The Pennsylvania State University
The Graduate School
Department of Plant Pathology and Environmental Microbiology
CHARACTERIZATION OF Pythium and Phytopythium SPECIES FREQUENTLY
FOUND IN IRRIGATION WATER
A Thesis in
Plant Pathology
by
Carla E. Lanze
© 2015 Carla E. Lanze
Submitted in Partial Fulfillment
of the Requirement
for the Degree of
Master of Science
August 2015
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The thesis of Carla E. Lanze was reviewed and approved* by the following
Gary W. Moorman
Professor of Plant Pathology
Thesis Advisor
David M. Geiser
Professor of Plant Pathology
Interim Head of the Department of Plant Pathology and Environmental Microbiology
Beth K. Gugino
Associate Professor of Plant Pathology
Todd C. LaJeunesse
Associate Professor of Biology
*Signatures are on file in the Graduate School
iii
ABSTRACT
Some Pythium and Phytopythium species are problematic greenhouse crop pathogens.
This project aimed to determine if pathogenic Pythium species are harbored in greenhouse
recycled irrigation water tanks and to determine the ecology of the Pythium species found in
these tanks. In previous research, an extensive water survey was performed on the recycled
irrigation water tanks of two commercial greenhouses in Pennsylvania that experience frequent
poinsettia crop loss due to Pythium aphanidermatum. In that work, only a preliminary
identification of the baited species was made. Here, detailed analyses of the isolates were
conducted. The Pythium and Phytopythium species recovered during the survey by baiting the
water were identified and assessed for pathogenicity in lab and greenhouse experiments. The
Pythium species found during the tank surveys were: a species genetically very similar to P. sp.
nov. OOMYA1702-08 in Clade B2, two distinct species of unknown identity in Clade E2, P.
coloratum or one of the very closely related species such as P. diclinum, P. middletonii, an
unknown species in Clade B2, an isolate somewhat similar to P. sp. nov. OOMYA1646-08 (E2),
P. rostratifingens, and an unknown species in Clade A. In addition, three Phytopythium species
were found: Phytopythium litorale, Ph. helicoides, and Ph. chamaehyphon. Many of these
species are considered weak pathogens and some display resistance to the Oomycete fungicide,
mefenoxam. Of the baited isolates, seven expressed resistance (Ph. helicoides, Clade E2-2
unknown, P. middletonii, P. sp. nov. OOMYA1646-08 (E2),) with three displaying high
resistance (P. coloratum, P. rostratifingens, Clade A unknown). Seven expressed sensitivity (Ph.
helicoides, Clade B2 unknown, P. sp. nov. OOMYA1646-08 (E2), Ph. chamaehyphon) with
three displaying high sensitivity (Clade E2-1 unknown, P. coloratum, Clade E2-2 unknown). In a
lab experiment, using Pelargonium X hortorum seeds germinated on moistened filter paper,
some of the baited isolates were pathogenic. However in another test using small pots containing
pasteurized, peat-based soilless potting mix, none of the baited isolates were pathogenic on
geranium seedlings. It was assessed whether or not these isolates that were frequently obtained
by baiting interfere with known pathogenic Pythium species, P. aphanidermatum, P. irregulare,
and P. cryptoirregulare, in disease development. Some of the isolates slowed or promoted plant
disease in the lab test using geranium seedlings on moistened filter paper, but these results were
unable to be reproduced in the greenhouse experiments under more natural production
conditions. At the end of the greenhouse experiments, root sections were plated in order to
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recover isolates. It was found that in the co-inoculated plants, P. irregulare and P.
cryptoirregulare were almost always the only species recovered from the roots. The baited
isolates were still recovered from the roots in the control plants. Lastly, a simulation of the
greenhouse ebb and flood irrigation system was set up to determine if P. aphanidermatum can
coexist with representatives of the frequently baited isolates in recycled irrigation water tanks. P.
aphanidermatum was not recovered from any of the tanks or on the roots of plants the tanks
watered. We conclude that there is an array of Pythium and Phytopythium species that reside in
greenhouse irrigation systems, and that P. aphanidermatum is not one of those species. Thus,
treating irrigation water with chlorine or other chemicals to remove Pythium spp. may not be
necessary in greenhouses where potted plants are irrigated with recycled water. We also
conclude that the highly virulent species Pythium irregulare and Pythium cryptoirregulare have
attributes that allow them to dominate the niche of plant roots over those species frequently
found in the irrigation water.
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TABLE OF CONTENTS
List of Tables……………………………………………………………………………..…….vii
List of Figures………………………………………………………………………………….viii
Acknowledgements…………………………………………………………………………...…ix
Chapter 1. LITERATURE REVIEW…………………………………………….………………1
The Genera Pythium and Phytopythium……………………………….…………...………....….1
Pythium in Greenhouses………………………………………………………………………….2
Pythium Evolution & Ecology………………………………...………………………………… 6
Objectives……………………………………………………………………………………….10
Literature cited……………………………………………………………....……………...…..11
Chapter 2. IDENTIFICATION AND CHARACTERIZATION OF Pythium AND
Phytopythium SPECIES IN TWO COMMERCIAL GREENHOUSE RECYCLING
IRRIGATION WATER SYSTEMS……………………………………………………..…......20
Abstract……….…………………………………………………………………………...……20
Introduction………………………………………………………………………………...…...20
Materials and Methods……………………………………………………………………….....23
Baiting and Biological Characterization…………………………………………..…....23
Mefenoxam Resistance………………………………………………………………....24
Genetic characterization……………………………………………………………..….25
Results………………………………………………………………………………………......26
Discussion………………………………………………………………...……………....….....47
Acknowledgements………………………………………………………………...…………...49
Literature cited……………………………………………………….……………...………….50
Chapter 3. PATHOGENICITY OF THE SPECIES OF Pythium AND Phytopythium
FREQUENTLY FOUND IN RECYCLED IRRIGATION WATER AND THEIR
INTERACTIONS WITH Pythium aphanidermatum, P. irregulare, AND
P. cryptoirregulare………………………………………………………………………….…..56
Abstract…………………………………………………………………………………..……..56
Introduction…………………………………………………………….……………………….56
Materials and Methods……………………………………………………………………...…..58
Results………………………………………………………………………………………......62
Discussion………………………………………………………………………………..…..…65
Acknowledgements….…………………………………………………………………...……..67
Literature cited…….……………………………………………………………………...….…68
Chapter 4. AQUATIC SURVIVAL OF Pythium aphanidermatum,
Phytopythium helicoides AND Pythium coloratum……………………………….....................71
Abstract……..………………………………………………………………………………..…71
vi
Introduction……...…………………………………………………………………………...…71
Materials and Methods……………………………………………………………………….....72
Results………………………………………………………………………………………......74
Discussion…………………………………………………………………………………........76
Acknowledgements…………………………………………………………………………......77
Literature cited……………………………………………………………………………….....78
CONCLUSION……………………………………………………………………………...….80
Appendix: Supplementary Data……………………………………………………….………..83
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LIST OF TABLES
Table 2-1. The cardinal temperatures of the baited isolates, their daily growth rates, and colony
morphology on PCA....................................................................................................................39
Table 2-2. The means in µm of structures of baited Pythium species. …………………………45
Table 2-3. Results of the poison plate assay. …………………………………………………..46
Table 3-1. Pathogenicity on geranium seedlings grown on filter paper moistened with soluble
fertilizer and co-inoculation results.……………………………………..……………………...64
Table 3-2. Combined results of the root isolations from the co-inoculation experiments…..….64
Table 4-1. Experimental setup for ebb and flow experiment………………………………..….73
Table 4-2. The number of hours the tanks had water temperatures between 25° and 30°
C.………………………………………………………………………………………………..75
Table 4-3. Isolates from baits during the experiment……………………………………...........75
Table A-1. Initial morphological observations of the isolates. ………………………………...86
Table A-2. A summary of the Pythium isolates baited from greenhouse irrigation tanks……...91
Table A-3. The cardinal temperatures of the isolates, mean daily growth rates
at 25C on PCA, and colony morphology on PCA……………………………………….….…..92
Table A-4. Full results of the poison plate assay…………………………………………....…..94
Table A-5. A list of the isolates used for detailed microscopic identification………………….97
Table A-6. The average water temperature during the week the isolates were initially baited
from the two greenhouses, compared to their cardinal temperatures…………………..........….98
Table A-7. Average water temperature (°C) for 7 day periods ending on the sampling date in
two commercial greenhouses………………………………………………………………...…99
Table A-8. Isolate pathogenicity and co-inoculating results……………...……..…………….100
Table A-9. The representative isolates used in the greenhouse pathogenicity and co-inoculation
tests……………………………………………....………………………………….…………101
Table A-10. A list of the isolates used for the lab soil pathogenicity tests…………………....103
Table A-11. Average weekly tank temperatures in the tank isolate survival tests………..…...104
viii
LIST OF FIGURES
Figure 2-1. A maximum likelihood analysis concatenated gene tree of the
ITS and COII regions with 1000 bootstraps……………………………..……………………..33
Figure 2-2. A new species analysis of the isolates of unknown identity in Clade E2…….....…34
Figure 2-3. A portion of the new species analysis for the Clade B2 unknown isolate……..…..35
Figure 2-4. A selection of sequence alignments from our baited isolates and their
most closely related species…………………………………………………………………….36
Figure 2-5. The Pythium and Phytopythium species baited from greenhouse E,
displayed by the number of isolates baited per month………………………………….………37
Figure 2-6. The Pythium and Phytopythium species baited from greenhouse S,
displayed by the number of isolates baited per month………………………………………….37
Figure 2-7. Pythium colony morphologies…………………………………………….…..……38
Figure 2-8. Clade E2-1 unknown characteristics…………………………………………..…...40
Figure 2-9. Pythium coloratum characteristics………………………………………….…..….40
Figure 2-10: Phytopythium helicoides characteristics……………………………………….….41
Figure 2-11: Clade E2-2 unknown characteristics………………………………………….......41
Figure 2-12. Pythium middletonii characteristics………………………………………….……42
Figure 2-13. Clade B2 unknown characteristics…………………………………………..……42
Figure 2-14. Pythium sp. nov. OOMYA1646-08 (E2) characteristics…………………….……43
Figure 2-15. Pythium rostratifingens characteristics………………………………………..….43
Figure 2-16. Phytopythium chamaehyphon characteristics……………………………………..44
Figure 2-17. Clade A unknown characteristics………………………………………………....44
Figure 3-1. The experimental setup of the soilless pathogenicity tests…………………………60
Figure 3-2. The laboratory potting mix pathogenicity test setup……………...…………..……61
Figure 3-3. The greenhouse experimental setup………………………………………….…….62
Figure 4-1. The experimental setup………………………………….…………………………73
Figure A-1. ITS and cox sequences from a representative isolate of each species baited….…105
ix
ACKNOWLEDGEMENTS
Firstly, I would like to thank my advisor, Dr. Gary Moorman, for his omnipresent support during
this project. Next, I acknowledge my committee and any professor at Penn State whom I have
interacted with during my time here. All of these people have contributed to my success as a
graduate student and I am very thankful for the time they have taken out of their schedule to talk
with me. I would especially like to thank Miss Jessie Edson, our former laboratory technician,
and Miss Sara Getson, an undergraduate researcher of the lab, for their technical support on these
projects. I also thank Dr. Maria Burgos-Garay, our former graduate student and postdoc, who
contributed to some of this work and helped get me acquainted in the laboratory. I thank Ms.
Sara May for kindly letting me use her microscope and camera. Finally I would like to
acknowledge the USDA-ARS Specialty Crops Research Initiative Grant (SCRI Project #: 201051181-21140): “Integrated management of zoosporic pathogens and irrigation water quality for a
sustainable green industry” for making this research project possible.
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Chapter 1
LITERATURE REVIEW
The Genera Pythium and Phytopythium
Pythium Pringsh. is a genus of organisms in the class Oomycota and kingdom
Chromalveolata, subgroup-Stramenopiles/Heterokontophyta (7). Pythium has a cosmopolitan
distribution and there are over 130 described species in the genus (24; 49; 70). Oomycetes,
belonging to the Heterokont grouping Pseudofungi (55), are fungus-like in their morphology,
reproductive strategies, and in their mode of plant infection (38). Oomycetes used to be classified
in the kingdom Fungi before genetic tools were available. Thus, it was regarded that these
similarities between Fungi and Oomycetes were merely superficial and result from convergent
evolution (4). Indeed, there are substantial differences between true Fungi and Oomycetes
including cell wall composition and vegetative state ploidy (42). A pairing of morphological
differences with analysis of small subunit rRNA and protein-encoding genes verified that
Oomycetes were not in the kingdom Fungi (5). Nonetheless, convergent evolution was not a
comprehensive explanation as to why the fungi and Oomycetes are so similar. Phylogenetic
techniques have revealed that the Oomycetes received several of their genes associated with a
plant-pathogenic lifestyle from the fungi, through horizontal gene transfer (41; 54).
Pythium derives from the Greek “pythein” – to cause rot (45), and it is quite aptly named.
Many species of Pythium are plant pathogenic, and notoriously cause damping-off on a wide
variety of host seedlings. Seeds infected with virulent Pythium species fail to germinate and
seedlings infected will collapse. If Pythium infects the plant after the seedling stage, the plant
may develop other adverse effects such as a drastic size reduction (1). Generally, Pythium
establishes its infection by releasing pectin-degrading hydrolytic enzymes in the outer root cells.
These cells lose their adhesion to each other and Pythium proceeds deeper into the root tissue (9).
Different species of Pythium vary in which degradative enzymes they release, in what amount
they are produced, and at what time they are released. This may be due to the interaction of the
host’s response to that specific species (14). A class of candidate effectors has been discovered in
the genome of the pathogen Pythium ultimum, named YxSL[RK] effectors for their repeating
amino acid motif, but nothing else is known about these potential effectors besides their place in
the genome (32).
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Pythium species utilize two life cycle stages that establish them as persistent pathogens.
From sexual reproduction, an overwintering structure (oospore) is produced. The oospore has a
thick cell wall and can survive in the soil for years, even amidst drought, freezing, and parasitism
from antagonistic microorganisms (33). Wet soils favor oospore germination in P.
aphanidermatum (63). From vesicles formed outside of the asexually produced sporangia, most
species of Pythium release zoospores. Zoospores are noteworthy for their ability to swim and for
the increase in inoculum they bring (72). Pythium species have been called 'water molds' because
of their zoospores and because many of the diseases they cause are most severe at high soil
moisture content. Pythium includes both homothallic and heterothallic species, although the
majority of species are homothallic; i.e. the majority of Pythium species can recombine their
genes and produce sexual structures by a single, clonal isolate (43). Sexual recombination occurs
after the antheridium attaches itself and transfers its nucleus to the oogonium. The resulting
oospore can germinate, produce a hypha, and then directly infect the plant, or it can produce
zoospores that can swim away and germinate near and infect a new plant. The sporangium can
directly germinate and infect the plant or produce infectious zoospores. Pythium will often kill
the plant it infects in order to absorb nutrients from the dead, more easily digestible cells (1; 2;
70).
Recently, it was discovered and confirmed that the species in Clade K of Pythium
(according to the categorization of the genus by Lévesque and De Cock, (39)) are genetically
quite distinct from the rest of the genus. Members of this clade also have a morphology that is
somewhat of an intermediary between Phytophthora and Pythium. Species in this grouping have
been given the new genus name Phytopythium (6; 17). They have ovoid to lemon-shaped
sporangia with a papilla and the sporangia proliferate internally, like Phytophthora, but
zoospores are released from a vesicle that forms externally from the sporangium, like Pythium.
Phytopythium helicoides is a problematic pathogen in greenhouses that use ebb-and-flood
watering systems (34; 68).
Pythium in Greenhouses
My research is funded by the USDA Specialty Crops Research Initiative Grant (SCRI
Project #: 2010-51181-21140): “Integrated management of zoosporic pathogens and irrigation
water quality for a sustainable green industry.” “Sustainable green industry” is a buzz-phrase,
3
because in the near future climate change and the depletion of non-renewable resources loom.
Earth is on the verge of a freshwater crisis. By 2050, it is predicted that 67% of Earth’s
population will experience water scarcity. Three-quarters of freshwater use is tied up in
agricultural production (73), therefore it ought to be the duty of appropriate scientists to help
agricultural workers in implementing environmentally friendly, water conserving practices. The
pumping of groundwater requires an increasingly higher energy input as the water supply is
depleted. Although the use of fertilizer makes farming much more land efficient, the production
of nitrogen fertilizers requires a high energy input (35) and the release of excess fertilizer into the
environment is considered a form of pollution. Recycled irrigation water seems a promising part
of the solution to these problems. Not only will its use conserve water and fertilizer, it will also
aid in the prevention of fertilizer runoff. The use of recycled irrigation water has become
compulsory in some agricultural systems due to government mandates (28). A major problem
stemming from the use of recycled irrigation water, however, is its ability to harbor and spread
pathogenic inoculum, especially zoosporic and aquatic organisms such as Pythium (12).
This research will focus on the pathogenic species: Pythium irregulare Buis., Pythium
cryptoirregulare Garzón, Yánez and Moorman, and Pythium aphanidermatum (Edson) Fitzp. as
they are regarded as constituting the major Pythium pathogens in Pennsylvania greenhouses (46).
