survival of the pathogen (Hardham 2005). Additionally they produce stromata which
are hyphal aggregations (Crone et al. 2012), and lignotubers which are cells enclosed
within a thick lignified wall (Jung et al. 2013). Both of these structures contribute to
survival of the pathogen. Phytophthora species also produce asexual motile
zoospores that swim through water to plant roots (Hardham and Shan 2009). They
encyst on the root surface and germinate to initiate new infections. Infections can also
3
initiate by growth of hyphae from the roots of one plant to another (Hardham and
Shan 2009).
P. cinnamomi is considered to be a necrotrophic pathogen. In the plant it causes cells
to lyse by degrading the cell wall and then feeds on the contents released from the
burst cells (Hardham 2005). This results in maceration of the tissue resulting in a
brown lesion on the root. However recent research has shown that P. cinnamomi can
exist within plants asymptomatically without causing lesions (Crone et al. 2013c,
Jung et al. 2013). Within these plants the pathogen can complete all stages of its life
cycle producing the various sexual and asexual reproductive and survival structures.
This has significant biosecurity implications as it means that inoculum can be
transported undetected.
Additionally, P. cinnamomi is considered to be a relatively recent introduction to
Australia. Plant pathogens often cause little damage at their centre of origin having
developed a natural balance through co-evolution with their host plants (Brasier
2008b). However, problems arise when pathogens are introduced to other regions of
the world. Additionally the pathogen population is more diverse at the centre of
origin. The greatest diversity of P. cinnamomi is in Papua New Guinea suggesting
that this potentially could be its centre of origin (Dobrowolski et al. 2003).
1.3 Management of Phytophthora diseases
Devastating effects have also been seen in natural ecosystems and on plant species
diversity from Phytophthora caused diseases, and huge losses of CO2 absorption
4
accounted for by the death of many older established trees have been reported (Fisher
et al. 2012). Not only does extensive management and control need to be given in
forest areas already affected with P. cinnamomi, but also management in reducing the
spread of the pathogenic oomycete into other unaffected areas is essential. For
example, hygiene policies and restricted access of dieback infested areas are just two
on the management procedures utilized by the Alcoa mine site in Western Australia
for minimizing the spread and impact caused by P. cinnamomi (Colquhoun and
Elliont 2000). These hygiene procedures in place at Alcoa include manual wash down
of all vehicles, and cleaning of all equipment and footwear when entering and exiting
dieback infested areas.
Other current management procedures for control of Phytophthora diseases include
attempts at pathogen elimination by susceptible and diseased vegetation removal
(Dunstan et al. 2010), and the use of chemicals such as phosphite (Hardy et al. 2001).
Phosphite acts by induction of the host defence system (Eshraghi et al. 2011), and it
can also directly inhibit growth of the pathogen (Wilkinson et al. 2001, Wong et al.
2008, King et al. 2010). Studies have shown treatment with chemicals such as
phosphite to leaves and stems of P. cinnamomi infected plants may be effective as an
ongoing control of dieback disease in native tree species of Western Australia (Scott
et al. 2013b). However, plants species have been shown to vary in their responses to
phosphite. Some species require high concentrations for induction of defence systems
whereas others can be induced by exposure to low concentrations (Barrett et al.
2002). This variability limits the effectiveness of phosphite as a control measure
within native forest ecosystems (Shearer et al. 2004c).
5
Management of the disease is complicated by the recent discovery that some plant
species are asymptomatic to Phytophthora infection. For example, P. capsici and P.
ramorum were found to colonize different weed species asymptomatically (FrenchMonar et al. 2006, Shishkoff 2012). Also, 12 genera of annual and herbaceous plants
previously not known to contain species that can act as a host P. cinnamomi without
showing symptoms were found (Crone et al. 2013a). P. cinnamomi was shown to
produce a range of propagules such as chlamydospores, oospores, and stromata within
these asymptomatic hosts. These can act as a source of inoculum for further disease
outbreaks (Crone et al. 2013b). Additionally, woody plant species can also be
infected with P. cinnamomi without showing symptoms (Jung et al. 2013).
1.4 Biological Control
Presently the only chemical for management of Phytophthora diseases in native
ecosystems is the use of phosphite (Hardy et al. 2001, Cahill et al. 2008), and in
recent years considerable success has been achieved with biological control of plant
pathogenic microorganisms. Many microorganisms (actinomycetes, bacteria and
fungi) that colonise plant tissues are antagonistic to pathogenic microorganisms and
can protect the plant from the pathogen (Alabouvette et al. 2006, Rosenblueth and
Martinez-Romero 2006, Franco et al. 2007a, van der Lelie et al. 2009, Zamioudis and
Pieterse 2012). Colonisation of black pepper vine with endophytic Pseudomonas
species resulted in 90% reduction in lesion lengths and 60% of plantlets free from
infection caused by P. capsici (Aravind et al. 2012). In another study the combined
application of endophytic Fusarium oxysporum and Bacillus firmus resulted in an
6
86.2% reduction in the density of the pathogenic nematode Radopholous similis in
banana plants, (Mendoza and Sikora 2009).
1.5 Mechanism of Action of Endophytes
Biological control agents protect plants against pathogens by induction of systemic
resistance (Choudhary and Johri 2009), and production of antibiotics to directly
inhibit the pathogen. For example, the elicitation of 2,3-Butadeniol by B. subtilis
GB03 has been shown to induce systemic resistance in Arabidopsis plants against
Erwinia carotovora (Ryu et al. 2004). Also, Pyrrolnitrin, produced by Burkholderia
and Pseudomonas species, has the ability of inhibiting Rhizoctonia solani, Botrytis
cinerea, and Verticillium dahliae (Raaijmakers et al. 2002).
Additionally, competing with pathogens for nutrients and space, and production of
hydrolytic enzymes to degrade the host cell wall are also methods utilized by
endophyes in resistance against plant pathogens (Rosenblueth and Martínez-Romero
2006, Schulz et al. 2006, Thakore 2006). Rhizospheric P. putida has been shown to
control F. oxysporum in tomato plants by preventing the pathogen from sequestering
iron and inhibiting its proliferation via production of siderophores (Bashan and de
Bashan 2005). Also, Pseudomonas species have been shown to produce hydrolytic
enzymes such as chitinase and lamarinase within carnation plants, which lyse cells of
pathogenic Fusarium oxysporum f. sp. dianthi (Ajit et al. 2006).
Endophytes also promote faster growth of the host plant through the production of
phytohormones (Lodewyckx et al. 2002, Guo et al. 2008b, Ryan et al. 2008). For
7
example, phytohormones such as auxins, cytokinins, and gibberellins produced by
endophytes can stimulate growth in plant tissues (Tanimoto 2005, Weyens et al.
2009). Gibberellin producing Paecilomyces formosus was shown to significantly
enhance shoot length by 6.9% in normal growth conditions, and 4.5% under high
salinity stress conditions in cucumber plants (Khan et al. 2012). Additionally, rice
shoot growth enhancement has been attributed to gibberellin production by
endophytic Chrysosporium pseudomerdarium (Hamayun et al. 2009).
Many studies have found certain bacterial endophyte species to confer pathogen
resistance against various plant pathogens. For example, many Pseudomonas species
have been found as successful biocontrol agents against different plant pathogens.
Two P. fluorescens strains with different secondary metabolite productions were
found to provide successful biocontrol against P. cactorum when dual inoculated into
strawberry plants (Agusti et al. 2011). Additionally, combination of different P.
putida strains were observed to enhance disease suppression of Fusarium oxysporum
by approximately 50% in radish plants (de Boer et al. 2003). However, studies have
shown certain Pseudomonas species, such as P. putida, to establish at higher levels
and have a higher biocontrol effect when inoculated into the rhizosphere in
comparison to plant stem and leaf tissue (Molina et al. 2000). Therefore, although
different endophytes may show resistance against different pathogens, further
research is needed on their effectiveness as biocontrol agents within different plant
tissues.
8
1.6 The Aims of this Study
The aims of this study were to: a) identify whether isolated endophytes differ between
sites and between infested and non-infested areas; b) determine if endophytes differ
between plant host species; and c) evaluate culturable endophytic bacteria from native
plant species for biocontrol activity against Phytophthora cinnamomi.
1.7 Experimental Strategy
The strategy followed in this study was to isolate endophytic bacteria from native
plant species by plating on agar and testing them for production of antibiotics that
inhibit P. cinnamomi in vitro, and for inhibition of P. cinnamomi lesion development
in planta. Plants were chosen at random for isolation of endophytes. Because P.
cinnamomi can persist in infested sites for long periods of time, and because plants
can therefore be subjected to successive cycles of infection, it is important that a
biological control agent will not be negatively affected by infection of the host by P.
cinnamomi. For this reason we looked at endophytes from plants in infested areas and
in adjacent non-infested areas. This approach for the isolation of biocontrol agents has
been used widely and has resulted in the isolation of effective biocontrol agents from
many species (Clay 1989, Barka et al. 2002a, Franco et al. 2007a, Kim et al. 2007,
Liu et al. 2009, Baldauf et al. 2011, Filho et al. 2013, Yau et al. 2013). We focused
exclusively on culturable bacterial endophytes isolated from plants within P.
cinnamomi infested and non-infested areas, because we need to be able to grow the
bacterium to be able to use it as a biocontrol agent. Although the culturable
9
endophytes probably represent only a small proportion of the total endophytic
population (Ikeda et al. 2010), their ability to be cultured and inoculated into different
plant species means they could potentially be used as biocontrol agents.
10
Chapter 2
2.0 Diversity of Endophytic Bacteria in Native Australian
Plant Species
2.1 Introduction
All plants contain endophytic bacteria and fungi that live within the plant tissues
without causing any apparent negative effects (Hallmann et al. 1997, Hardoim et al.
2008). The positive benefits that many of these endophytes confer onto their host
have been well documented (Selosse et al. 2004). For example, endophytic bacteria
have been shown to directly promote plant growth and to increase resistance to both
abiotic stress such as salinity and drought, and biotic stress such as pathogen infection
(Glick 2012). Additionally, endophytes have been observed to enhance plant growth
and yield on a variety of agricultural crops (Sturz et al. 2000, Kennedy et al. 2004,
Ryan et al. 2008).
Endophytes are often effective as biological control agents against plant diseases (De
Jonghe et al. 2005, Newman and Reynolds 2005, Nielsen et al. 2006, Franco et al.
2007b, Hultberg et al. 2010). For example, rhizobacterial strains isolated from
watermelon roots reduced infection of Didymella bryoniae by up to 70% when
applied to watermelon seeds before planting (Nga et al. 2010). As a first approach to
develop a biological control agent for control of P. cinnamomi in native ecosystems,
we need to identify what species of microbial endophytes are found in native plant
species. To be an effective biocontrol agent in native ecosystems the organism must
be capable of colonising multiple species of plants that are commonly found in native
11
forest (Pal and Gardener 2006), such as the jarrah forest areas of south Western
Australia.
In addition, we also need to determine how infection by P. cinnamomi affects the
diversity of endophytes within the plant. Studies on endophyte diversity in diseased
plants have shown that some endophyte species disappear from the host plants,
resulting in changes to endophyte diversity within the plant (Lian et al. 2008,
Kowalski and Drozynska 2011, Douanla-Meli et al. 2013, Jami et al. 2013). Because
P. cinnamomi persists in infested sites, plants may be subjected to successive cycles
of infection (McDougall et al. 2002, Auesukaree et al. 2004). It’s highly desirable
therefore that the biocontrol agent is not easily displaced following infection of the
host plant by a pathogen, but continues to confer long term protection on the host.
Additionally, a biocontrol agent having the ability to colonise potential host species
under different environmental conditions would be highly desirable. Therefore, we
need to look at endophyte diversity in the same host species at different sites.
The aim of this study was to determine if differences were present in culturable
bacterial endophyte diversity between different sites, P. cinnamomi infested and noninfested areas, and between plant host tissues. To achieve this aim three sites were
chosen. Each site consisted of a healthy and an adjacent P. cinnamomi infested area.
The endophyte diversity in root, stem, and leaf tissues from different species in each
area were assessed. This study was limited to bacterial endophytes for reasons of
time. Fungal endophytes take much longer to grow (Strobel and Daisy 2003)and
because of this were not included in this study.
12
2.2 Materials and Methods
2.2.1 Study Sites
A total of three woodland and forest sites were sampled during late summer and early
autumn (March and May) of 2013 for plant tissue samples for the purpose of
bacterial endophyte extraction. These study sites were chosen with knowledge that
they were infested with Phytophthora cinnamomi. Each site comprised of a P.
cinnamomi infested woodland area and an adjacent healthy woodland area. One of the
sites was located on the Murdoch University campus. The two additional jarrah
(Eucalyptus marginata) forest graveyard sites were located approximately 68km
south-east from Perth in forest areas directly adjacent to Albany highway (Figure 2.1).
=Murdoch
=Jarrah
graveyards
Figure 2.1: Murdoch and jarrah graveyard sites (courtesy Google Earth,
2013).
13
2.2.1.1 Murdoch Woodland Site
The Murdoch woodland study site (32°04’35.46”S 115°49’52.72”E) on the Murdoch
University campus consisted of a medium density upper story comprised mostly of
Eucalyptus marginata trees (Figure 2.2). The mid-story of the woodland contained a
large variety of small tree and large shrub species, mostly dominated by Banksia
attenuata and B. menziesii (Figure 2.2). Additionally, the smaller understory plants
consisted of many different species, most of them including grasses and small
herbaceous plants. A main difference between the infested and non-infested Murdoch
woodland study areas included the infested area had a higher observed number of
unhealthy looking trees. The infested area was reported as being infested with P.
cinnamomi in 2010. Overall, the Murdoch study site was of a medium density,
comprised of a majority of native Australian and Western Australian plant species.
Figure 2.2: Healthy study site within Murdoch University woodlands.
14
The location of the infested and non-infested sampling areas at this study site is
shown in Figure 2.3. Infested and non-infested areas are represented by red and
yellow rectangles respectively, and are adjacent to each other. Plant tissue samples
were collected from the infested study area located approximately 25 m from
Farrington road, and also from the adjacent non-infested area in which the majority of
plants appeared healthy.
Figure 2.3: Infested (red) and non-infested (yellow) sampling areas within
the Murdoch woodland site (Courtesy Google Earth, 2013).
2.2.1.2 Jarrah Graveyard Sites
The two additional study sites included jarrah forest graveyard areas, located adjacent
to Albany highway south of the Perth metro region (32°23’41.02”S 116°15’53.96”E;
and 32°23’10.12”S 116°15’19.42”E respectively). The infested graveyard areas were
located approximately 20 metres from the road, with healthy adjacent areas observed
directly beside the infested forest areas (Figure 2.4a and 2.4b). The two jarrah
graveyard sites were located approximately 2km apart.
15
A
B
Figure 2.4: Infested (red) and non-infested (yellow) sampling areas within
Jarrah graveyard sites 1 (A) and 2 (B) located off Albany highway
(courtesy Google Earth, 2013).
These graveyard sites consisted primarily of E. marginata and Corymbia calophylla,
with the two infested areas containing a very limited number of other species. There
were very few indicator species such as Banksia grandis or Xanthorrhoea gracilis
present in the infested areas of each site, and much of the ground was covered by
small grass plants. Most of the E. marginata and C. calophylla trees within the
infested areas were of poor health, and the density of plants and trees within the area
was low (Figure 2.5).
16
Figure 2.5: Infested sampling area within the jarrah forest graveyard site 1.
In comparison, the non-infested areas within jarrah forest graveyard sites 1 and 2
were of medium density and were comprised of a larger variety of different plant
species. The dominant tree species present within the healthy areas were still E.
marginata and Corymbia calophylla (Figure 2.6) however, diversity of mid and
lower-storey plants were also observed as well as a number of indicator species such
as B. grandis, X. gracilis, and Persoonia longifolia.
17
Figure 2.6: Non-infested sampling area within the jarrah forest graveyard site 1.
For this study we did not confirm the presence of P. cinnamomi in any of the infested
areas, however, P. cinnamomi is known to persist in infested sites for very long
periods (McDougall et al. 2002, Crone et al. 2013a).
2.2.3 Sample Collection
A variety of different plant species were used for the collection of plant tissue
samples for the purpose of this study. Trees and plants specimens were chosen at
random within the designated infested or healthy study sites. Within the infected sites,
specimens were chosen based on their visible health characteristics. That is, trees with
unhealthy canopies and bare branches were chosen to be most representative of the
diversity of morphotypes within an infected forest area (Figure 2.7).
18
Figure 2.7: Example of an unhealthy B. attenuata specimen sampled
within the infested sampling area of the Murdoch woodlands site.
2.2.3.1 Plant Species Used
A variety of plant and tree species were utilized for sample collection within the
Murdoch woodland site. These specimens included plants and trees of varying sizes,
such as established trees like E. marginata and grass trees and shrubs such as
Xanthorrhoea preissii and Hibbertia hypericoides respectively. All plants and trees
selected within infested and non-infested areas were also at least 5 m apart.
Additionally, plants within the Murdoch woodland study site were identified using
“Flora of Murdoch University” (Dell and Bennett 1986).
Trees used for sample collection within the two jarrah forest graveyard sites consisted
only of E. marginata and C. calophylla species. These jarrah forest graveyard sites
were of very low species diversity, thus it was determined that only these two species
19
were to be sampled for consistency between the infested and non-infested sampling
areas. The trees sampled within these forest areas were identified by their leaf and
fruit characteristics. Additionally, as the infested jarrah forest graveyard areas
appeared to be of poor health, trees were selected randomly and were all
approximately 10-20 m apart from each other. In the adjacent non-infested sampling
areas of the two jarrah forest graveyard sites however, plants and trees were of a
much greater density, and tree specimens were sampled at least 5 m apart.
2.2.3.2 Plant Tissues Sampled
Stem and leaf samples were cut from the plants using secateurs. For collection of root
tissue, the roots were exposed by digging and a section of rot tissue was removed
using secateurs. All cutting and digging implements were washed with methanol
between plants to prevent cross contamination. The samples were placed in clean zip
lock bags for transport to the laboratory, and these samples were processed as quickly
as possible (usually within one day). Leaf, root, and stem or bark samples were
collected for both plant and tree specimens within infested and non-infested sampling
areas. Root samples were collected by digging at the base of the tree or shrub, and
collecting roots growing directly from the stem of that specimen. It was however
difficult on occasions to ensure all root samples were representative of the plant
specimen being sampled, thus roots were taken within the closest possible proximity
to the plant or tree specimen.
20
2.2.4 Comparison of Endophyte Isolation Methods
Two methods of mortar and pestle grinding were compared in order to determine the
most efficient protocol for isolation of bacterial endophytes from within plant tissue
samples. These methods included the use of a small electronic pestle in which the
surface sterilized leaf, stem, or root tissue sample was ground in a sterile eppendorf
tube. Additionally, a larger sterile ceramic mortar and pestle was used in which the
surface sterilized plant tissue sample was ground for 2-3 minutes down to a paste by
hand.
2.2.5 Bacterial Endophyte Isolation from Plant Tissues
Endophytes were isolated from separate leaf, root, and stem samples from all plant
and tree specimens sampled. All bacterial endophytes and surface rinse controls were
plated onto LB agar media (Sambrook et al. 1989a) containing 50 μg/ml of
cycloheximide (Sigma, St Louis, MO) to inhibit fungal growth, and incubated at a
controlled temperature of 25° for 2 days in the dark.
Leaf, root, and stem samples were rinsed separately with deionized water, and washed
briefly with a 70% ethanol. Samples were then rinsed again with sterile water and
soaked in a 2% hypochlorite solution for 2 minutes in order to sterilize the surface of
the plant tissue. These tissue samples were then rinsed twice with sterile water to
remove traces of the hypochlorite solution from the surface of the plant tissue, and
200μl of the second sterile water rinse was pipetted onto LB medium plates and
incubated at 25° for 2 days in the dark. This plating of plant sample surface rinsed
water served as a control to ensure surface microorganisms were removed from the
leaf, root and stem samples.
21
Mortar and pestles for the grinding of plant tissues were autoclaved at 121° for 3
hours. Once washing and surface sterilization of the plant tissue samples were
completed, the samples were placed into the sterilized mortars along with 3 ml of
sterile 0.75% saline solution. The tissue was then ground vigorously for 2-3 minutes
with the sterile pestles, and the resulting liquid was transferred to a sterile bijoux
bottle. For isolation of endophytic bacteria 200 µl were spread plated onto LB agar.
Each of the resulting colonies from the spread of plant tissue liquids were streaked
onto LB agar medium, and kept at 4° C until future use.
2.2.6 Test of Optimum Bacterial Endophyte Growth Conditions
Two incubation temperatures as well as two different agar media were compared in
order to determine the most optimum growth conditions for recovering the highest
number of endophytic bacterial colonies extracted from within leaf, stem, and root
tissue samples. Ground tissue samples were plated on both LB and TSA media and
incubated in the dark at 25° C to determine which agar medium resulted in the
greatest amount of bacterial growth. Leaf, stem, and root tissue samples from both E.
marginata and B. attenuata were again used for bacterial isolations.
Isolated bacterial endophytes from plant tissue samples were grown on LB agar
medium in the dark at 25° C and 37° C, to determine which temperature resulted in
the larger amount of bacterial growth. Leaf, stem, and root tissue samples from both
E. marginata and B. attenuata were used for endophytic bacteria isolation, and the
25° C and 37° C temperatures were compared over a 3 day incubation period.
22
2.2.7 Morphotype Identification
For comparison of morphotypes, isolates were patch plated onto LB agar plates with
50 μg/ml cyclohexamide and incubated at 25° for 2 days in the dark. Patch plating
involved the transfer of a single colony of each streak plate isolate (as described in
section 2.2.5) from the streak plate onto LB agar medium, using a sterile wire loop.
To detect any influence of agar batch on colony morphology, one isolate was included
in every patch plate as a control.
2.2.8 Data Analysis
Statistical analyses undertaken on the morphological data obtained for this study was
performed using IBM SPSS Statistical software version 22.
