*Warsaw University of Life Sciences, Warsaw, Poland
**Plant Breeding and Acclimatization Institute –
National Research Institute, Radzików, Poland
***Poznań University of Life Sciences, Poznań, Poland
SUSCEPTIBILITY OF FLINT AND DENT MAIZE EARS
TO FUSARIUM SPECIES1
*M. Wit, **R. Warzecha, *E. Mirzwa-Mróz, *E. Jabłońska,
**P. Ochodzki, ***A. Waśkiewicz and *W. Wakuliński
Abstract
Ear rot susceptibility of Zea mays var. indentata (dent maize) and Z. mays var.
indurata (flint maize) was tested with the silk channel and nail punch inoculation
method, in relation to Fusarium spp., weather conditions and kernel starch component content. In a three-year field experiment (2007–2009), ears of Z. mays var.
indentata showed a significantly higher degree of infection. Total starch concentration in kernels of this variety ranged from 70 to 89% and did not differ statistically
from starch content in flint kernels (62–85%). In contrast, concentration of
amylose responsible for kernel hardness differed significantly and was higher in
endosperm of Z. mays var. indurata (25–29%) than in Z. mays var. indentata
(20–22%). Isolates of Fusarium graminearum, F. culmorum and F. subglutinans were
found the most aggressive, regardless of growing season and inoculation method
applied.
Key words: ear rot, Fusarium, maize
Introduction
Maize (Zea mays) is, along with wheat and rice, one of the major cereal grains,
with estimated annual global production in 2010 of over 817 million tons and a
cultivation area of approximately 160 million hectares (World... 2011). During the
The work was financed by the Ministry of Science and Higher Education, project No. N N310
376933.
1
Phytopathologia 60: 35–45
© The Polish Phytopathological Society, Poznań 2011
ISSN 2081-1756
36
M. Wit et al.
last decade, the cereal has also become an increasingly important crop in Poland
(Księżak and Bojarczuk 2010). Zea mays var. indentata (dent) and Z. mays var.
indurata (flint) are among the most common types of maize cultivated in Europe
(Hallauer et al. 2010). They differ in various properties such as plant growth, ear
number per stem, ear size, and vegetation period, but particularly in morphology
and anatomy of kernels. Kernels of flint type are characterized by a hard outer endosperm layer enclosing the soft endosperm, while dent maize does not contain
the mentioned layer at the kernel top (Dickerson 2003).
An important disadvantage of maize is its susceptibility to Fusarium pathogens
(White 1999). They attack plants at different developmental stages, leading to root
rot, seedling blight, stalk rot and ear rot, which is considered the most important
disease of this crop worldwide. Ear rot caused by Fusarium results in yield decrease,
poor grain quality and contamination by various mycotoxins (Logrieco et al. 2002).
Data on susceptibility of Z. mays var. indentata and Z. mays var. indurata to ear rot are
confusing. Löffler et al. (2010) reported higher disease severity on maize with flint
than dent endosperm type, opposite results were presented by Doko et al. (1996)
as well as Czembor and Ochodzki (2009), while Hennigen (2000) did not note any
significant differences in cob infection degree between genotypes of flint and dent
maize.
Etiology of the disease is complex, but in practice the principal causative factors
are members of the Discolor and Liseola sections of Fusarium genus. The diverse
symptomatology associated with these two groups of fungi is emphasized by different disease names, i.e. pink and red ear rot. Red (or Gibberella) ear rot is caused
mostly by F. graminearum and F. culmorum, while F. verticillioides, F. subglutinans and
F. proliferatum are considered etiological factors of pink ear rot. Maize ears are also
infected with F. avenaceum, F. croockwellense, F. culmorum, F. equiseti, F. poae, F. temperaum, and F. sporotrichioides (Bottalico 1998, Hussein et al. 2002, Logrieco et al.
2002, Dorn et al. 2009, Scauflaire et al. 2011). However, the mentioned fusaria are
observed seldom and they are usually reported as maize stalk pathogens (Lew et al.
1997). Their occurrence increases the risk of corn contamination by secondary metabolites (Logrieco et al. 2003).