Pythium aphanidermatum is a frequently isolated greenhouse crop pathogen and is considered
the main causal agent of root rot (78). Pythium irregulare also causes root rot (15) as well as
crown rot (19). Pythium cryptoirregulare, a species split from the P. irregulare complex (21)
also causes root rots. Pythium aphanidermatum has a broad host range and can even cause
infection in nematodes (69) as well as in deep human wounds (13); although in Pennsylvania
greenhouses it is most commonly found infecting poinsettias (46). Pythium aphanidermatum is
often implicated in causing root rot in plants grown hydroponically (67) or in rockwool (47). In
one survey, Pythium species have been found to be very common in greenhouse recycled
irrigation water, but were not identified to the species level (28). Another study found the weak
pathogens Pythium rostratum and P. dissotocum in greenhouse water sources. Both species were
able to colonize plant roots (53).
Our lab has done a survey of recycled irrigation water tanks in two Pennsylvania
greenhouses and baited many weak Pythium pathogens and some unidentified Pythium species
4
(10). The DNA sequences of some of these isolates do not match any known Pythium species
when their ITS-1, ITS-2, and cox I & II regions were compared (using BOLD ITS database and
BLAST for the cox regions). One commercial greenhouse our lab works with has experienced
massive losses of poinsettia plants due to P. aphanidermatum. Much to our lab’s chagrin, after
three years of baiting and filtering the water supply and sampling floors in two commercial
greenhouses, P. aphanidermatum has yet to be isolated; leading our lab to think P.
aphanidermatum is not an aquatic organism and not endemic to those greenhouses. The baiting
method used creeping bentgrass (Agrostis stolonifera L. 'Penn Eagle') blades, which has been
found to be the most effective bait method to date for Pythium zoospore colonization (74).
Perhaps the lack of successful baiting should not be surprising though, because in one study of
Pythium in hydroponic systems, clade B (species with non-inflated, filamentous sporangia; also
known as Pythium group F according to Plaats-Niterink (90)) was most commonly found and P.
aphanidermatum only represented 5% of the species found (22). A possible hypothesis as to why
we have not yet found P. aphanidermatum in the water is that the other Pythium species suppress
P. aphanidermatum. Some bacteria suppress Pythium due to competition for unsaturated fatty
acids (71). Nevertheless, it appears that there are several different Pythium species coexisting in
recycled irrigation water. A better understanding of these aquatic Pythium species’ ecology may
lead to a greater understanding of how to control pathogenic Pythium in water tanks.
There are four recognized ways to manage pathogens in water: cultural practices (i.e.
sanitation), physical practices (filtration), chemical control (such as fungicides applied to plants
or treatments applied to water), and biological control (64). One way biological control can be
effective for controlling Pythium diseases is by using a compost which contains high levels of
available organic matter, which will support suppressive bacterial communities (25). There are
three ways microbes can act as antagonists. One way is space competition in the rhizosphere.
Microorganisms can compete for physical space, or for the availability of carbon, nitrogen,
and/or iron. Another method of antagonism is producing secondary metabolites that are
antibiotics. The third way antagonism can happen is direct parasitism. Despite the promise of
biological control, a successful application of biocontrol methods to contaminated irrigation
systems has yet to be achieved (3).
5
UV radiation water treatment is effective at removing pathogens from the water, but the
system is not very economical. In addition, it can be challenging to prevent the introduction of
contaminated water to the treated water. Furthermore, UV radiation will also kill possible
beneficial microbial communities in the system (50). Some methods of filtration, including slow
sand filtration (77) can be effective at removing Pythium, but not small bacteria or viruses. There
are also issues associated with clogging and limitations to the amount of water that can be
filtered. Filtration by constructing wetlands is an exciting prospect, but the maintenance is high,
the clogging problem has not been completely resolved (76), and the system would have to be
indoors to be used during freezing weather. Heat treating the water gives a very similar story. It
is effective, but very expensive and can greatly reduce the number of beneficial microbes. It is
currently in use in Dutch greenhouses, largely because environmental protection laws require
greenhouses to have closed systems (23).
Chemical treatments are more effective and economical ways to manage plant pathogens
in irrigation water than the aforementioned physical practices. Treating water systems with
chlorine is very effective at eliminating plant pathogens, especially Pythium, and algae. The price
of a chlorination system is moderate and requires moderate upkeep, especially to prevent
phytotoxic effects. Chlorinating the water can produce harmful byproducts, especially those
caused by reacting with fertilizer (20). Mefenoxam is the most commonly used chemical against
Pythium in commercial greenhouses. In a study from 1996-2001, it was found that nearly 75% of
the Pythium diseased greenhouse samples submitted to the Pennsylvania Plant Disease Clinic
were infected with the species P. aphanidermatum and P. irregulare. Of the P. aphanidermatum
and P. irregulare isolates, 40% were mefenoxam-resistant (46). Therefore it is obvious that
chemical control is not the solution for greenhouse disease eradication.
The economic threshold is not established for managing plant pathogens in irrigation
water, therefore making investments on sanitation equipment or chemical regimes is not yet
known to increase profits for a grower (26). Cultural disease prevention practices require a low
financial input and are essential to maintaining a healthy crop. Practices that can have a
tremendous impact on crop health include purchasing pathogen-free or resistant plants and
sanitizing equipment and soil that comes into contact with the plants. The soil makeup can also
be carefully chosen to retain less water if a certain crop is particularly susceptible to Pythium
6
disease (56). The best management strategy for plant pathogens in recycled irrigation water may
be a combination of two or more of the control categories, but this may not be economical (28).
In my experiments, I will explore aspects of biological and chemical control. Using the
as yet uncharacterized species of Pythium that were obtained by baiting water in commercial
greenhouses, I sought isolates that delay or promote plant symptom development caused by the
three plant pathogenic species P. irregulare, P. cryptoirregulare, or P. aphanidermatum. I
sought isolates of Pythium that may provide some amelioration of disease, and collected data on
the mefenoxam resistance of all isolates. Some plant pathogenic isolates are mefenoxam resistant
and non-pathogenic isolates may be quite sensitive. The use of mefenoxam may worsen disease
development by eliminating a non-pathogenic species that is inhibiting or competing with the
pathogenic species.
Pythium Evolution & Ecology
By studying small subunit rRNA, some scientists determined that the evolution of plant
pathogenicity in Oomycetes is not common, and that the original Oomycetes were likely
saprotrophic (36). Other scientists however, assert that plant pathogenicity is an ancestrally
derived characteristic and the earliest Oomycete groups were marine microorganisms that
obligately parasitized algae, nematodes, and crustaceans and that saprotrophism is the derived
trait (7). However from an evolutionary standpoint, becoming an obligate parasite entails a
significant loss of biochemical pathways and gaining them back in order to live saprotrophically
would be extremely improbable. It is hypothesized that Oomycetes may have come to land with
the nematodes that they parasitized (8). Then, horizontal gene transfer from bacterial and fungal
plant pathogens may have helped the Oomycetes to become plant pathogenic (54). The earliest
fossil evidence of Oomycetes shows that they were cosmopolitan root and stem plant pathogens
during the Carboniferous period. These ancient Oomycetes had a potential haustorium, which
suggests a biotrophic lifestyle (65).
According to the most recent, comprehensive analysis of the genus done by Lévesque and
De Cock, Pythium is divided into clades A-K (39). It would appear that those in clade A are the
most basal species in the genus because they include marine organisms that parasitize algae.
Furthermore clade K contains species from the newly named genus Phytopythium, which are
7
somewhat of an intermediary between Pythium and Phytophthora (17). Phytophthora is more
divergent than Pythium, and Phytophthora are believed to have given rise to the downy mildew
obligate biotrophs (16), which are evolutionary dead ends (62). However Lévesque and De Cock
(39) made the opposite conclusion than I do, regarding clade K as most basal (39). The clades’
evolutionary paths are still nebulous (58). Nevertheless, one of the most interesting discoveries
in sequencing the ITS and cox I regions in Pythium is that the morphological similarities such as
sporangia morphology often corresponded with genetic similarities (58; 70).
Some scientists hypothesize that plant pathogens eventually evolve to become
commensals because the pathogen dies if it kills the plant (its food source)(44). Pythium early in
infections is a biotroph but later in the infection is necrotrophic; which means it kills plant cells
before extracting nutrients. If it has to kill cells to obtain nutrition, this would prevent it from
evolving into less pathogenic strains (31). The necrotrophic stage also allows Pythium to live
saprophytically, which is problematic for growers. Pythium lacks many genes needed for
breaking down plant cell wall carbohydrates and cutin, thus focusing on the utilization of starch
and sucrose (32). These degradative enzymes possessed by Pythium ultimum allow it to be a
destructive pathogen as well as a successful saprophyte.
An objective of my research was to determine the ecological roles of the multiple
Pythium isolates that are commonly found in water. In the case of carrot cavity spot, nonpathogenic isolates of Pythium are often found in lesions alongside pathogenic strains, but not
believed to influence disease development. It was noted that during the infection phase, it is not
understood how these non-pathogenic and pathogenic species interact in disease (66). On parsnip
and parsley, 11 different Pythium species were found on the roots of diseased plants. In lab
pathogenicity tests, the species varied in their virulence (52). By co-inoculating plants with the
pathogenic and baited, non-pathogenic strains, I attempted to better understand the ecology of
the rhizosphere by isolating Pythium from the plant’s roots to determine if the non-pathogenic
species were still present in the rhizosphere, or if the pathogenic isolate dominated.
Both pathogenic and non-pathogenic Pythium species were isolated from healthy alfalfa
roots, (37) indicating that some species do act as commensals. Nonetheless, relatively nonpathogenic isolates may still cause root lesions (75). In carrot cavity spot usually only one
species was isolated from a single lesion. Rarely though, more than one species was isolated
8
from a single lesion (40). On soybean seedlings, uncommonly more than one Pythium species
was isolated from roots (79). Therefore, it is possible that there are different ecological niches of
these isolates. Non-pathogenic species may remain on roots, not causing disease, and eventually
get outcompeted or coexist with pathogenic species. The pathogenic species may not be able to
totally dominate the environment due to presence of antagonistic microorganisms or the
establishment of non-pathogenic Pythium on the roots. Perhaps the more pathogenic species have
a greater nutrient requirement and thus are more easily out-competed.
The question why many different Pythium species exist in the same area is challenging. It
is difficult to determine if the species are occupying different niches, or are mainly competing for
space. Gause’s law of competitive exclusion is the traditional ecology viewpoint that states only
one species will dominate a niche (61). Gause’s law explains the presence of multiple Pythium
species in water tanks or plant roots, assuming that the species differ in their nutrient
requirements, growth rates, etc. A more modern ecological theory proposed to replace Gause’s
Law is the Unified Neutral Theory of Biodiversity and Biogeography (57). The Neutral Theory
is based on findings that suggest species abundance distributions display universal patterns. The
Neutral Theory proposes that species on the same trophic level have equal likelihoods of death,
immigration, speciation, and birth, therefore stochastic processes are involved in determining
which species will occupy an open space in the ecosystem (29). Plant pathogens and saprotrophs
are on the same trophic level in the soil food web (30). In some fish, the “lottery hypothesis” is
applied in which two species compete for space and when space becomes available, the closest
fish to the area will colonize and persist in that space (48). Some scientists have applied this
lottery concept to microbial ecology with the added facet that although colonization is random,
the possession of certain functional genes is the prerequisite for microbes existing in a certain
niche (11). If this concept was applied to Pythium in recycled irrigation water, the presumption
would be that if all the Pythium species found in irrigation tanks shared the same functional
genes that allowed colonization of the tank water, that their presence and abundance in a certain
tank is due to random chance. This is a null model to test my hypothesis that certain Pythium
species possess characteristics allowing them to outcompete or suppress other Pythium species.
In the Neutral Theory, the model used to determine the abundance of species is a zerosum multinomial distribution dependent on the community size, immigration rate, speciation
9
rate, and the size of adjacent communities (29). When a community of soil arbuscular
mycorrhizal fungi was studied, it was found that the community’s species abundance fit a zerosum multinomial distribution. While this is in support of the Neutral Theory, it was found that
the most important factor that determines species composition is soil pH (18), which is indicative
of Gause’s niche theory. Another group studying microbial communities in wastewater proposed
that neutral processes are the core of species abundance (51). I hypothesize that a similar
situation applies to Pythium in recycled irrigation water. Factors such as temperature, pH, and
the presence of antagonistic microorganisms will be the major factors in determining tank
community structure, while random chance will contribute to species abundance and differences
between communities in different tanks. On plant roots however, I hypothesize that the most
abundant species are the ones best at utilizing the available nutrients.
It has been noted that learning the diversity and roles of aquatic Pythium species (59) and
understanding the biology of many aquatic plant pathogens is an area of research that needs
attention (27). Furthermore, it is proposed that Fungi and Oomycetes play a role in aquatic
carbon cycling and their ecology needs to be a new focal point of aquatic ecology research (60).
Ideally, this ecological information I gather about Pythium community structure in recycled
irrigation water tanks will help inform the respective scientific community and horticulturalists
of biological and abiotic factors that can reduce yield losses due to Pythium diseases. My
research also may also inform of whether these species have an affinity more towards plant roots
or water. This may in turn also provide some suitable information to aquatic ecologists.
10
Objectives
1) To characterize the community of Pythium species that inhabit the recycled irrigation water
greenhouse tanks of two commercial Pennsylvania greenhouses.
A 34-week intensive baiting of several water tanks in two commercial greenhouses where crop
losses due to P. aphanidermatum have historically occurred yielded many other species of
Pythium. I identified the baited isolates using both molecular identification and morphological
observations, determined their sensitivity to mefenoxam, and identified the cardinal temperatures
of the isolates.
2) To assess the pathogenicity of the Pythium and Phytopythium species found in the recycled
irrigation water tanks of two Pennsylvania greenhouses and to determine if the species residing
in water tanks interfere with or enhance disease development in highly virulent Pythium species.
3) To determine if the baited species or highly virulent species dominate the plant roots when coinoculated.
If neutral ecological forces are responsible for Pythium species that occupy the same niche, it is
predicted that the commonly baited species and the highly virulent Pythium species will be
recovered equally as often from plant roots, when they are co-inoculated.
4) To determine if the commonly baited species: Phytopythium helicoides and Pythium
coloratum colonize bentgrass baits before P. aphanidermatum.
It is possible that the very prolific zoospore forming commonly baited species (Phytopythium
helicoides and Pythium coloratum ) simply colonize the baits before P. aphanidermatum.
11
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20
Chapter 2
IDENTIFICATION AND CHARACTERIZATION OF Pythium AND Phytopythium
SPECIES IN TWO COMMERCIAL GREENHOUSE RECYCLING IRRIGATION
WATER SYSTEMS
Abstract
Commercial greenhouses producing potted plants in Pennsylvania (PA) have incurred
significant crop losses due to Pythium aphanidermatum. Thorough greenhouse and plant material
screening in some of these greenhouses has failed to reveal the source of the pathogen. In
cooperation with two PA commercial greenhouses, their recycled water tanks were baited and
checked for Pythium weekly for 34 weeks. This amounted to a total of 128 tank samplings. P.
aphanidermatum was not recovered from any of the baits; however nine other species of Pythium
and three species of Phytopythium were discovered, representing Clades A, B, E, and K. The
species found during the tank baiting were: an isolate with close similarity to Pythium. sp. nov.
OOMYA1702-08 in clade B2, two distinct species of unknown identity in Clade E2, P.
coloratum or one of the very closely related species (e.g. P. diclinum), P. middletonii, an
unknown species in Clade B2, an isolate with similar genetic identity to P. sp. nov.
OOMYA1646-08 (E2), P. rostratifingens, and an unknown species in Clade A. The
Phytopythium species found were: Ph. litorale, Ph. helicoides, and Ph. chamaehyphon. Many of
these species are considered weak pathogens and some display resistance to the Oomycete
fungicide active ingredient, mefenoxam. Of the baited isolates, seven expressed resistance
(including isolates in Phytopythium. helicoides, Clade E2-2 unknown, P. middletonii, P. sp. nov.
OOMYA1646-08 (E2),) with three displaying high resistance (P. coloratum, P. rostratifingens,
and Clade A unknown.) Seven expressed sensitivity (including isolates in Ph. helicoides, Clade
B2 unknown, P. sp. nov. OOMYA1646-08 (E2), and Ph. chamaehyphon) with three displaying
high sensitivity (including isolates in Clade E2-1 unknown, P. coloratum, and Clade E2-2
unknown).
Introduction
Due to the awareness of the environmental impacts of fertilizer runoff from agricultural
systems, some state governments have mandated the use of recycled irrigation water in
commercial greenhouses (25). An added benefit to these mandates is conserving water in plant
21
production, but this benefit comes at a cost. Plant pathogens may spread throughout the crop via
the recycled water. The broad-spectrum, zoosporic species in the genus Pythium pose such a
threat (3; 14; 26).
Pythium, from the Greek “pythein” – to cause rot (34), is a genus of organisms in the
class Oomycota and kingdom Chromalveolata (10). Some species of Pythium are plant
pathogens and have the potential to cause significant losses of greenhouse crops, especially P.
aphanidermatum (Edson) Fitz., P. ulitmum Trow, P. irregulare (Buis.) and P. cryptoirregulare
(Garzón, Yánez, and Moorman) (37). Pythium species are generally considered to be waterborne
and therefore are thought to be harbored in or dispersed via irrigation water (56) largely due to
the zoosporic phase of their lifecycle (26). Thus monitoring of irrigation water for Pythium can
provide knowledge needed to manage a Pythium disease outbreak (20). Recently, it was
discovered and confirmed that the species in Clade K of Pythium are genetically quite distinct
from the rest of the genus. Species in this grouping have been given the new genus name
Phytopythium (9; 15). Phytopythium helicoides is a problematic pathogen in greenhouses that use
ebb-and-flood watering systems (30; 52).