Tests performed on the morphotype data included a non-parametric Kruskal-Wallis
test in order to determine whether the distribution of morphotypes differed between
the six study sites. As a Levene’s test for homogeneity of variances was undertaken
on the data, it could not be assumed that morphotype samples within each site had
approximately the same distribution, hence a non-parametric Kruskal-Wallis test was
performed. Additionally, a non-parametric Mann-Whitney test was undertaken on the
morphotype data in order to determine whether the distributions of morphotypes
across healthy and infected sites differed.
23
2.3 Results
Prior to isolation of the various bacterial morphotypes isolated from leaf, root and
stem plant tissue samples, testing of isolation procedures for the bacterial endophytes
was required.
2.3.1 Test of Isolation Procedures
In order to determine whether bacterial endophyte isolations were in fact most likely
those occurring within the plant tissue samples, sterile water used to rinse the surface
of the sterilized plant tissue samples was plated onto LB agar medium. If no bacterial
growth occurred on these sterile rinse water control plates then it was concluded that
bacterial colonies isolated from plating the plant tissue extractions were endophytes
that reside within the plant tissue (Figure 2.8). Therefore, each of the isolates in which
no growth occurred on the control plate, were determined to have originated from
within the leaf, stem, or root plant tissue samples, meaning the sterilization protocol
was adequate.
A
B
Figure 2.8: Growth of leaf isolated bacterial colonies on LB agar medium
supplemented with 50μg/ml cyclohexamide. No growth occurred on the
sterile rinse water control plate (A), and growth of leaf isolate colonies
(B).
24
2.3.2 Comparison of Bacterial Endophyte Isolation Methods
No bacterial endophytes were isolated onto LB agar following isolation by the
electronic pestle method. However, the larger ceramic mortar and pestle method
however resulted in observed endophytic bacterial colony growth on LB agar after
each leaf, stem, or root tissue sample was ground and plated. Thus, it was determined
using sterilized ceramic mortar and pestles by hand were most efficient for the
isolation of bacterial endophytes from plant tissue samples.
2.3.3 Test of Optimum Bacterial Endophyte Growth Conditions
A large number and diversity of bacterial endophytes grew on LB medium, compared
to TSA. Therefore, it was determined that LB medium was to be used for plating out
all endophytes isolated from plant tissue.
Since there were no observed differences in number and diversity of bacterial
endophytes between 25° C and 37° C, it was determined to grow all isolated bacterial
endophytes at 25° C because the optimum growth temperature for P. cinnamomi is
25° C.
2.3.4 Test for Morphotype Patch Plate Consistency
Once bacterial endophytes had been isolated from plant tissue samples and were
streaked out onto LB agar, they were patch plated onto fresh LB agar plates to
compare all morphotypes present between study sites and infested and non-infested
areas. In order to ensure consistency between patch plate medium batches and
morphotypes, one particular morphotype (morphotype 9) was plated onto all of the
patch plates. Results from this test allowed for the conclusion that there were no
25
discrepancies between morphotypes over the various patch plates as this morphotype
did not vary in morphology between batches (Figure 2.9).
Figure 2.9: Morphotype patch plates for extracted plant tissue bacterial
endophytes. Arrows indicate the same morphotype (morphotype 9) plated
twice to ensure consistency between plating.
2.3.5 Bacterial Endophyte Morphotypes Isolated from Each Study Site
A total of 252 isolates were recovered from the tissue samples across all sites, and the
number of isolations from the Murdoch woodland site was higher (n=100) than from
either of the jarrah forest graveyard sites (n=74, n=78 for GS 1 and GS 2 respectively)
(Table 2.1). In comparing the number of isolations from the infested and non-infested
areas, the infested areas of the jarrah forest graveyard sites yielded a greater number
of isolates compared to the non-infested areas. However, the reverse trend was
observed for the Murdoch woodland site.
The isolates were grouped into morphotypes on the basis of colony morphology on
LB agar, and a total of forty eight morphotypes were recorded. A greater number of
morphotypes occurred at the Murdoch woodland site (n=69) compared to the jarrah
forest graveyard sites (n=36, n=32 for GS1 and GS2 respectively) (Table 2.1), and the
26
infested areas of the graveyard sites yielded a greater number of morphotypes (n=25)
compared to the non-infested areas (n=19).
For the Murdoch site, a higher number of isolations were made from root tissue
(n=52) compared to the leaf and stem tissues (n=24, n=24 respectively) (Table 2.1).
This was the case for roots from both the non-infested and infested areas. The number
of isolations did not differ between the leaf and stem tissues from either area of the
Murdoch woodland site. For the jarrah forest graveyard sites, numbers of isolates
from all tissue samples from infested and non-infested areas of both sites were
comparable, with the exception of the root samples from the infested areas from
which a greater number of isolates were recovered.
27
Table 2.1: Number of bacterial isolates and morphotypes for each study site, sampling area, and plant tissue type.
LEAF
Murdoch
GS 1
GS 2
STEM
ROOTS
Number
of
isolations
Number of
morphotypes
Number of
isolations
Number of
morphotypes
Number of
isolations
Number of
morphotypes
Total
Isolations
Total
Morphotypes
Noninfested
Infested
12
7
14
11
28
16
54
34
12
11
10
7
24
17
46
35
Noninfested
Infested
8
3
10
2
15
6
33
11
10
5
10
8
21
12
41
25
Noninfested
Infested
11
3
10
3
12
7
33
13
13
6
12
4
20
9
45
19
TOTAL
68
35
66
35
120
67
252
137
28
2.3.6 Association Between Bacterial Endophyte Morphotypes and Study Sites
Six of the morphotypes (1, 11, 19, 20, 21, 22) were unique to a single site although
they were found in both the infested and non-infested areas of the site (Table 2.2). All
morphotypes were represented by multiple isolations and none of these six were
specific to a tissue type.
Twenty seven morphotypes were unique to an area. They were found at only one site
and only in either the non-infested or infested area of that site. There was no
specificity in regard to the tissue in which these morphotypes were found. For
example, morphotype 5 occurred only in the infested area of the Murdoch woodland
site, although it was recovered from both leaf and stem tissue. However, it must be
noted that the number of isolations of these morphotypes is so low that it’s difficult to
attach any significance to their limited distribution.
A limited number of morphotypes were found at high frequency across the sites. In
particular, morphotype 33 (sixty two isolations) was isolated at high frequency from
all tissue types from jarrah forest graveyard sites 1 and 2. In contrast only two
isolations of morphotype 33 were recovered from the non–infested area of the
Murdoch woodland site. Other morphotypes recovered at high frequency included 2
and 44 (twelve isolations each), and 9 (twenty six isolations). There does not appear
to be any specificity with respect to site, area or tissue type. A possible exception is
morphotype 44, which displayed a slight preference for non-infested areas. For
example, whilst two isolations of morphotype 44 were made from the infested area of
the Murdoch woodland site, ten isolations were made from the non-infested area of
jarrah graveyard site 2 (Table 2.2).
29
Table 2.2: The frequency of occurrence of each morphotype within each site,
sampling area, and plant tissue. (NI= non infested, I= infested; L, R, S= leaf,
root, and stem respectively).
Morphotype
MU NI
L
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
S
1
R
1
4
MU I
L
S
1
1
1
1
1
1
4
1
1
GS1 NI
R
2
3
L
S
R
GS1 I
L
S
GS2 NI
R
L
S
R
2
GS2 I
L
S
R
1
1
1
1
4
1
1
1
5
1
1
1
2
1
1
2
1
2
2
3
3
1
1
1
1
1
1
1
1
2
1
5
2
1
1
3
1
1
4
1
2
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
7
3
2
3
1
1
1
1
1
1
6
8
5
4
1
3
5
6
9
4
1
3
1
2
2
1
1
2
1
1
1
2
1
1
1
2
1
2
1
2
2
2
2
1
1
2
1
2
2
5
2
1
1
30
4
1
1
2.3.6.1 Analyses of Bacterial Endophyte Morphotype Distribution at Different
Sites
It was apparent that different bacterial endophyte morphotypes varied in their
distributions across the three study sites, and within each infested and non-infested
area. Therefore, it was of interest to determine statistically whether there was a
significant difference of morphotype distributions across the sampling sites. As the
Levene’s test had a significant result (P<0.05), equality of variance for the
morphotype samples between each of the study site groups could not be assumed.
Hence a non-parametric Kruskal Wallis test was performed to compare whether the
distributions of morphotypes between the three study sites were different. Results
from this test proved significant (P<0.05), and it could be concluded that the different
Murdoch and jarrah forest graveyard sites had different distributions of bacterial
endophyte morphotypes present.
In addition to determining whether there was a significant difference between
bacterial endophyte morophytpe distributions between sites, it was also of interest to
ascertain whether morphotype distributions differed according to infested and noninfested sampling area.. A non-paramentric Mann Whitney test was performed on the
morphotype data. As stated previously the assumption for equality of variances could
not be met and a non-parametric analysis was performed. Results from this test
proved significant (P<0.05). Therefore it was concluded that infested and non-infested
sampling areas had significantly different bacterial endophyte morphotype
distributions.
31
2.3.7 Association Between Bacterial Endophyte Morphotypes and Plant Host
Species
The Murdoch woodland site contained a greater diversity of plant species and hence a
greater number of species were sampled (Table 2.3), and the jarrah forest graveyard
sites in contrast were predominantly E. marginata and C. calophylla. Of the forty
eight morphotypes identified, twenty five were recovered from a single host species
suggesting a degree of host specialization. However, because the frequencies of
isolation were so low, any such interpretations must be treated with caution.
A number of morphotypes showed no apparent host specificity. These were recovered
from multiple hosts in sufficiently high frequencies to have some confidence in the
results. For example, morphotype 9 was recovered from eleven different host species
across the three sites. Similarly morphotypes 12, 19 and 28 were also recovered from
multiple host species (Table 2.3).
Comparison of a single host species such as E. marginata across the different sites
shows there are differences in the endophyte populations between the different sites
(Table 2.3). Morphotypes 3, 4, 5, 12, 17, and 34 were isolated from E. marginata
trees at the Murdoch woodland site, but not from either of the jarrah forest graveyard
sites. Morphotypes 39, 40, 46, 47, and 48 are unique to E. marginata trees at the
jarrah forest graveyard site 1, whilst morphotypes 41 and 44 were found at both jarrah
graveyard sites but not the Murdoch site.
Similarly in comparing morphotypes from C. calophylla at the jarrah forest graveyard
sites (C. calophylla was not present at the Murdoch site), there were no morphotypes
specific to C. calophylla at either site. The morphotypes that do occur in C.
32
callophylla also occur in E. marginata at both these sites. Some morphotypes, for
example morphotype 45, were found only in C. calophylla from jarrah forest
graveyard site 2 (Table 2.3) however, low frequencies of isolation make interpretation
difficult.
33
Table 2.3: Frequency of isolation of morphotypes from different host plant
species. (GS1 and GS2= jarrah graveyard site 1 and 2 respectively).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Corymbia calophylla
GS2
Eucalyptus marginata
Corymbia calophylla
Eucalyptus marginata
Leucopogon spp.
Anigozanthos spp.
Allocasuarina fraseriana
Adenanthos sericeus
Unknown spp.
GS1
Melaleuca spp.
Macrozamia riedlei
Hibbertia hypericoides
Hakea spp.
Eucalyptus marginata
Banksia menzeisii
Banksia ilicifolia
Banksia attenuata
Allocasuarina humulis
Murdoch
1
5
3
1
2
1
1
1
2
1
1
1
2
4
3
4
1
1
1
2
1
1
6
2
1
2
3
4
1
1
1
1
2
1
1
1
1
1
1
2
1
1
2
1
2
1
1
2
1
2
2
1
1
1
1
1
1
2
1
1
1
2
1
1
4
2
1
1
2
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
2
1
1
1
1
21
6
20
12
1
2
2
2
1
3
1
1
34
37
38
39
40
41
42
43
44
45
46
47
48
1
35
7
2
2
1
1
Corymbia calophylla
1
Eucalyptus marginata
Murdoch
Corymbia calophylla
Eucalyptus marginata
Leucopogon spp.
Anigozanthos spp.
Allocasuarina fraseriana
Adenanthos sericeus
Unknown spp.
Melaleuca spp.
Macrozamia riedlei
Morphotype
Hibbertia hypericoides
Hakea spp.
Eucalyptus marginata
Banksia menzeisii
Banksia ilicifolia
Banksia attenuata
Allocasuarina humulis
Table 2.3: Frequency of isolation of morphotypes from different host plant species.
(GS1 and GS2= jarrah graveyard site 1 and 2 respectively).
GS1
2
1
GS2
1
1
2
1
1
3
4
10
1
1
2.4 Discussion
Two hundred and fifty four isolations were made and these were grouped into forty
eight morphotypes. The number of isolates and morphotypes from roots was higher
than from leaves or stems, and twenty seven morphotypes were specific to an area of
the forty eight identified. Additionally, site had a significant influence on morphotype
diversity within a host species and there were no obvious differences in morphotypes
present between infested and non-infested areas, or between tissues.
2.4.1 Extraction of Bacterial Endophytes
The most common method for isolation of culturable endophytes is by plating
macerates of surface sterilized tissue on agar medium (Lodewyckx et al. 2002,
Andreote et al. 2009). Agar media most commonly used are LB agar and Tryptone
Soy Agar. Different results can be obtained by modifying the medium with the
addition of plant tissue or soil extracts, antioxidative stress compounds such as
pyruvate or catalase, or by changing environmental the conditions (Bishop and Slack
1987). Examples of recalcitrant groups that can only be isolated by using modified
media are Verrucomicrobium and Acidobacteria (Andreote et al. 2009). In this study
we did not use modified media to additional groups of bacteria as the approach we
have used is one that has previously led to isolation of endophytes with biocontrol
capabilities (Barka et al. 2002b, Melnick et al. 2008).
The surface sterilization method can affect the recovery of endophytes, as there is
inevitably some penetration of the sterilizing agents into the tissue with consequent
36
negative effects on internal endophytes (Andreote et al. 2009). Additionally, the
chemistry of the plant tissue samples can influence recovery of endophytes (Arnold
2007). For example, plant tissues contain substances such as phenolics and lignins
that inhibit the outgrowth of organisms within the tissue (Arnold and Herre 2003).
This is frequently encountered with phytopathogens, where a pathogenic species can
be detected in tissues by PCR or ELISA but cannot be grown out of the tissue on an
agar medium (O'Brien et al. 2009).
In some cases extensive washing of the tissue is required to achieve growth of the
pathogen. Inhibition of in vitro growth of endophytes from Theobroma cacao by leaf
extracts has been reported by Arnold et al. (2003a), and their results demonstrates
host chemistry can influence the composition of the endophyte population in the
plant. Old leaves differ from young leaves in that they contain increased
concentrations of anti-pathogen defence compounds such anthocyanins, lignins and
phenolics (Arnold and Herre 2003). Therefore, culturable endophytes isolated from
native plant species in this study will not be representative of all endophytes present
within their host plant tissues.
That the results of agar plating to investigate endophyte diversity do not give a
representative picture of endophyte diversity is reinforced by the results from other
studies that use of culture independent molecular methods. These methods include TRFLP, pyrosequencing, Denaturing Gradient Gel Electrophoresis (DGGE) and
Automated Ribosomal DNA Restriction Analysis (ARDRA) (Andreote et al. 2009).
Examination of bacterial endophytes in grapevine by DGGE and culturing was
undertaken by West et al. (2010), where a number of bacterial genera were detected
37
by DGGE but could not be isolated from the tissue. Similarly, Hou et al. (2010) could
only detect endophytes in Zizyphus jujube by PCR and DGGE but not by culturing,
while Manter et al. (2010) identified five common genera of bacterial endophytes in
potato that could not be cultured. Based on the results of these studies we would fully
expect that the present study did not detect all species of endophytes in the plant
species examined. However we were interested only in the culturable species as our
overall aim is to develop a biocontrol strategy for P. cinnamomi.
In the present study, the number of morphotypes per host species ranged from one in
Allocasuarina humulis, to twenty nine in E. marginata (Table 2.4). By comparison,
Jasim et al. (2013) found a total of just four morphotypes within the rhizome tissue of
Zingiber officinale. In general, the numbers reported by different studies range quite
widely with different values being quoted for the same species by different studies. In
some cases no endophytes could be isolated. For example, Ferreira et al. (2008)
compared endophytes in several species of eucalypt, but did not recover any
endophytes from four particular eucalypt species. Another study showed although
endophytic bacteria were detected in seedlings of Z. jujube using molecular methods,
they could not be cultured from the tissues (Hou et al. 2010).
The current study found different morphotypes could be isolated from the same plant
species at different sites. An explanation to this is that plant genotype can affect
endophyte diversity and a number of studies have shown this. For example, a
comparison of species of endophytic bacteria in four poplar hybrids showed
significant differences in both the number of species and the genera of bacteria
recovered (Ulrich et al. 2008). Host genotype influences in endophyte diversity have
38
also been reported for willow (Cambours et al. 2005) and citrus (Araujo et al. 2002).
The endophytic state is a symbiotic state that is maintained by an interaction of
protein effectors from both the endophyte and the host (Eaton et al. 2011, Koeck et al.
2011, Subramoni et al. 2011). These exist in a delicate balance that can easily be
upset by changes in the biotic or abiotic environment causing a shift to pathogenic
growth. Many species of bacteria that colonise plants such as the plant pathogenic
bacteria Xanthomonas oryzae (Ferluga and Venturi 2009), and Xanthomonas
campestris (Zhang et al. 2007) contain quorum sensing LuxR proteins that respond
only to plant proteins by binding to these plant proteins. In the biocontrol strains
Pseudomonas fluorescens Pf-5 and Pseudomonas fluorescens CHAO, production of
the PsoR protein both positively and negatively regulates expression of many genes
including those for the synthesis of compounds such as 2,4 diacetylphloroglucinol
(DAPG) and pyoluteorin (PLT), that antagonize the growth of plant pathogens and
affect genes for iron acquisition which is important for rhizophere colonisation
competency (Subramoni et al. 2011). The PsoR protein is a member of the LuxR
superfamily and requires interaction with an activating plant molecule (Zhang et al.
2007). Additionally, Subramoni et al. (2011) have speculated that variation in the
production of this activating compound may be the basis of the dependency of
rhizosphere colonisation by P. fluorescens on host genotype. Plant genotypes would
be expected to differ with respect to the effectors they produce and also with the types
and amounts of antimicrobials such as phytoalexins as part of the defence response
(Subramoni et al. 2011).
Just as the ability to establish a mutualistic endophytic interaction depends on the host
genotype, it also depends on the microbial genotype. Isolates of a given species will
39
show variation in the production of various secondary metabolites that promote
growth of the plant and induce the defence responses (Kliebenstein et al. 2005). Thus
Aravind et al. (2009), in screening endophytic bacteria from Theobroma cacao for
use as potential biocontrol agents against P. capsici, found that only three out of
twenty isolates of Pseudomonas were useful. Baldauf et al. (2011) reported
considerable variation in the ability of the fungal endophyte Neotyphodium
coenophialum to protect tall fescue against black cutworm infection. Similarly, a
strong effect of endophyte genotype was reported for control of the fungal pathogen
Rhizoctonia solani and the oomycete Pythium spp. by the bacterial species Bacillus
subtilis, Pseudomonas, and Streptomyces (Broadbent et al. 1971). Thus, in the present
study we observed differences in the ability of different isolates from the same
morphotype to inhibit growth of P. cinnamomi in vitro (Chapter 3). This suggests
genotypic variation between isolates of the same morphotype.
The plants in this study were located at three different sites. We wanted to determine
whether the endophytes we isolated were highly specific for a given host plant
species. Additionally, we wanted to see how much variation in morphotypes occurred
between sites, as previous studies have shown that site can have a significant
influence on endophyte diversity. This is because most of the endophytes in a plant
are acquired by horizontal transmission from adjacent plants, or from the rhizosphere
(Lodewyckx et al. 2002, Izumi et al. 2008). Arnold et al. (2003a) reported that
similarities in the fungal endophyte populations in Theobroma cacao decreased as a
curvilinear function of the distance between the sites. Additionally, they found that
endophyte population within a plant was more similar to other plant species at the
same site than to those of the same species at a distal site (Arnold et al. 2003a).
40
Similar findings were reported for the composition of methylobacterium species in
Arabidopsis thaliana (Knief et al. 2010a). Again these differed from site to site but
the species composition at any one site was more similar to other plant species at that
site than to A. thaliana at other sites (Knief et al. 2010a).
A common route for endophytes to enter the plant is by infection of the roots and
subsequent migration up to the aerial parts (Hardoim et al. 2008). For example, Ji et
al. (2008) studied infection of mulberry by a strain of Bacillus subtilis tagged with the
green fluorescent protein (GFP). The bacterium entered the root system through the
cracks formed at the lateral root junctions and the zone of differentiation and
elongation, and the bacterial cells were able to develop and transfer mainly within the
intercellular spaces of different tissues (Ji et al. 2008). The population of the GFPlabeled inoculant was larger and more stable in leaves than in roots and stems, and the
bacterium could be recovered from the roots stems and leaves 42 days after infection
(Ji et al. 2008).
However not all bacteria that colonise the roots will migrate to the aerial parts of the
plant. A study by Germaine et al. (2004) on colonisation of poplar by endophytic
Pseudomonas strains, observed that only two of three strains infected into the roots
were subsequently recovered from the aerial parts. Concentrations of these strains in
the leaves, stems, and sap were lower (103 -104 CFU/g) than in the roots (105 – 106
CFU/g), and the ability of these strains to colonise the aerial parts was correlated with
the production of cellulose implying an active colonization rather than a passive
infection (Germaine et al. 2004). This is supported by the observation that endophytic
bacteria can infect intact roots to enter the plant and that many endophytic bacterial
41
species produce hydrolytic enzymes such as pectinases and cellulases (Lodewyckx et
al. 2002). A similar concentration gradient from roots to upper plant tissues was
observed for endophyte application in banana (Harish et al. 2009). Additionally, in A.
thaliana, although the endophytic bacterial communities in the leaves and roots share
many species in common, the structures of the communities are different
(Bodenhausen et al. 2013).