The incidence of Fusarium species varies in particular cropping seasons and is
strongly influenced by environmental conditions (Wit et al. 2007, 2009, Dorn et al.
2009, Scauflaire et al. 2011). High temperature generally promotes occurrence of
F. graminearum, whose optimum temperatures for growth (25°C; Campbell and
Lipps 1998) and inoculum production (25–29°C; Sutton 1982) exceed the corresponding optima of F. avenaceum or F. culmorum (Brennan et al. 2003).
The purpose of this study was to analyze disease severity after plant inoculation
with Fusarium species recognized in Poland as the cause of maize ear rot, in relation
to weather conditions and kernel starch component content.
Susceptibility of flint and dent maize ears...
37
Materials and methods
The pathogenicity test was set up in a three-factorial design with two replications (maize variety, Fusarium species, inoculation method) and performed during
three growing seasons (2007–2009) in an experimental field of the Plant Breeding
and Acclimatization Institute (PBAI) at Radzików.
Plant material
Two maize botanical varieties were used: Zea mays var. indentata and Z. mays var.
indurata.
Fungal strains
Seven Fusarium isolates (F. avenaceum, F. culmorum, F. equiseti, F. graminearum,
F. proliferatum, F. subglutinans and F. verticillioides) were used. They had been isolated
from rotten maize cobs and stored in the culture collection of the Department of
Plant Pathology, Warsaw University of Life Sciences (SGGW). Isolates were grown
on PDA in Petri dishes for 10 days at 22oC. Aerial mycelium was scraped, suspended in distilled water and filtered through cheesecloth. Concentration of suspension was adjusted to 106 cfu per 1 ml.
Plant inoculations and disease severity rating
Eight plants per replication were inoculated at R1 maize growth stage (Ritchie
et al. 1993) using the silk channel and nail punch method (Chungu et al. 1996).
Disease severity ratings were performed at the end of the growing season according to a 6-degree scale (0 – no symptoms, 5 – very severe infection with over 50% of
kernels damaged) as described by Wit et al. (2009).
Kernel analysis
Amylose and starch analysis was performed with the Amylose/Amylopectin
Assay Kit (Megazyme) according to the procedure recommended by the producer
and adapted to low volumetric samples.
Environmental data and statistics
Temperature and rainfall were recorded by the automatic meteorological station located at PBAI Radzików. The data obtained were analyzed statistically using
Statgraphics 4.0 for Windows.
38
M. Wit et al.
Results
A significant influence of growing season on disease severity was found. The
lowest infection degree of maize ears occurred in 2009, while there were no significant differences between 2007 and 2008 seasons. Statistical differences in infection degree at = 0.05 between 2009 and previous years were found for isolates of
three fungus species: F. avenaceum, F. culmorum and F. graminearum (Table 1).
The highest aggressiveness was exhibited by isolates of F. graminearum, F. culmorum and F. subglutinans, regardless of the applied inoculation methods (Fig. 1). DisTable 1
Mean infection degree of maize cobs
Year
Mean
2007
2.68 a
2009
1.90 b
2008
Mean
2.65 a
Fusarium
avenaceum
Fusarium
culmorum
2.07 a
3.02 a
2.37 a
1.08 b
1.84 c
3.05 a
2.18 b
2.75 b
Fusarium Fusarium
Fusarium
Fusarium
Fusarium
equiseti graminearum proliferatum subglutinans verticillioides
2.14 a
4.18 a
2.26 a
2.89 a
2.13 a
1.64 a
1.84 b
2.20 a
2.88 a
1.63 a
1.92 a
1.90 c
4.00 a
3.34 a
2.10 a
2.18 c
Numbers followed by the same letter are not significantly different at
3.48 a
3.08 b
= 0.05.
Fig. 1. Aggressiveness of Fusarium species towards maize cobs inoculated
by silk channel (SCIM) and nail punch (NPIM) methods
1.92 a
1.89 c
Susceptibility of flint and dent maize ears...
39
Fig. 2. Distribution of maize infection degree after infection with Fusarium culmorum,
F. graminearum and F. subglutinans, mean values for three years (2007–2009)
tribution of plant infection degree was species specific for the pathogens in
question, in spite of mean pathogenicity of F. culmorum and F. subglutinans not being
statistically different (Fig. 2, Table 1).