A water survey was conducted in cooperation with two Pennsylvania commercial
greenhouses that recycle irrigation water during the production of potted floricultural crops and
had experienced crop losses resulting from disease initiated by Pythium (12), but only
preliminary identifications were completed. One objective of the current research was to clearly
identify Pythium from the irrigation tanks of these greenhouses in Pennsylvania in that survey.
A great deal of research reports the presence of plant pathogens including Oomycetes in
water (24). Pythium spp. have been baited from freshwater sources (2; 5; 40; 51). Pythium
species were found in a survey of the water that enters a Colorado greenhouse, but not species
responsible for the disease (43). A large number of Pythium species were found in a Virginia
greenhouse irrigation system (14). Pythium irregulare was discovered in an irrigation water
sample submitted to the Plant Disease Clinic at Penn State (37). Phytopythium litorale was
frequently baited from vegetable irrigation ponds in Georgia, and was pathogenic on squash (41).
An extensive literature review reported 14 species and 3 groups of Pythium to be present in ebband-flow or hydroponic systems (24).
22
Mefenoxam (methyl N-(2,6-dimethylphenyl)-N-(methoxyacethyl)-d-alaninate) is a
phenylamide fungicide that is used to control diseases caused by Oomycetes (45). The mode of
action of phenylamides is the inhibition of ribosomal RNA polymerization, thus deactivating the
pathogen (45). Resistance to this class of fungicides has been reported many times (45).
Resistance in Phytophthora has been associated with a single-nucleotide polymorphism in RNA
polymerase I (45). In Pythium ultimum however, there is evidence that their large number of
ABC transporters act to pump out mefenoxam in resistant isolates (28). Propamocarb and
mefenoxam come from different chemical classes and are among the most effective fungicides
for controlling Pythium (32). Despite having different modes of action, Pythium isolates exhibit
resistance to both compounds (32). Another objective of this study is to determine whether
resistance to mefenoxam exists in the community of Pythium species that are frequently
recovered from recycled irrigation water tanks. The rationale for this study is twofold. Firstly,
these data will help provide a better understanding of if some of our isolates are clonal (13; 32).
Secondly, this will give us information on whether applying mefenoxam might inhibit non-plant
pathogenic species of Pythium present in water that may be competing with the plant pathogenic
species. We hypothesize that the Pythium isolates from baiting differ in their sensitivity to
mefenoxam from the pathogenic species of Pythium and therefore mefenoxam may play a role in
their interaction through selection.
Some species of Pythium are found more commonly associated with water than with
plants. P. aphanidermatum is found in Pythium Clade A, which contains species pathogenic to
marine algae. Clade B contains species with non-inflated, filamentous sporangia (32) (Group F
according to the system of Plaats-Niterink (56).) In a study of Pythium in hydroponic systems,
Clade B species were most commonly found and P. aphanidermatum only represented 5% of the
species found (23). Both Clade A and B contain waterborne Pythium species, and both have been
isolated from a lake (5). Four new Pythium species belonging to Clade B have been found in
Japanese freshwater (54). Overall it appears that Clade A species, other than P. aphanidermatum,
and Clade B species are found more often than P. apanidermatum in water (23; 39; 51). These
commonly found species are not notable pathogens and may only cause diseases on a certain
crop or under particular environmental conditions (56). Therefore we anticipated finding a large
number of Pythium species from Clades A & B in our survey, including P. aphanidermatum.
23
Materials & Methods
The two commercial greenhouses in Pennsylvania cooperating in this research produce
plants all year. Greenhouse S (40°49'31.30"N, 76°48'18.17"W; less than 1 ha under glass) and E
(40°13'21.56"N, 76°16'13.60"W; approximately 6.5 ha under glass) have similar production
practices, starting crops from their own cuttings or seedlings or from purchased rooted and
unrooted cuttings or seedlings. Bench ebb and flood irrigation systems are used in E and cement
floor ebb and flood is used in both greenhouses to irrigate crops, collect excess water, and return
it to the tanks for use in the next irrigation cycle. The water tanks are filled from onsite wells.
The excess irrigation water is passed through a screen or fabric to remove coarse particulate
matter before it returns to the tank. Both greenhouses grow bedding plants, potted
chrysanthemums and poinsettias as major crops. Various water soluble fertilizers are added to
the irrigation water
Baiting and Biological Characterization
Continuous baiting of the tanks was conducted as follows (12). Blades of creeping
bentgrass (Agrostis stolonifera L. 'Penn Eagle') were placed between pieces of fiberglass screen
and positioned in the recycling irrigation water tanks when they were in active use. Water
temperatures in each tank were recorded continuously (HOBO Water Temp Pro v2 sensors,
Onset Computer Corp., Bourne, MA ). The average water temperature for each tank is noted in
Table A-7 in the Appendix. Continuous baiting was conducted for 27 weeks (March-June and
September-December, 2011) of one tank (designated C) in greenhouse S and in one to four tanks
(designated L, 32 weeks; R, 33 weeks; SB, 15 weeks; and G, 21 weeks) in greenhouse E (MarchDecember, 2011) for a total of 128 samplings. Two bait holders were deployed per tank. One
was attached to an anchor at the bottom (designated B) of the tank (> 3 meters depth) along with
the temperature sensor and the other was loosely attached to the anchor rope by a floating ring
that allowed the bait holder to always float at the top water surface (designated T) in the tank.
After being deployed for 7 days, the bait holders were sent by the grower via overnight express,
to the laboratory and fresh leaf blades were deployed. Blades were plated on NARF (clarified
20% V8 juice agar amended with nystatin, ampicillin, rifampicin, and fluazinam) (38) in 60 X 15
mm petri plates. Plates were incubated in the dark at 21°C. Mycelium was transferred into new
NARF plates to obtain a pure culture and to water agar (WA) for initial microscopic observations
24
(see Table 2-2.) During initial observations, a single hyphal tip was cut and used to continue the
culture. This abovementioned work was done by the greenhouse growers, Gary Moorman, and
Maria Burgos (12). The rest of the work was done by me.
In the current research, the cardinal temperatures of selected isolates, representing the full
diversity of isolates, were determined on potato dextrose agar (PDA). Plugs of colonized agar
were taken from the new mycelia growth and placed on two plates of PDA. They were incubated
for 24 hours in the dark at temperatures ranging from 5°C to 45°C in 5°C intervals. After 24
hours, mycelial growth was marked under a dissecting microscope and measured with Sylvac
digital calipers (Crissier, Switzerland). The measurement was automatically entered into the
computer spreadsheet using Gage Wedge software (TAL Technologies, Inc., Philadelphia, PA).
This experiment was repeated 1 or more times, depending on the isolate. The daily growth rate of
the isolates was measured using the same procedure as above, except that this was done on
potato-carrot agar (PCA) at 25°C, a standard procedure in Pythium identification (56). On these
plates, the colony morphology was observed for each isolate (see Figure 2-4). The cardinal
temperature, daily growth rate, and colony morphology data are in Table 3. The microscopic
pictures were taken with an Olympus DP26 camera and the CelSens dimension software using an
Olympus CKX41 inverted microscope. Parts were measured using the software ImageJ (50) and
can be found in Table 4. A list of the representative isolates used for microscopic measurements
can be found in Table A-5, in the Appendix.
Mefenoxam Resistance
Fungicide resistance was determined for mefenoxam (Subdue MAXX; 21.3%
mefenoxam; Syngenta, Greensboro, NC) using a poison plate method (37). Pythium cultures
were maintained on potato dextrose agar (PDA). In the poison plate tests, 20% V8 juice agar
(100mL of clarified V8 juice diluted in 400 mL of distilled water; V8, Campbell's Soup Co.,
Camden, NJ) was used as the control agar. The experimental agar was 20% V8 juice agar
amended with a discriminating concentration of 100 ppm mefenoxam (45.5μL/.05g Subdue
MAXX added to molten 500mL V8 agar) for a dose of 100 μg/ml. For each isolate on PDA, a
size 3 cork borer (5.5 mm diameter) was used to remove mycelial plugs from a young culture. A
plug was placed on each of two 60 mm diameter plates of V8 agar free of fungicide and two
plates of V8 agar amended with mefonoxam. The date and time of transfer were recorded. The
25
plates were incubated in the dark at 25°C. Following incubation, the edge of the inoculum block
and the edge of the mycelium was marked in 2 arbitrary places. The length of growth was
measured using Sylvac digital calipers (Crissier, Switzerland). The measurement was
automatically entered into the computer spreadsheet using Gage Wedge software (TAL
Technologies, Inc., Philadelphia, PA). The average growth rates per hour were calculated and the
percent growth rate on the fungicide plates as compared to growth on fungicide-free agar was
calculated. If this number is below 50% then the isolate is considered sensitive to the fungicide,
as previously described (12; 37). Growth of <10% was considered highly sensitive (HS); 4048.5%, moderately sensitive (MS); 48.5-51.5%, intermediate (I); 51.5-60%, moderately resistant
(MR); and >90%, highly resistant (HR.) This experiment was replicated at least 2 or more times
per isolate.
Genetic characterization
For amplification of the internal transcribed spacer region (ITS1, 5.8, and ITS2 DNA ),
the universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’
TCCTCCGCTTATTGATAGC-3’) were used. The cytochrome oxidase gene was amplified
using FM 59 (TTTATGGTCAATGTAGTGAAA) and FM 55
(GGCATACCAGCTAAACCTAA) (12). The PCR master mix used to carry out the ITS direct
PCR reactions contained 2 μl of (10x) PCR buffer standard, 0.5 μl of dNTP (10 mM), 1 μl of
each ITS1 and ITS4 (5 mM), 15.4 μl of sterile distilled water, and 0.1 μl of Taq polymerase,
totaling 20 μl in each reaction tube. Colonies on NARF agar were gently scraped with a pipette
tip and swirled into the reaction tubes. The thermocycling protocol, electrophoresis, and DNA
visualization is described in Burgos-Garay (2013) (12). Cox I and II amplification followed the
methods of Garzón et al (2007) (22). Cycling parameters were those described by Martin (2000)
(33). DNA sequences were edited using Sequencher (11).
Once the forward and reverse sequences were obtained and edited, the genes were
searched on The National Center for Biotechnology Information’s Basic Local Alignment Search
Tool (NCBI BLAST) (29) and The Barcode of Life Data System (BOLD) (46). Isolates were
then grouped when their identity results were identical or off by one to two nucleotides. For the
phylogenetic analysis, Pythium and Phytophythium species ITS sequences in BOLD (46) with
similarities to the DNA sequences obtained for the baited isolates were added to one dataset. The
26
sequences were aligned using MAFFT v7.147.b’s (31) G-INS-I option. The genes were
concatenated using SequenceMatrix (55). The maximum likelihood (ML) and 1000 bootstrap
analyses for both individual gene and concatenated trees were performed using the CIPRES
Science Gateway (36) tool “Genetic Algorithm for Rapid Likelihood Inference” (GARLI 2.01 on
XSEDE). Consensus trees were made using the CIPRES tool “Consense.” The results were
converted into Phylip format using the CIPRES tool “NCLconverter” and opened in the program
Molecular Evolutionary Genetics Analysis (MEGA) Version 6 (53). The identities of some of the
types were still unknown. Therefore, two more analyses using the same methods were performed
to confirm that some of the types appear to be new species. This was made possible by including
the baited species’ sequences with the sequences of all other species in their clade. The cutoff
value for species identity was 98% on both the ITS and cox genes. If the percent similarity value
was below 98% for either gene, then it was classified as an unknown species. A polyphasic
approach that integrated the DNA with biological and morphological characteristics was utilized
to make a clear species delineation. The representative isolates used in the analyses can be found
in the Appendix, in Table A-2. The cox and ITS sequences of representatives isolates from each
species are in Figure A-1 in the Appendix. Isolates that follow have been deposited in the CBS
Fungal Biodiversity Centre: Clade A unknown (S9.26.11.CB), CBS 140047; Pythium sp. nov.
OOMYA1646-08 (E2) (E10.31.11RT), CBS 140048; Clade E2-1 unknown (E6.16.11LT), CBS
140049; Clade E2-2 unknown (E10.4.11LT), CBS 140050; Clade B2 unknown (E7.14.11SBB),
CBS 140051; and Pythium middletonii (E8.30.11LT), CBS 140052.
Results
Figure 2-1 is a phylogenic tree that elucidates which of the baited strains are specific
species of Pythium or Phytopythium and which strains are undescribed species. If a name on the
tree does not have an isolate code after the name or if it has parenthesis after the code, this
indicates it is the strain that we baited. P. aphanidermatum was not recovered from any of the
baits; however nine other species of Pythium and three species of Phytopythium were discovered,
representing Clades A, B, E, and K (Table 2-1). The Pythium species found during the tank
baiting were: isolates identical to or high similarity to... P. sp. nov. OOMYA1702-08 in clade
B2, two distinct species of unknown identity in Clade E2, P. coloratum or one of the very closely
related species (e.g. P. diclinum), P. middletonii, an unknown species in Clade B2, isolates
27
identical to P. sp. nov. OOMYA1646-08 (E2), P. rostratifingens, and an unknown species in
Clade A. The Phytopythium species recovered were: P. litorale, P. helicoides, and P.
chamaehyphon.
Isolates of Phytopythium litorale and Pythium sp. nov. OOMYA1702-08 (B2) were
quickly lost in culture and therefore did not receive any morphological characterization. All of
the isolates that lasted in culture were used for the morphological characterization. Clade E2-1
unknown, Pythium coloratum, Clade E2-2 unknown, Pythium sp. nov. OOMYA1646-08 (E2),
and Clade A unknown isolates were found in tanks in both of the greenhouses. Clades A, B2, E,
and K include all the species baited, and species from each clade were found in both
greenhouses. Figures 2-4 and 2-5 display the number of isolates that were baited from each
greenhouse during each month of sampling. Phytopyhtium helicoides was baited only from
greenhouse E and most often in July and was also baited in June and August. P. sp. nov.
OOMYA 1702-08 (B2) was only baited one time from greenhouse E in March. Clade E2-1
unknown was baited from greenhouse E in June, July, and August; and from greenhouse S in
September 2013. P. coloratum was frequently baited in greenhouse E from March-August and
from greenhouse S throughout the sampling time (March-June and October-December.) Clade
E2-2 unknown was recovered from greenhouse E in July and October and in greenhouse S in
September 2011 and 2013. P. middletonii was isolated only from greenhouse E in September.
The Clade B2 unknown strain was baited frequently in greenhouse E in May-August. P. sp. nov.
OOMYA 1646-08 (E2) was found in greenhouse E in November and December and in
greenhouse S in December. P. rostratifingens was baited from greenhouse E in November and
December. Clade A unknown was baited from greenhouse E in April and Greenhouse S in May,
September 2011 and 2013. P. litorale was baited once from greenhouse S in March. P.
chamaehyphon was baited from greenhouse S in September-December.
Our baited isolate most closely related to Pythium sp. nov. OOMYA1702-08 (99.72%
ITS similarity) has two nucleotide differences in the ITS region and one gap in the cox region
where a base is in Pythium sp. nov. OOMYA1702-08 (Figure 2-4.) This isolate looked unusual
for a Pythium and did not last in our labs long-term Pythium culture storage protocol. In fact it
was just as closely related to Lagenidium caudatum as it was to Pythium sp. nov. OOMYA170208. Therefore it is possible that this species was a Lagenidium or an intermediary between
28
Lagenidium and Pythium. Unfortunately the isolate did not last in culture, so its mention is
merely for a complete survey. In our Phytopythium litorale isolate, the coxI region differs by two
base pairs from OOMYA1379-08 , Ph. litorale (Figure 2-4.) This isolate was also lost in culture
and is concluded to have been Phytopythium litorale.
The Clade E2-1 unknown isolates are most closely related to OOMYA235-07, Pythium
marsipium (84.67% ITS similarity,) but their ITS regions differ by 77 nucleotides, while their
cox1 regions differ by 49 nucleotides. Figure 2-2 displays an in depth phylogenetic analysis of
the entire Pythium Clade E2, showing that these isolates group as highly divergent species from
Pythium marsipium. The sporangia for the species can be globose, which was observed in our
Clade E2-1 unknown isolates (Figure 2-8.). But, a defining characteristic of P. marsipium is the
asymmetrically utriform sporangium, which were not observed in our isolates. We observed
sporangia proliferating internally just as observed in P. marsupium. Although oospores were rare
in this isolate, aplerotic oopores were found as is the case in P. marsupium, and oospores with
one ornament was observed which is not described in P. marsupium. As for the cardinal
temperatures, the minimum was higher and maximum was lower in the isolate from the PA
greenhouses as compared to what is reported in the literature for P. marsupium. The daily growth
rate reported in the literature is 12 mm and the isolate obtained here grew at about 13.5 mm/day.
The colony morphology is reported as radiate and that is what was observed. The oogonia of our
isolate are slightly larger than those of P. marsupium and the discharge tubes are shorter (2; 113).
Our Clade E2-1 unknown isolates and P. marsupium are clearly genetically and morphologically
distinct.
Our P. coloratum isolates from both greenhouses have ITS sequences 100% identical to
OOMYA1380-08 P. coloratum. The cox I regions differ by two nucleotides. P. coloratum is in
Clade B2, and closely related isolates can be difficult to differentiate. Not only do many
members of this clade look almost identical with their non-inflated or slightly inflated
filamentous sporangia, but P. coloratum, P. lutarium, P. marinum, and P. dissotocum all have
identical ITS sequences; P. diclinum differing by 1 bp. P. dissotocum is common in greenhouse
crops, but the cox I region of our isolates matches P. coloratum more closely (32). The cardinal
temperatures of most of our isolates were: 5°C, 30°C, and 35°C; which is identical to those
reported for P. coloratum and P. lutarium (7; 56). However, the daily growth rate reported for P.