Endophytes also colonise the plant from the phylloplane (Ji and Wilson 2003).
Arnold et al. (2003a) estimate that 10,000 fungal propagules are deposited on leaf
surfaces each day, and a number of these consistently enter the leaves and persist as
endophytes within the tissue. Similarly, the phylloplane is rich in bacterial species
that can also enter the plant and establish endophytic relationships (Hallmann et al.
1997). Entry can occur through natural openings such as stomata or lenticels, or can
be actively mediated by the production of hydrolytic enzymes (Lodewyckx et al.
2002). Additionally, endophytes can also be transferred between plants by insects
(Johnson et al. 1993).
2.4.2 Differences of Bacterial Endophytes Between Study Sites
In this study we observed significant differences in endophyte diversity between
infested areas and non-infested areas. Infection of a plant results in changes in the
expression of defence genes, MAPK signaling pathways, and quorum sensing protein
genes and would therefore be expected to influence the balance between microbial
and host effector proteins that maintains the endophytic state (Thatcher et al. 2005).
In consequence, we would expect to see a change in the diversity of endophytes, as
some endophytes would be affected by infection more than others.
42
This is supported by the results of a number of studies on endophyte diversity in
infected plants. A comparison of fungal endophytes in healthy and Huanglongbing
(HBL) affected Citrus limon revealed significant differences in endophyte diversity
(Douanla-Meli et al. 2013). Of the twenty phylotypes detected in healthy plants, only
five were detected in diseased plants (Douanla-Meli et al. 2013). Similar effects were
reported for infection of Pinus nigra with the pathogen Dothistroma septosporum,
which causes red needle blight disease (Kowalski and Drozynska 2011), as healthy
and infected plants showed significant differences in fungal endophyte diversity. In
addition, bacterial endophytes also respond to infection. Fusarium infection of banana
plantlets resulted in significant changes in the diversity of bacterial endophytes (Lian
et al. 2008).
Although a number of trees sampled in the present study were within P. cinnamomi
infested areas, it is not known whether these trees were actually infected and suffering
from dieback disease. Where possible, we did sample from trees that did not look
healthy but these symptoms can be induced by abiotic processes such as drought.
However, they were from sites known to be infested by P. cinnamomi, and in the case
of the jarrah forest graveyard sites, the pathogen has been present for over 50 years.
Consequently, it is very likely that the plants sampled were infected by P. cinnamomi.
Future studies, should also bait the soils for P. cinnamomi whilst collecting
endophytes to confirm the presence of P. cinnamomi. Analysis of plant samples for
the presence of the pathogen could be carried out but this is only of significance if the
result is positive. It is easy to miss the pathogen as infection may result from only a
limited area of the root system (Weste and Marks 1987). In addition, techniques for
43
detection of P. cinnamomi such as baiting and plating frequently have a low success
rate (O'Brien et al. 2009).
Lastly, it was of interest to determine whether these endophytes isolated from infested
and non-infested areas were capable of inhibiting the growth of P. cinnamomi in vitro.
This is discussed in the next chapter.
44
Chapter 3
3.0 Screening of Endophytes for Antibiotic Production
3.1 Introduction
A number of studies have demonstrated that biological control is a viable option for
control of plant diseases. Park et al. (2013) found disease severity was reduced by
65% in chili pepper plants infected with P. capsici, when inoculated with antibiotic
producing B. vallismortis BS07. Additionally, a study by Hakizimana et al. (2011)
found selected bacterial endophytes isolated from Persea americana trees capable of
inhibiting the pathogen P. cinnamomi both within in vitro and in vivo assays, whilst
greenhouse and field studies have found bacterial endophytes capable of inhibiting the
pathogen Verticillium dahliae in cotton plants (Li et al. 2012).
Mechanisms by which endophytes protect plants against pathogens include
competitive inhibition, induction of systemic resistance within the host plant,
activation of host plant genes stimulating plant growth or production of
phytohormones, and production of secondary metabolites that are inhibitory to plant
pathogens (Coombs et al. 2004, Sessitsch et al. 2004, Hardoim et al. 2008). However,
a number of studies have shown the production of antibiotics as a key factor in plant
pathogen suppression (Raaijmakers et al. 2002). This has been shown by inactivation
of antibiotic production by mutation, enhancement of antibiotic production, and the
45
fact that cell free culture supernatants give equivalent levels of control to the intact
organism (Raaijmakers et al. 2002).
The production of antibiotics by endophytes is important in plant protection because
the antibiotics can betransported to areas of the plant where pathogens are present but
endophytes may not colonise (Brader et al. 2014). For example, a study conducted by
Ji and Wilson (2003) showed inoculation of plants with a phenazine producing
Pseudomonas strain on foliar surfaces resulted in the transportation of the phenazine
antibiotic to the rhizosphere. Similarly, production and transportation of antibiotics to
the rhizosphere, such as fengycin and surfactin (McSpadden Gardener 2004, Stein
2005) has been shown by bacterial species such as Bacillus subtilis, that have an
antagonistic effect against soil borne plant pathogens (Cazorla et al. 2007).
This ability of antibiotics to be transported to other areas of the host plant is important
because in developing a biological control strategy for P. cinnamomi, biocontrol
endophytes would be applied to foliar surfaces from which they would colonise the
internal tissues of the plant. However P. cinnamomi is a root infecting pathogen, and
it may be limited to only part of the root, as infection of a small part is sufficient to
trigger disease (Weste 1994). Therefore it is possible that the endophyte may not
colonise the same part of the plant as the pathogen. However, transport of an
endophyte produced antibiotic to the root system would potentially have the effect of
inhibiting the pathogen.
Therefore, we undertook in vitro confrontation assays to identify potential antibiotic
producing bacterial endophytes. Each of the two hundred and fifty two isolates was
46
tested in an in vitro confrontation assay against P. cinnamomi, in order to observe
potential antagonism.
47
3.2 Materials and Methods
3.2.1 Isolate of P. cinnamomi Used
The Phytophthora isolate used for the purpose of this study was P. cinnamomi W15
793, GenBank number JX113308. This isolate was mating type A2, and accessed
through the Murdoch University collection. The isolate was maintained on PDA
plates at 40 C. It was periodically transferred onto fresh PDA plates (Fig 3.1).
Figure 3.3: LB media plate containing Phytophthora cinnamomi 793.
3.2.2 Preparation of Endophyte Isolates Used
Endophytes used in the in vitro confrontation assays were isolated from the tissues of
different native plant species (Chapter 2). Isolates were taken directly from cultures
streaked on LB agar medium, for use in the assays.
48
3.2.2 Comparison of Media for Optimal Pathogen and Bacterial Isolate
Growth
Growth of P. cinnamomi and the endophyte isolates were compared on a variety of
different agar media to determine the most efficient medium to be used for the
confrontation assays against the pathogen. Media used for comparisons included
Cornmeal (Bocobo and Benham 1949), V8 juice (1:5 dilution Campbell’s V8
vegetable juice with 15g DIFCO agar, adjusted to pH 5.5 with NaOH), Czapek Dox
(DIFCO), Potato Dextrose (PDA), half strength Potato Dextrose (Ainsworth 1968),
and LB agar (Sambrook et al. 1989b).
All media were made following standard procedures, with the exception of the V8
juice medium, which was adjusted to a pH of 5.5 using 50% NaOH prior to
autoclaving. Bacterial endophyte morphotypes were patch plated onto each agar
medium, as well as onto LB agar medium in order to ensure consistency between agar
batches and determine whether each agar type had an effect on endophytic bacterial
growth. Additionally, P. cinnamomi colonized PDA plugs (5mm diameter) were
placed mycelial surface down in the centre of each media plate type, including LB
agar medium as a control, to determine the most efficient agar medium for growth of
P. cinnamomi 793.
Patch plates of bacterial endophytes and P. cinnamomi were grown at 25°C for 3
days. Plates were then checked daily for growth and compared to the LB agar control
plates to determine which agar medium was similar in efficiency to support growth of
both the endophytic bacteria, as well as P. cinnamomi 793. Patch plates were
replicated three times for each agar medium.
49
3.2.3 In Vitro Confrontation Assays of P. cinnamomi and Bacterial
Endophyte Isolates
Confrontation assays were undertaken as described by Haesler et al. (2008) on PDA
plates between all two hundred and fifty two endophyte isolates and P. cinnamomi, in
order to determine whether any of the endophytes could inhibit the growth of P.
cinnamomi. Each confrontation assay was replicated three times for each isolate so an
average could be made from resulting inhibition zones between the bacterial isolate
and P. cinnamomi. Additionally, in order to ensure consistency between isolate
appearance and batches of PDA, the same isolate was plated on each of the different
confrontation assay plates. Each confrontation plate was comprised of four different
endophytic bacterial isolates (Figure 3.2).
Figure 3.4: Confrontation plate assay showing four different bacterial
endophyte isolates on the edges of a Potato Dextrose Agar plate, with a
Phytophthora cinnamomi agar plug placed in the centre.
The four isolates were streaked on to each confrontation plate and incubated at 25°C
for 3 days. 5-7 day old culture of PDA plugs containing the hyphae of P. cinnamomi
were then placed in the centre of each confrontation plate, and incubated in the dark at
25°C for 5-7 days (Figure 3.2). After this final incubation period, inhibition zones
50
were then measured for each isolate, and the mean was taken between each of the
three replicates for each isolate to determine the average inhibition zone produced.
3.2.4 Data Analysis
Statistical analyses undertaken on the confrontation assay inhibition zone data
obtained for this study were performed using IBM SPSS Statistical software version
22.
Tests performed on the plate confrontation assay data included a linear regression on
log transformed zone of inhibition and morphotype to determine whether any
predictive relationship was present between bacterial morphotype and inhibition zone.
Additionally, a linear regression on log transformed inhibition zone and sampling area
was undertaken to observe whether a predictive relationship was present between
zone of inhibition and forest sampling areas. Lastly, a multiple linear regression was
performed on log transformed mean inhibition zone versus plant species and whether
each sampling area was infested or not in order to determine if mean inhibition zones
could be predicted by plant species and forest areas sampled within this study. In
order to satisfy assumptions for normality and homoscedasticity, inhibition zone and
mean inhibition zone data for all bacterial isolates were log transformed.
51
3.3 Results
Prior to undertaking in vitro confrontation assays on bacterial endophyte isolates
against P. cinnamomi, a comparison of different media types was performed in order
to determine optimum growth conditions for both the isolates and P. cinnamomi.
3.3.1 Media Comparisons for Optimum Growth of Endophyte Isolates and
P. cinnamomi
Bacterial endophyte isolates and P. cinnamomi 793 were patch plated onto Czapek
Dox, PDA, half strength PDA, V8, and CMA to determine which medium supported
the most efficient growth of both bacterial isolates and the P. cinnamomi. All agar
media comparisons were compared with the LB agar medium, which was used as a
control for observing differences between bacterial endophyte colonies on the
separate media types in comparisons.
CMA and V8 agar media supported less growth of bacterial endophyte colonies, and
P. cinnamomi grew slower on these two media types in comparison with PDA.
Czapek Dox and half strength PDA media supported the growth of more bacterial
endophyte isolates than on CMA and V8 agar media. However, there were still a
small number of bacterial endophyte isolates which did not grow on these two media
types even though they were observed on LB agar.
It was concluded from the comparison of the different media that PDA provided
optimal growth conditions for bacterial isolates and P. cinnamomi. All endopytes
52
grew on PDA, and P. cinnamomi growth covered the PDA plate within 5 days,
compared with the other five agar types where it took longer to grow. Additionally,
endophyte colony appearances did not appear to differ between the LB and PDA agar
media. Thus, PDA was used for the in vitro confrontation assays between isolated
endophytes and P. cinnamomi.
53
3.3.2 In Vitro Confrontation Plate Assays of Endophytic Bacterial Isolates
Against P. cinnamomi
The confrontation plate assays indicated a number of endophyte isolates were capable
of inhibiting the growth of P. cinnamomi on PDA, and some were not (Figure 3.3).
Additionally, endophyte isolates capable of suppressing P. cinnamomi growth on
PDA belonged to a variety of different morphotypes (Figure 3.3). For example, the
‘red’ endophyte on the right (Figure 3.3b) is moderately inhibitory to P. cinnamomi
whilst the almost ‘colourless’ endophyte on the left-hand side (Figure 3.3b) is not
inhibitory and is being ‘overrun’ by P. cinnamomi.
A
B
C
D
Figure 3.3: In vitro confrontation plate assays between different bacterial
endophyte isolates and P. cinnamomi. Different isolates show different
levels of inhibition (A, B, C, D).
54
Mean Inhibition Zones (cm)
0
167BS-1
107BR…
107BL-2
114BR…
107BL-3
108BR…
163BR…
110BR…
133BR…
110BS-1
128BS-2
111BL-1
117BL-1
123BR…
124BR…
132BS-2
132BS-3
133BS-1
133BS-2
141BS-1
157BR…
165BR…
172BL-1
111BR…
118BS-1
129BR…
136BR…
139BL-2
114BR…
116BR…
140BR…
140BS-2
145BR…
159BL-1
163BL-1
116BR…
119BR…
121BR…
121BR…
125BR…
147BR…
149BL-1
160BL-1
163BR…
167BR…
167BR…
168BL-1
172BR…
120BL-1
120BS-1
123BS-1
145BR…
150BL-1
151BR…
122BS-1
122BR…
127BL-2
136BL-1
138BR…
139BL-1
140BL-1
140BR…
145BR…
100BR-1
108BS-1
140BR-1
100BR-2
103BR-1
118BR-1
120BS-2
121BR-2
121BS-1
124BR-1
128BR-2
130BR-2
153BR-1
155BR-1
171BL-1
100BL-1
100BL-2
100BL-3
104BL-1
101BL-1
106BL-1
101BS-1
101BS-2
101BS-3
101BR-1
101BR-2
107BR-2
109BR-1
110BR-1
110BS-2
110BS-3
111BR-1
117BR-1
120BR-1
125BR-1
127BR-1
131BR-2
132BR-1
144BR-2
146BL-3
149BS-1
149BS-2
150BL-2
154BR-1
156BR-2
157BR-2
162BL-1
162BR-1
166BR-2
166BR-3
170BL-1
172BR-1
173BR-1
103BR-2
107BL-1
129BR-1
103BR-3
131BL-3
104BR-1
143BR-1
144BR-3
145BR-4
167BL-1
Mean Inhibition Zones (cm)
3
2.5
A
2
1.5
1
0.5
0
1
13 14 15
2
16
17
3 4
18
5
19
6
7
8
9
20
21
22
55
23
10
2
24
25
26
27
11
12
Isolate numbers within each morphotype group
3
2.5
B
1.5
1
0.5
Isolate numbers within each morphotype group
28
Figure 3.4: Mean inhibition zones (cm) and standard error bars of endophyte isolates against P. cinnamomi on in vitro confrontation
assays. Isolates are within their morphotype groups (shaded green and pink).
175BL-1
175BS-1
176BS-1
177BL-1
177BS-1
130BR-3
131BL-1
131BL-2
131BS-1
144BR-1
156BR-1
161BR-1
164BS-1
168BR-1
168BR-2
170BR-1
131BR-1
140BS-1
132BS-1
135BR-1
151BS-1
134BR-1
141BR-1
141BR-2
142BS-1
146BL-1
147BL-1
148BS-1
154BS-1
155BS-1
172BS-1
173BR-2
179BR-1
143BS-1
143BS-2
157BR-1
158BR-1
162BS-1
165BR-1
171BR-2
147BR-1
165BR-2
171BR-1
174BR-1
177BR-1
166BR-1
148BR-1
150BR-1
178BL-1
178BR-1
178BS-1
179BL-1
179BS-1
180BL-1
180BS-1
181BL-1
181BL-2
181BS-1
181BR-1
146BR-1
146BR-2
149BR-1
149BS-3
Mean Inhibition Zones (cm)
125BS-1
127BL-1
128BL-1
128BL-2
128BS-1
128BR-1
129BL-1
130BR-1
141BL-1
141BR-3
142BR-1
143BR-2
144BL-1
146BL-2
146BS-1
151BL-1
152BL-1
152BR-1
152BS-1
153BL-1
153BS-1
154BL-1
154BR-2
154BS-2
155BS-2
156BR-3
156BS-1
159BL-2
159BR-1
159BS-1
160BL-2
160BR-1
160BS-1
161BL-1
161BS-1
163BL-2
163BR-3
163BS-1
164BL-1
164BR-1
164BS-2
165BS-1
165BS-2
166BL-1
166BR-4
166BS-1
167BL-2
168BL-2
168BS-1
169BL-1
169BS-1
170BR-2
170BS-1
171BL-2
171BS-1
172BS-2
173BL-1
173BR-3
173BS-1
174BL-1
174BL-2
174BR-2
174BS-1
Mean Inhibition Zones (cm)
3
2.5
C
2
1.5
1
0.5
0
29 30 31 32
34
33
3
Isolate numbers within each morphotype group
2.5
2
D
1.5
1
0.5
0
35
36
37
Isolate numbers within each morphotype group
38
39 40
56
41
42
43
44
45 46
47 48
Figure 3.4: (Continued) Mean inhibition zones (cm) and standard error bars of endophyte isolates against P. cinnamomi on in vitro
confrontation assays. Isolates are within their morphotype group (shaded green and pink).
There was variation in mean zones of inhibition between bacterial endophyte isolates
(Figure 3.4). For example, two different isolates (170BR-2 and 171BL-1) resulted in
mean inhibition zones of 2.5cm (±0.08), which was the largest mean inhibition zone
measured (Figure 3.4a and 3.4c). In comparison, a number of isolates resulted in no
inhibition zones (Figure 3.4).
As well as variation between mean inhibition zones for each isolate, variation in the
size of zones within each morphotype was also observed. The size of the zones
produced by different isolates belonging to the same morphotype varied from no
inhibition zone to large inhibition zones (Figure 3.4). For example, morphotype 9
(Figure 3.4a) has four isolates with mean inhibition zones ranging from 0.5 to 1.2cm
and the remaining twenty-five isolates have either less than 0.1cm or no observed
zone of inhibition. Also morphotype 33 (Figure 3.4c and 3.4d) has two isolates
producing mean zones of 2.5cm (±0.08) and 2cm (±0.15) respectively, and the
remaining fifty nine isolates produced mean inhibition zones of 0.8cm or less.
Not all morphotypes had such large variability between all the isolates belonging to
that morphotype. For example, bacterial isolates belonging to morphotype 44
produced inhibition zones between 0 and 0.2cm (Figure 3.4d). Additionally, bacterial
isolates belonging to morphotype 42 were all observed to produce no inhibition zones
against P. cinnamomi, except for one isolate (147BR-1) that had a mean zone of just
0.1cm (±0.03) (Figure 3.4d).
57
Additionally, a number of morphotypes were observed to have more isolates capable
of inhibiting P. cinnamomi on PDA compared to others. For example, morphotypes
19 and 38 each have the same number of isolates (n= 12) (Figure 3.4b and 3.4d).
Morphotype 19 had a total of seven isolates capable of producing inhibition zones
(Figure 3.4b). In comparison, morphotype 38 had four isolates resulting in zones of
inhibition (Figure 3.4d). Similarly, morphotype 20 resulted in three isolates having an
observed zone of inhibition, whilst morphotype 41 had no bacterial isolates that were
inhibitory.
As differences were observed between isolates within the same morphotype and
between morphotypes themselves in regards to mean inhibition zones, it was of
interest to determine statistically whether any predictive relationship between
endophyte morphotype and zones of inhibition for each isolate existed. Therefore, a
linear regression was performed on these two variables. As stated previously, the
assumptions for this test could not be met and thus a log transformation on mean
inhibition zones for each isolate was performed. Results from this analysis were nonsignificant (P>0.05), and it was concluded that morphotype had no predictive
relationship on log transformed mean zone of inhibition for each isolate.
3.3.3 Association Between Mean Inhibition Zones and Sampling Area
Results from in vitro confrontation assays between bacterial endophyte isolates and P.
cinnamomi also included observed differences between mean zones of inhibition and
sampling area. Total mean inhibition zones for all morphotypes were observed to
differ when compared across each of the sampling areas (Table 3.1). For example,
58
large mean inhibition zones were observed from specific morphotypes contained in
one area, and no inhibition zones were produced from the same specific morphotypes
within other areas (Table 3.1).
Morphotype 19 occurred throughout all three study sites and within all of the
sampling areas within those sites. This morphotype gave a mean inhibition zone of
0.99cm (±0.17) within the Murdoch non-infested sampling area, but produced mean
zones between 0 and 0.07cm for all other sampling areas (Table 3.1). Additionally,
morphotype 24 produced mean inhibition zones of 0.3cm (±0.17) and 0.28cm (±0.12)
within the Murdoch infested and non-infested areas respectively (Table 3.1).
However, this morphotype resulted in zones between only 0 and 0.1cm within
infested and non-infested jarrah graveyard areas.
In addition, mean inhibition zones produced by different isolates appeared different
according to the sampling areas they were isolated from. For example, the infested
sampling area within the jarrah graveyard site 2 had the largest mean inhibition zone
of 2.47cm (±0.09), whereas the largest mean inhibition zone observed within the noninfested area of that same site was just 0.13cm (± 0.02). Similarly, the Murdoch
infested and non-infested areas had similar largest mean inhibition zones between
them (1.57cm (±0.03) and 1.53cm (±0.32) respectively) (Table 3.1); however these
were much larger when compared to the jarrah graveyard site 1 infested and noninfested areas (0.53cm (±0.23) and 0.18cm (±0.03), respectively).
59
Morphotypes that inhibited P. cinnamomi varied in number between the sampling
areas. The non-infested area of jarrah graveyard site 2 had two morphotypes
(morphotypes 9 and 12) that did not produce inhibition zones (Table 3.1). The noninfested area of Jarrah graveyard site 1, had five morphotypes (morphotypes 9, 19, 22,
28, 39, 40, 47, and 48) that did not produce any zones of inhibition (Table 3.1).