Zea mays var. indentata was generally more susceptible to the tested Fusarium
spp. than Z. mays var. indurata, although host response depended on the applied inoculation technique. Significant differences in disease severity between maize varieties were noted after silk cob inoculation with all Fusarium species with the
exception of F. equiseti (Fig. 3). The nail punch method differentiated susceptibility
of maize varieties only to F. verticillioides (Fig. 4).
In particular years, kernels of both maize varieties contained a similar level of
starch, but amylose concentration was significantly higher in endosperm of Z. mays
var. indurata (Table 2).
Fig. 3. Reaction of Zea mays var. indentata and Z. mays var. indurata after silk channel
inoculation with seven Fusarium species
40
M. Wit et al.
Fig. 4. Reaction of Zea mays var. indentata and Z. mays var. indurata after nail punch
inoculation with seven Fusarium species
Starch and amylose content in kernels of Zea mays var. indentata
and Z. mays var. indurata (%)
Compound
Starch
Amylose
indurata
77.90 a
27.62 a
2007
indentata
indurata
22.90 b
29.88 a
76.54 a
2008
85.64 a
indentata
indurata
21.35 b
25.97 a
89.00 a
Numbers followed by the same letter are not significantly different at
62.05 a
2009
Table 2
indentata
70.23 a
20.61 b
= 0.05.
Discussion
During the study years (2007–2009) Z. mays var. indentata was more susceptible
to tested Fusarium spp. than Z. mays var. indurata. Recently, a similar conclusion but
restricted to two Fusarium species (F. graminearum and F. verticillioides) and based on
a one-year study was presented by Czembor and Ochodzki (2009). Several factors
may influence maize resistance to ear rot. Available records emphasize the role of
kernel structure, especially thickness of pericarp, wax and aleurone layers (Cle-
Susceptibility of flint and dent maize ears...
41
ments et al. 2004). The hard kernel coat, typical of flint maize, can make this variety less susceptible to fungal infection than dent maize (Bennetzen and Hake
2009). Moreover, as we found, flint kernels contain more amylose. The content of
this endosperm component correlated with physical properties of kernels, mainly
their hardness (Robutti et al. 2000) and in consequence with the susceptibility to
biological damage (Pearson et al. 2010).
Among the Fusarium species tested, the highest aggressiveness was shown by
members of the Discolor section, especially F. graminearum. The fungus is reported a
common, destructive pathogen of cereals, including maize, and several factors may
contribute to its pathogenic properties. Researchers have in particular stressed the
role of some enzymes such as pectinases, cellulases and xylanases, which cause
fragmentation of cellulose microfibrils and facilitate spread of the pathogen in
plant tissue (Wanjiru et al. 2002). Plant colonization by F. graminearum hyphae can
also be promoted by toxins of this fungus (mostly trichothecenes). However, their
production is not necessary during the course of infection (Desjardins et al. 1996).
On the other hand, a molecular approach indicates involvement in pathogenesis of
Tri5 gene products of the trichothecene biosynthetic gene cluster (Bai et al. 2001),
MAP kinase genes (Hou et al. 2002, Urban et al. 2003) and the carbamoyl-phosphate synthase 1 gene CPS1 (Lu et al. 2003). Maize infections as serious as those
caused by F. graminearum or F. culmorum have also been reported for F. subglutinans.
In this case, knowledge regarding pathogenesis is less advanced, but the fungus
has been commonly isolated worldwide (Logrieco et al. 1993, Wit et al. 2007,
2009, Tamburic-Ilincic and Schaafsma 2009, Goertz et al. 2010). Intriguing is the
weak pathogenicity of F. verticillioides, a species taxonomically very close to
F. subglutinans. Although the occurrence of highly aggressive strains has been noted
in the F. verticillioides population (Iglesias et al. 2010), their low frequency is evidence that low pathogenicity is favoured in the process of natural selection. This
could be explained by the endophytic properties of F. verticillioides, which often colonizes maize without symptoms, both the plant and the fungus benefiting from
such coexistence (Lee et al. 2009).