29
coloratum is 20 mm, and 18 mm for P. lutarium, a rate that none of our isolates approach. The
daily growth rate of P. diclium is 19 mm, which wass also not close to our isolates. P. marinum
is a marine organism, with an optimum growth temperature of 15-20° C, which was not found in
any of our isolates (21). There was some variability in the cardinal temperatures among our
isolates, but their cardinal temperatures match those of P. lutarium (7), and their optima were
slightly higher than has been reported for P. dissotocum (56). The daily growth rate from P.
dissotocum is 13 mm, which is close to that of many of our strain isolates. The lack of thick
walled oospores is not incongruous with these isolates being P. coloratum. It is certainly possible
that not all of the isolates in the P. coloratum strain are the same species, but determining this is
very difficult.
Our Clade E2-2 unknown isolates differ from their nearest relation, P. middletonii
(96.2% ITS similarity (OOMYA177-07)) by 32 and 13 nucleotides from the ITS and cox I
regions respectively. Figure 2-2 displays an in depth phylogenetic analysis of the entire Pythium
Clade E2, showing that these isolates group as different species from P. middletonii The
morphology of Clade E2-2 unknown isolates is similar to P. middletonii because of their
internally proliferating globose and limnioform sporangia (56). The cardinal temperatures of
Pythium middletonii are: 5°, 30°, and 35°; and a daily growth rate of approximately 9 mm. The
daily growth rates of Clade E2-2 unknown isolates is lower than that of P. middletonii and the
optimal and upper limits are one category lower than Clade E2-2 unknown. In this case, the
genetic differences are what delineate a new species.
Our Clade B2 unknown isolates are most closely related to Pythium pachycaule (95.9%
ITS similarity, OOMYA064-07) and differ by 25 and 27 nucleotides from the ITS and coxI
regions respectively. P. apleroticum and P. aquatile are also closely related species. The cardinal
temperatures are close for our isolates and these two species, with only the optimum temperature
being higher in our isolates than in P. aquatile. Based on morphology, our isolates match more
closely with P. apleroticum and Pythium pachycaule due to their possession of filamentous, noninflated sporangia, aplerotic oospores, terminal and intercalary oogonia, and diclinous antheridia.
P. aquatile on the other hand sometimes has slightly inflated sporangia and monoclinous
antheridia. However a globose sporangial vesicle was found, like those in P. aquatile. The
aplerotic character of the oospore is more pronounced in Clade B2 unknown than in P. aquatile.
30
The daily growth rates are approximately 13-22 mm (except one with 1.27 mm) which contain
the daily growth rates of both species. P. aquatile has a rosette pattern, which was only observed
on the isolate with the slow daily growth rate (56). P. apleroticum has a radiate growth pattern
(27), which matches our isolates. No information was found on yellow oogonia in either species.
P. pachycaule has been isolated from a river (49). Our isolate’s oogionia were larger with thicker
walls than P. pachycaule and lacked the characteristic oogonial stalk pachycaulous development
(7). Therefore we conclude that both genetically and morphologically, this baited strain is a
previously unclassified Pythium species.
Our putative Phytophthium helicoides isolate differs from the Ph. helicoides isolate,
OOMYA1420-08 by three base pairs in the ITS region (99.6 % ITS similarity) and two base
pairs in the cox I region. The cardinal temperatures and morphology are concordant with each
other, particularly one to four antheridia/oogonium, proliferating papillate sporangia, terminal
oogonia, and aplerotic oospores. The colony pattern is radiate, which is also the pattern of all of
our putative Ph. helicoides isolates. However the daily growth rate is 34 mm (56), which is quite
higher than those rates of our Ph. helicoides isolates. Our oogonia also are smaller.
Our P. middletonii baited isolates sequences match 100% to the ITS region, but differ
from the cox I region by eight nucleotides. Their cox I area is still most closely related to P.
middletonii than any other species. The cardinal temperatures of our strain were: 5°, 30°, and
35°; and a daily growth rate of approximately 9 mm are concordant with P. middletonii’s
cardinal temperatures but was lower than the reported daily growth rate. Our strain has the
hypogynous antheridia just like P. middletonii (56). Both P. middletonii and our isolates have
globose or limnioform internally proliferating sporangia. Our isolate’s oogonial diameter is
within the range for P. middletonii, though our isolates have a slightly thicker oospore wall than
what is reported for P. middletonii (56). We did not observe any inconsistencies between the
morphology of our isolates and P. middletonii. The difference in the cox I gene is interesting and
may suggest that our isolates are in the process of speciation.
Our isolates most closely related to Pythium sp. nov. OOMYA1646-08 (99.88% ITS
similarity) differ from it’s ITS region by only one base pair, but from its cox I region by 13 base
pairs. A Blast search shows that a close relation is Pythium carolinianum, but their morphology
is not concordant because P. carolinianum are noted for their lack of oogonia and both have a
31
different method of proliferation. (56). Pythium carolinianum is reported to have a radiate
growth pattern and a daily growth rate of 10 mm on Difico-CMA (1), which matches with our
isolates. Genetically and morphologically it is apparent that Pythium sp. nov. OOMYA1646-08
(E2) isolates are not P. carolinianum. Genetically they could be Pythium sp. nov. OOMYA164608, or an entirely new species.
The sequences of our Pythium rostratifingens isolates match the ITS and cox I regions of
P. rostratifingens OOMYA1699-08 100%. Their morphologies are concordant with one to four
antheridia, and mostly two per oogonium and the lack of zoospores. However, with our baited
isolates, the minimum temperature was higher and maximum temperature was lower than
reported. The daily growth rate of our isolates was slightly slower than that reported for P.
rostraifingens (9mm) and the colony morphology (chrysanthemum) was the same (17).
The sequences of our Phytopythium chamaehyphon isolates match the ITS and cox I
regions of Ph. chamaehyphon OOMYA088-07 100%. The sporangia of our baited isolates are
the same as Ph. chamaehyphon, however we did not observe any oogonia. Our minimum growth
temperature was higher than reported. Our daily growth rate was lower than reported (22mm)
and the colony morphology is the same (radiate) (56).
Our Clade A unknown isolates are most closely related to OOMYA1376-08, Pythium
chondricola (98.88% ITS similarity.) Our isolates differ from them by six base pairs in the ITS
region and 18 base pairs in the cox I region. P. chondricola is genetically close to Pythium
adhaerens and Pythium porphyrae, both genetically and morphologically. P. chondricola has
only been found in The Netherlands and Pythium porphyrae in Japan (32). A distinguishing
feature of P. chondricola is its lower cardinal temperatures than P. porphyrae, and our baited
isolates have higher cardinal temperatures than those. Another distinguishing feature of P.
chondricola is the presence of aplerotic oospores (16). We only observed plerotic oospores in
our baited isolates, though it is possible aplerotic oospores still could have been present. P.
adhaerens’ sporangia do not differ from the vegetative hypha, which is not the case in Clade A
unknown’s filamentous sporangia. Furthermore the sporangial vesicles resemble those of P.
angustatum, another closely related species that is in Clade B. However the daily growth rate of
P. angustum (9mm) is substantially different from those of Clade A unknown (56). Perhaps our
strain is a new species which is an intermediary between Clades A and B of Pythium.
32
Clade E2-1 unknown isolates were sensitive to mefenoxam. P. coloratum isolates
displayed a lot of variation in their resistance, having at least one isolate in each designation.
However most of the isolates (n=23) were sensitive and eight were highly resistant. Four Ph.
helicoides isolates were moderately sensitive and one was intermediate. Most Clade E2-2
unknown isolates were sensitive, with one isolate being moderately resistant. The one isolate of
P. middletonii was resistant. Clade B2 unknown isolates were sensitive to mefenoxam. Most P.
sp. nov. OOMYA1646-08 (E2) isolates were resistant, with one being sensitive. The P.
rostratifingens isolates were resistant. Ph. chamaehyphon isolates were sensitive, with two being
intermediate. Clade A unknown isolates were resistant. Our P. aphanidermatum isolate was
resistant while P. cryptoirregulare and P. irregulare were sensitive. Of the baited isolates, seven
strains expressed resistance with three displaying high resistance. Seven strains expressed
sensitivity with three displaying high sensitivity.
33
Figure 2-1. A maximum likelihood analysis concatenated gene tree of the ITS and cox I regions
with 1000 bootstraps. Phytophthora cinnamomi represents the outgroup. This analysis
incorporates clade groupings (according to Lévesque and De Cock (32))of Pythium sp. baited
from irrigation water tanks in two commercial greenhouses in Pennsylvania and species included
sequences from the Oomycetes barcoding effort (OOMYA- labeled isolates) (47) that most
closely match the baited isolate ITS and cox regions. Our isolates are written in grey.
34
Figure 2-2. A new species analysis of the isolates of
unknown identity in Clade E2. Species labeled OMYA
are from the Oomycete barcoding effort (47). The
clades noted are from the molecular phylogeny of
Pythium (32).
35
Figure 2-3. A portion of the new species analysis for the Clade B2 unknown isolate.
OMYA-labeled species are from the Oomycete barcoding effort (47).
36
Figure 2-4. A selection of sequence alignments from our baited isolates and their most closely
related species. OMYA-labeled species are from the Oomycete barcoding effort (47).
14
Greenhouse E Isolates
37
Number of Isolates Baited
12
Pythium sp. nov OOMYA1702-08 (B2)
10
Clade E2-1 unknown
8
Pythium coloratum
Phytopythium helicoides
6
Clade E2-2 unknown
Pythium middletonii
4
Clade B2 unknown
Pythium sp. nov OOMYA1646-08 (E2)
2
Pythium rostratifingens
Clade A unknown
0
Month Baited
Figure 2-5. The Pythium and Phytopythium species baited from greenhouse E, displayed by the
number of isolates baited per month.
8
Greenhouse S Isolates
Number of Isolates Baited
7
6
5
Clade E2-1 unknown
4
Pythium coloratum
3
Phytopythium litorale
Clade E2-2 unknown
2
Pythium sp. nov OOMYA1646-08 (E2)
1
Phytopythium chamaehyphon
0
Clade A unknown
Month Baited
Figure 2-6. The Pythium and Phytopythium species baited from greenhouse S, displayed by the
number of isolates baited per month.
38
A
B
C
D
Figure 2-7. Pythium colony morphologies. Chrysanthemum (A), rosette (B), no pattern (C), radiate (D).
39
Table 2-1. The cardinal temperatures of the baited isolates and their daily growth rates and
colony morphology on PCA.
Isolate designation
(number of isolates)
Clade E2-1 unknown (3)
Pythium coloratum (37)
Phytopythium helicoides
(9)
Clade E2-2 unknown (6)
Pythium middletonii (1)
Clade B2 unknown (4)
Pythium sp. nov.
OOMYA1646-08 (E2) (6)
Pythium rostratifingens
(3)
Phytopythium
chamaehyphon (7)
Clade A unknown (4)
Minimum
Growth
Temp (°C)
9,5
5,9,15
Optimum
Growth
Temp (°C)
35,30
30,35,25
Maximum
Growth
Temp (°C)
40,35
35,40,30
Daily
Growth
(mm)
13.11
11.74
Colony
Morphology
15,5
35
40
20.00
5,15
5
9,5,15
5
30
25
35,30
25,30,35
35,40
30
40,35
30,35
12.64
9.76
14.39
10.48
9,5
25
30
6.31
Ra
Ra
Ra,R
Ra, Ra/C,
R/C
C
9,15,5
30
35
15.13
Ra, Ra/R
9,5,15
30,35
35,40
0.08
C/Ra, R/Ra,
Ra
Ra/N
Ra
Ra,R,N,R/Ra,
Ra/C
Ra,N
9
35,30
40
10.55
Pythium
aphanidermatum (2)
9
30
35
15.44
N
Pythium irregulare (1)
25
35
20.77
Ra/N
Pythium cryptoirregulare 5
(1)
Ra-radiate, R-rosette, N-no pattern, C-chrysanthemum, a slash indicates an intermediary form
with the more dominant feature listed first. If more than one number or abbreviation is listed, the
most commonly occurring one is listed first. OMYA-labeled designations are from the Oomycete
barcoding effort (47). The expanded table can be found in the Appendix, Table A-3.
40
A
B
Figure 2-8. Clade E2-1 unknown characteristics. Terminal oogonium with
diclinous antheridium (arrow) and aplerotic oospore (A), empty intercalary
sporangium (B), internally proliferating sporangium (C), proliferating
sporangium (D), sporangium with released and germinating zoospores (E).
C
D
E
A
B
C
D
Figure 2-9. Pythium coloratum characteristics. Globose/ellipsoid sporangia (A), aplerotic
oogonium (B), possible appresoria (C), filamentous sporangia (D).
41
A
B
C
Figure 2-10. Phytopythium helicoides characteristics. Limoniform papillate sporangia (A),
aplerotic oospore (B), release of zoospores (C).
A
B
C
D
Figure 2-11. Clade E2-2 unknown characteristics. Zoospore discharge (A), aplerotic oospore
(B), internally proliferating sporangia (C), mass of zoospores emerging from a grass blade
(D).
42
A
B
C
D
Figure 2-12. Pythium middletonii characteristics. Intercalary and terminal aplerotic
oospores with hypogynous antheridia (arrow) (A) Intercalary internally proliferating
sporangium (B), globose terminal sporangia (C), limoniform shaped intercalary
sporangium (D).
A
B
C
D
Figure 2-13. Clade B2 unknown characteristics. Yellow oogonium with aplerotic oospore
(A), filamentous sporangia (B), Pyriform sporangium (C), globose sporangium (D).
43
A
B
C
Figure 2-14. Pythium sp. nov. OOMYA1646-08 (E2) characteristics. Aplerotic oospore &
terminal oogonium with diclinous antheridium (arrow) (A), externally proliferating limoniform
sporangium (B), intercalary oogonium with plerotic oospore with monoclinous hypogynous
antheridium (arrow) (C).
A
B
Figure 2-15. Pythium rostratifingens characteristics. Intercalary globose
sporangium (A) and plerotic oospore with diclinous antheridium (arrow)
(B).
44
Figure 2-16. Phytopythium chamaehyphon characteristics. Globose, terminal sporangia.
A
C
B
D
Figure 2-17. Clade A unknown characteristics. Filamentous, slightly inflated sporangia (A),
globose sporangium (B), plerotic oospore with antheridium (C), Sporangial vesicle (D).
45
Table 2-2. The means in µm of structures of baited Pythium and Phytopythium species.
Isolate
designation
Oogonia
diameter
Oospore Hyphae
wall
width
thickness
Clade E2-1
unknown
40.5
(1)
4.5±.5
(4)
4.3±1.3
(3)
Pythium
coloratum
17.4±5.7
(4)
2.2±.8
(6)
0.4±.1
(2)
Pythium sp. nov. 18.7±1.1
(7)
OOMYA164608 (E2)
2.7±.6
(6)
3.7±1.6
(3)
Phytopythium
helicoides
11.5
(1)
Clade E2-2
unknown
1.3
(1)
Discharge
tube
length X
width
4.4 x 2.7
(1)
3.3±1.3 x
4.6±1.8
(4)
20.35±1.3 2.3±.1
(3)
(3)
3.1 (1)
5.3±1.2 x
4.6±.7 (3)
Pythium
middletonii
21.2±.6
(3)
2.65±.2
(3)
2.75±.75
(3)
Clade B2
unknown
30.2±1.4
(3)
4.35±1.3
(3)
Pythium
rostratifingens
17±.3 (2)
2.4±.1
(2)
Clade A
unknown
4 x 2.8 (1)
1.5 (1)
2.2±.1
(2)
15.5±1.1
(1)
1.4±.6
(2)
3.3±.4
(2)
25.9±4.3
(7)
34.1 x 48.3
14.4±8.3 x
41.7±13.2
(9)
28.7±5.4
(8)
2.3±.2
(3)
Phytopythium
chamaehyphon
Sporangia Antheridia Zoospore
diameter Width
diameter
X length
3.6 x 3.8
(1)
9.8±1.5
(5)
3.4±.5
(5)
7.15±1.2
(5)
15.9 x 23.6
25.3±3.2 x 2 (1)
26.2±4.1
(5)
23.5±16.8
(2)
25.6±2.8
(5)
28±1.3
(7)
7.95±.7
(4)
12 x 6.9
(1)
28±1.8 (4)
20.3±2.9
(2)
15.4 (1)
9.1±1.3
(5)
9.1 (1)
6.5±.5
(2)
24.6±2.9
(4)
8.8±7.2
(6)
7.7±.8 (4)
9.5±.3
(4)
3.9 (1)
Only sporangia diameter is given for globose sporangia ± =standard deviations. Isolates with
OMYA-labeled designations are from the Oomycete barcoding effort (47). Parenthesis after the
measurements indicate number of parts measured. A list of representative isolates can be found
in Table A-5, in the Appendix.
9.7 (1)
46
Table 2-3. Results of the poison plate assay
Isolate designation (number of isolates)
Clade E2-1 unknown (4)
Pythium coloratum (39)
Phytopythium helicoides (9)
Clade E2-2 unknown (6)
Pythium middletonii (1)
Clade B2 unknown (5)
Pythium sp. nov. OOMYA1646-08 (E2)
(6)
Pythium rostratifingens (3)
Phytopythium chamaehyphon (7)
Clade A unknown (5)
Pythium aphanidermatum (1)
Pythium cryptoirregulare (1)
Pythium irregulare (1)
Number of isolates in each Fungicide
Response Classification
HS S
MS I
MR R
HR
1
3
2
23 2
2
1
1
8
4
1
4
1
4
1
1
4
1
1
5
3
2
1
2
2
3
1
2
1
1
This table displays the number of isolates in each group and the mean values
categorized by response to mefenoxam. Highly sensitive (HS), moderately sensitive
(MS), intermediate (I), moderately resistant (MR), Highly resistant (HR.) OMYAlabeled designation are from the Oomycete barcoding effort (47). The full dataset can
be found in Table A-4.