Many morphotypes are represented by one or two isolates from a single area. In some
cases where a morphotype is represented by multiple isolates from different areas, we
find that isolate(s) from one area (often a single isolate) was highly inhibitory in
contrast to other isolates of the same morphotype from different sites, for example,
morphotypes 2, 19, 20, 33, 35.
Additionally, results from morphotype 22 showed isolates from three of the five areas
in which it was detected were highly inhibitory (Table 3.1). Unfortunately the
morphotype 22 isolates from the GS1 site failed to grow in the confrontation assays.
Also, of the five areas in which morphotype 33 was detected, isolates from two areas
(Murdoch non-infested, GS2 infested) were highly inhibitory in contrast to
morphotype 33 isolates from the other areas (Table 3.1).
60
Table 3.1: Mean inhibition zones (cm ± standard error for each morphotype
against P. cinnamomi within each sampling area (0 indicates no growth, blank
indicates morphotype was not present within that area; NI = non-infested, I =
infested with P. cinnamomi).
Mean Inhibition Zone (±SE) in cm
Morphotype
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
MU-NI
MU-I
1.53 (±0.32)
0.15 (±0.06)
0.33 (±0.31)
0.29 (±0.25)
0.77 (±0.77)
0.63 (±0.58)
0.28 (±0.14)
0.52 (±0.5)
0.13 (±0.03)
0.13 (±0.08)
0.17 (±0.09)
0.03 (±0.03)
0
0
0.73 (±0.73)
0.02 (±0.02)
0
0
0
0
0
0.02 (±0.02)
0
0.47 (±0.09)
0
0.30 (±0.17)
1.57 (±0.03)
0
0.34 (±0.17)
0.17 (±0.07)
0.13 (±0.13)
0.47 (±0.23)
0
0.99 (±0.17)
0.53 (±0.2)
0.90 (±0.11)
0.28 (±0.12)
0
GS1-NI
GS1-I
GS2-NI
0.02 (±0.02)
0
GS2-I
2.47 (±0.09)
0
0.03 (±0.03)
0.03 (±0.02)
0
0
0.17 (±0.03)
0
0
0
0
0
0.03 (±0.03)
0.07 (±0.07)
0.03 (±0.03)
0.67 (±0.03)
0.1 (±0.1)
0.01 (±0.01)
0.05 (±0.01)
0.4 (±0.09)
0.01 (±0.01)
0
0.18 (±0.03)
0.09 (±0.03)
0.03 (±0.02)
0.53 (±0.23)
0.08 (±0.04)
0.03 (±0.03)
0.12 (±0.06)
0
0
0.56 (±0.29)
0.04 (±0.03)
0
0.03 (±0.03)
0
0.02 (±0.02)
0.13 (±0.02)
0
0.08 (±0.05)
0
0
61
0
0
0
3.3.4 Analysis of Mean Inhibition Zone, Sampling Area, and Host Plant
Species
As differences were observed between mean inhibition zones for each morphotype
and the sampling area in which they occurred, it was of interest to determine
statistically whether any predictive relationship was present between morphotype
mean inhibition zones and sampling area. Thus a linear regression was performed on
these two variables. Due to assumptions not having been met for the requirements of
this test, a log transformation was carried out on morphotype mean inhibition zones.
Results from this analysis were non-significant (P>0.05), thus it was concluded
sampling area had no predictive relationship on log transformed mean inhibition zone
for each endophyte morphotype.
Additionally, a linear regression was undertaken on log transformed mean inhibition
zones for each isolate and plant host species, to determine whether any predictive
relationship was present between the two variables. However, results from this
analysis were non-significant (P>0.05), and it was concluded plant host species had
no predictive relationship on log transformed mean inhibition zone for each
endophyte isolate.
62
3.4 Discussion
Of the two hundred and fifty two isolates, one hundred and forty four produced
inhibition zones against the pathogen, and these isolates belonged to twenty nine of
the forty eight morphotypes. Differences in inhibition zone measurements were seen
between and within morphotypes, and it was determined that sampling area, bacterial
morphology, and host plant species had no significant influence on the inhibition
zones produced by the endophytic bacterial isolates.
3.4.1 Diversity of Endophytic Isolates within Morphotypes
Difference in zone widths produced by the endophytes in this study demonstrates that
although certain isolates appear to have the same morphology, they behave differently
in vitro against P. cinnamomi. Therefore, bacterial endophytes isolated from P.
cinnamomi infested and non-infested areas within the Murdoch and jarrah graveyard
sites may represent morphologically similar species, representing strains with
potentially different genotypes. Differences observed in biocontrol and plant growth
promoting activities exhibited by endophytic bacteria are attributed to genotype
(Germaine et al. 2004, Ryan et al. 2008). For example, variation in biocontrol
efficiency was shown by Bardin et al. (2013), for B. subtilis isolates against B.
cinerea.
Further evidence on the importance of endophyte genotype and its role in conferring
resistance to the host plant has been shown by a number of studies linking the
presence of particular genes within the endophyte genotype as a determinant for
63
resistant abilities expressed by the plant host (Barzanti et al. 2007). An example of
this was shown in a study undertaken by Taghavi et al. (2005), where separate groups
of poplar plants were inoculated with two different strains of Burkholderia cepacia.
One of these B. cepacia strains included a gene for toluene resistance, and the other
strain did not. It was found poplar plants inoculated with the strain containing the
toluene resistance gene within its genotype conferred a higher level of resistance to
toluene to its host plant (Taghavi et al. 2005).
The different zone widths produced by the endophyte isolates within the present study
suggest genotypic variability between strains of the same morphotype. Isolates
producing an inhibitory zone within in vitro assays would be expressing genes for
production of secondary metabolites or compounds allowing for their inhibition
against P. cinnamomi, which may not necessarily be expressed within the host plant
(Coombs et al. 2004). This genotypic variation across endophyte species can be
acquired by horizontal transmission of transferrable genetic elements (Ryan et al.
2009), such as plasmids, in bacteria that may be present within the rhizosphere. For
example, Kaneko et al. (2010) found terminases, capsid proteins, and tail proteins to
be present within two independent putative prophages on the Azospirillum sp. B510
chromosome. These genetic traits show evidence of a horizontally transferred element
(Kaneko et al. 2010).
3.4.2 Ability of In Vitro Antagonistic Isolates as Potential Biocontrol
Agents
The in vitro confrontation assays carried out in this study using endophytic bacterial
isolates and P. cinnamomi 793 demonstrated the antagonistic effects of these isolates
64
against the plant pathogen. Such antagonistic effects are due to the production of
antibiotics (Sessitsch et al. 2004, Bouizgarne 2013).
Production of secondary metabolites such as antibiotics by endophytes have been
shown as a factor in biocontrol application in plants susceptible to phytopathogens
(Whipps 2001). For example, a study conducted on endophytic actinobacteria, known
to be prolific producers of secondary metabolites, found 64% of isolates had
antifungal activity against Gaeumannomyces graminis var. tritici (Ggt) within in vitro
confrontation assays (Coombs et al. 2004). Production of phenazine antibiotics by
Pseudomonas aureofaciens has been shown to be an important component in
biocontrol of Ggt by this bacterium (Delaney et al. 2001). More recent studies have
reported that the production of fengycin antibiotics are important for the biocontrol
activity of B. subtilis and Bacillus amyloliquefaciens (Sun et al. 2013, Wang et al.
2013).
However, antibiotics production by endophytes is not the only mechanisms of
protection against disease. (Compant et al. 2005a). For example, disruption of a gene
responsible for pyrrolnitrin production (an antifungal agent)in an isolate of B. cepacia
resulted in only slightly less inhibition against Fusarium moniliforme when compared
with the parent endophyte strain (Mendes et al. 2007). Thus it was concluded that
most antifungal activity against F. moniliforme by this endophytic isolate was related
to other antagonistic mechanisms such as defence induction, degradation of the
pathogen cell wall, inactivation of pathogen toxins, promotion of plant growth
(Mendes et al. 2007).
65
In vitro production of antibiotics by endophytes and their effects against plant
pathogens does not suggest endophytic isolates will produce these inhibitory
metabolites within the plant (Coombs et al. 2004). Additionally, it cannot be assumed
that secondary metabolites produced by endophytes will affect potential
phytopathogens in natural environments, as a variety of other factors need to be
considered (Sessitsch et al. 2004). Such factors include host plant genotype,
environmental conditions, and presence of other microbiota within the internal plant
tissues (Bulgarelli et al. 2013).
To elaborate, the variety of mechanisms utilized by endophytes in aiding the host
plants’ resistance against disease causing agents is more complex than the simple
production of antibiotics or other compounds with the potential to inhibit pathogen
growth (Whipps 2001). For example, methods including competitive inhibition, the
induction of systemic resistance in the plant, stimulation of plant growth, and the
capability of some endophyte strains to activate host genes resulting in phytohormone
production may all have a role in effective biocontrol against plant pathogens
(Compant et al. 2005a, Choudhary and Johri 2009, Monte 2010).
Additionally, these mechanisms are not directly observed by endophytic bacteria
against phytopathogens from only carrying out in vitro assays. For example, the
complex interactions between different endophyte strains, as well as combined effects
of endophyte strains in activating host defense responses occurs within the host plant
(Kobayashi and Palumbo 2000). Thus, isolates which did not produce an inhibition
zone against P. cinnamomi does not necessarily mean they are ineffective potential
biocontrol gents, as their abilities against plant pathogens when inoculated within a
66
plant is not fully understood. Therefore, to identify endophytes that are capable of
protecting plants against pathogens such as P. cinnamomi requires determination of
whether the endophytes are able to inhibit phytopathogen infection when inoculated
within a plant. This is further examined in Chapter 4.
67
Chapter 4
4.0 Screening Endophytes for in planta Inhibition of
Phytophthora cinnamomi
4.1 Introduction
In Chapter 3, a number of the endophytes were shown to inhibit activity against P.
cinnamomi in vitro, an implication that that these endophytes may be useful as
biocontrol agents against P. cinnamomi. However, in vitro inhibition does not always
translate to effective biocontrol in the plant. To be an effective biocontrol agent the
endophyte has to colonise the plant, and also be able to produce the inhibitory
substance within that plant. Neither of these conditions can be assumed and often
endophytes capable of inhibiting pathogen growth in vitro are not always effective at
protecting the plant against colonisation by the pathogen.
In addition, many bacteria protect plants by mechanisms other than antibiotic
production, such as induction of host plant defences, detoxification of fungal toxins,
degradation of fungal cells walls, and promotion of plant growth (Kloepper and Ryu
2006, Ryan et al. 2008). Such organisms may be effective biocontrol agents, although
in the type of confrontation assay used in chapter 2 they would not appear as
antagonistic to P. cinnamomi. Therefore, the only way to identify effective biocontrol
agents is to use an in planta assay.
This chapter describes the screening of the endophytes for in planta biocontrol
activity against P. cinnamomi. The two assays used for this were the under-bark
68
inoculation assay, and the lupin (Lupinus angustifolius) rollmop assay (Stasikowski
2012).
69
4.2 Materials and Methods
Two in planta assays were used to screen the different endophyte morphotypes for
biocontrol activity towards P. cinnamomi were evaluated in this study. These were an
under-bark stem inoculation assay, and a lupin rollmop assay.
4.2.1 Preparation of Morphotype Representatives for in planta Assays
For, this experiment one representative of each morphotype described in Chapter 2
was used. Each randomly chosen representative isolate for each of the forty eight
morphotypes were inoculated separately into 10ml of autoclaved liquid nutrient
medium (DIFCO) in sterile McCartney bottles. These were then incubated for 2 days
in the dark at 25°C.
After growth of these liquid cultures, 1ml of each of the forty eight morphotypes were
centrifuged at 5000rpm for 3 minutes. After removal of the supernatant, the bacterial
cells were re-suspended in 1ml of sterile water, and then further diluted into 9ml of
sterile water to be used in inoculation of the L. angustifolius seedlings for the in vivo
experiment.
4.2.2 P. cinnamomi 793 Growth Conditions
Agar plugs from the stock P. cinnamomi 793 LB plates were transferred onto PDA
medium plates and incubated for 5 days in the dark at 25°C. These P. cinnamomi
plates were the source of inoculum for the under-bark stem inoculation assay, and the
lupin rollmop assay.
70
4.2.3 Underbark Stem Inoculation Method
Approximately 1-2cm diameter green stems were cut from E. marginata trees within
the jarrah graveyard sites to be used for the under-bark stem inoculation assay trials.
All stems were cut to approximately the same length, and each end was sealed with
candle wax to prevent loss of moisture from the tissue. Stems were inoculated with a
streaked colony of the endophyte morphotype and a P. cinnamomi agar plug (5mm
diameter) mycelial surface down, approximately 3cm apart, and this was wrapped
with Parafilm to prevent contamination. One stem for each treatment was placed into
trays (30x50cm) and incubated in the dark for 5 days at 25°C within sealed containers
to maintain humidity and prevent desiccations. Stems were checked daily for the
appearance of lesions. Each stem was replicated eight times. The controls consisted of
stems inoculated with P. cinnamomi only.
4.2.4 The Lupin Rollmop Assay.
Seeds of L. angustifolius, supplied by Murdoch University, were germinated by
incubating in the dark for 2 days at 25°C on damp paper towel and deionized water
within containers. Germinated seeds with visible shoot growth (Figure 4.1) were
selected for use in the rollmop assays.
Figure 4.1: Germinated L. angustifolius seeds after 2 days incubation.
71
Rollmop construction for in vivo assays between morphotypes and P. cinnamomi
were constructed as described by Stasikowski (2012). Damp bench coat, two paper
towels, and three pieces of large filter paper were placed within plastic sheets,
resulting in one rollmop. Ten germinated L. angustifolius seeds were placed on each
of the rollmops for the in vivo assays (Figure 4.2). These are considered not to be true
replicates but to be pseudo-replicates. Each rollmop was then replicated three times.
Rollmops were incubated at 25°C for a total of 7 days, and were placed under lights
(150μE) on a 16 hour day cycle. The rollmops were also placed randomly on shelving
under the lights, in order to minimise any potential positional bias.
Figure 4.2: Placement of L. angustifolius seeds on constructed rollmop.
4.2.4.3 Endophyte Effects on Seedling Growth
After two days incubation under lights, 150μL of morphotype culture was inoculated
onto the leaves of each of the ten pseudo-replicates L. angustifolius seedlings within
each rollmop, and each rollmop was replicated three times. Controls consisted of noninoculated seedlings. Seedlings inoculated with morphotype 2 were included in every
group in order to check for variation between batches of rollmops. The inoculated
seedlings were incubated for a further six days. Each of the seedlings were then
placed in a drying cabinet for 2 days at 60°C, and dry weights were taken for the
72
whole seedling. The dry weights of all morphotype inoculated seedlings were
compared with the two controls.
4.2.4.4 Endophyte Effects on Lesion Lengths Caused by P. cinnamomi Infection
Rollmops were constructed, incubated and inoculated with morphotypes as described
in section 4.2.4. Three days after inoculation with the endophyte morphotype, each
seedling was inoculated with P. cinnamomi by placing an agar plug (prepared as
described in section 4.2.2) on the roots on the seedlings (Figure 4.3). The rollmops
were then incubated for a further four days. These rollmops were checked daily for
lesions on the roots and at the end of the incubation period lesions were measured.
Controls consisted of non-inoculated and morphotype 2 inoculated rollmop seedlings,
to check consistency between rollmop groups tested.
Figure 4.3: Inoculation of L. angustifolius seedling roots contained in
rollmops with agar plugs of P. cinnamomi 793.
73
4.2.5 Data Analysis
Statistical analyses undertaken on the data obtained for lesion lengths and dry weight
resulting from the rollmop in vivo assays. All analyses were performed using IBM
SPSS Statistical software version 22.
Data obtained for mean lesion lengths and mean dry weights, for all of the
morphotypes as well as the morphotype 2 replicated control were log transformed.
This transformation was necessary in order to satisfy the assumption of
homoscedasticity for all analyses performed. Additionally, it must be noted that all
outliers of dry weights resulting in zero (from seedlings which failed to grow within
the rollmops), were removed in order to eliminate potential bias of results from the
analyses undertaken.
Initial analyses undertaken were ANOVA tests on data from each of the two controls
for both lesion lengths and dry weights, in order to determine there were no
significant differences between groups of rollmops tested. Additionally, ANOVA
tests were performed on all log transformed mean lesion length and dry weight data
obtained for all of the morphotypes tested, in order to determine whether any
significant differences were present between log transformed mean lesion lengths and
dry weights for the controls and each of the morphotypes.
Also, separate multiple linear regression analyses were undertaken on log transformed
mean lesion lengths and dry weights against sampling area health, plant species, and
interactions between the two latter variables, to determine whether any predictive
relationships were present and whether an interaction existed between sampling area
74
health and plant species. Lastly, a linear regression was also performed between log
transformed mean lesion lengths and dry weights, in order to observe whether and
significant predictive relationship was present between the two variables of interest.
4.2.6 Identification of Significant Morphotypes
The six morphotypes found to significantly reduce lesion length were identified using
DNA sequencing. A BLAST search was then performed on sequences obtained for
each of the morphotypes to identify closest matching genera.
75
4.3 Results
4.3.1 Preliminary trial of the Underbark Stem Inoculation Assay for in planta
inhibition of P. cinnamomi.
Smaller lesion lengths were observed for stems inoculated with bacterial isolates
derived from Adenanthos sericeus plant tissue, representing morphotypes 1, 28, 22,
and 36, and P. cinnamomi, compared with the control of stems inoculated with P.
cinnamomi only (Figure 4.4). Lesion lengths in endophyte inoculated stems were
different for some isolates when compared to the control, and the length of a number
of lesions appeared to stop in the presence of the inoculated bacterial isolate (Figure
4.4c).
A
B
C
Figure 4.4: Under bark stem inoculation assays conducted on excised stems of E.
marginata inoculated with P. cinnamomi only as the control (A), P. cinnamomi
challenged with isolate 140BS-1 (B), and P. cinnamomi challenged with isolate
140BL-2 (C).
In stems inoculated with P. cinnamomi alone the mean lesion length was 4.52 cm
(Table 4.1). Of the six morphotypes tested for their effect on P. cinnamomi in planta,
five stimulated the pathogen as they caused an increase in mean lesion length
compared to P. cinnamomi alone. The one exception was isolate 140BL-2 with a
mean lesion length of 4.2 cm. In some cases, for example 140BL-1 and 140BS-1, the
lesion lengths were almost double that observed with P. cinnamomi alone. None of
76
the morphotypes could be said to have inhibited lesion development by P.cinnamomi
in this assay.
Table 4.1: Mean lesion length (cm ± standard error) of under bark stem
inoculation assays between selected endophyte isolates and P. cinnamomi 793.
Mean Lesion Length1 cm (±SE)
7.43 (±1.16)
4.29 (±0.79)
5.60 (±1.15)
5.06 (±1.02)
7.86 (±1.00)
6.54 (±1.00)
4.52 (±0.94)
Isolate Number
140BL-1
140BL-2
140BR-1
140BR-2
140BS-1
140BS-2
Control (P. cinnamomi alone)
L, R, and S indicate leaf, root, and stem plant tissue from which the isolate was derived.
1= Mean of 10 replicates
This preliminary trial showed that the under-bark stem inoculation assay is a
cumbersome assay to use, especially where large numbers of morphotypes have to be
tested. A major drawback is the lack of uniformity of the stems, as getting sufficient
numbers of stems of the same stage of growth and thickness is very difficult. This
introduces considerable variability into the assay. It was therefore determined that the
lupin rollmop assay would be a more appropriate assay to use to screen the
morphotypes for inhibition of P. cinnamomi, as the assay would not suffer from the
variability of the under-bark stem inoculation assay.
4.3.2 The Lupin Rollmop Assay for Effect on Dry Weight by Morphotypes
Dry weights were measured for each of the seedlings within the morphotype
inoculated and non-inoculated rollmops (Figure 4.5). Means of these pseudo-replicate
seedlings were taken.
77
0.14
0.1
0.08
0.06
0.04
0.02
0
Control
Mean Dry Weight (grams)
0.12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Morphotype
Figure 4.5: Mean dry weight and standard error of L. angustifolius seedlings after inoculation with endophyte morphotypes.
78
Mean dry weights for seedlings within three of the rollmops representing
morphotypes 1, 7, and 18 were 0.092, 0.101, and 0.107 grams respectively (Figure
4.5). These mean dry weights were larger than the control, which had a mean dry
weight of 0.073 grams. However, error bars calculated for the mean dry weight data
show an overlap between these three morphotypes and the control, therefore these
results should be viewed with caution. Similarly, morphotypes 5, 16, 32, and 43 had
mean dry weights of 0.061, 0.06, 0.062, and 0.061 grams respectively, which were
smaller again in comparison with the control (Figure 4.5).
In order to determine whether there was a statistically significant relationship between
morphotype and mean dry weight of L. angustifolius seedlings, ANOVA analyses
were undertaken across morphotype and control groups. The initial ANOVA tests
performed on mean dry weight data for the non-inoculated control seedlings, and the
morphotype 2 control seedlings were done in order to determine there was no
significant difference across control groups. Homogeneity of variances was assumed
for both ANOVA analyses (P>0.01), and results from these tests allowed for the
conclusion that no significant difference was present across either of the control
groups (P<0.05). That is, mean dry weight did not significantly differ across the noninoculated and morphotype 2 control groups, and results from further analyses
undertaken on the mean dry weight data were reported with confidence.
To determine whether significant differences in mean dry weight were present across
each of the endophyte morphotypes, an ANOVA analysis was performed.
Homogeneity of variances was assumed after the mean dry weight data was log
transformed (P>0.01). Results from this ANOVA was significant (P<0.05), and it
79
could be concluded that log transformed mean dry weight differed across the
categories of morphotypes. Tukey HSD post hoc tests were then undertaken on the
log transformed mean dry weight data in order to determine which of the morphotype
categories were significantly different from the non-inoculated control group. These
post hoc tests resulted in the conclusions that log transformed mean dry weight for
morphotypes 5, 12, 16, 28, 35, and 38 were significantly different from the noninoculated control.