A substantial impact on expression of Fusarium pathogenicity is also exerted by
environmental conditions. In 2009 the reason of low severity of ear rot caused by
F. graminearum, F. culmorum and F. avenaceum was probably temperature. Although
mean temperatures during the growing season were very similar, and even the
month-to-month temperature fluctuation was low (Fig. 5), August of 2009 was
characterized by the highest number of days with temperature below 19oC (Fig. 6).
On the other hand, it is known that the optimum temperature for growth of the
fungi considered is 20–25oC (Pettitt et al. 1996, Campbell and Lipps 1998). The remaining species tested are more eurythermal, and temperature changes had no noticeable influence on their pathogenicity during 2007–2009.
Ear rot epidemics are generally related to rain and humid weather (Sutton
1982, Vigier et al. 1997, Stewart et al. 2002). The minimum water activity (aw) for
Fusarium germination and growth is around 0.85–0.90. Fusarium verticillioides and
F. proliferatum develop at aw = 0.88, while F. culmorum and F. graminearum develop at
42
M. Wit et al.
Month
July
August
September
o
2007
o
2008
o
2009
temp. ( C)
rain (mm)
temp. ( C)
rain (mm)
temp. ( C)
rain (mm)
19.3
58.2
18.8
51.4
18.5
81.4
19.1
13.6
94.8
48.0
19.5
12.7
62.5
93.4
20.0
15.4
138.6
12.8
Fig. 5. Daily rainfall (black bars) and temperature (gray area) in experimental fields of the PBAI at
Radzików in 2007–2009 and table of mean temperature and sum of rainfall in particular months
Fig. 6. Temperature distribution in August 2007–2009
aw = 0.90 (Hope et al. 2005). Rain also promotes dispersal of Fusarium spores and
contributes to their long-distance transmission (Goswami and Kistler 2004). During the study (2007–2009), water supply was not a limiting factor for fusaria development, which might be the reason for the lack of a relationship at a detectable
level between precipitation and ear rot severity.
Streszczenie
PODATNOŚĆ KOLB KUKURYDZY PASTEWNEJ I ZWYKŁEJ
NA PORAŻENIE PRZEZ FUSARIUM SPP.
Fuzarioza kolb kukurydzy jest zaliczana do najgroźniejszych chorób w uprawie
tej rośliny. Etiologia choroby jest złożona, a wśród czynników sprawczych wymienia się szereg gatunków Fusarium, w tym szczególną rolę odgrywają przedstawiciele
sekcji Discolor i Liseola. W trakcie trzyletnich (2007–2009) inokulacyjnych badań
Susceptibility of flint and dent maize ears...
43
polowych, realizowanych na terenie pól doświadczalnych IHAR w Radzikowie,
stwierdzono zróżnicowaną podatność odmian kukurydzy na fuzariozę kolb, jak też
patogeniczność uwzględnionych gatunków Fusarium. Niezależnie od sezonu wegetacyjnego oraz metody inokulacji roślin istotnie silniejszemu porażeniu ulegały
kolby kukurydzy pastewnej (Zea mays var. indentata) niż zwykłej (Z. mays var. indurata). Średni stopień porażenia tych odmian w okresie czteroletnim wynosił odpowiednio 2,7 i 2,1 w przyjętej 6-stopniowej skali.
Spośród siedmiu gatunków Fusarium uwzględnionych w badaniach największą
patogeniczność wykazywały F. graminearum, F. culmorum oraz F. subglutinans.
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Authors’ addresses:
Marcin Wit M.Sc., Dr. Ewa Mirzwa-Mróz, Emilia Jabłońska, Dr. hab.
Wojciech Wakuliński, Department of Plant Pathology, Warsaw University of
Life Sciences, ul. Nowoursynowska 159, 02-787 Warszawa, Poland, e-mail:
[email protected]
Dr. Roman Warzecha, Dr. Piotr Ochodzki, Plant Breeding and
Acclimatization Institute – National Research Institute, Radzików, 05-870
Błonie, Poland
Dr. Agnieszka Waśkiewicz, Department of Chemistry, Poznań University of
Life Sciences, ul. Wojska Polskiego 75, 60-625 Poznań, Poland
Accepted for publication: 12.05.2011