47
Discussion
Each of the two greenhouses has a characteristic community of Pythium, however many
of the species were found in both greenhouses. Some seasonal patterns seem to exist in the baited
isolates, but because of the lack of uniformity of the baiting times, we did not choose to look into
these patterns any further. Table A-6 in the Appendix displays the cardinal temperatures of these
species and the temperatures at which they were baited. None of the species were baited at their
optimum growth temperature, but always baited at higher temperature than their minimum
growth temperature and at lower temperatures than their maximum growth temperature.
Pythium/Phytopythium from only from Clades A, B, E, and K (32) were found in the two
commercial greenhouses, and they were present in both greenhouses. In a lake survey in
Germany, Pythium clades A, B, and K were also found, with the addition of clades J and F. The
latitude of the Lake Constance survey area is (9°11’20” E, 47°41’48” N) (40) The latitude of
Greenhouse S is (40°49'31.30"N, 76°48'18.17"W) and Greenhouse E (40°13'21.56"N,
76°16'13.60"W). Certainly clades A, B, and K include species that live in aquatic ecosystems,
with B2 species being especially prevalent. Either this is the sole reason they were found in our
water baiting, or they live in waters associated with greenhouse crops. More extensive
greenhouse surveying will be necessary to clarify.
Pythium aphanidermatum, P. irregulare, and P. cryptoirregulare are species known to
have caused crop losses in the two greenhouses in seasons prior to when the extensive water
sampling survey was conducted. Those species were not obtained during the baiting. Most of the
isolates baited are not regarded as major pathogens in greenhouse crops. However all of the
species we have isolated except for Phytopythium chamaehyphon and Pythium rostraifingens
have been reported to cause plant diseases in laboratory tests. Some of these cases appear to be
quite temperature dependent (8; 30; 41; 42; 58). Pythium diclinum is moderately pathogenic in
lab pathogenicity tests (4; 60) and P. coloratum is pathogenic on some root crops (19; 57).
Phytopythium helicoides, P. diclinum, Ph. litorale, and Ph. chamaehyphon were all baited from
Tennessee streams, suggesting their lifestyle could be mainly aquatic (51). P. diclium is
commonly found in freshwater, irrigation water and in association with plant roots (5; 35; 48). P.
middletonii has been found in irrigated soils (6).
48
None of the baited isolates have been found causing crop losses in samples submitted to
the Penn State Plant Disease Clinic (Gary Moorman, personal communication.) However, some
of the baited isolates are mefenoxam resistant; suggesting the use of fungicides has genetic
consequences on the non-targeted organisms, or it can be a quality that these species possess and
hypothetically could be transferred to pathogenic species through hybridization (39). Pythium
species differ in their sensitivity to mefenoxam and therefore it is recommended to using a
combination of fungicides in rotation when attempting to control Pythium disease (59).
Resistance can often come from the wild relatives of a pathogen (18) therefore it is troubling to
see high resistance present in some of these environmentally-baited species.
It is possible that the baiting technique used was not ideal for detecting P.
aphanidermatum, however this method has successfully captured P. aphanidermatum from an
aquatic source (5). It is still unclear whether these species in the water tanks pose a problem to
greenhouse growers or only as secondary pathogens. Therefore we aim to assess the
pathogenicity of these isolates and further study their ecology in water tanks.
It is very probable that the Pythium species commonly found in aquatic environments are
better adapted for that lifestyle, perhaps by reproducing more often asexually. P. dissotocum and
Pythium catenulatum zoospores have been shown to greatly differ in their survival in air dried
silica sand soil, with P. dissotocum unable to survive and P. catenulatum surviving up to 16 days
(44). P. dissotocum is the species almost indistinguishable from P. coloratum, which was our
most commonly baited isolate. Therefore it is likely adapted to live in aquatic environments,
whereas species like P. catenulatum are better adapted at surviving on land. The categorization
of a Pythium as lentic (in still or slowly moving water), part of the periphyton (attached to
submerged substrates), or soil-borne should be done on a species-by-species basis. Phylogenetic
analyses do not provide answers to Pythium ecology. P. aphanidermatum, is in Clade A with
algal parasites (periphyton) but some of these species can also be land plant parasites (32). Yet in
the work here, P. aphanidermatum does not appear to be well adapted to or be a long term
resident in the aquatic environment.
49
Acknowledgements
I would like to thank Miss Jesse Edson for her technical contributions to this project and Dr.
David Geiser for his inputs on the phylogenetic analysis. I also thank Dr. Maria Burgos-Garay
for her work on this project and Sara May for letting me use her microscope camera. This project
was funded by the USDA-ARS Specialty Crops Research Initiative Grant (SCRI Project #: 201051181-21140): “Integrated management of zoosporic pathogens and irrigation water quality for a
sustainable green industry”.
50
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56
Chapter 3
PATHOGENICITY OF THE SPECIES OF Pythium AND Phytopythium FREQUENTLY
FOUND IN RECYCLED IRRIGATION WATER AND THEIR INTERACTIONS WITH
Pythium aphanidermatum, P. irregulare, AND P. cryptoirregulare
Abstract
The first objective of this study was to assess the pathogenicity of Pythium and
Phytopythium species found in the greenhouse recycled irrigation water tanks of two
Pennsylvania greenhouses. The second objective was to determine if the species residing in
water tanks interfere with disease development in pathogenic Pythium species. The third
objective was to determine if the non-pathogenic or pathogenic species dominate the plant roots
when co-inoculated. In a lab test using geranium (Pelargonium X hortorum) seedlings grown on
filter paper moistened with fertilizer, most of the baited species frequently found in irrigation
water were found to be pathogenic. We can presume that an environment more like the soilless
pathogenicity tests, such as a hydroponic greenhouse could experience seedling losses to these
environmental Pythium. However in a second lab test using small pots with a pasteurized peatbased potting mix, none of the baited species were pathogenic. When fertilizer was added to this
experiment, Ph. helicoides and Clade B2 unknown caused seedling mortality. In greenhouse
experiments, none of the baited isolates caused plant disease, nor did Pythium aphanidermatum,
Pythium cryptoirregulare, or Pythium irregulare. In co-inoculation tests using geranium
seedlings grown on filter paper moistened with fertilizer, Clade E2-1 unknown, Clade A
unknown, and P. coloratum were found to slow disease progress. Isolates of P. coloratum, B2
unknown, E2-1 unknown, P. rostratifingens, and Phytopythium chamaehyphon demonstrated
disease promoting properties. None of these interactions were observed in the greenhouse
experiments. At the end of the greenhouse experiments, co-inoculated plant roots were plated on
agar and the known pathogenic species P.cryptoirregulare, and P. irregulare were found
overwhelmingly more often on plant roots than baited species. Gause’s law of competitive
exclusion best explains the occurrence of Pythium species on plant roots.
Introduction
Pythium, from the Greek “pythein” – to cause rot (15), is a genus of organisms in the
class Oomycota and kingdom Chromalveolata (5). Some species of Pythium, especially P.
57
aphanidermatum (18), are plant pathogens and have the potential to cause significant losses of
greenhouse crops. It is generally assumed that Oomycetes are waterborne and therefore may be
harbored in or dispersed via irrigation water (30). Thus monitoring of irrigation water for
Pythium can provide knowledge needed to manage a Pythium disease outbreak (10). As noted in
Chapter 2, several Pythium and Phytopythium species were baited from the irrigation tanks of
two Pennsylvania greenhouses. According to Penn State Plant Disease Clinic records and
experience working with these two greenhouses (Moorman, pers. com.), these species are not
responsible for disease outbreaks in these greenhouses. One objective of the present work was to
assess the pathogenicity of all of these isolates, in order to gauge the risk of using recycled
irrigation water. This study also assesses if these baited isolates can suppress disease
development caused by Pythium aphanidermatum, P. irregulare, and P. cryptoirregulare,
species known to have caused losses in these greenhouses. In the case of carrot cavity spot and
on alfalfa roots, non-pathogenic isolates of Pythium are often found in lesions alongside
pathogenic species, but not believed to influence disease development (14). It was noted that
during the infection phase, it is not understood how these non-pathogenic and pathogenic species
interact (29). The interaction between soil organisms such as fungi and oomycetes is not well
understood, but is important in disease development (17). On parsnip and parsley, 11 different
Pythium species were found on the roots of diseased plants. In lab pathogenicity tests, the species
varied in their virulence (22). By co-inoculating plants with the pathogenic and non-pathogenic
species, a better understanding of the ecology these species in the rhizosphere is sought. By reisolating Pythium from the plant’s roots, the objective was to determine if the non-pathogenic
species are still present in the rhizosphere, or if the pathogenic isolate dominated.
It is possible that there are different ecological niches of these isolates. Non-pathogenic
species may remain on roots, not causing disease, and eventually get outcompeted or coexist
with pathogenic species. The pathogenic species may not be able to totally dominate the
environment due to presence of antagonistic microorganisms or the establishment of nonpathogenic Pythium on the roots. Perhaps the more pathogenic species have a greater nutrient
requirement and thus are more easily out-competed.
The question why many different Pythium species exist on plant roots is challenging. It is
difficult to determine if the species are occupying different niches, or are mainly competing for
58
space. Gause’s law of competitive exclusion is the traditional ecology viewpoint that states only
one species will dominate a niche (28). Gause’s law explains the presence of multiple Pythium
species in a place, assuming that the species differ in their requirements, growth rates, etc. and
therefore actually are occupying different niches. A more modern ecological theory proposed to
replace Gause’s law is the Unified Neutral Theory of Biodiversity and Biogeography (27). The
Neutral Theory is based on findings that suggest species abundance distributions display
universal patterns. The Neutral Theory proposes that species on the same trophic level have
equal likelihoods of death, immigration, speciation, and birth, therefore stochastic processes are
involved in determining which species will occupy an open space in the ecosystem (12). In some
fish, the “lottery hypothesis” is applied in which two species compete for space and when space
becomes available, the closest fish to the area will colonize and persist in that space (19). Some
scientists have applied this lottery concept to microbial ecology with the added facet that
although colonization is random, the possession of certain functional genes is the prerequisite for
microbes existing in a certain niche (7). If this concept was applied to Pythium communities
associated with plant roots, the presumption would be that if all the Pythium species found on
roots shared the same functional genes that allowed colonization of the roots, that their presence
and abundance on a certain plant’s roots is due to random chance.
Besides early colonization, environmental conditions, and random chance, there are some
reasons as to why some plant roots may lack a diversity of Pythium species. There are several
mycoparasitic Pythium species that will target other Pythium species. One such mycoparasite is
Pythium oligandrum, which will also induce the plant to defend against other Pythium species
(6). In this study, we aim to discover if the Pythium species that make up the community in
greenhouse irrigation water tanks have any antagonistic properties against pathogenic Pythium
species, have any advantage over the pathogenic species in root survival and to assess their
pathogenicity.
Materials and Methods
The pathogenicity of selected isolates representative of species frequently found in
irrigation water was tested on Pelargonium X hortorum 'White Orbit' or 'Maverick White'
geranium seedlings. White flowered cultivars tend to be more susceptible to disease, (20)
therefore making these varieties good model host plants to employ. Five seeds were placed on
59
sterile filter paper moistened with 15 mL of 300 ppm nitrogen water soluble fertilizer (15% N,
15% P2O5; 15% K2O; Peters Fertilizer) in plastic dishes. The dishes were placed under
fluorescent growth lights with a 12 hour day/night cycle at room temperature. After a week of
growth, the seedlings were inoculated. Isolates were grown on water agar with 10, 1 cm long
segments of creeping bentgrass (Agrostis stolonifera L. 'Penn Eagle') blades that had been boiled
for 10 min. in distilled water. After 3 days of incubation in the dark at 25°C, a grass blade
colonized by the isolate was placed on the root of the seedling. Two dishes were set up for each
isolate and the experiment was repeated. The experimental setup was a randomized block design.
Disease progression over a one-week period was recorded. Different ratings were given for
browning of the roots, browning of the stem, and collapse of the seedling. In co-inoculation tests,
Pythium aphanidermatum (P128; originally isolated from chrysanthmums in greenhouse E in
2003), P. irregulare (P84; originally isolated from a hydroponic production system in a
commercial greenhouse in 2001) and P. cryptoirregulare (P123; originally isolated from
impatiens in greenhouse E in 2003) were used because of their known pathogenicity on geranium
seedlings. Disease progression was said to be slowed or promoted if the first day of infection
happened later or sooner in the co-inoculation dishes than in the control. First symptoms were
usually observed 1-2 days after inoculation. The same methods were used in the co-inoculation
lab tests. The temperatures were recorded for each test (StowAway; Onset Computer). For the
co-inoculation tests, one grass blade colonized with a baited isolated was placed on top of the
plant roots and one blade colonized with a pathogenic isolate was placed over that.
60
Figure 3-1. Geranium (Pelargonium X hortorum) seedlings germinated on sterile filter paper
moistened with 15 mL of 300 ppm nitrogen water soluble fertilizer (15% N, 15% P2O5; 15%
K2O; Peters Fertilizer) in plastic dishes. Seedlings were inoculated by placing a 1 cm long
segment of creeping bentgrass (Agrostis stolonifera L. 'Penn Eagle') blade that had been boiled
for 10 min. in distilled water and colonized by the isolate on the root of the seedling.
Pathogenicity was tested in potting mix in the laboratory as follows. Farard #2 potting
mix was steam pasteurized for 60 minutes. Approximated 75 cc of this mix was placed in each
4.7 cm (2.25 inch) square plastic flower pot. Five Pelargonium X hortorum 'Maverick White'
geranium seeds were planted at the perimeter of each pot and watered thoroughly. The pots were
placed in an opaque, black plastic flat, covered with a clear plastic dome, and another opaque
black plastic flat was inverted over the clear dome in order to foster good seed germination.
After 48 hr, the inverted flat was removed and the dome covered flat of potted seedlings was
placed under fluorescent lights (12 hr day/night cycle) at room temperature (20 -22°C) in the
laboratory. Autoclaved rye (Secale cereale L.), seeds were sprinkled on the surface of water
61
agar. While one plate was not inoculated, each isolate to be tested was inoculated onto the center
of other plates. The plates were incubated at room temperature for approximately 7 days.
Approximately 7 days after seeding, pots with 5 seedlings were chosen or seedlings from extra
pots were transplanted to obtain 5 seedlings per pot. In the center of each pot, a 7 mm diameter
instrument was used to make a hole to the bottom of the pot. Two incubated, non-inoculated rye
seeds were placed in the hole of each check pot and two colonized seeds were place in the hole
of each pot of inoculated plants and all of the pots were watered lightly. Five check pots and five
pots per test isolate were incubated at room temperature under the flurorescent lights in one
laboratory and an additional five check pots and five pots per test isolate were incubated in
another laboratory. In addition to the non-inoculated check plants, Pythium aphanidermatum
isolates P128 and P220 (originally isolated from tomatoes grown in a commercial greenhouse
hydroponic system in 2014) were included as standard comparisons. The number of infected
seedlings in each pot was recorded. The pots were arranged in a completely random manner
across three greenhouse benches.
Figure 3-2. The laboratory potting mix pathogenicity test setup.
Isolates were also tested in a greenhouse experiment to verify their pathogenicity singly
and in co-inoculations. The tests ran in late autumn 2013 and spring 2014. In the autumn
greenhouse temperatures ranged from 14 to 20° C, with a mean temperature of 17.6° C in
November and 15.5° C in December. In the spring the temperatures ranged from 15 to 30° C
with averages of 18.8° C in March, 20.9° C in April, and 22.8° C in May. Inoculum was grown
for a week at 25° C in the dark on potato dextrose agar and then homogenized with sterile
62
distilled water (100 ml of sterile water per 100 X 15 mm plate). Ten milliliters of the
homogenate was placed on the potting soil (Fafard #2) surface in 10 cm diameter round plastic
pots each containing one, 6 week old geranium. There were 30 pots each for P. aphanidermatum,
P. irregulare, P. cryptoirregulare, and the control. Each baited isolate was inoculated alone in
10 pots and it was co-inoculated with P. aphanidermatum, P. irregulare, P. cryptoirregulare (5
pots for each treatment.) The experimental design was completely random across three
greenhouse benches. After the experiment, the height of every plant was measured and analyzed
using the Sidak Method and Bonferroni Method in a General Linear Model ANOVA in SAS
(25). Five, 2 cm long root segments were placed on water agar and Pythium or Phytopythium
isolated from the agar. This was done on two plant each for every replicate, except the negative
controls and the positive control plants with the highly virulent species, which got 5 plants each
both experiments. Identification was done by sequencing the ITS region of the isolates obtained
(see Chapter 2 for sequencing details).
Figure 3-3. The greenhouse experimental setup.
Results
In the laboratory fertilizer dish experiments shown in Figure 3-1 the Clade E2-1
unknown, Pythium coloratum, Clade E2-2 unknown, Clade B2 unknown, and Phytopythium
chamaehyphon strains contained isolates that were both pathogenic and non-pathogenic.