In order to investigate these results further, T-tests were performed on log
transformed mean dry weight for all six significant morphotypes. Results from these
one-tailed analyses proved non-significant (P>0.05 across all categories). That is,
morphotypes 5, 12, 16, 28, 35, and 38 were significantly different from the noninoculated control, but the T-tests show that the log transformed mean dry weights of
these morphotypes were not significantly greater than this control, they were actually
less than the control. Therefore, results from all the above analyses indicate that the
morphotypes did not result in an increased growth and larger mean dry weight of L.
angustifolius seedlings.
4.3.3 Relationship Between Mean Dry Weight of L. angustifolius Seedlings,
Sampling Area, and Number of Plant Species Each Morphotype is Present
Within
It was also of interest to determine if mean dry weight differed in regards to whether a
sampling area was infested or not, and whether the number of plant species each
morphotype was identified within had any relationship with mean dry weight
recorded for L. angustifolius seedlings within the in vivo assays.
80
Mean Dry Weight (grams)
30
25
20
15
10
5
0
1
Infested
2
Non-Infested
3
Both
Morphotypes present in each sampling area type
Figure 4.6: Mean dry weight and standard error of L. angustifolius
seedlings inoculated with morphotypes present across infested, noninfested, and both sampling areas.
Mean dry weight appeared similar for morphotypes present within infested, noninfested, and both sampling areas. Morphotypes present within infested, non-infested,
and both sampling areas had a mean dry weight of 0.073, 0.074, and 0.073 grams
respectively (Figure 4.6). Again it was of interest to determine whether each of the
three sampling area categories, and the number of plant species each morphotype was
present within had any predictive relationship on mean dry weight of L. angustifolius
seedlings.
In order to satisfy the assumption of homoscedasticity as before, log transformed
mean dry weight data were analysed using a multiple linear regression to determine
whether there was a relationship between sampling areas, number of plant species for
each morphotype, and an interaction between both independent variables on log
transformed mean dry weight data. Results from this analysis proved non-significant
81
across all categories (P>0.05), allowing for the conclusion that sampling area, and
numbers of plant species represented by each morphotype did not have a predictive
relationship on log transformed mean dry weight of L. angustifolius rollmop
seedlings.
4.3.4 The Lupin Rollmop Assay for Morphotype Effect on Lesion Lengths by P.
cinnamomi
Mean lesion lengths for a number of endophyte inoculated seedlings appeared smaller
than the control (morphotype 2) inoculated seedlings (Figure 4.7).
A
B
Figure 4.7: Lesions produced by P. cinnamomi in L. angustifolius
seedlings. Lesions were larger for morphotype 2 (A) compared with
morphotype 40 (B).
82
35
25
20
15
10
5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Control
Mean Lesion Length (mm)
30
Morphotype
Figure 4.8: Mean lesion length of L. angustifolius rollmop seedlings caused by P. cinnamomi 793 after inoculation with endophyte
morphotypes.
83
Morphotypes 14, 34, 35, 41, and 47 had mean lesion lengths of 15.9, 13.9, 14.7, 15.5,
and 15.5mm, respectively (Figure 4.8). These lengths were smaller in comparison to
the control, which had a mean lesion length of 23.3mm. Additionally, error bars of
these five mean lesion lengths for these morphotypes do not appear to overlap with
the control, whereas most of the other mean lesion lengths for the remaining
morphotypes do (Figure 4.8). The largest mean lesion length observed was 29.3mm,
which was greater than the control and represented morphotype 10.
In order to determine whether there was a significant relationship between mean
lesion length of L. angustifolius seedlings caused by P. cinnamomi, and the
morphotype they were inoculated with, analyses were performed on morphotype and
control groups. Initial ANOVA tests were undertaken for the control seedlings that
were inoculated with P. cinnamomi only, and the morphotype 2 inoculated control
seedlings. This was done in order to determine there were no significant differences
across control groups. Homogeneity of variances was assumed for both ANOVA
analyses (P>0.01). Results from the ANOVA analysis on the mean lesion lengths of
seedlings inoculated with P. cinnamomi only allowed for the conclusion that there
was no significant difference across this control group.
However, results from the ANOVA analysis on the morphotype 2 control group
determined there was a significant difference between mean lesion lengths of this
control group. A Tukey HSD post hoc analysis further confirmed that the fourth
replicate of the morphotype 2 control group was significantly different from the other
four morphotype 2 control replicate groups. Therefore, further results of analyses
performed on mean lesion lengths across each of the morphotype categories should be
84
viewed with caution, as one of the morphotype 2 control group replicates was
significantly different from the others. Due to time constraints however, a repeat of
the significantly different batch could not be repeated.
To determine whether significant differences in mean lesion length were present
across each of the morphotypes, an ANOVA analysis was performed. Mean lesion
length data was log transformed in order to assume homogeneity of variances
required for this analysis (P>0.01). Results from this ANOVA was significant
(P<0.05), and it could be concluded that log transformed mean lesion length differed
across the categories of morphotypes. Tukey HSD post hoc tests were then
undertaken on the log transformed mean lesion length data in order to determine
which of the morphotype categories were significantly different from the control
group of seedlings inoculated with P. cinnamomi only. These post hoc tests resulted
in the conclusions that log transformed mean lesion length for morphotypes 14, 34,
35, 40, 41, and 47 were significantly different from the P. cinnamomi inoculated only
control.
However, as detailed previously, the morphotype 2 control replicate 4 group was
significantly different from the other replicate control groups. As morphotype 40
occurred within the replicate 4 group, it should be noted that the significant result of
this morphotype be viewed with caution. It cannot therefore be stated whether the
significantly different result of the morphotype 2 control group was a result of bias,
thus morphotype 40 cannot confidently be concluded that it is significantly different
to the control group of seedlings inoculated with P. cinnamomi only.
85
4.3.5 Relationship Between Mean Lesion Length of L. angustifolius Seedlings
Caused by P. cinnamomi, Sampling Area, and Number of Plant Species Each
Morphotype is Present Within
It was also of additional interest to determine if mean lesion length recorded differed
in regards to whether a sampling area was infested or not, and whether the number of
plant species each morphotype was identified within had any relationship with mean
dry weight recorded for L. angustifolius seedlings within the in vivo assays.
Mean Lesion Length (mm)
30
25
20
15
10
5
0
1
Infested
2
Non-Infested
3Both
Morphotypes present in each sampling area type
Figure 4.9: Mean lesion length and standard error of L. angustifolius
seedlings inoculated with P. cinnamomi 793 and morphotypes present
across infested, non-infested, and both sampling areas.
Mean lesion length appeared similar again for morphotypes present within infested,
non-infested, and both sampling areas, with mean lesion length of 21.9, 20.9, and
22.0mm, respectively (Figure 4.9). Additionally, it was of interest to determine
whether each of the three sampling area categories, and the number of plant species
each morphotype was present within had any predictive relationship on mean lesion
length of L. angustifolius seedlings.
86
Mean lesion length data were log transformed in order to satisfy the assumption of
homoscedasticity, and analysed using a multiple linear regression to determine
whether there was a relationship between sampling areas, number of plant species for
each morphotype, and an interaction between both independent variables on log
transformed mean lesion length data. Results from this analysis proved nonsignificant across all categories (P>0.05), allowing for the conclusion that sampling
area, and numbers of plant species represented by each morphotype did not have a
predictive relationship on log transformed mean lesion length of L. angustifolius
seedlings.
Lastly, it also must be noted that most of the forty eight different morphotypes
identified within this study were present within more than one plant tissue type.
Therefore, as each morphotype was represented by just one isolate for the purpose of
testing within in vivo assays, differences between plant tissue type could not be
determined in regards to mean dry weight and lesion lengths in L. angustifolius
seedlings, as only forty eight of the two hundred and fifty two isolates were tested for
the in vivo assays.
4.3.6 Relationship Between Mean Dry Weight and Mean Lesion Length of L.
angustifolius Seedlings
In addition to determining whether morphotype, sampling area morphotypes were
present in, and number of plant species each morphotype was present within, had a
significant relationship on either mean dry weight or mean lesion lengths in L.
angustifolius seedlings, it was also of interest to determine whether any predictive
relationship was present between mean dry weight and mean lesion length data.
87
A linear regression was performed on log transformed mean dry weight and log
transformed mean lesion length to determine whether any predictive relationship was
present between the two variables. Results from this analysis were significant
(P<0.05), and it was concluded that a predictive relationship was present between log
transformed mean dry weight and lesion length.
4.3.7 Identification of the Six Endophyte Morphotypes Shown to Significantly
Reduce Lesion Lengths in L. angustifolius Seedlings Caused by P. cinnamomi
Differences in morphology can be seen between each of the six endophyte
morphotypes found to significantly reduce lesion length in P. cinnamomi infected L.
angustifolius seedlings (P<0.05) (Figure 4.10).
34
14
41
47
35
40
Figure 4.10: Differences in endophyte morphology of the six significant
morphotypes found to reduce lesion length caused by P. cinnamomi in L.
angustifolius seedlings.
The six morphotypes were analysed using DNA sequencing, however identification
could not be determined for morphotype 47, most likely due to a mixed culture.
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Results from DNA sequencing show that five of the six morphotypes belong to the
genera Chrysobacterium, Lysinibacillus, Pseudomonas, and Bacillus (Table 4.2).
Table 4.2: DNA sequencing results of morphtoypes found to significantly reduce
lesion length caused by P. cinnamomi.
Morphotype
Sequence Length
Identical Sites %
14
887
100
Morhotype
Identity
Chrysobacterium
34
759
90
Lysinibacillus
35
715
99.7
Pseudomonas
40
773
97.2
Bacillus
41
882
100
Bacillus
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4.4 Discussion
The in vivo assays found six significant morphotypes out of the total forty eight tested
significantly reduced P. cinnamomi induced lesion lengths in L. angustifolius
seedlings, and that no predictive relationship was present between whether a sampling
area was infested, number of plant species each morphotype was present within, and
mean dry weight and mean lesion length. However, a significant predictive
relationship was present between mean lesion length and mean dry weight of
morphotype inoculated L. angustifolius rollmop seedlings, indicating that
morphotypes that influence the growth of seedlings also have an influence on
resulting lesion lengths caused by P. cinnamomi.
One of the major differences between the under-bark inoculation and lupin rollmop
assays was that in the former, both the morphotype and P. cinnamomi were inoculated
simultaneously, whereas seedlings in the latter were inoculated with the pathogen
three days after being colonized with the morphotype. Studies have shown that
simultaneous inoculation masks biocontrol activity (Wilhelm et al. 1998, Ran et al.
2005). This may be related to the fact that in stems inoculated with the morphotypes,
a second cut was made in the stem in order to inoculate the morphotype. In contrast
stems inoculated with P. cinnamomi alone did not receive this second cut. It may be
that this second cut somehow resulted in the enhanced lesion development.
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4.4.1 Ability of Endophyte Morphotypes to Enhance Growth and Reduce Lesion
Lengths in L. angustifolius Seedlings
A number of mechanisms exhibited by endophytic bacteria are known to enhance
growth in plants (Hardoim et al. 2008, Forchetti et al. 2010, Glick 2012). These
methods involve affecting plant metabolism and increasing the plants’ tolerance to
stress (Compant et al. 2005a). Promotion of plant growth by affecting plant
metabolism allows for substances such as plant hormones and solubilized compounds
such as iron and phosphorus to be readily available to the host plant, thus increasing
the plants’ tolerance to stress and resulting in enhanced plant growth (Bashan Y et al.
2008). For example, a study undertaken by Esitken et al. (2006) on sweet cherry trees
inoculated with Pseudomonas and Bacillus strains, found the resulting increase in
phosphorus, nitrogen, and potassium within the plant tissue from the bacterial
inoculation to be correlated with increased growth and yield of the plant. Similarly,
phytohormones such as auxins, cytokinins, and gibberellins produced by endophytes
can stimulate growth in plant tissues such as roots in Arabidopsis plants (Tanimoto
2005, Weyens et al. 2009).
Additionally, significant results of a predictive relationship between mean lesion
length and dry weight of L. angustifolius seedlings is not unexpected, as the beneficial
mechanisms employed by endophytes can have positive effects in both plant growth
promotion and biocontrol against plant pathogens. In some cases, the effect of
enhancing plant physiology caused by endophytes, strengthens the plants defence
against pathogens (Compant et al. 2005a). For example, a study undertaken by
(Compant et al. 2005b), found Burkholderia phytofirmans PsJN to strengthen cell
walls in the exodermis of Vitis vinifera plants, leading to the induction of host defense
responses against plant pathogens.
91
Although this study undertaken on endophytes isolated from P. cinnamomi infested
and non-infested areas found isolated morphotypes to have no significant influence on
seedling mean dry weight, total lengths of L. angustifolius seedlings were not
measured. To elaborate, endophyte morphotype inoculation of plants cannot
confidently be concluded that they had no influence on seedling growth, as only one
factor of plant growth, the mean dry weight and not seedling length, was recorded.
For example, bacterial endophytes are shown to be associated with faster root
development in plants. This can be seen in a study conducted by (Taghavi et al.
2009), who found root development in poplar plants inoculated with Enterobacter
species occurred at a much faster rate over a period of two weeks, in comparison with
non-inoculated poplar plants. Therefore, as the present study recorded the mean dry
weight of endophyte inoculated L. angustifolius seedlings only seven days post
inoculation, effects on the overall weight of the seedlings from varying plant growth
promotion mechanisms may not have yet established due to the shorter period of time.
Similarly, varying mechanisms exhibited by endophytes have an effect on plant
pathogen inhibition (Enya et al. 2007, Franco et al. 2007b). Antibiotic production,
production of toxic molecules such as hydrogen cyanide, hydrolytic enzymes such as
extracellular chitinase and laminarinase, and competition for nutrients and
colonization sites have all been found as biocontrol mechanisms utilized by
endophytes (Bashan and de Bashan 2005). For example, Pseudomonas species have
been shown to produce hydrolytic enzymes such as chitinase and lamarinase within
carnation plants, which lyse cells of pathogenic Fusarium oxysporum f. sp. dianthi
(Ajit et al. 2006). However, productions of enzymes have only been assumed as
92
playing a role in biocontrol, confidence in proof is yet to be determined. Additionally,
rhizospheric P. putida has been shown to control F. oxysporum in tomato plants by
preventing the pathogen from sequestering iron and inhibiting its proliferation via
production of siderophores (Bashan and de Bashan 2005), and P. syringae are known
to competitively inhibit colonization of Botrytis cinerea in post harvest apples (Zhou
et al. 2001).
However, biocontrol techniques employed by the endophyte morphotypes within this
study were not known, as further molecular testing was not undertaken. Additionally,
only one representative isolate was chosen for each morphotype for testing within the
in vivo assays. Therefore, the varying abilities of each of the endophyte isolates
belonging to each of the forty eight different morphotypes is not known, as genetic
differences resulting in different biocontrol abilities may be present within the
different isolate strains.
4.4.2 Morphotypes Tested in In Vivo Assays
As only one isolate for each morphotype was tested within the in vivo assays, effects
against P. cinnamomi is not known for every endophyte isolated. Endophyte isolates
belonging to the same species have been shown to exhibit different molecular
techniques and abilities in biocontrol against plant pathogens. For example, a study
undertaken by de Boer et al. (2003) found both P. putida strains WCS358 and RE8
have been shown to suppress the pathogenic effects of F. oxysporum in radish plants.
However, when the two P. putida strains were mixed and inoculated into radish
plants, disease suppression of F. oxysporum was enhanced by 50%, in comparison
with 30% suppression in single strain treatments (de Boer et al. 2003). The combined
93
effect of these two bacterial strains suggest different biocontrol mechanisms are being
expressed by each of the strains, thus enhancing the overall disease suppression in
radish plants.
Different genes present within different endophyte strains can also influence the
growth promotion and biocontrol effects within a plant (Hol et al. 2013).
Additionally, interactions between separate endophyte species can lead to different
genes being expressed by the bacteria (Combes-Meynet et al. 2011). For example,
Garbeva et al. (2011) found expression of genes responsible for secondary metabolite
production in P. fluorescens Pf0-1 was affected by the presence of Bacillus,
Brevundimonas and Pedobacter species within confrontation assays. These changes
in gene expression from the presence of other endophytes and pathogens can result in
changes in the host plant defense responses (Hol et al. 2013).
Similarly, it has been shown that genes involved in biocontrol traits within ten
different P. fluorescens strains vary (Loper et al. 2012). An example on the effects in
different gene expression between P. fluorescens strains is shown in a study by Agusti
et al. (2011), where two P. fluorescens strains with different secondary metabolite
productions were found to have an enhanced control against P. cactorum in
strawberry plants when inoculated together. Thus the importance of genetic
differences within separate endophyte strains can have an enhanced effect on plant
pathogen resistance by the host plant.
To reiterate, the unknown antagonistic abilities of all endophyte isolates within this
study cannot be known, as all isolates were not tested within in vivo assays against P.
94
cinnamomi. Therefore, although six of the morphotypes were found to significantly
reduce lesion length in L. angustifolius seedlings, differences in genetic traits and
hence antagonistic abilities against P. cinnamomi for all two hundred and fifty two
isolates cannot be concluded.
95
Chapter 5
5.0 Endophytes and their Future Prospects as Biocontrol
Agents
5.1 Discussion
This study aimed to develop a biocontrol strategy for use in native ecosystems to
control dieback diseases of plant and tree species. Investigation of endophytic bacteria
from native plant and tree species in jarrah forest areas was undertaken, and further
analysis of isolates resulted in identification of six endophytes, that were capable of
significantly reducing length of lesions caused by P. cinnamomi in roots of L.
angustifolius seedlings. All endophyte isolates were tested for antibiotic production in
an in vitro plate confrontation assays against P. cinnamomi, and only two of the six
morphotypes reducing lesion lengths also had the ability to inhibit P. cinnamomi in
vitro. The strategy of isolation and analysis of endophytes utilized in this study is one
commonly used to identify biocontrol agents against plant diseases (Long et al. 2008,
El-Tarabily et al. 2009, Melnick et al. 2011).
Plant host species and environmental factors differ throughout separate native
ecosystems. In order for a biocontrol agent to be effective, it must not have a strict
host or environment specificity. As vegetation within an area can repeatedly undergo
cycles of infection with P. cinnamomi, endophytes must also remain viable within
potentially infected plant hosts. Results from this study indicate many of the
96
endophyte isolates fulfill these criteria, as there was no significant difference in
endophyte diversity between infested and non-infested areas, and no significant
association between endophyte and host plant species. These results are consistent
with those reported in the literature, showing endophytes within different host plant
species can vary (Arnold et al. 2003b, Knief et al. 2010b).
Endophytes of a host plant can be acquired through inheritance from the parents, or
transferred via horizontal transmission from neighbouring plants (Rodriguez et al.
2009). Horizontal transmission results in a more diverse endophyte community within
older plants (Arnold et al. 2003b), and is important from the view of biocontrol as it
means potential biocontrol endophytes can be transmitted between and become
established within a variety of plants. This biocontrol strategy would therefore be self
perpetuating, and the length of time a biocontrol agent remains viable within host
plant tissues will determine how long the biocontrol effect would last. Few studies
have been undertaken on how long endophytes persist in host tissues. One however
includes a biocontrol bacterium being transmitted and conferring resistance for at
least two generations within grapevine (Barka et al. 2002b). Another study showed
the biocontrol agent Phlebiopsis gigantean as persisting within spruce stumps for six
years post inoculation (Vainio et al. 2001).
Due to diversity of plant species and varying environmental conditions, use of
mixtures of organisms for biocontrol have been considered. For example, mixtures of
bacteria were more effective than single isolates for P. capsici infection resistance in
pepper plants (Kim et al. 2008). Combinations of Trichoderma species were found to
be more effective than single isolates against F. oxysporum (Akrami et al. 2011).
97
Additionally, other studies have shown mixtures of bacteria and/or fungi have
resulted in improved disease control of varying plant pathogens (Szczech and Shoda
2004, Liu et al. 2011, Stockwell et al. 2011). Differences in mechanisms used by the
endophytes to combat the pathogen are likely to be the cause for microorganism
mixtures to have an enhanced ability of conferring resistance to the plant host
(Kloepper and Ryu 2006, Rosenblueth and Martínez-Romero 2006, Guo et al. 2008a,
Ryan et al. 2008).
The strategy of biocontrol against P. cinnamomi using colonization of endophytic
bacteria into plants would be a viable approach in disease control in native
ecosystems and rehabilitation of previously mined areas. Mining companies in
Western Australia, such as Alcoa, are required to re-vegetate previously mined sites.
This is accomplished through broadcast seeding of native tree and understory plants
(Craig et al. 2010). However, germinated seedlings can remain vulnerable to disease
and environmental stresses, resulting in losses of plant density within that area. Thus,
exploiting beneficial mutualistic relationships between endophytes and host plants
would result in enhanced resistance to infection with pathogens such as P.
cinnamomi, and also promote growth and reduce vulnerability of seedlings.
Therefore, application of endophytes to seeds and seedlings of re-vegetated areas
could facilitate the rehabilitation process, and aid in the long term survivability of
plants.
Additionally, application of biocontrol agents would be beneficial in nurseries, in
protection of plants against pathogens such as P. cinnamomi (and probably other
Phytophthora species). Nurseries often have high levels of contamination with
98
Phytophthora diseases, with recent studies on Western Australian nurseries showed
10% of plants were infected with Phytophthora, and 25% with Pythium species
(Davison et al. 2006). Similar levels of contamination are also seen within nurseries
throughout the USA and Europe (Schwingle et al. 2007, Moralejo et al. 2009). It is
the transport of these plants contaminated with Phytophthora from nurseries to home
gardens that serve as a main route for this pathogens’ spread. Having a biocontrol
agent that could reduce contamination of Phytophthora would be effective at
lessening the spread from nurseries.
In conclusion, this study has shown the diversity of endophytes from native
Australian plants differs with site, but not with host plant species or infestation of the
sampling area within a site with P. cinnamomi. Lack of specificity for endophytes in
native plants was shown as the same endophyte morphotypes were found throughout
different host plant species and separate sites. The strategy of a biocontrol agent
implies the agent could easily colonise the plant and be transferred to adjacent plants
of the same or different species. We have shown some of the endophytes isolated in
this study were capable of inihibiting P. cinnamomi on in vitro plate confrontation
assays, suggesting the production of antibiotics by these antagonistic endophytes.
This mechanism of resistance is highly desirable in a biocontrol agent as the antibiotic
can be transferred to other areas of the plant that are not colonized by the endophyte
species. Additionally, some of the endophytes isolated had the ability to inhibit P.
cinnamomi in planta. However, although some of the endophytes in this study were
found to exhibit desirable biocontrol traits, a number of challenges remain. For
example, long term persistence of the biocontrol agent needs to be evaluated under
field conditions, and horizontal transfer of these agents needs to be investigated as to
99
whether they confer resistance against P. cinnamomi within different plant host
species.
100
6.0 References
Agusti, L., Bonaterra, A., Moragrega, C., Camps, J. and Montesinos, E. (2011).
Biocontrol of root rot of strawberry caused by Phytophthora cactorum with a
combination of two Pseudomonas fluorescens strains. Journal of Plant Pathology
93(2): 363-372.
Ainsworth, G. C., Ed. (1968). Plant Pathologists Pocketbook. Kew, Commonwealth
Mycological Institute.
Ajit, N. S., Verma, R. and Shanmugam, V. (2006). Extracellular chitinases of
fluorescent pseudomonads antifungal to Fusarium oxysporum f. sp. dianthi causing
carnation wilt. Current microbiology 52(4): 310-316.
Akrami, M., Golzary, H. and Ahmadzadeh, M. (2011). Evaluation of different
combinations of Trichoderma species for controlling Fusarium rot of lentil. African
Journal of Biotechnology 10(14): 2653-2658.
Alabouvette, C., Olivain, C. and Steinberg, C. (2006). Biological control of plant
diseases: the European situation. European Journal of Plant Pathology 114(3): 329341.
Andreote, F. D., Azevedo, J. L. and Araujo, W. L. (2009). Assessing the diversity of
bacterial communities associated with plants Brazilian Journal of Microbiology
40(3): 417-432.
Araujo, W. L., Marcon, J., Maccheroni, W. J., van Elsas, J. D., van Vuurde, J. W. L.
and Azevedo, J. L. (2002). Diversity of endophytic bacterial populations and their
interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68: 49064914.
Aravind, R., Kumar, A. and Eapen, S. (2012). Pre-plant bacterisation: a strategy for
delivery of beneficial endophytic bacteria and production of disease-free plantlets of
black pepper (Pipernigrum L.). Archives Of Phytopathology And Plant Protection
45(9): 1115-1126.
Aravind, R., Kumar, A., Eapen, S. J. and Ramana, K. V. (2009). Endophytic bacterial
flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation,
identification and evaluation against Phytophthora capsici. Letters in Applied
Microbiology 48(1): 58-64.
Arnold, A. E. (2007). Understanding the diversity of foliar endophytic fungi:
progress, challenges, and frontiers. Fungal Biology Reviews 21(2–3): 51-66.
Arnold, A. E. and Herre, E. A. (2003). Canopy cover and leaf age affect colonization
by tropical fungal endophytes: ecological pattern and process in Theobroma cacao
(Malvaceae). Mycologia 95(3): 388-398.
101
Arnold, A. E., Mejia, L. C., Kyllo, D., Rojas, E. I., Maynard, Z., Robbins, N. and
Herre, E. A. (2003). Fungal endophytes limit pathogen damage in a tropical tree.
Proceedings of the National Academy of Sciences of the United States of America
100(26): 15649-15654.
Auesukaree, C., Homma, T., Tochio, H., Shirakawa, M., Kaneko, Y. and Harashima,
S. (2004). Intracellular phosphate serves as a signal for the regulation of the PHO
pathway in Saccharomyces cerevisiae. J Biol Chem 279(17): 17289-17294.
Baldauf, M. W., Mace, W. J. and Richmond, D. S. (2011). Endophyte-mediated
resistance to black cutworm as a function of plant cultivar and endophyte strain in tall
fescue. Environmental Entomology 40(3): 639-647.
Barber, P. A., Paap, T., Burgess, T. I., Dunstan, W. and Hardy, G. E. S. J. (2013). A
diverse range of Phytophthora species are associated with dying urban trees. Urban
Forestry & Urban Greening, In press.
Bardin, M., Troulet, C. and Nicot, P. C. (2013). The efficacy of biocontrol against
Botrytis cinerea on tomato depends on the strain of the pathogen. International
Congress of Plant Pathology. Beijing.
Barka, E. A., Gognies, S., Nowak, J., Audran, J.-C. and Belarbi, A. (2002). Inhibitory
effect of endophyte bacteria on Botrytis cinerea and its influence to promote the
grapevine growth. Biological Control 24(2): 135-142.
Barrett, S. R., Shearer, B. L. and Hardy, G. E. S. (2002). Root and shoot development
in Corymbia calophylla and Banksia brownii after the application of the fungicide
phosphite. Australian Journal of Botany 50(2): 155-161.
Barzanti, R., Ozino, F., Bazzicalupo, M., Gabbrielli, R., Galardi, F., Gonnelli, C. and
Mengoni, A. (2007). Isolation and characterization of endophytic bacteria from the
nickel hyperaccumulator Plant Alyssum bertolonii. Microbial Ecology 53(2): 306-316.
Bashan Y, Puente M, Bashan L and P, H. (2008). Environmental uses of plant
growth-promoting bacteria. Plant–microbe interactions. Ait Barka E and C. C.
Trivandrum, Kerala, India, Research Signpost: 69-93.
Bashan, Y. and de Bashan, L. (2005). Plant growth-promoting. Encyclopedia of soils
in the environment 1: 103-115.
Beakes, G., Glockling, S. and Sekimoto, S. (2012). The evolutionary phylogeny of the
oomycete fungi. Protoplasma 249(1): 3-19.
Bishop, A. L. and Slack, S. A. (1987). Effect of inoculum dose and preparation, strain
variation, and plant growth conditions on the eggplant assay for bacterial ring rot.
American Potato Journal 64(5): 227-234.
Bocobo, F. C. and Benham, R. W. (1949). Pigment production in the differentiation
of Trichophyton mentagrophytes and Trichophyton rubrum. Mycologia 41(3): 291302.
102
Bodenhausen, N., Horton, M. W. and Bergelson, J. (2013). Bacterial communities
associated with the leaves and the roots of Arabidopsis thaliana. PloS one 8(2).
Bouizgarne, B. (2013). Bacteria for plant growth promotion and disease management.
Bacteria in Agrobiology: Disease Management, Springer: 15-47.
Brader, G., Compant, S., Mitter, B., Trognitz, F. and Sessitsch, A. (2014). Metabolic
potential of endophytic bacteria. Current Opinion in Biotechnology 27(0): 30-37.
Brasier, C. M. (2008). The biosecurity threat to the UK and global environment from
international trade in plants. Plant Pathology 57: 792-808.
Broadbent, P., Baker, K. F. and Waterworth, Y. (1971). Bacteria and actinomycetes
anatagonistic to fungal root pathogens in Australian soils. Australian Journal of
Biological Science 24: 925-944.
Bulgarelli, D., Schlaeppi, K., Spaepen, S., van Themaat, E. V. L. and Schulze-Lefert,
P. (2013). Structure and functions of the bacterial microbiota of plants. Annual
Review of Plant Biology 64(1): 807-838.
Burgess, T. I., Webster, J. L., Ciampini, J. A., White, D., Hardy, G. E. S. and Stukely,
M. J. (2009). Re-evaluation of Phytophthora species isolated during 30 years of
vegetation health surveys in Western Australia using molecular techniques. Plant
Disease 93(3): 215-223.
Cahill, D. M., Rookes, J. E., Wilson, B. A., Gibson, L. and McDougall, K. L. (2008).
Phytophthora cinnamomi and Australia’s biodiversity: impacts, predictions and
progress towards control. Australian Journal of Botany 56(4): 279-310.
Cambours, M. A., Nejad, P., Granhall, U. and Ramstedt, M. (2005). Frost related
dieback of willows. Comparison of epiphytically and endophytically isolated bacteria
fromdifferent Salix clones, with emphasis on ice nucleation activity, pathogenic
properties and seasonal variation. Biomass Bioenergy 28: 15-27.
Cazorla, F. M., Romero, D., Pérez-García, A., Lugtenberg, B. J. J., Vicente, A. d. and
Bloemberg, G. (2007). Isolation and characterization of antagonistic Bacillus subtilis
strains from the avocado rhizoplane displaying biocontrol activity. Journal of Applied
Microbiology 103(5): 1950-1959.
Choudhary, D. K. and Johri, B. N. (2009). Interactions of Bacillus spp. and plants–
With special reference to induced systemic resistance (ISR). Microbiological
research 164(5): 493-513.
Clay, K. (1989). Clavicipitaceous endophytes of grasses: their potential as biocontrol
agents. Mycological Research 92: 1-12.
Colquhoun, I. J. and Elliont, P. E. (2000). Management of Phytophthora cinnamomi
during bauxite mining in Eucalyptus marginata forest-a special case. Diseases and
Pathogens of Eucalypts. P. J. Keane, G. A. Kile, F. D. Podger and B. N. Brown.
Victoria, CSIRO: 477-486.
103
Combes-Meynet, E., Pothier, J. F., Moënne-Loccoz, Y. and Prigent-Combaret, C.
(2011). The Pseudomonas secondary metabolite 2, 4-diacetylphloroglucinol is a
signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth
promotion. Molecular Plant-Microbe Interactions 24(2): 271-284.
Compant, S., Duffy, B., Nowak, J., Clément, C. and Barka, E. A. (2005a). Use of
plant growth-promoting bacteria for biocontrol of plant diseases: Principles,
mechanisms of action, and future prospects. Applied and Environmental Microbiology
71(9): 4951-4959.
Compant, S., Reiter, B., Sessitsch, A., Nowak, J., Clément, C. and Ait Barka, E.
(2005b). Endophytic colonization of Vitis vinifera L. by plant growth-promoting
bacterium Burkholderia sp. Strain PsJN. Applied and Environmental Microbiology
71(4): 1685-1693.
Coombs, J. T., Michelsen, P. P. and Franco, C. M. M. (2004). Evaluation of
endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in
wheat. Biological Control 29(3): 359-366.
Craig, M. D., Hobbs, R. J., Grigg, A. H., Garkaklis, M. J., Grant, C. D., Fleming, P.
A. and Hardy, G. E. S. J. (2010). Do thinning and burning sites revegetated after
bauxite mining improve habitat for terrestrial vertebrates? Restoration Ecology 18(3):
300-310.
Crone, M., McComb, J. A., O'Brien, P. A. and Hardy, G. E. S. J. (2013a). Annual and
herbaceous perennial native Australian plant species are symptomless hosts of
Phytophthora cinnamomi in the Eucalyptus marginata (jarrah) forest of Western
Australia. Plant Pathology 62(5): 1057-1062.
Crone, M., McComb, J. A., O'Brien, P. A. and Hardy, G. E. S. J. (2013b). Assessment
of Australian native annual/herbaceous perennial plant species as asymptomatic or
symptomatic hosts of Phytophthora cinnamomi under controlled conditions. Forest
Pathology 43(3): 245-251.
Crone, M., McComb, J. A., O’Brien, P. A. and Hardy, G. E. S. (2012). Survival of
Phytophthora cinnamomi as oospores, stromata and thick-walled chlamydospores in
roots of symptomatic and asymptomatic annual and herbaceous perennial species
Fungal Biology 117: 112-123.
Davison, E., Drenth, A., Kumar, S., Mack, S., Mackie, A. and McKirdy, S. (2006).
Pathogens associated with nursery plants imported into Western Australia.
Australasian Plant Pathology 35(4): 473-475.
de Boer, M., Bom, P., Kindt, F., Keurentjes, J. J., van der Sluis, I., Van Loon, L. and
Bakker, P. A. (2003). Control of Fusarium wilt of radish by combining Pseudomonas
putida strains that have different disease-suppressive mechanisms. Phytopathology
93(5): 626-632.
104
De Jonghe, K., De Dobbelaere, I., Sarrazyn, R. and Höfte, M. (2005). Control of
Phytophthora cryptogea in the hydroponic forcing of witloof chicory with the
rhamnolipid-based biosurfactant formulation PRO1. Plant Pathology 54(2): 219-226.
Delaney, S. M., Mavrodi, D. V., Bonsall, R. F. and Thomashow, L. S. (2001). phzO, a
gene for biosynthesis of 2-hydroxylated phenazine compounds in Pseudomonas
aureofaciens 30-84. Journal of Bacteriology 183(1): 318-327.
Dell, B. and Bennett, I. J. (1986). The flora of Murdoch University : a guide to the
native plants on campus. Western Australia, Murdoch University.
Dobrowolski, M. P., Tommerup, I. C., Shearer, B. L. and O'Brien, P. A. (2003). Three
clonal lineages of Phytophthora cinnamomi in Australia revealed by microsatellites.
Phytopathology 93(6): 695-704.
Douanla-Meli, C., Langer, E. and Mouafo, F. T. (2013). Fungal endophyte diversity
and community patterns in healthy and yellowing leaves of Citrus limon. Fungal
Ecology 6(3): 212-222.
Dunstan, W., Rudman, T., Shearer, B., Moore, N., Paap, T., Calver, M., Dell, B. and
Hardy, G. S. J. (2010). Containment and spot eradication of a highly destructive,
invasive plant pathogen (Phytophthora cinnamomi) in natural ecosystems. Biological
Invasions 12(4): 913-925.
Eaton, C. J., Cox, M. P. and Scott, B. (2011). What triggers grass endophytes to
switch from mutualism to pathogenism? Plant Science 180(2): 190-195.
El-Tarabily, K. A., Nassar, A. H., Hardy, G. E. S. J. and Sivasithamparam, K. (2009).
Plant growth promotion and biological control of Pythium aphanidermatum, a
pathogen of cucumber, by endophytic actinomycetes. Journal of Applied
Microbiology 106(1): 13-26.
Enya, J., Shinohara, H., Yoshida, S., Tsukiboshi, T., Negishi, H., Suyama, K. and
Tsushima, S. (2007). Culturable leaf-associated bacteria on tomato plants and their
potential as biological control agents. Microbial Ecology 53(4): 524-536.
Eshraghi, L., Anderson, J., Aryamanesh, N., Shearer, B., McComb, J., Hardy, G. E. S.
and O’Brien, P. A. (2011). Phosphite primed defence responses and enhanced
expression of defence genes in Arabidopsis thaliana infected with Phytophthora
cinnamomi. Plant Pathology 60(6): 1086-1095.
Esitken, A., Pirlak, L., Turan, M. and Sahin, F. (2006). Effects of floral and foliar
application of plant growth promoting rhizobacteria (PGPR) on yield, growth and
nutrition of sweet cherry. Scientia Horticulturae 110(4): 324-327.
Ferluga, S. and Venturi, V. (2009). OryR is a LuxR-family protein involved in
interkingdom signaling between pathogenic Xanthomonas oryzae pv. oryzae and rice.
J. Bacteriol 191: 890-897.
105
Ferreira, A., Quecine, M. C., Lacava, P. T., Oda, S., Azevedo, J. L. and Araujo, W. L.
(2008). Diversity of endophytic bacteria from Eucalyptus species seeds and
colonization of seedlings by Pantoea agglomerans. FEMS Microbiology Letters
287(1): 8-14.
Filho, R. L., de Souza, R. M., Ferreira, A., Quecine, M. C., Alves, E. and de Azevedo,
J. L. (2013). Biocontrol activity of Bacillus against a GFP-marked Pseudomonas
syringae pv. tomato on tomato phylloplane. Aust. Pl. Pathol. In press.
Fisher, M. C., Henk, D. A., Briggs, C. J., Brownstein, J. S., Madoff, L. C., McCraw,
S. L. and Gurr, S. J. (2012). Emerging fungal threats to animal, plant and ecosystem
health. Nature 484(7393): 186-194.
Forchetti, G., Masciarelli, O., Izaguirre, M., Alemano, S., Alvarez, D. and Abdala, G.
(2010). Endophytic bacteria improve seedling growth of sunflower under water stress,
produce salicylic acid, and inhibit growth of pathogenic fungi. Current Microbiology
61(6): 485-493.
Franco, C., Michelsen, P., Percy, N., Conn, V., Listiana, E., Moll, S., Loria, R. and
Coombs, J. (2007). Actinobacterial endophytes for improved crop performance.
Australasian Plant Pathology 36(6): 524-531.
French-Monar, R. D., Jones, J. B. and Roberts, P. D. (2006). Characterization of
Phytophthora capsici associated with roots of weeds on Florida vegetable farms.
Plant Disease 90(3): 345-350.
Garbeva, P., Silby, M. W., Raaijmakers, J. M., Levy, S. B. and de Boer, W. (2011).
Transcriptional and antagonistic responses of Pseudomonas fluorescens Pf0-1 to
phylogenetically different bacterial competitors. The ISME journal 5(6): 973-985.
Germaine, K., Keogh, E., Garcia-Cabellos, G., Borremans, B., van der Lelie, D.,
Barac, T., Oeyen, L., Vangronsveld, J., Moore, F. P., Moore, E. R. B., Campbell, C.
D., Ryan, D. and Dowling, D. N. (2004). Colonisation of poplar trees by gfp
expressing bacterial endophytes. FEMS Microbiology Ecology 48(1): 109-118.
Giraud, T., Gladieux, P. and Gavrilets, S. (2010). Linking the emergence of fungal
plant diseases with ecological speciation. Trends in ecology & evolution 25(7): 387395.
Glick, B. R. (2012). Plant growth-promoting bacteria: Mechanisms and applications.
Scientifica 2012: 1-15.
Grant, M. (2003). The distribution and impact of Phytophihora cinnamomi Rands in
the south coast region of Western Australia. Phytophthora in Forests and Natural
Ecosystems: 34.
Guharoy, S., Bhattacharyya, S., Mukherjee, S., Mandal, N. and Khatua, D. (2006).
Phytophthora melonis associated with fruit and vine rot disease of pointed gourd in
India as revealed by RFLP and sequencing of ITS region. Journal of Phytopathology
154(10): 612-615.
106
Guo, B., Wang, Y., Sun, X. and Tang, K. (2008). Bioactive natural products from
endophytes: a review. Applied Biochemistry and Microbiology 44(2): 136-142.
Haesler, F., Hagn, A., Frommberger, M., Hertkorn, N., Schmitt-Kopplin, P., Munch,
J. C. and Schloter, M. (2008). In vitro antagonism of an actinobacterial Kitasatospora
isolate against the plant pathogen Phytophthora citricola as elucidated with ultrahigh
resolution mass spectrometry. Journal of Microbiological Methods 75(2): 188-195.
Hakizimana, J., Gryzenhout, M., Coutinho, T. and van den Berg, N. (2011).
Endophytic diversity in Persea americana (avocado) trees and their ability to display
biocontrol activity against Phytophthora cinnamomi. Proceedings VII World Avocado
Congress 2011.
Hallmann, J., Quadt-Hallmann, A., Mahaffee, W. F. and Kloepper, J. W. (1997).
Bacterial endophytes in agricultural crops. Canadian Journal of Microbiology 43(10):
895-914.
Hamayun, M., Khan, S., Iqbal, I., Na, C.-I., Khan, A., Hwang, Y.-H., Lee, B.-H. and
Lee, I.-J. (2009). Chrysosporium pseudomerdarium produces gibberellins and
promotes plant growth. The Journal of Microbiology 47(4): 425-430.
Hansen, E. (2008). Alien forest pathogens: Phytophthora species are changing world
forests. Boreal Environ Res 13(Suppl A): 33-41.
Hardham, A. R. (2005). Phytophthora cinnamomi. Molecular Plant Pathology 6(6):
589-604.
Hardham, A. R. and Shan, W. (2009). Cellular and molecular biology of
Phytophthora plant interactions. The Mycota. H. Deising. Berlin Heidelberg,
Springer Verlag.
Hardoim, P. R., van Overbeek, L. S. and Elsas, J. D. v. (2008). Properties of bacterial
endophytes and their proposed role in plant growth. Trends in Microbiology 16(10):
463-471.
Hardy, G. E. S. J., Barrett, S. and Shearer, B. L. (2001). The future of phosphite as a
fungicide to control the soilborne plant pathogen Phytophthora cinnamomi in natural
ecosystems. Australasian Plant Pathology 30(2): 133-139.
Harish, S., Kavino, M., Kumar, N., Balasubramanian, P. and Samiyappan, R. (2009).
Induction of defense-related proteins by mixtures of plant growth promoting
endophytic bacteria against Banana bunchy top virus. Biological Control 51(1): 1625.
Hol, W. G., Bezemer, T. M. and Biere, A. (2013). Getting the ecology into
interactions between plants and the plant growth-promoting bacterium Pseudomonas
fluorescens. Frontiers in plant science 4.
107
Hou, X., Li, Z., Han, D., Huang, Q. and Ran, L. (2010). Presence of indigenous
endophytic bacteria in jujube seedlings germinated from seeds in vitro. Frontiers of
Agriculture in China 4(4): 443-448.
Hultberg, M., Alsberg, T., Khalil, S. and Alsanius, B. (2010). Suppression of disease
in tomato infected by Pythium ultimum with a biosurfactant produced by
Pseudomonas koreensis. BioControl 55(3): 435-444.
Ikeda, S., Okubo, T., Anda, M., Nakashita, H., Yasuda, M., Sato, S., Kaneko, T.,
Tabata, S., Eda, S., Momiyama, A., Terasawa, K., Mitsui, H. and Minamisawa, K.