63
Phytopythium helicoides and Clade A unknown strains contained pathogenic isolates and
Pythium rostratifingens isolates were non-pathogenic. In single isolate inoculations, symptoms
generally appeared approximately one to two days after inoculation. The selected Clade E2-1
unknown isolates slowed disease progression of P. irregulare and promoted disease progression
with P. aphanidermatum. P. coloratum isolates slowed disease progression with all pathogens
and promoted disease progression with P. irregulare and P. aphanidermatum. Clade B2
unknown isolates promoted disease with P. aphanidermatum. P. rostratifingens, and Ph.
chamaehyphon isolates promoted disease development with P. irregulare. Clade A unknown
isolates slowed disease progression with P. irregulare and P. cryptoirregulare. All of this
information can be found in Table 3-1, with an expanded table in the Appendix, Table A-8.
The laboratory potting mix pathogenicity tests included two isolate representatives from
every baited species (see Table A-9 in the Appendix for a listing of the isolates). None of the
baited species caused visible symptoms, and P220 P. aphanidermatum consistently caused
seedling mortality. Symptoms as a result of P220 inoculation generally appeared four days after
inoculation but sometimes were exhibited after two days and the maximum number of plants
dying was reached by day seven or eight. P128 P. aphanidermatum did not consistently cause
disease. This may be the result of it extended time in culture and a reduction of pathogenicity.
None of the plants died in either greenhouse experiments, including the positive controls.
Again, the reduction of pathogenicity in isolate P128 may have been a factor. The heights of
each group were compared using the Sidak Method and Bonferroni Method in a General Linear
Model ANOVA (P≤0.05), and no significant differences were found among any of the treatment
groups. The greenhouse experiments did yield data on the species that existed on the geranium
roots after the experiment. Overwhelmingly, P. irregulare, and P. cryptoirregulare were
recovered more often from the plant roots than the baited isolates. The control plants showed that
both the baited isolates and the pathogenic isolates were recovered from co-inoculations with all
of the isolates except Phytophthium helicoides. P. middletonii, Clade B2 unknown, and Clade A
unknown strains were not recovered from the co-inoculated plant roots. P. coloratum was
recovered 35.4% of the time, Clade E2-1 unknown 4.4%, Clade E2-2 unknown 5.3%, P. sp. nov.
OOMYA1646-08 (E2) 13.3%, P. rostratifingens 7%, and Phytophthium. chamaehyphon 9.1%.
64
Table 3-1. Pathogenicity on Pelargonium X hortorum geranium seedlings grown on filter paper
moistened with soluble fertilizer (300 ppm N; 15% N, 15% P2O5; 15% K2O) and co-inoculation
results.
Isolate designation
(number of isolates)
Clade E2-1 unknown
Pythium coloratum
Phytopythium helicoides
Clade E2-2 unknown
Clade B2 unknown
Pythium rostratifingens
Phytopythium
chamaehyphon
Clade A unknown
Pathogenic Co-inoc with
P. irregulare
(-)(+)
S
(-)(+)
S, P
(+)
(-)(+)
(-)(+)
(-)
P
(-)(+)
P
Co-inoc with P. Co-inoc with P.
cryptoirregulare apahanidermatum
P
S
S, P
(+)
S
S
P
S- slowed disease progression, P-promoted disease progression. (+) = pathogenic, (-) = nonpathogenic. If more than one category is listed, it means isolates within the strain gave different
results.
Table 3-2. Combined results of the root isolations from the co-inoculation experiments.
Isolate
% of times
pathogenic
isolate
recovered
95.6
Clade E2-1 unknown
64.6
Pythium coloratum
100
Phytopythium helicoides
95.7
Clade E2-2 unknown
100
Pythium middletonii
100
Clade B2 unknown
Pythium sp. nov. OOMYA1646-08 86.7
(E2)
93
Pythium rostratifingens
90.9
Phytopythium chamaehyphon
100
Clade A unknown
% of times
baited isolate
recovered
Were both
species
recovered?
4.4
35.4
0
5.3
0
0
13.3
yes
yes
no
yes
yes
yes
yes
7
9.1
0
yes
yes
yes
% pathogenic- % of the time P. irregulare and P. cryptoirregulare were recovered. %
baited- % of the time the baited isolates were recovered. Note: The sample size for P.
helicoides is 1.
65
Discussion
As noted in Chapter 2, most of the isolates we obtained through baiting have been
reported pathogenic in lab tests (3; 13; 21; 22; 31). Often these isolates were classified as
moderately pathogenic because they only caused 60% mortality of the plants. The fertilizer lab
tests performed were a very simplified and perfect environment for Pythium disease
development. The seedlings were one week old, the filter paper was constantly moist, the roots
were exposed, and the corresponding microbial community was probably very small (1). In the
lab tests using geranium seedlings on moistened filter paper, we found pathogenic activities in all
of the isolates evaluated except P. rostratifingens, which is concordant with the literature (9). To
our knowledge, this is the first report of Phytopyhtium chamaehyphon being classified as
pathogenic in any system. We can presume that an environment more like the filter paper
pathogenicity tests, such as a hydroponic greenhouse could experience seedling losses to these
environmental Pythium. It is therefore advised for hydroponic greenhouses to either start their
seedlings in a potting mix, or have a sterilizing system implemented in their water system.
When the experiment was redesigned and rerun with one-week old seedlings in
pasteurized, peat-based potting mix, the baited isolates did not infect plants. This system is more
complex than the filter paper system and probably has a more complex microbial community
present in the potting mix. This experiment was repeated with 300pm fertilizer, and in this
instance, Ph. helicoides and Clade B2-unknown caused disease symptoms. Ph. helicoides has
already been reported as a greenhouse pathogen, (13) therefore growers still face a possible risk
from these species lurking in their waters. Concerning the greenhouse experiment positive
controls not causing disease, it is possible that the temperatures were not conducive to disease or
the pathogenic isolates had been kept in culture too long and lost pathogenicity. The loss of
pathogenicity in Fungi is not uncommon (8), but to our lab’s knowledge has not been reported in
the literature for Pythium.
The known pathogenic species, P. irregulare and P. cryptoirregulare in the greenhouse
experiments, were isolated much more frequently from the geranium roots than any of the baited
isolates at the end of the experiments (approximately 2.5 months). Pythium dissotocum and P.
irregulare were most commonly found on the roots of greenhouse-grown Kummerowia
stipulacea (Korean clover) (16). In our experiment Pythium coloratum isolates had the highest
66
recovery percentage of all the water tank isolates, and P. irregulare was also highly recovered. A
study found P. dissotocum in greenhouse water sources, and it was able to colonize plant roots
but did not cause obvious disease symptoms (23). Perhaps Pythium Clade B2 isolates have a
niche both on greenhouse plant roots and in greenhouse water tanks. From the previous chapter,
it was shown that P. irregulare and P. cryptoirregulare have higher growth rates than the baited
isolates, with the exception of Phytopythium helicoides, Clade B2 unknown, and some isolates of
Phytophtyium chamaehyphon. Despite this, in the co-inoculated plants with Phytopythium
helicoides and Clade B2 unknown, only P. irregulare and P. cryptoirregulare were recovered.
Therefore it would not appear that growth rate on agar is not a good indicator of recovering a
specific species. The recovered Pythium species were not equal in the co-inoculated plants,
therefore the Neutral Theory does not explain why different Pythium species are found together
on plant roots; instead Gause’s law of competitive exclusion best explains the results. This
means that the pathogenic species are better adapted to colonize the plant roots, out-competing
the isolates that were obtained by baiting the water. It has been shown that species of Pythium
will differ in their zoospore encystment on plant roots, depending on the host plant (24). This
may be a factor in the association of the pathogenic isolates with plant roots.
Interestingly, we found that P. irregulare and P. cryptoirregulare can remain on plant
roots without causing disease symptoms. On alfalfa seedlings, post-emergence damping-off
cause by P. irregulare was most severe at 16°C and 21°C (11), so perhaps the temperature was
not favorable for disease development. Or it could be similar to our observations with Pythium
ultimum, which is a highly pathogenic species that is found in abundance in agricultural soils
across the globe without causing disease (2). In the case of cotton, plants inoculated with P.
irregulare did not have any final height differences than the control plants, but yielded 11-14%
less seed (26). Therefore we should not be too surprised by the lack of obvious disease
symptoms, as it may have manifested itself in flower and seed production, which was not
measured in these studies. In the case of P. aphanidermatum, it was discovered that the culture
used for inoculation (P128) had lost pathogenicity. It also exhibited a much slower growth rate in
culture. It did not survive on the plant roots when they were sampled at the end of the
experiments. Soil pH can be a limiting factor of long-term Pythium survival in soil, as some
species may fail to form oospores (4). This does not appear to be the case for the low rate of
67
recovery in the water isolates because in the control plants with only the baited isolate, it was
recovered at the end of the experiment.
We have provided laboratory-generated data on the pathogenicity of Pythium and
Phytopythium species frequently found in greenhouse recycled irrigation water tanks. It would be
ideal to repeat these experiments in a greenhouse setting in order to properly assess if these
species could potentially pose a problem to greenhouse growers.
The baited isolates that slowed disease progression in lab tests did not prevent the death
of the seedlings in the greenhouse. There is the possibility that in the lab filter paper experiments,
the baited isolates colonized the roots first which prevented the highly pathogenic Pythium
species from gaining more access to root space; accounting for the retardation in disease
development. In the greenhouse experiment, it was apparent that the highly pathogenic species
were highly associated with the root systems, perhaps indicating that they are better procurers of
space in the long run and can easily overcome initial colonization by other species. More
research is required to clearly define the interactions among species of Pythium and their
possible influence on plant disease development. It is likely that the conditions in the two
different experiments were very different. The temperatures differed greatly and it is likely that
the lab tests had a much simpler microbial community present as compared to the greenhouse
experiment. In the soilless potting mix pathogenicity tests, none of the baited isolates were
pathogenic, except when this test was done with fertilizer and then Ph. helicoides and Clade B2
unknown were pathogenic. Therefore hydroponic producers and flood-floor production
greenhouse growers may need to treat their recycled water if they commonly encounter disease
caused by Ph. helicoides or Clade B2 unknown species.
Acknowledgements
I would like to thank Miss Jessie Edson and Miss Sara Getson for their help in the greenhouse
experiments. This project was funded by the USDA-ARS Specialty Crops Research Initiative
Grant (SCRI Project #: 2010-51181-21140): “Integrated management of zoosporic pathogens
and irrigation water quality for a sustainable green industry”.
68
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Pythium dissotocum Drechsler in lettuce. Summa Phytopathologica 37(1):52-58.
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Barton, R. 1958. Occurrence and establishment of Pythium in soils. Transactions of the
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Beakes, G. W., S. L. Glockling, and S. Sekimoto. 2012. The evolutionary phylogeny of
the oomycete "fungi". Protoplasma 249(1):3-19 doi:10.1007/s00709-011-0269-2.
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Benhamou, N., G. le Floch, J. Vallance, J. Gerbore, D. Grizard, and P. Rey. 2012.
Pythium oligandrum: an example of opportunistic success. Microbiology-Sgm 158(Pt
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Burke, C., P. Steinberg, D. Rusch, S. Kjelleberg, and T. Thomas. 2011. Bacterial
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doi:10.1073/pnas.1101591108.
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Dahmen, H. 1983. Technique for Long-Term Preservation of Phytopathogenic Fungi in
Liquid Nitrogen. Phytopathology 73(2):241 doi:10.1094/Phyto-73-241.
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De Cock, A. W. A. M., and C. A. Lévesque. 2004. New species of Pythium and
Phytophthora. Studies in Mycology(50):481-487.
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Fisher, P., and C. Smith. 2007. Monitoring Pathogens & Algae In Irrigation Water. Pages
8. Meister Media Worldwide, Willoughby.
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Hancock, J. G. 1991. Seedling and rootlet diseases of forage alfalfa caused by Pythium
irregulare. Plant Disease 75(7):691-694.
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Hubbell, S. P. 2001. The unified neutral theory of biodiversity and biogeography. Vol.
32. Princeton University Press, Princeton, N.J.
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13.
Kageyama, K., T. Aoyagi, R. Sunouchi, and H. Fukui. 2002. Root rot of miniature roses
caused by Pythium helicoides. Journal of General Plant Pathology 68(1):15-20
doi:10.1007/PL00013047.
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Larkin, R. P., English, J. T., Mihail, J. D. 1995. Identification, distribution, and
comparative pathogenicity of Pythium spp associated with alfalfa seedlings. Soil Biology
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Merriam-Webster.com. 2014. Pythium. in: Merriam-Webster.
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Mihail, J. D., L. F. Hung, and J. N. Bruhn. 2002. Diversity of the Pythium community
infecting roots of the annual legume Kummerowia stipulacea. Soil Biology and
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Mirkova, E., Maneva, S. 2007. Effect of four soil-borne fungi on mortality of greenhouse
carnation separately or in combination. Plant Science 44(4):323-327.
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Moorman, G. W., S. Kang, D. M. Geiser, and S. H. Kim. 2002. Identification and
characterization of Pythium species associated with greenhouse floral crops in
Pennsylvania. Plant Disease 86(11):1227-1231.
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71
Chapter 4
AQUATIC SURVIVAL OF Pythium aphanidermatum, Phytopythium helicoides AND
Pythium coloratum
Abstract
This experiment was performed in order to explore possible reasons why after an
exhaustive baiting of two greenhouse recycled irrigation water tanks, several Pythium species
were found but a species responsible for frequent disease losses in the greenhouses, Pythium
aphanidermatum, was not found. Thirty liter tanks with trays containing plants above the tanks
were set up to simulate the ebb and flood watering method used in these greenhouses. Tanks
were co-inoculated with Pythium aphanidermatum, the pathogen known to cause crop losses but
not found in the baiting survey, and two species commonly found in the irrigation tanks:
Phytopythium helicoides and Pythium coloratum. The tanks were baited weekly for 17 weeks
and plants checked occasionally for Pythium growth on the roots. P. aphanidermatum was not
recovered from any of the experimental or control tanks or any of the plants, while Ph. helicoides
and P. coloratum were successfully recovered from both tanks and geranium roots. It is
hypothesized that the reason for this result is that P. aphanidermatum is not one of the Pythium
species commonly found living in lentic environments.
Introduction
The Oomycete plant pathogen Pythium aphanidermatum (Edson) Fitz., causes losses in
both field (3) and greenhouse crops (2), and is most often found on poinsettias in Pennsylvania
greenhouses (13). During the continuous baiting of two commercial greenhouse recycled
irrigation water tanks P. aphanidermatum, which has been known to cause crop losses in both
greenhouses, was not recovered from the greenhouse’s water (6). Numerous other species of
Pythium were frequently isolated. This raised the question as to whether P. aphanidermatum is
truly aquatic and can persist in the recycled irrigation water tanks of greenhouses after a disease
event and if the water tanks provide the source of inoculum.
In a survey of Pythium in hydroponic systems, Clade B2 species (according to Lévesque
(12); Pythium group F, according to Plaats-Niterink (18)) that is, species with non-inflated,
filamentous sporangia including P. coloratum, were most commonly found and P.
aphanidermatum only represented 5% of the species found (9). Based on this report, water was
72
inoculated with only P. aphanidermatum, or with P. aphanidermatum and two species
commonly found in Pennsylvania greenhouse irrigation tanks, Pythium coloratum (Clade B2)
and Phytopythium helicoides. Ph. helicoides has been baited from a stream (15) and found
infecting greenhouse crops (19). The occurrence of Oomycetes in greenhouse water sources is
documented (7; 8) as well as in environmental sources (11; 14; 15). Wet soils are reported to
favor oospore germination in P. aphanidermatum (16). It has been postulated that Oomycetes
evolved from marine organisms (5). But since coming to land, some Oomycetes have become
adapted to soil environments and are no longer truly aquatic. The objective of this work was to
develop an understanding of whether water used for pot plant production is a harbor for P.
aphanidermatum in order to better understand the disease ecology of Pythium root rot.
Materials and Methods
Sixteen 30L tanks of tap water amended with 100 ppm N soluble fertilizer (15% N, 15%
P2O5; 15% K2O; Peters Fertilizer) were inoculated with ten, 3 cm long segments of creeping
bentgrass (Agrostis stolonifera L. 'Penn Eagle') blades colonized with selected isolates that were
frequently obtained from irrigation water (P. coloratum S5.2.11CB and Phytopythium helicoides
E7.20.11LT) and/or Pythium aphanidermatum (P128; originally isolated from chrysanthemums
in a commercial greenhouse in 2003).See Table 4-1 for a description of the inoculation
treatments. The experimental design was randomized and only preformed one time. Water
cultures with fertilizer were inoculated with a grass blade and checked with a hemocytometer to
confirm zoospore production in each isolate. Each tank had a pump that delivered water every
day from 1000 to 1015 hr. into above trays containing ten geranium plants (Pelargonium X
hortorum 'Maverick White') each. The tanks were continuously baited with 3 cm long segments
of bentgrass blades and Pythium isolated from the baits weekly by plating the blades on water
agar. The experiment lasted for 120 days. The plants were changed every 6 weeks with 2 week
old seedlings and the roots of two plants from each tank were plated on agar. Each tank’s water
was checked for pH and conductivity levels weekly. Temperature monitors were in most of the
tanks (HOBO U22 Water Temp Pro v2 logger; Onset Computer Corp., Bourne, MA) , in some of
the pots, and measuring the air (HOBO Pro Series temperature sensors; Onset Computer Corp.).
A dissolved oxygen sensor (HOBO Dissolved Oxygen Logger; Onset Computer Corp.) was in
one tank and a conductivity logger (HOBO U24 Conductivity Logger; Onset Computer Corp.) in
73
another tank for continuous readings. The recovered Pythium species grew out on water agar and
were then transferred to a water culture and identified based on morphology using an inverted
microscope.