(2010). Community and genome-based views of plant-associated bacteria: plant–
bacterial interactions in soybean and rice. Plant and Cell Physiology 51(9): 13981410.
Izumi, H., Anderson, I. C., Killham, K. and Moore, E. R. B. (2008). Diversity of
predominant endophytic bacteria in European deciduous and coniferous trees.
Canadian Journal of Microbiology 54(3): 173-179.
Jami, F., Slippers, B., Wingfield, M. J. and Gryzenhout, M. (2013). Greater
Botrysphgaeriaceae diversity in healthy than associated diseased Acacia karoo trees
tissues. Australasian Plant Pathology 42(4): 421-430.
Jasim, B., Joseph, A. A., John, C. J., Mathew, J. and Radhakrishnan, E. (2013).
Isolation and characterization of plant growth promoting endophytic bacteria from the
rhizome of Zingiber officinale. 3 Biotech: 1-8.
Ji, P. and Wilson, M. (2003). Enhancement of population size of a biological control
agent and efficacy in control of bacterial speck of tomato through salicylate and
ammonium sulfate amendments. Applied and Environmental Microbiology 69(2):
1290-1294.
Ji, X. L., Lu, G. B., Gai, Y. P., Zheng, C. C. and Mu, Z. M. (2008). Biological control
against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis
strain. FEMS Microbiology Ecology 65(3): 565-573.
Johnson, K. B., Stockwell, V. O., Burgett, D. M., Sugar, D. and Loper, J. E. (1993).
Dispersal of Erwinia amylovora and Pseudomonas fluorescens by honey bees to apple
and pear blossoms. Phytopathology 83: 478-484.
Jung, T., Colquhoun, I. J. and Hardy, G. E. S. J. (2013). New insights into the survival
strategy of the invasive soilborne pathogen Phytophthora cinnamomi in different
natural ecosystems in Western Australia. Forest Pathology 43: 266-288.
Jung, T., Stukely, M., Hardy, G. S. J., White, D., Paap, T., Dunstan, W. and Burgess,
T. (2011). Multiple new Phytophthora species from ITS Clade 6 associated with
natural ecosystems in Australia: evolutionary and ecological implications. Persoonia:
Molecular Phylogeny and Evolution of Fungi 26: 13.
108
Kaneko, T., Minamisawa, K., Isawa, T., Nakatsukasa, H., Mitsui, H., Kawaharada,
Y., Nakamura, Y., Watanabe, A., Kawashima, K., Ono, A., Shimizu, Y., Takahashi,
C., Minami, C., Fujishiro, T., Kohara, M., Katoh, M., Nakazaki, N., Nakayama, S.,
Yamada, M., Tabata, S. and Sato, S. (2010). Complete genomic structure of the
cultivated rice endophyte Azospirillum sp. B510. DNA Research 17(1): 37-50.
Kennedy, I. R., Choudhury, A. T. M. A. and Kecskés, M. L. (2004). Non-symbiotic
bacterial diazotrophs in crop-farming systems: can their potential for plant growth
promotion be better exploited? Soil Biology and Biochemistry 36(8): 1229-1244.
Khan, A. L., Hamayun, M., Kang, S.-M., Kim, Y.-H., Jung, H.-Y., Lee, J.-H. and Lee,
I.-J. (2012). Endophytic fungal association via gibberellins and indole acetic acid can
improve plant growth under abiotic stress: an example of Paecilomyces formosus
LHL10. BMC microbiology 12(1): 3.
Kim, H. Y., Choi, G. J., Lee, H. B., Lee, S. W., Lim, H. K., Jang, K. S., Son, S. W.,
Lee, S. O., Cho, K. Y., Sung, N. D. and Kim, J. C. (2007). Some fungal endophytes
from vegetable crops and their anti-oomycete activities against tomato late blight.
Letters in Applied Microbiology 44(3): 332-337.
Kim, Y. C., Jung, H., Kim, K. Y. and Park, S. K. (2008). An effective biocontrol
bioformulation against Phytophthora blight of pepper using growth mixtures of
combined chitinolytic bacteria under different field conditions. European Journal of
Plant Pathology 120(4): 373-382.
King, M., Reeve, R., Van der Hoek, M., Williams, N., McComb, J., O’Brien, P. A.
and Hardy, G. E. S. (2010). Defining the phosphite-regulated transcriptome of the
plant pathogen Phytophthora cinnamomi. Molecular Genetics & Genomics 284: 425435.
Kliebenstein, D. J., Rowe, H. C. and Denby, K. J. (2005). Secondary metabolites
influence Arabidopsis/Botrytis interactions: variation in host production and pathogen
sensitivity. The Plant Journal 44(1): 25-36.
Kloepper, J. W. and Ryu, C.-M. (2006). Bacterial endophytes as elicitors of induced
systemic resistance. Microbial root endophytes, Springer: 33-52.
Knief, C., Ramette, A., Frances, L., Alonso-Blanco, C. and Vorholt, J. A. (2010). Site
and plant species are important determinants of the Methylobacterium community
composition in the plant phyllosphere. Isme Journal 4(6): 719-728.
Kobayashi, D. and Palumbo, J. (2000). Bacterial endophytes and their effects on
plants and uses in agriculture. Microbial endophytes. Marcel Dekker, New York: 199236.
Koeck, M., Hardham, A. R. and Dodds, P. N. (2011). The role of effectors of
biotrophic and hemibiotrophic fungi in infection. Cellular microbiology 13(12): 18491857.
109
Kowalski, T. and Drozynska, K. (2011). Mycobiota in needles and shoots of Pinus
nigra following infection by Dothistroma septosporum. Phyton-Annales Rei
Botanicae 51(2): 277-287.
Li, C. H., Shi, L., Han, Q., Hu, H. L., Zhao, M. W., Tang, C. M. and Li, S. P. (2012).
Biocontrol of Verticillium wilt and colonization of cotton plants by an endophytic
bacterial isolate. Journal of Applied Microbiology 113(3): 641-651.
Lian, J., Wang, Z. F. and Zhou, S. N. (2008). Response of endophytic bacterial
communities in banana tissue culture plantlets to Fusarium wilt pathogen infection.
Journal of General and Applied Microbiology 54(2): 83-92.
Liu, B., Qiao, H., Huang, L., Buchenauer, H., Han, Q., Kang, Z. and Gong, Y. (2009).
Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and
potential mode of action. Biological Control 49: 277-285.
Liu, Y., Chen, Z., Liu, Y., Wang, X., Luo, C., Nie, Y. and Wang, K. (2011).
Enhancing bioefficacy of Bacillus subtilis with sodium bicarbonate for the control of
ring rot in pear during storage. Biological Control 57(2): 110-117.
Lodewyckx, C., Vangronsveld, J., Porteous, F., Moore, E. R. B., Taghavi, S.,
Mezgeay, M. and der Lelie, D. v. (2002). Endophytic bacteria and their potential
applications. Critical Reviews in Plant Sciences 21(6): 583-606.
Long, H. H., Schmidt, D. D. and Baldwin, I. T. (2008). Native bacterial endophytes
promote host growth in a species-specific manner; phytohormone manipulations do
not result in common growth responses. PLoS One 3(7): e2702.
Loo, J. A. (2009). Ecological impacts of non-indigenous invasive fungi as forest
pathogens. Biological Invasions 11(1): 81-96.
Loper, J. E., Hassan, K. A., Mavrodi, D. V., Davis II, E. W., Lim, C. K., Shaffer, B.
T., Elbourne, L. D., Stockwell, V. O., Hartney, S. L. and Breakwell, K. (2012).
Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity
and inheritance of traits involved in multitrophic interactions. PLoS genetics 8(7)
Manter, D. K., Delgado, J. A., Holm, D. G. and Stong, R. A. (2010). Pyrosequencing
reveals a highly diverse and cultivar-specific bacterial endophyte community in
potato roots. Microbial Ecology 60(1): 157-166.
McDonald, V., Pond, E., Crowley, M., McKee, B. and Menge, J. (2007). Selection for
and evaluation of an avocado orchard soil microbially suppressive to Phytophthora
cinnamomi. Plant and Soil 299(1-2): 17-28.
McDougall, K. L., Hardy, G. E. S. and Hobbs, R. J. (2002). Distribution of
Phytophthora cinnamomi in the northern jarrah (Eucalyptus marginata) forest of
Western Australia in relation to dieback age and topography. Australian Journal of
Botany 50: 107-114.
110
McDougall, K. L., Hobbs, R. J. and Hardy, G. E. S. J. (2005). Distribution of
understorey species in forest affected by Phytophthora cinnamomi in south-western
Western Australia. Australian Journal of Botany 53(8): 813-819.
McSpadden Gardener, B. B. (2004). Ecology of Bacillus and Paenibacillus spp. in
agricultural systems. Phytopathology 94(11): 1252-1258.
Melnick, R. L., Suárez, C., Bailey, B. A. and Backman, P. A. (2011). Isolation of
endophytic endospore-forming bacteria from Theobroma cacao as potential biological
control agents of cacao diseases. Biological Control 57(3): 236-245.
Melnick, R. L., Zidack, N. K., Bailey, B. A., Maximova, S. N., Guiltinan, M. and
Backman, P. A. (2008). Bacterial endophytes: Bacillus spp. from annual crops as
potential biological control agents of black pod rot of cacao. Biological control 46(1):
46-56.
Mendes, R., Pizzirani-Kleiner, A. A., Araujo, W. L. and Raaijmakers, J. M. (2007).
Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical
characterization of Burkholderia cepacia complex isolates. Applied and
environmental microbiology 73(22): 7259-7267.
Mendoza, A. and Sikora, R. (2009). Biological control of Radopholus similis in
banana by combined application of the mutualistic endophyte Fusarium oxysporum
strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic
bacteria Bacillus firmus. BioControl 54(2): 263-272.
Molina, L., Ramos, C., Duque, E., Ronchel, M. C., Garc a, J. M., Wyke, L. and
Ramos, J. L. (2000). Survival of Pseudomonas putida KT2440 in soil and in the
rhizosphere of plants under greenhouse and environmental conditions. Soil Biology
and Biochemistry 32(3): 315-321.
Monte, E. (2010). Understanding Trichoderma: between biotechnology and microbial
ecology. International Microbiology 4(1): 1-4.
Moralejo, E., Pérez‐ Sierra, A., Alvarez, L., Belbahri, L., Lefort, F. and Descals, E.
(2009). Multiple alien Phytophthora taxa discovered on diseased ornamental plants in
Spain. Plant Pathology 58(1): 100-110.
Newman, L. A. and Reynolds, C. M. (2005). Bacteria and phytoremediation: new
uses for endophytic bacteria in plants. Trends in Biotechnology 23(1): 6-8.
Nga, N. T. T., Giau, N., Long, N., Lübeck, M., Shetty, N. P., De Neergaard, E., Thuy,
T. T. T., Kim, P. and Jørgensen, H. J. L. (2010). Rhizobacterially induced protection
of watermelon against Didymella bryoniae. Journal of applied microbiology 109(2):
567-582.
Nielsen, C., Ferrin, D. and Stanghellini, M. (2006). Efficacy of biosurfactants in the
management of Phytophthora capsici on pepper in recirculating hydroponic systems.
Canadian journal of plant pathology 28(3): 450-460.
111
O'Brien, P. A., Williams, N. and Hardy, G. E. S. J. (2009). Detecting Phytophthora.
Critical Reviews in Microbiology 35(3): 165-181.
Pal, K. K. and Gardener, B. M. (2006). Biological control of plant pathogens. The
plant health instructor 2: 1117-1142.
Park, J.-W., Balaraju, K., Kim, J.-W., Lee, S.-W. and Park, K. (2013). Systemic
resistance and growth promotion of chili pepper induced by an antibiotic producing
Bacillus vallismortis strain BS07. Biological Control 65(2): 246-257.
Raaijmakers, J., Vlami, M. and de Souza, J. (2002). Antibiotic production by bacterial
biocontrol agents. Antonie van Leeuwenhoek 81(1-4): 537-547.
Ran, L. X., Li, Z. N., Wu, G. J., Loon, L. C. and Bakker, P. A. M. (2005). Induction
of systemic resistance against bacterial wilt in Eucalyptus urophylla by fluorescent
Pseudomonas spp. European Journal of Plant Pathology 113(1): 59-70.
Rea, A., Burgess, T., Hardy, G. S. J., Stukely, M. and Jung, T. (2011). Two novel and
potentially endemic species of Phytophthora associated with episodic dieback of
Kwongan vegetation in the south‐ west of Western Australia. Plant Pathology 60(6):
1055-1068.
Rodriguez, R., White Jr, J., Arnold, A. and Redman, R. (2009). Fungal endophytes:
diversity and functional roles. New Phytologist 182(2): 314-330.
Rosenblueth, M. and Martinez-Romero, E. (2006). Bacterial endophytes and their
interactions with hosts. Mol Plant Microbe Interact 19: 827-837.
Rosenblueth, M. and Martínez-Romero, E. (2006). Bacterial endophytes and their
interactions with hosts. Molecular plant-microbe interactions : MPMI 19(8): 827-837.
Ryan, R. P., Germaine, K., Franks, A., Ryan, D. J. and Dowling, D. N. (2008).
Bacterial endophytes: recent developments and applications. FEMS Microbiology
Letters 278(1): 1-9.
Ryan, R. P., Monchy, S., Cardinale, M., Taghavi, S., Crossman, L., Avison, M. B.,
Berg, G., van der Lelie, D. and Dow, J. M. (2009). The versatility and adaptation of
bacteria from the genus Stenotrophomonas. Nature Reviews Microbiology 7(7): 514525.
Ryu, C.-M., Farag, M. A., Hu, C.-H., Reddy, M. S., Kloepper, J. W. and Paré, P. W.
(2004). Bacterial volatiles induce systemic resistance in Arabidopsis. Plant
Physiology 134(3): 1017-1026.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: A laboratory
manual. 2nd Edition. Cold Spring Harbor Laboratory Press, New York.
Schulz, B. J., Boyle, C. J. and Sieber, T. N. (2006). Microbial root endophytes,
Springer.
112
Schwingle, B., Smith, J. and Blanchette, R. (2007). Phytophthora species associated
with diseased woody ornamentals in Minnesota nurseries. Plant Disease 91(1): 97102.
Scott, P., Burgess, T. I. and Hardy, G. E. S. (2013a). Globalisation of Phytophthora.
Phytophthora a Global Perspective. K. Lamour, CABI.
Scott, P. M., Burgess, T., Barber, P., Shearer, B., Stukely, M., Hardy, G. S. J. and
Jung, T. (2009). Phytophthora multivora sp. nov., a new species recovered from
declining Eucalyptus, Banksia, Agonis and other plant species in Western Australia.
Persoonia 22: 1.
Scott, P. M., Dell, B., Shearer, B. L., Barber, P. A. and Hardy, G. E. S. J. (2013b).
Phosphite and nutrient applications as explorative tools to identify possible factors
associated with Eucalyptus gomphocephala decline in South-Western Australia.
Australasian Plant Pathology 42(6): 701-711.
Selosse, M.-A., Baudoin, E. and Vandenkoornhuyse, P. (2004). Symbiotic
microorganisms, a key for ecological success and protection of plants. Comptes
Rendus Biologies 327(7): 639-648.
Sessitsch, A., Reiter, B. and Berg, G. (2004). Endophytic bacterial communities of
field-grown potato plants and their plant-growth-promoting and antagonistic abilities.
Canadian Journal of Microbiology 50(4): 239-249.
Shearer, B., Crane, C. and Cochrane, A. (2004a). Quantification of the susceptibility
of the native flora of the South-West Botanical Province, Western Australia, to
Phytophthora cinnamomi. Australian Journal of Botany 52(4): 435-443.
Shearer, B. L., Crane, C. E., Barrett, S. and Cochrane, A. (2007). Phytophthora
cinnamomi invasion, a major threatening process to conservation of flora diversity in
the South-west Botanical Province of Western Australia. Australian Journal of
Botany 55(3): 225-238.
Shearer, B. L., Crane, C. E. and Cochrane, A. (2004b). Quantification of the
susceptibility of the native flora of the South-West Botanical Province, Western
Australia, to Phytophthora cinnamomi. Australian Journal of Botany 52(4): 435-443.
Shearer, B. L., Crane, C. E. and Fairman, R. G. (2004c). Phosphite reduces disease
extension of a Phytophthora cinnamomi front in Banksia woodland, even after fire.
Australasian Plant Pathology 33(2): 249-254.
Shishkoff, N. (2012). Susceptibility of some common container weeds to
Phytophthora ramorum. Plant Disease 96(7): 1026-1032.
Stasikowski, P. (2012). Biochemical effects of phosphite on the phytopathogenicity of
Phytophthora cinnamomi Rands. PhD Thesis. Murdoch University.
Stein, T. (2005). Bacillus subtilis antibiotics: structures, syntheses and specific
functions. Molecular Microbiology 56(4): 845-857.
113
Stockwell, V., Johnson, K., Sugar, D. and Loper, J. (2011). Mechanistically
compatible mixtures of bacterial antagonists improve biological control of fire blight
of pear. Phytopathology 101(1): 113-123.
Strobel, G. and Daisy, B. (2003). Bioprospecting for microbial endophytes and their
natural products. Microbiology and Molecular Biology Reviews 67(4): 491-502.
Sturrock, R. N., Frankel, S. J., Brown, A. V., Hennon, P. E., Kliejunas, J. T., Lewis,
K. J., Worrall, J. J. and Woods, A. J. (2011). Climate change and forest diseases.
Plant Pathology 60(1): 133-149.
Sturz, A. V., Christie, B. R. and Nowak, J. (2000). Bacterial endophytes: potential
role in developing sustainable systems of crop production. Critical Reviews in Plant
Sciences 19(1): 1-30.
Subramoni, S., Gonzalez, J. F., Johnson, A., Péchy-Tarr, M., Rochat, L., Paulsen, I.,
Loper, J. E., Keel, C. and Venturi, V. (2011). Bacterial subfamily of LuxR regulators
that respond to plant compounds. Applied and environmental microbiology 77(13):
4579-4588.
Sun, H., Wang, Q., Wang, X. Q., Li, Y. L., Hu, B., Zheng, J. Q. and Li, Y. (2013).
Antifungal mechanism of Bacillus amyloliquefaciens dlt3 against sugar beet root rot.
International Congress of Plant Pathology. Beijing.
Szczech, M. and Shoda, M. (2004). Biocontrol of Rhizoctonia damping‐ off of
tomato by Bacillus subtilis combined with Burkholderia cepacia. Journal of
Phytopathology 152(10): 549-556.
Taghavi, S., Barac, T., Greenberg, B., Borremans, B., Vangronsveld, J. and van der
Lelie, D. (2005). Horizontal gene transfer to endogenous endophytic bacteria from
poplar improves phytoremediation of toluene. Applied and Environmental
Microbiology 71(12): 8500-8505.
Taghavi, S., Garafola, C., Monchy, S., Newman, L., Hoffman, A., Weyens, N., Barac,
T., Vangronsveld, J. and van der Lelie, D. (2009). Genome survey and
characterization of endophytic bacteria exhibiting a beneficial effect on growth and
development of poplar trees. Applied and Environmental Microbiology 75(3): 748757.
Tanimoto, E. (2005). Regulation of root growth by plant hormones-roles for auxin
and gibberellin. Critical Reviews in Plant Sciences 24(4): 249-265.
Thakore, Y. (2006). The biopesticide market for global agricultural use. Industrial
Biotechnology 2(3): 194-208.
Thatcher, L. F., Anderson, J. P. and Singh, K. B. (2005). Plant defence responses:
what have we learnt from Arabidopsis? Functional Plant Biology 32(1): 1-19.
114
Ulrich, K., Ulrich, A. and Ewald, D. (2008). Diversity of endophytic bacterial
communities in poplar grown under field conditions. FEMS Microbiology Ecology
63(2): 169-180.
Vainio, E., Lipponen, K. and Hantula, J. (2001). Persistence of a biocontrol strain of
Phlebiopsis gigantea in conifer stumps and its effects on within‐ species genetic
diversity. Forest Pathology 31(5): 285-295.
van der Lelie, D., Taghavi, S., Monchy, S., Schwender, J., Miller, L., Ferrieri, R.,
Rogers, A., Wu, X., Zhu, W., Weyens, N., Vangronsveld, J. and Newman, L. (2009).
Poplar and its bacterial endophytes: coexistence and harmony. Critical Reviews in
Plant Sciences 28(5): 346-358.
Wang, P. P., Guo, Q. G., Li, S. Z., Lu, X. Y., Zhang, X. Y. and Ma, P. (2013). The
pleiotropic regulatory gene degQ regulates the fengycins production and biofilm
formation of Bacillus subtilis NCD-2. International Congress of Plant Pathology.
Beijing.
West, E. R., Cother, E. J., Steel, C. C. and Ash, G. J. (2010). The characterization and
diversity of bacterial endophytes of grapevine. Canadian Journal of Microbiology
56(3): 209-216.
Weste, G. (1994). Impact of Phytophthora species on native vegetation of Australia
and Papua New Guinea. Australasian Plant Pathology 23(4): 190-209.
Weste, G. and Marks, G. C. (1987). The biology of Phytophthora cinnamomi in
Australian forests. Annual Review of Phytopathology 25: 207-229.
Weyens, N., van der Lelie, D., Taghavi, S., Newman, L. and Vangronsveld, J. (2009).
Exploiting plant–microbe partnerships to improve biomass production and
remediation. Trends in Biotechnology 27(10): 591-598.
Whipps, J. M. (2001). Microbial interactions and biocontrol in the rhizosphere.
Journal of Experimental Botany 52(suppl 1): 487-511.
Wilhelm, E., Arthofer, W., Schafleitner, R. and Krebs, B. (1998). Bacillus subtilis an
endophyte of chestnut (Castanea sativa) as antagonist against chestnut blight
(Cryphonectria parasitica). Plant cell, tissue and organ culture 52(1): 105-108.