Figure 4-1. The experimental setup
Tank
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Inoculations
Ph. helicoides
P. aphanidermatum + Ph. helicoides
P. aphanidermatum + Ph. helicoides + P. coloratum
P. aphanidermatum + P. coloratum
P. aphanidermatum + Ph. helicoides + P. coloratum
No inoculum
P. coloratum
P. aphanidermatum + P. coloratum
P. aphanidermatum
P. aphanidermatum + Ph. helicoides + P. coloratum
P. aphanidermatum + Ph. helicoides + P. coloratum
P. aphanidermatum + Ph. helicoides
P. coloratum
No inoculum
Ph. helicoides
P. aphanidermatum
Table 4-1. Experimental setup for ebb and flow experiment. Species are Phytopythium
helicoides- E7.20.11LT, P. coloratum S5.2.11CB, P. aphanidermatum P128. For each isolate is
listed, 10 colonized creeping bentgrass blades were added to the tank.
74
Results
The weekly temperature averages for the tanks can be found in the Appendix in Table A10. These temperatures are very similar to those recorded in the commercial greenhouses at the
time of the initial baiting for detection of Pythium (see Table A-7 in the Appendix) The number
of hours the tanks were between 25 and 30° C can be found in Table 4-2 and ranged between 0471 hours. The cardinal temperatures for the isolates we used in this experiment are: P
aphanidermatum 9° C, 35° C, 40° C, Ph. helicoides15° C, 35° C, 40° C, and P. coloratum 5° C,
30° C, 35° C. Thus, the tanks did not have the optimum temperatures for any of these isolates, as
the temperatures never reached 30°. At some points during the experiment, the tank temperatures
fell below 15°C, which was the minimum growth temperature of Ph. helicoides. The mean water
pH of the tanks ranged from 6.95 to 7.51. The mean electrical conductivity of the water in the
tanks ranged from 1.06 to 2.42 mS/cm. The mean conductivity for tank 3 was 2.269 mS/cm. The
starting dissolved oxygen content in tank 2 was 8.07mg/L and the ending was 8.43 mg/L.
Table 4-3 displays the species isolated from the tanks during the experiment. More than
one isolate of the same species was often recovered from the tanks. P. aphanidermatum was not
recovered from any of the tanks or plant roots, even the tanks in which it was the only species
inoculated. P. coloratum was the isolate most frequently recovered from the tanks, and was
recovered over 4 months after inoculation. Ph. helicoides was recovered up until October 30th
from the tanks and plant roots (Table 4-2). P. coloratum apparently contaminated adjacent tanks.
P. coloratum did not contaminate tanks 15 & 16, and it was not in a tank proximal to those tanks.
Therefore, it is likely the mode of contamination was by splashing when water returned to the
tank from the trays.
75
Tank
Tank 1
Tank 2
Tank 4
Tank 6
Tank 8
Tank 9
Tank 10
Tank 12
Tank 13
Tank 14
Tank 16
Phytopyhtium helicoides
P. aphanidermatum + Ph. helicoides
P. aphanidermatum + P. coloratum
No inoculum
P. aphanidermatum + P. coloratum
P. aphanidermatum
P. aphanidermatum + Ph. helicoides + P. coloratum
P. aphanidermatum + Ph. helicoides
P. coloratum
No inoculum
P. aphanidermatum
Hours between
25° and 30° C
25.5
0
29.3
339.2
325.8
340.8
471.2
323.7
131
176.7
290
Table 4-2. The number of hours the tanks had water temperatures between 25 and 30 degrees C.
Tank
8 9 10
c
c
c
c
c
c
c
c
c
h
h
h
c
1 2 3 4 5 6 7
11 12 13 14 15 16
Date
c c c
c
8/19
c
c
c
c
8/20
h
c
c
c
h
8/21
c c c h c
c
h c
8/28
c c
c
9/4
c
h
c c
h
h
P9/8
h
c
c
c
9/11
c h c
c
9/18
c c
h c
9/25
c
c
c
10/2
c c
h
10/9
c c
h
10/16
c
c c
10/23
c c c c
h
10/30
P11/1
c c c c
11/7
c
c c c
11/13
c c c c
11/20
c c c c c c
12/4
c c c c c
c
c
12/11
P12/14
Table 4-3. Isolates from baits during the experiment. Phytopythium helicoides- E7.20.11LT (h),
Pythium coloratum S5.2.11CB (c), Pythium aphanidermatum P128 (a).
76
Discussion
P. aphanidermatum was never recovered from any of the tanks. P. coloratum was very
frequently recovered. In fact, P. coloratum ended up contaminating the tanks containing no
Pythium and a tank that contained only P. aphanidermatum (Tank 9.) However, this
contamination of Tank 9 occurred towards the end of the experiment and for most of the
experiment, nothing was recovered from the baits in Tank 9. It is possible that the baiting
technique used was not ideal for detecting P. aphanidermatum, however this method has
successfully captured P. aphanidermatum from an aquatic source (4). The tank temperatures did
not reach the optimum temperatures of these isolates and in fact reached temperatures below the
minimum growth temperature for Ph. helicoides in the latter half of the experiment. On the other
hand, the average temperatures in this experiment were very similar to those recorded in the
commercial greenhouse tanks during the original continuous baiting that was done (see Table...
in the Appendix) by Burgos (cite her dissertation). Tanks 1-5 were next to the greenhouse
cooling system, and the temperature data logs show they remained colder than the other tanks.
Nevertheless, it does not seem this temperature difference prevented Ph. helicoides and P.
coloratum from being recovered.
It is apparent that during this experiment, the P. aphanidermatum isolate (P128)
employed was not readily baited, had lost its pathogenicity, or is not truly aquatic and does not
survive in water. P. aphanidermatum did not colonize the plant roots, but both of the other
isolates were found associated with the roots. More experiments need to be performed with
other, more pathogenic isolates of P. aphanidermatum to further assess the question of whether
P. aphanidermatum can survive in water, such as attempting to detect it’s presence by filtration.
It is theorized that Oomycetes evolved from marine parasites (5). Pythium and
Phytophthora produce zoospores, which implies an aquatic aspect of their lifestyle. However,
many of the downy mildews that evolved from Phytophthora and subsequently Pythium (17)
rarely form zoospores and obligate parasites of plants (1). Therefore, it is possible that plant
pathogenic oomycetes such as P. aphanidermatum have an ecology more closely tied to their
lifestyle than to their ancestry. Hong (10) noted that rather than a pathogen being waterborne, the
type of irrigation could merely be making the crop more susceptible to diseases from that
pathogen and it is present but actually not waterborne. This may be the case with P.
aphanidermatum in pot plant production utilizing ebb and flood irrigation.
77
Acknowledgements
I would like to thank Miss Sara Getson for her help in the setup of this experiment. This project
was funded by the USDA-ARS Specialty Crops Research Initiative Grant (SCRI Project #: 201051181-21140): “Integrated management of zoosporic pathogens and irrigation water quality for a
sustainable green industry”.
78
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Burgos-Garay, M. L. 2013. Effect of heterotrophic bacerial communities on Pythium spp.
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Lévesque, C., A. , and A. W. A. M. De Cock. 2004. Molecular phylogeny and taxonomy
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Moorman, G. W., S. Kang, D. M. Geiser, and S. H. Kim. 2002. Identification and
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Pennsylvania. Plant Disease 86(11):1227-1231.
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Nechwatal, J., A. Wielgoss, and K. Mendgen. 2008. Diversity, host, and habitat
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Stanghellini, M. E. 1973. Effect of soil water potential on disease incidence and oospore
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80
CONCLUSION
This research has revealed that there is a community of Pythium and Phytopythium
species that inhabit the irrigation water tanks of two commercial Pennsylvania greenhouses that
recycle their water for potted plant production. Importantly, the three plant pathogens that have
been known to cause crop losses in those greenhouses, P. aphanidermatum, P. irregulare, and P.
cryptoirregulare, were not detected across a total of 128 samplings involving intensive baiting in
the two greenhouses. The isolates that were frequently recovered have not been found infecting
symptomatic plants submitted to the Penn State’s Plant Disease Clinic from those greenhouses.
In a test to determine if the commonly baited species colonized the baits before an isolate of P.
aphanidermatum when co-inoculated, it was discovered that P. aphanidermatum was never
recovered from the inoculated tanks nor did that isolate become associated with geranium roots
in the experiment. It appears that P. aphanidermatum does not have an aquatic lifestyle.
The Pythium species found during the tank surveys were identified as: isolates almost
genetically identical to P. sp. nov. OOMYA1702-08 in Clade B2, two distinct species of
unknown identity in Clade E2, P. coloratum or one of the very closely related species such as P.
diclinum, P. middletonii, an unknown species in Clade B2, a species closely related to Pythium
sp. nov. OOMYA1646-08 (E2), P. rostratifingens, and an unknown species in Clade A. In
addition, three Phytopythium species were recovered through baiting: Ph. litorale, Ph. helicoides,
and Ph. chamaehyphon. Although some isolates were pathogenic on geranium seedlings grown
on fertilizer moistened filter paper, none of these isolates were pathogenic on geraniums in tests
using pasteurized, peat based soilless potting mix. However, when fertilizer was added to this
experiment, Ph. helicoides and Clade B2 unknown caused seedling mortality. In laboratory coinoculation tests using geraniums on fertilizer moistened filter paper, isolates from Clade E2-1
unknown, Clade A unknown, and P. coloratum were found to slow disease progress i.e. delay the
date until first symptom appearance. Isolates of P. coloratum, B2 unknown, E2-1 unknown, P.
rostratifingens, and Ph. chamaehyphon demonstrated disease promoting properties. However the
co-inoculating results were not observed in a greenhouse experiment using peat based potting
soil. Of the baited isolates, seven expressed varying levels of resistance to mefenoxam, including
isolates identified as Ph. helicoides, Clade E2-2 unknown, P. middletonii, and Pythium sp. nov.
OOMYA1646-08 (E2), with three displaying high resistance including isolates identified as P.
81
coloratum, P. rostratifingens, Clade A unknown. Seven expressed sensitivity, including isolates
identified as Ph. helicoides, Clade B2 unknown, Pythium sp. nov. OOMYA1646-08 (E2), and
Ph. chamaehyphon) with three displaying high sensitivity including isolates identified as Clade
E2-1 unknown, P. coloratum, and Clade E2-2 unknown. Isolates that are not sensitive to
mefenoxam indicate that resistance can exist in Pythium species and that using mefenoxam has
had effects on environmental species.
The pathogenicity and co-inoculation tests were set up in a large greenhouse experiment
in order to replicate the results in a natural setting. Unfortunately, no disease symptoms were
observed on any of the greenhouse plants. However, after the experiments, geranium roots were
tested for Pythium and it was discovered that most of the isolates inoculated remained on the
roots. In the co-inoculated plants, the species P. irregulare and P. cryptoirregulare were
recovered at a significantly higher frequency more often than the tank baited isolates. P.
irregulare and P. cryptoirregulare appear to out-compete the tank baited isolates for space on
plant roots. The recovered Pythium were not recovered with equal frequencies in the coinoculated plants, therefore the Neutral Theory does not explain why different Pythium species
are found together on plant roots; instead Gause’s law of competitive exclusion best explains our
results. This means that the pathogenic species are potentially better adapted to colonize the plant
roots, out-competing the aquatic isolates.
Overall it appears that these experiments dealt with two types of Pythium species, the
first type being the known pathogens P. irregulare, P. cryptoirregulare, and P. aphanidermatum
that are more adapted to live with soil and plants; and the water tank baited isolates that are
better adapted to an aquatic environment, perhaps as saprophytes and occasionally as root
commensals. Some of the water species displayed resistance to the Oomycete fungicide
mefenoxam. This could indicate that they are often found in association with greenhouse crops,
or that they already possessed the genetic capabilities for resistance.
It does not appear that the Pythium and Phytopythium species frequently found in
greenhouse water tanks play a role in reducing disease by other species of Pythium, or
suppressing P. aphanidermatum in the water system. It is definitely possible for severe Pythium
diseases to be spread by recycled irrigation water, but the water does not appear to be an initial
source of the inoculum in pot plant production. Treating irrigation water with chlorine or
82
fungicides to remove harmful Pythium spp., may be a futile effort, because the recirculating
water found in irrigation tanks may not be where they are harbored.
83
APPENDIX
SUPPLEMENTARY DATA
84
Characteristics Key
1) Zoospores released. Dpi=days past inoculation in water culture
2) Very few zoospores
3) Short discharge tube
4) Sporangia globose, ellipsoidal, or irregular
5) Sporangia globose, ellipsoidal, or irregular and catenulate
6) Sporangia just globose
7) Sporangia filamentous, inflated
8) Sporangia filamentous, non-inflated
9) Sporangia slightly inflated
10) Sporangia inflated parts + pyriform elements
11) Sporangia club-like
12) Sporangia spherical
13) Sporangia sometimes in sympodial succession arising from immediately below a
sporangium
14) Sporangia proliferating
15) Sporangia proliferating internally
16) Sporangia proliferating externally
17) Sporangia not proliferating
18) Sporangia on average 30 x 25 µm
19) Oospores often irregularly shaped
20) Oogonia intercalary
21) Oogonia terminal
22) Oospores aplerotic
23) Oospores plerotic or nearly so
24) Oogonia produced in single cultures
25) Oogonia not or scarcely produced in single cultures
26) Oogonial wall smooth or occasionally with few projections
27) Oogonial projections cylindrical, irregular
28) Oospore walls up to 3µm thick
85
29) Oogonial stalks straight
30) Oogonia containing a single oospore
31) Oogonia on average 21 µm
32) Oogonia on average 30 µm
33) Oogonia 35-40µm
34) Oospores uncolored
35) Many spherical smooth sporangia or oospore and no antheridia
36) Antheridia monolinous
37) Antheridia diclinous
38) Antheridia hypogynous
39) Antheridia stalks unbranched
40) Antheridia often intercalary
41) Antheridia making apical contact with oogonia
42) Antheridia 1-3 per oogonium
43) Antheridia absent
44) Antheridia entwining the oogonial stalk and base
45) Tiny, terminal spherical structures
86
Table A-1. Initial morphological observations of the isolates.
Isolate
designation
Isolate number
Pythium sp. nov.
OOMYA170208 (B2)
Clade E2-1
unknown
E3.28.11LB
Pythium
coloratum
E6.16.11LT
E6.16.11GB
E7.14.11GT
E7.20.11GT
E7.27.11GT
E8.2.11GT
E8.9.11GT
E8.16.11GT
S3.22.11CB
S3.22.11CT
E3.22.11RB
E3.28.11RB
E3.28.11RT
E4.4.11RB
E4.4.11RT
S4.5.11CB
S4.11.11CT
S4.11.11CB
E4.11.11RB
E4.11.11RT
S4.18.11CB
S4.18.11CT
E4.18.11RT
S4.27.11CB
S4.27.11CT
S5.2.11CB
S5.2.11CT
E5.2.11LT
E5.9.11LT
S5.9.11CT
S5.16.11CB
S5.16.11CT
S5.25.11CT
S6.6.11CB
Zoospore Morphological
release
Characteristics
date
Possible
Characteristics
4
12
6,14
4,25
6,16
4
4
10
10
4
2, 9
19-20, 24,30,36
4
7
21,42
7 21,42
8
8
8
7
7
7
7
4
7
12
8
8
8
87
Phytopythium
litorale
Phytopythium
helicoides
S6.6.11CT
E6.16.11GT
E6.23.11LB
E6.23.11LT
E6.23.11GB
E6.23.11GT
E7.8.11LB
E7.8.11LT
E8.9.11LT
E8.16.11LT
S10.11.11CT
S10.18.11CB
S10.18.11CT
S10.25.11CT
S11.1.11CB
S11.8.11CB
S11.15.11CB
S11.15.11CT
S11.22.11CT
S11.29.11CT
S12.6.11CT
S12.13.11CBP
S12.13.11CT
S12.13.11CTP
S3.28.11CT
E6.16.11RB
E7.1.11RT
E7.1.11RB
E7.1.11GB
E7.1.11GT
E7.8.11RB
E7.8.11RT
E7.14.11RBa
E7.14.11RT
E7.14.11SBT
E7.20.11LT
E7.20.11SBT
E7.20.11RB
E8.2.11SBT
E8.9.11SBB
8
8
4,12
4
3-4,8,13,20,24,26,28-30,33,37,41-42
8
8
8
10,14
24, 26
8
9,21,22,24,26
9,24,26
8
4
4
4,8
8
8
4
8
8
6 20, 37
4
4,21,22,24,42
4,21-22,24,26
4,17,21,24,26,37,39
4,17,21,22,24,26,30,37,39,42
4
3,4,12
20-22,24,26,37,42
4,15,21,22,24,26
21,22,24,25,42
3,4,15,22,24,42
4,15
4,24,42
4,23,24,26,42
8
15
15
88
Clade E2-2
unknown
Pythium
middletonii
Clade B2
unknown
Pythium sp. nov.