Wilkinson, C. J., Shearer, B. L., Jackson, T. J. and Hardy, G. E. S. (2001). Variation
in sensitivity of Western Australian isolates of Phytophthora cinnamomi to phosphite
in vitro. Plant Pathology 50(1): 83-89.
Wong, M., McComb, J. A., Hardy, G. E. S. and O’Brien, P. A. (2008). Phosphite
induces expression of a putative proteophosphoglycan gene in Phytophthora
cinnamomi. Australasian Plant Pathology 38(3): 235-241.
115
Yau, J. A., Dianez, F., Marin, F., Carretero, F. and Santos, M. (2013). Screening and
evaluation of potential biocontrol fungi and bacteria foliar endophytes against
Phytophthora capsici and Phytophthora parasitica on pepper. Journal of Food
Agriculture & Environment 11(2): 490-495.
Zamioudis, C. and Pieterse, C. M. J. (2012). Modulation of host immunity by
beneficial microbes. Molecular Plant-Microbe Interactions 25(2): 139-150.
Zhang, L. Y., Wang, J. L. and Fang, R. (2007). A proline iminopeptidase gene
upregulated in planta by a LuxR homologue is essential for pathogenicity of
Xanthomonas campestris pv. campestris. Mol. Microbiol 65: 121-136.
Zhou, T., Chu, C.-L., Liu, W. T. and Schaneider, K. E. (2001). Postharvest control of
blue mold and gray mold on apples using isolates of Pseudomonas syringae.
Canadian Journal of Plant Pathology 23(3): 246-252.
116
7.0 Appendix
Table 7.1: Data for each endophyte isolate, showing mean in vitro assay inhibition zones against P. cinnamomi, and significance in
reducing lesion lengths caused by P. cinnamomi in in vivo assays.
MU
Infested
100BL-1
Eucalyptus marginata
L
3
0.00
Significantly Reduce Lesion
Length
✕
MU
Infested
100BL-2
Eucalyptus marginata
L
4
1.00
✕
MU
Infested
100BL-3
Eucalyptus marginata
L
5
0.07
MU
Infested
100BR-1
Eucalyptus marginata
R
1
0.77
MU
Infested
100BR-2
Eucalyptus marginata
R
2
0.63
MU
Infested
101BL-1
Melaleuca
R
6
0.00
MU
Infested
101BR-1
Melaleuca
S
9
0.00
MU
Infested
101BR-2
Melaleuca
S
9
0.00
MU
Infested
101BS-1
Melaleuca
L
7
0.77
✕
MU
Infested
101BS-2
Melaleuca
R
8
0.00
✕
MU
Infested
101BS-3
Melaleuca
S
8
0.03
MU
Infested
103BR-1
Eucalyptus marginata
R
2
1.00
MU
Infested
103BR-2
Eucalyptus marginata
R
10
0.00
MU
Infested
103BR-3
Eucalyptus marginata
R
11
0.03
MU
Infested
104BL-1
Eucalyptus marginata
R
5
0.00
MU
Infested
104BR-1
Eucalyptus marginata
L
12
0.03
MU
Infested
106BL-1
Eucalyptus marginata
L
6
0.73
Site
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
117
✕
✕
✕
MU
Infested
107BL-1
Banksia attenuata
L
10
0.13
Significantly Reduce Lesion
Length
✕
MU
Infested
107BL-2
Banksia attenuata
L
14
0.53
✓**
MU
Infested
107BL-3
Banksia attenuata
L
15
0.00
MU
Infested
107BR-1
Banksia attenuata
R
13
0.00
MU
Infested
107BR-2
Banksia attenuata
R
9
0.00
MU
Infested
108BR-1
Allocasuarina humulis
S
16
0.10
MU
Infested
108BS-1
Allocasuarina humulis
S
1
0.63
MU
Infested
109BR-1
Banksia attenuata
R
9
0.00
MU
Infested
110BR-1
Hakea
R
9
0.00
MU
Infested
110BR-2
Hakea
S
17
0.00
MU
Infested
110BS-1
Hakea
R
18
0.03
MU
Infested
110BS-2
Hakea
S
9
0.00
MU
Infested
110BS-3
Hakea
S
9
0.00
MU
Infested
111BL-1
Banksia attenuata
L
19
0.07
MU
Infested
111BR-1
Banksia attenuata
R
9
0.00
MU
Infested
111BR-2
Banksia attenuata
R
20
1.53
MU
Infested
114BR-1
Unknown spp.
S
14
0.00
✕
MU
Infested
114BR-2
Unknown spp.
R
21
0.63
✕
MU
Infested
116BR-1
Banksia attenuata
R
22
0.60
MU
Infested
116BR-2
Banksia attenuata
R
23
0.97
MU
Infested
117BL-1
Hibbertia hypercoides
L
19
0.00
MU
Infested
117BR-1
Hibbertia hypercoides
R
9
0.03
MU
Infested
118BR-1
Macrozamia riedlei
S
2
0.03
MU
Infested
118BS-1
Macrozamia riedlei
R
20
0.07
MU
Infested
119BR-1
Banksia ilicifolia
R
24
0.73
MU
Infested
120BL-1
Banksia attenuata
R
25
0.07
Site
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
118
✕
✕
✕
Site
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Significantly Reduce Lesion
Length
MU
Infested
120BR-1
Banksia attenuata
L
9
0.03
MU
Infested
120BS-1
Banksia attenuata
S
26
0.53
MU
Infested
120BS-2
Banksia attenuata
R
2
0.50
MU
Non-infested
121BR-1
Banksia attenuata
R
24
0.00
MU
Non-infested
121BR-2
Banksia attenuata
R
2
0.00
MU
Non-infested
121BR-3
Banksia attenuata
R
24
0.00
MU
Non-infested
121BS-1
S
2
0.13
MU
Non-infested
122BR-1
R
28
0.10
MU
Non-infested
122BS-1
Banksia attenuata
Allocasuarina
fraseriana
Allocasuarina
fraseriana
S
27
0.00
MU
Non-infested
123BR-1
Banksia menzeisii
R
19
0.13
MU
Non-infested
123BS-1
Banksia menzeisii
S
26
0.00
MU
Non-infested
124BR-1
Banksia attenuata
R
2
0.27
MU
Non-infested
124BR-2
Banksia attenuata
R
19
0.43
MU
Non-infested
125BR-1
Hibbertia hypericoides
R
9
0.00
MU
Non-infested
125BR-2
Hibbertia hypericoides
R
24
0.00
MU
Non-infested
125BS-1
Hibbertia hypericoides
S
29
0.03
MU
Non-infested
127BL-1
Anigozanthos
L
29
0.07
MU
Non-infested
127BL-2
Anigozanthos
L
28
0.00
MU
Non-infested
127BR-1
Anigozanthos
R
9
0.47
MU
Non-infested
128BL-1
Banksia attenuata
L
30
0.03
MU
Non-infested
128BL-2
Banksia attenuata
L
30
0.70
✕
MU
Non-infested
128BR-1
Banksia attenuata
R
32
0.00
✕
MU
Non-infested
128BR-2
Banksia attenuata
R
2
0.00
MU
Non-infested
128BS-1
Banksia attenuata
S
31
0.00
✕
MU
Non-infested
128BS-2
Hibbertia hypericoides
S
18
0.00
✕
119
✕
✕
Site
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Significantly Reduce Lesion
Length
MU
Non-infested
129BL-1
Hibbertia hypericoides
L
33
0.00
MU
Non-infested
129BR-1
Hibbertia hypericoides
R
10
0.23
MU
Non-infested
129BR-2
Hibbertia hypericoides
R
20
0.90
MU
Non-infested
130BR-1
Eucalyptus marginata
R
33
0.00
MU
Non-infested
130BR-2
Eucalyptus marginata
R
2
0.00
✕
MU
Non-infested
130BR-3
Eucalyptus marginata
R
34
0.00
✓***
MU
Non-infested
131BL-1
Macrozamia riedlei
L
35
0.00
MU
Non-infested
131BL-2
Macrozamia riedlei
L
35
0.33
MU
Non-infested
131BL-3
Macrozamia riedlei
L
11
0.77
MU
Non-infested
131BR-1
Macrozamia riedlei
R
36
0.00
MU
Non-infested
131BR-2
Macrozamia riedlei
R
9
0.00
MU
Non-infested
131BS-1
S
35
0.17
MU
Non-infested
132BR-1
R
9
0.00
MU
Non-infested
132BS-1
S
37
0.63
MU
Non-infested
132BS-2
S
19
0.03
MU
Non-infested
132BS-3
Macrozamia riedlei
Allocasuarina
fraseriana
Allocasuarina
fraseriana
Allocasuarina
fraseriana
Allocasuarina
fraseriana
S
19
0.13
MU
Non-infested
133BR-1
Eucalyptus marginata
R
17
0.87
MU
Non-infested
133BS-1
Eucalyptus marginata
S
19
1.30
MU
Non-infested
133BS-2
Eucalyptus marginata
S
19
1.53
MU
Non-infested
134BR-1
Eucalyptus marginata
R
38
0.00
MU
Non-infested
135BR-1
Eucalyptus marginata
R
37
0.03
MU
Non-infested
136BL-1
Banksia attenuata
L
28
0.13
MU
Non-infested
136BR-1
Banksia attenuata
R
20
0.10
MU
Non-infested
138BR-1
Hakea
R
28
0.00
120
✕
✕
✕
✕
✕
Site
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
MU
Non-infested
139BL-1
Leucopogon
L
28
0.07
MU
Non-infested
139BL-2
Leucopogon
L
20
0.63
MU
Non-infested
140BL-1
Adenanthos sericeus
L
28
0.10
MU
Non-infested
140BR-1
Adenanthos sericeus
R
1
0.00
MU
Non-infested
140BR-2
Adenanthos sericeus
R
28
0.00
MU
Non-infested
140BR-3
Adenanthos sericeus
R
22
0.97
MU
Non-infested
140BS-1
Adenanthos sericeus
S
36
0.00
MU
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
Non-infested
140BS-2
Adenanthos sericeus
S
22
0.83
Infested
141BL-1
Eucalyptus marginata
L
33
0.00
Infested
141BR-1
Eucalyptus marginata
R
38
2.23
Infested
141BR-2
Eucalyptus marginata
R
38
2.57
Infested
141BR-3
Eucalyptus marginata
R
33
0.00
Infested
141BS-1
Eucalyptus marginata
S
19
1.83
Infested
142BR-1
Eucalyptus marginata
R
33
0.00
Infested
142BS-1
Eucalyptus marginata
S
38
0.30
Infested
143BR-1
Eucalyptus marginata
R
12
0.00
Infested
143BR-2
Eucalyptus marginata
R
33
0.00
Infested
143BS-1
Eucalyptus marginata
S
39
0.13
Infested
143BS-2
Eucalyptus marginata
S
40
0.07
Infested
144BL-1
Corymbia calophylla
L
33
0.03
Infested
144BR-1
Corymbia calophylla
R
35
0.00
121
Significantly Reduce Lesion
Length
✕
✕
✕
✓*
Site
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Infested
144BR-2
Corymbia calophylla
R
9
0.03
Infested
144BR-3
Corymbia calophylla
R
12
0.00
Infested
145BR-1
Corymbia calophylla
R
26
0.00
Infested
145BR-2
Corymbia calophylla
R
22
1.53
Infested
145BR-3
Corymbia calophylla
R
28
0.00
Infested
145BR-4
Corymbia calophylla
R
12
0.00
Infested
146BL-1
Eucalyptus marginata
L
38
2.47
Infested
146BL-2
Eucalyptus marginata
L
33
0.03
Infested
146BL-3
Eucalyptus marginata
L
9
0.03
Infested
146BR-1
Eucalyptus marginata
R
46
0.10
Infested
146BR-2
Eucalyptus marginata
R
46
0.03
Infested
146BS-1
Eucalyptus marginata
S
33
0.03
Infested
147BL-1
Corymbia calophylla
L
38
0.20
Infested
147BR-1
Corymbia calophylla
R
42
0.03
Infested
147BR-2
Corymbia calophylla
R
24
0.17
Infested
148BR-1
Eucalyptus marginata
R
44
0.03
Infested
148BS-1
Eucalyptus marginata
S
38
0.00
Infested
149BL-1
Eucalyptus marginata
L
24
0.00
Infested
149BR-1
Eucalyptus marginata
R
47
0.00
122
Significantly Reduce Lesion
Length
✕
✕
✓**
Site
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Infested
149BS-1
Eucalyptus marginata
S
9
0.00
Infested
149BS-2
Eucalyptus marginata
S
9
0.30
Infested
149BS-3
Eucalyptus marginata
S
48
0.13
Infested
150BL-1
Eucalyptus marginata
L
26
0.00
Infested
150BL-2
Eucalyptus marginata
L
9
1.57
Infested
150BR-1
Eucalyptus marginata
R
44
0.00
Infested
151BL-1
Eucalyptus marginata
L
33
0.13
Infested
151BR-1
Eucalyptus marginata
R
26
0.00
Infested
151BS-1
Eucalyptus marginata
S
37
0.20
Non-infested
152BL-1
Corymbia calophylla
L
33
0.17
Non-infested
152BR-1
Corymbia calophylla
R
33
0.03
Non-infested
152BS-1
Corymbia calophylla
S
33
0.00
Non-infested
153BL-1
Eucalyptus marginata
L
33
0.00
Non-infested
153BR-1
Eucalyptus marginata
R
2
0.00
Non-infested
153BS-1
Eucalyptus marginata
S
33
0.17
Non-infested
154BL-1
Eucalyptus marginata
L
33
0.10
Non-infested
154BR-1
Eucalyptus marginata
R
9
0.40
Non-infested
154BR-2
Eucalyptus marginata
R
33
0.00
Non-infested
154BS-1
Eucalyptus marginata
S
38
0.00
123
Significantly Reduce Lesion
Length
✕
Site
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
GS
1
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Non-infested
154BS-2
Eucalyptus marginata
S
33
0.13
Non-infested
155BR-1
Eucalyptus marginata
R
2
0.00
Non-infested
155BS-1
Eucalyptus marginata
S
38
2.07
Non-infested
155BS-2
Eucalyptus marginata
S
33
0.00
Non-infested
156BR-1
Corymbia calophylla
R
35
0.57
Non-infested
156BR-2
Corymbia calophylla
R
9
0.00
Non-infested
156BR-3
Corymbia calophylla
R
33
0.23
Non-infested
156BS-1
Corymbia calophylla
S
33
0.03
Non-infested
157BR-1
Eucalyptus marginata
R
41
0.07
Non-infested
157BR-2
Eucalyptus marginata
R
9
0.00
Non-infested
157BR-3
Eucalyptus marginata
R
19
1.40
Non-infested
158BR-1
Eucalyptus marginata
R
41
0.00
Non-infested
159BL-1
Eucalyptus marginata
L
22
0.97
Non-infested
159BL-2
Eucalyptus marginata
L
33
0.17
Non-infested
159BR-1
Eucalyptus marginata
R
33
0.27
Non-infested
159BS-1
Eucalyptus marginata
S
33
0.00
Non-infested
160BL-1
Eucalyptus marginata
L
24
0.20
Non-infested
160BL-2
Eucalyptus marginata
L
33
0.00
Non-infested
160BR-1
Eucalyptus marginata
R
33
0.27
124
Significantly Reduce Lesion
Length
✕
Site
GS
1
GS
1
GS
1
GS
1
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Non-infested
160BS-1
Eucalyptus marginata
S
33
0.83
Non-infested
161BL-1
Eucalyptus marginata
L
33
0.00
Non-infested
161BR-1
Eucalyptus marginata
R
35
0.00
Non-infested
161BS-1
Eucalyptus marginata
S
33
0.00
Infested
162BL-1
Eucalyptus marginata
L
9
0.07
Infested
162BR-1
Eucalyptus marginata
R
9
0.07
Infested
162BS-1
Eucalyptus marginata
S
41
0.00
Infested
163BL-1
Eucalyptus marginata
L
22
1.07
Infested
163BL-2
Eucalyptus marginata
L
33
0.00
Infested
163BR-1
Eucalyptus marginata
R
24
0.00
Infested
163BR-2
Eucalyptus marginata
R
16
0.17
Infested
163BR-3
Eucalyptus marginata
R
33
0.00
Infested
163BS-1
Eucalyptus marginata
S
33
0.00
Infested
164BL-1
Eucalyptus marginata
L
33
0.23
Infested
164BR-1
Eucalyptus marginata
R
33
0.17
Infested
164BS-1
Eucalyptus marginata
S
35
0.03
Infested
164BS-2
Eucalyptus marginata
S
33
0.03
Infested
165BR-1
Eucalyptus marginata
R
41
0.10
Infested
165BR-2
Eucalyptus marginata
R
42
0.10
125
Significantly Reduce Lesion
Length
✓***
✕
✕
Site
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Infested
165BR-3
Eucalyptus marginata
R
19
0.47
Infested
165BS-1
Eucalyptus marginata
S
33
0.00
Infested
165BS-2
Eucalyptus marginata
S
33
0.10
Infested
166BL-1
Corymbia calophylla
L
33
0.30
Infested
166BR-1
Corymbia calophylla
R
43
0.07
Infested
166BR-2
Corymbia calophylla
R
9
0.00
Infested
166BR-3
Corymbia calophylla
R
9
0.00
Infested
166BR-4
Corymbia calophylla
R
33
0.10
Infested
166BS-1
Corymbia calophylla
S
33
0.07
Infested
167BL-1
Corymbia calophylla
L
12
0.00
Infested
167BL-2
Corymbia calophylla
L
33
0.07
Infested
167BR-1
Corymbia calophylla
R
24
0.00
Infested
167BR-2
Corymbia calophylla
R
24
0.00
Infested
167BS-1
Corymbia calophylla
S
12
0.00
Infested
168BL-1
Corymbia calophylla
L
24
0.00
Infested
168BL-2
Corymbia calophylla
L
33
0.17
Infested
168BR-1
Corymbia calophylla
R
35
0.00
Infested
168BR-2
Corymbia calophylla
R
35
0.00
Infested
168BS-1
Corymbia calophylla
S
33
0.00
126
Significantly Reduce Lesion
Length
✕
✕
Site
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Infested
169BL-1
Eucalyptus marginata
L
33
0.00
Infested
169BS-1
Eucalyptus marginata
S
33
0.00
Infested
170BL-1
Eucalyptus marginata
L
9
0.00
Infested
170BR-1
Eucalyptus marginata
R
35
0.07
Infested
170BR-2
Eucalyptus marginata
R
33
0.67
Infested
170BS-1
Eucalyptus marginata
S
33
0.13
Infested
171BL-1
Eucalyptus marginata
L
2
0.57
Infested
171BL-2
Eucalyptus marginata
L
33
0.00
Infested
171BR-1
Eucalyptus marginata
R
42
0.00
Infested
171BR-2
Eucalyptus marginata
R
41
0.13
Infested
171BS-1
Eucalyptus marginata
S
33
0.17
Non-infested
172BL-1
Corymbia calophylla
L
19
0.97
Non-infested
172BR-1
Corymbia calophylla
R
9
0.10
Non-infested
172BR-2
Corymbia calophylla
R
24
0.00
Non-infested
172BS-1
Corymbia calophylla
S
38
0.07
Non-infested
172BS-2
Corymbia calophylla
S
33
0.13
Non-infested
173BL-1
Corymbia calophylla
L
33
0.13
Non-infested
173BR-1
Corymbia calophylla
R
9
0.00
Non-infested
173BR-2
Corymbia calophylla
R
38
0.07
127
Significantly Reduce Lesion
Length
✓***
✕
✕
Site
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Non-infested
173BR-3
Corymbia calophylla
R
33
0.00
Non-infested
173BS-1
Corymbia calophylla
S
33
0.17
Non-infested
174BL-1
Eucalyptus marginata
L
33
0.00
Non-infested
174BL-2
Eucalyptus marginata
L
33
0.03
Non-infested
174BR-1
Eucalyptus marginata
R
42
0.00
Non-infested
174BR-2
Eucalyptus marginata
R
33
0.00
Non-infested
174BS-1
Eucalyptus marginata
S
33
0.00
Non-infested
175BL-1
Corymbia calophylla
L
33
0.03
Non-infested
175BS-1
Corymbia calophylla
S
33
0.13
Non-infested
176BS-1
Corymbia calophylla
S
33
0.07
Non-infested
177BL-1
Eucalyptus marginata
L
33
0.10
Non-infested
177BR-1
Eucalyptus marginata
R
42
0.07
Non-infested
177BS-1
Eucalyptus marginata
S
33
0.00
Non-infested
178BL-1
Eucalyptus marginata
L
44
0.00
Non-infested
178BR-1
Eucalyptus marginata
R
44
0.00
Non-infested
178BS-1
Eucalyptus marginata
S
44
0.00
Non-infested
179BL-1
Eucalyptus marginata
L
44
0.10
Non-infested
179BR-1
Eucalyptus marginata
R
38
0.10
Non-infested
179BS-1
Eucalyptus marginata
S
44
0.20
128
Significantly Reduce Lesion
Length
✕
✕
Site
GS
2
GS
2
GS
2
GS
2
GS
2
GS
2
Infested/Noninfested
Isolate
Number
Host Plant Species
Host
Tissue
Morphotype
Number
Mean in vitro inhibition
Zone
Non-infested
180BL-1
Eucalyptus marginata
L
44
0.23
Non-infested
180BS-1
Eucalyptus marginata
S
44
0.10
Non-infested
181BL-1
Eucalyptus marginata
L
44
0.00
Non-infested
181BL-2
Eucalyptus marginata
L
44
0.13
Non-infested
181BR-1
Eucalyptus marginata
R
45
0.10
Non-infested
181BS-1
Eucalyptus marginata
S
44
0.13
L, R, S indicates leaf root and stem plant tissue
GS1= jarrah forest graveyard site 1
GS2= jarrah forest graveyard site 2
MU= Murdoch woodland site
 indicates significantly reduced lesion length
 indicates did not significantly reduce lesion length
* (P<0.05)
** (P<0.01)
*** (P<0.001)
129
Significantly Reduce Lesion
Length
✕
130