OOMYA164608 (E2)
E7.20.11LB
E7.27.11LT
E7.27.11SBB
S9.16.11 screen
S9.16.11 10
S9.16.11 60
S9.16.11 100
S9.16.11 270
E10.4.11LT
S10.4.11CB
E8.22.11LB
E8.30.11LT
E9.13.11LB
E9.13.11LT
E5.9.11RB
E5.9.11RT
E5.18.11RT
E5.26.11RB
E5.26.11RT
E6.1.11RB
E6.1.11RT
E6.8.11RB
E6.8.11RT
E6.16.11RT
E6.23.11RB
E6.23.11RT
E7.8.11SBB
E7.14.11SBB
E7.20.11SBB
E7.27.11SBT
E8.2.11SBB
E8.9.11SBT
E8.9.11RT
E3.22.11LT
E4.27.11RT
E10.31.11RT
E11.7.11RT
E11.29.11RT
E11.29.11RT poinsettia
E12.6.11RT
E12.6.11RT poinsettia
4
4,24,25
4
4, 13-14
4, 13-14
4
4, 13-14
4, 13-14
4,24,26,42
4
4
3,4,16,24,26,37
4,14,24,26,31
4,24,26,31
8
8
8
8
8
8
8,12
8
8
8
8
4,8,15
8
4,20-22,24,26,27
4
6
4
8
8,24,26,37,39,42
4, 36
4,17-18,21-22,24,26,30
4,24,26,41
4,24,26,37,39,41,42
4,26,42
4,22,26,37,42
4,21,24,26,30,36-39,42
4,24,26,30,36,37,39,42
38
89
Pythium
rostratifingens
Phytopythium
chamaehyphon
Clade A
unknown
E11.21.11SBT
poinsettia
E12.6.11RB
E12.6.11RB poinsettia
S3.28.11CB
S4.5.11CT
S9.22.11CB
S9.22.11CT
S9.26.11CT
S10.4.11CT
S10.11.11CBb
S11.1.11CT
S11.8.11CT
S11.22.11CB
poinsettia
S11.22.11CT
poinsettia
S11.29.11CT
poinsettia
S11.29.11CB
S12.6.11CB
S12.6.11CB
poinsettia
S12.13.11CB
E4.27.11LT
S5.31.11CB
S9.26.11CB
S9.16.11 Bay 15
P.
aphanidermatum
Other
E3.22.11RT
E4.18.11RB
E4.22.11RB
E5.2.11RB
E5.2.11RT
S5.9.11CB
E5.18.11RB
S5.25.11CB
E7.14.11RBb
E7.14.11LT
E7.20.11RT
E7.27.11RB
E7.27.11RT
4,24,26,37,42
24,42
26,26,42
4
4
4
6
24,26,36,42
14
4
24,26
4
24, 26
4,24,42
8, 43
4
4 14
4
4
4
4
4, 24, 26
8
8
7, 22, 24, 26, 29,30,34,40-42
4, 19-21, 36
8
17-18,2122,24,26,30,37
4,36
7
7
8
45
4,25,42
4
8
8
90
E8.2.11RB
E8.2.11LT
E8.9.11LB
E8.22.11GT
S10.11.11CBa
E10.31.11RB
S11.22.11CB
S11.29.11CB
poinsettia
S12.6.11CTP
4,15,24,26,44
8,17,23,24,26,32
10
10
35
4
4,24,26,41
8
4
4
A blank square indicates no data. The characteristic key below is based on those features
used by Plaat-Niterink (117)(117). Clade E2-2 unknown isolates S9.16.11 screen, S9.16.11
10, S9.16.11 60, S9.16.11 100, and S9.16.11 270 are from debris trapped on a coarse
screen through which return water was passed. P. aphanidermatum S9.16.11 Bay 15 was
an isolate from an infected poinsettia.
Zoospore release
time (Dpi=days
past inoculation
in water culture)
1 dpi
2dpi
3dpi
more
no zoozpores
maybe zoospores
91
Table A-2. A summary of the Pythium isolates baited from greenhouse irrigation tanks and a
listing of the representative isolates used in the phylogenetic analysis.
Species/ Isolate
Designation
Pythium sp. nov.
OOMYA1702-08
(B2)
Clade E2-1
unknown
Pythium
coloratum
Phytopythium
litorale
Phytopythium
helicoides
Clade E2-2
unknown
Pythium
middletonii
Clade B2
unknown
Pythium sp. nov.
OOMYA1646-08
(E2)
Pythium
rostratifingens
Phytopythium
chamaehyphon
Isolate/s used
Clad
in Phylogenetic e
Analysis
E3.28.11LB
B2
Number
Months Isolated
of Isolates from Greenhouse
S
1
none
Months Isolated
from Greenhouse E
E7.14.11GT
E2
8
September 2013
June- August
E6.23.11LB
S3.22.11CT
S3.28.11CT
B2
57
March-August
K
1
March-June
October-December
March
E7.20.11RB
K
16
none
June- August
E10.04.11LT
E2
12
July and October
E9.13.11LB
E2
4
September 2011 &
2013
none
E6.01.11RB
B2
19
none
May-August
E11.07.11RT
E2
9
March- April
October-December
December
E12.06.11RBP
E1
4
none
S9.26.11CT
K
10
November &
December
none
SeptemberDecember
March-April
A
6
May & September,
Clade A unknown S5.31.11CB
September 2013
All isolates are from 2011 unless specified as September 2013.
March
none
August & September
April
92
Table A-3. The cardinal temperatures of the isolates, mean daily growth rates at 25C on PCA,
and colony morphology on PCA.
Isolate
designation
Isolate number
Minimu
m
Growth
Temp (C)
Clade E2-1
unknown
E6.16.11LT
E7.20.11GT
F2:4
S4.27.11CB
E3.28.11RB
E3.28.11RT
E4.11.11RB
E4.11.11RT
E4.18.11RT
S10.11.11CT
S11.1.11CB
S11.1.11CT
S12.6.11CT
S4.11.11CB
S4.11.11CT
S4.27.11CT
S11.22.11CTP
S3.22.11CT
S11.29.11CT
S3.22.11CB
S10.18.11CB
S4.18.11CB
S4.5.11CT
S5.16.11CB
S12.13.11CB
E6.23.11LB
E6.23.11LT
E6.16.11GT
E5.9.11LT
E4.14.11RT
E4.4.11RB
E5.9.11RB
E7.8.11LT
S12.13.11CBP
S12.13.11CT
9
9
5
5
5
5
5
5
9
5
5
5
5
5
5
9
5
5
5
9
5
9
15
5
9
9
9
9
9
5
9
9
5
5
5
Pythium
coloratum
Optimu
m
Growth
Temp
(C)
30-35
35
35
30-35
30
30
30
30
35
30-35
30
30
30
30
30
30
30
30
30
30
30
30
30
30
25
30
30
35
30
30
30
30-35
30
30
30
Maximu
m
Growth
Temp (C)
Daily
Colony
Growt Morpholog
h (mm) y
35
40
40
35
35
35
35
35
40
35
35
35
35
35
35
40
35
35
35
35
35
35
35
35
30
35
35
35
35
35
35
40
35
35
35
13.42
13.74
12.16
13.92
13.75
12.62
10.66
9.36
5.57
13.63
13.71
14.31
13.33
14.87
14.97
10.93
13.02
14.31
10.18
12.20
11.48
9.65
13.99
11.48
10.31
12.53
8.97
8.95
8.26
12.79
10.35
5.21
11.74
9.79
11.61
Ra
Ra
Ra
Ra
Ra/SR
Ra/SR
N/R
N
R
Ra
Ra
R
Ra
Ra/R
Ra
N
R/Ra
R
Ra/SR
N
Ra/R
Ra/R
Ra
Ra/N
Ra/C
R/Ra
Ra/R
R
Ra/R
Ra/R
N
R
Ra/R
Ra/C
R
93
Phytopythium
helicoides
Clade E2-2
unknown
Pythium
middletonii
Clade B2
unknown
S12.13.11CTP
S4.18.11CT
E8.16.11LT
S5.2.11CT
S5.2.11CB
E7.14.11SBT
E7.20.11LT
E7.14.11RT
E7.1.11RT
E7.20.11RB
E7.8.11RT
E7.1.11GB
E7.1.11RB
E7.20.11SBT
E7.20.11LB
E10.4.11LT
S9.16.11 270
S9.16.11 10
F1:5:1
S1
E8.30.11LT
E6.23.11RT
E7.20.11SBB
E7.27.11SBT
E7.14.11SBB
E12.6.11RTP
Pythium sp.
nov.
E10.31.11RT
OOMYA1646 E11.29.11RT
-08 (E2)
E11.7.11RT
E3.22.11LT
E4.27.11RT
E11.29.11SBTP
Pythium
rostratifingens E11.21.11SBTP
E12.6.11RB
Phytopythium S9.26.11CT
chamaehypho S10.4.11CT
n
S12.6.11CB
S9.22.11CB
S11.29.11CB
S3.28.11CB
S9.22.11CT
C13
Clade A
5
5
5
5
5
5-15
15
15
15
5
15
15
5-15
5-15
5
5
15
5
5
5
5
30
30
30
30
30
35
35
35
35
35
35
35
35
35
30
30
30
30
30
30
25
35
35
35
35
35
40
40
40
40
40
40
40
40
40
35
40
35
35
35
35
30
14.34
14.51
12.84
13.32
10.95
19.06
23.72
20.99
21.01
21.00
20.80
15.64
20.01
17.75
12.65
12.57
11.13
13.29
13.55
12.66
9.76
Ra/SR
R/Ra
R/Ra
R/Ra
R
Ra
Ra
Ra
Ra
Ra
Ra
N
Ra
Ra
Ra
Ra
Ra
Ra
Ra
Ra
Ra
9
5-15
9
15
5
5
5
5
5
5
9
9
5
15
9
9
15
15
5
9
9
35
35
30
35
30-35
25
25
25
25
25
25
25
25
30
30
30
30
30
30
30
30
40
40
35
40
35
30
30
30
30
30
30
30
30
35
35
35
35
35
35
35
35
1.27
22.31
13.45
20.54
13.06
8.70
9.88
10.14
10.62
9.44
6.73
6.32
5.87
16.65
12.60
15.64
16.94
14.96
13.00
16.10
0.00
R
Ra
Ra
Ra
Ra
R/C
Ra
Ra/C
Ra
Ra/C
C
C
C
Ra
Ra
Ra
Ra
Ra
Ra
Ra/R
C/Ra
94
unknown
Not Yet
Sequenced
Other
C3
S2
S9.26.11CB
E5.16.11RB
C5
C6
C1
C14
C2
C12
P.
aphanidermatu
m
P. irregulare
P.
cryptoirregular
e
S9.16.11 Bay
15
5
15
9
9
15
5
15
15
9
9
9
30
35
30-35
30
30
35
30-35
25
35
25
35
35
40
35
35
35
40
35
35
40
35
40
0.00
0.04
0.27
9.60
0.00
13.90
0.31
0.10
13.98
0.22
10.55
Ra/C
R/Ra
Ra
Ra
Ra/SC
Ra
Ra/C
C/Ra
C/Ra
C/Ra
Ra/N
9
5
30
25
35
35
15.44
20.77
N
Ra/N
9
30-35
40
20.02
N
Ra-radiate, C-chrysanthmum, R-rosette, N-no pattern, S-slight pattern; if more than one pattern is
observed,
the pattern that most strongly visible is listed first in the classification.
s
Table A-4. Full results of the poison plate assay.
Isolate
designation
Isolate number
Clade E2-1
unknown
E6.16.11GB
E6.16.11LT
E7.20.11GT
F2:4
S4.27.11CB
E3.28.11RB
E3.28.11RT
E4.11.11RB
E4.11.11RT
E4.18.11RT
S10.11.11CT
S11.1.11CB
S11.1.11CT
S12.6.11CT
S4.11.11CB
S4.11.11CT
Pythium
coloratum
Growth with
Fungicide
mefenoxam/ growth
Sensitivity
without mefenoxam (%) Classification
22
S
19.5
S
20
S
5.5
HS
7.5
HS
30
S
32.5
S
35
S
14.5
S
23.5
S
36
S
40
S
34.5
S
23.5
S
39.5
S
23
S
95
Phytopythium
helicoides
Clade E2-2
unknown
Pythium
S4.27.11CT
S11.22.11CTP
S3.22.11CT
S11.29.11CT
S11.29.11CTP
S3.22.11CB
S10.18.11CB
S4.18.11CB
S11.22.11CBP
S4.5.11CT
S5.16.11CB
S12.13.11CB
E6.23.11LB
E6.23.11LT
E5.2.11LT
E6.16.11GT
E5.9.11LT
E7.8.11LT
S12.13.11CBP
S12.13.11CT
S12.13.11CTP
S4.18.11CT
E8.16.11LT
S5.2.11CT
S5.2.11CB
E4.14.11RT
E4.4.11RB
E7.14.11SBT
E7.20.11LT
E7.14.11RT
E7.1.11RT
E7.20.11RB
E7.8.11RT
E7.1.11GB
E7.1.11RB
E7.20.11SBT
E7.20.11LB
E10.4.11LT
S9.16.11 270
S9.16.11 10
S1
F1:5:1
E8.30.11LT
37
25
34
17.3
18.5
30.5
43.5
40.5
49.7
48.5
64
99.5
98
91.5
101
92
102
99
7.5
36
35.5
52.5
98
34.5
35
26
19.5
44
42.1
51
54.5
54.5
56
45.5
59
47.5
32.8
35
5.5
12
53.5
11
88
S
S
S
S
S
S
MS
MS
I
I
R
HR
HR
HR
HR
HR
HR
HR
HS
S
S
MR
HR
S
S
S
S
MS
MS
I
MR
MR
MR
MS
MR
MS
S
S
HS
S
MR
S
R
96
middletonii
Clade B2
unknown
Pythium sp. nov.
OOMYA1646-08
(E2)
Pythium
rostratifingens
Phytopythium
chamaehyphon
Clade A
unknown
Not Yet
Sequenced
Other
E6.23.11RT
E7.20.11SBB
E7.27.11SBT
E7.14.11SBB
E5.9.11RB
E12.6.11RTP
E10.31.11RT
E11.29.11RT
E11.7.11RT
E3.22.11LT
E4.27.11RT
E11.29.11SBTP
E11.21.11SBTP
E12.6.11RB
S9.26.11CT
S10.4.11CT
S12.6.11CB
S9.22.11CB
S11.29.11CB
S3.28.11CB
S9.22.11CT
E4.27.11LT
S9.26.11CB
S2
C13
C3
E5.16.11RB
C5
C6
C1
C14
C2
C12
E8.9.11LB
P128 2012
P.
aphanidermatum
P. irregulare
P.
cryptoirregulare
13-151A
S9.16.11 Bay 15
16
33.1
28.5
42.4
9.5
7
84.5
89.5
89.5
89.5
77
91
97
83.5
41
22.8
24
43
43.5
50.7
50.5
69.3
77.5
105.5
96
152.5
66.5
85
3.5
109.5
111
6
82.5
14.5
97
98.6
S
S
S
MS
S
S
R
R
R
R
R
HR
HR
R
S
S
S
MS
MS
I
I
R
R
HR
HR
HR
R
R
HS
HR
HR
HS
R
S
HR
HR
8
13
HS
S
37.5
1
S
HS
Highly sensitive (HS), moderately sensitive (MS), intermediate (I),
moderately resistant (MR), highly resistant (HR.)
97
Table A-5. A list of the isolates used for detailed microscopic identification.
Species
Clade E2-1 unknown
Pythium coloratum
Pythium sp. nov. OOMYA1646-08
(E2)
Phytopythium helicoides
Clade E2-2 unknown
Pythium middletonii
Clade B2 unknown
Pythium rostratifingens
Phytopythium chamaehyphon
Clade A unknown
Isolate
E6.16.11LT
E7.20.11GT
F2:4
E4.11.11RT
E6.16.11GT
S4.27.11CT
S10.11.11CT
S12.13.11CTP
E4.27.11RT
E11.29.11RT
E12.6.11RTP
S12.13.11CB
E7.1.11RT
E7.8.11RT
E7.14.11SBT
E7.20.11LT
E7.27.11SBT
E10.4.11LT
S9.16.11 10
S9.16.11 270
E10.4.11LT
E7.20.11LB
E8.30.11LT
E5.9.11RB
E7.14.11SBB
E7.20.11SBB
E7.27.11SBT
E11.21.11SBTP
E11.29.11SBTP
E12.16.11RB
S10.11.11CB
S11.29.11CB
C3
C13
S2
S9.26.11CB
98
Table A-6. The average water temperature during the week the isolates were initially baited
from the two greenhouses, compared to their cardinal temperatures.
Species
Average temperature of the water
during the week of initial baiting
isolation
Minimum
Growth
Temp
(°C)
NA
Optimum
Growth
Temp
(°C)
NA
Maximu
m Growth
Temp
(°C)
NA
15.96
Pythium sp.
nov.
OOMYA170208 (B2)
26.3-26.8
9,5
35,30
40,35
Clade E2-1
unknown
16-16.3, 18.1, 18.9-21.2, 22.4-26.1
5,9,15
30,35,25
35,40,30
Pythium
coloratum
NA
NA
NA
Phytopythium 20.5
litorale
35
40
Phytopythium 16.3, 18.3, 22.6, 25.6, 26.3, 26.8, 27.2, 15,5
29.8
helicoides
18.3-18.6, 24, 31.4
5,15
30
35,40
Clade E2-2
unknown
20.2, 22.3-22.8
5
25
30
Pythium
middletonii
20.6, 22.2, 23.3, 25.3-25.9, 26.5-26.7, 9,5,15
35,30
40,35
Clade B2
27.2, 29.3-29.8, 31.4
unknown
16.4, 17.4-17.8, 18.1-18.5, 19.7
5
25,30,35
30,35
Pythium sp.
nov.
OOMYA164608 (E2)
17.4-17.6, 18.5
9,5
25
30
Pythium
rostratifingens
9,15,5
30
35
Phytopythium 19.2, 20.4-20.5, 21.2-21.8, 22.6
chamaehyphon
16.4, 21.8, 25.2
9,5,15
30,35
35,40
Clade A
unknown
A range of numbers indicates that species were baited at a multitude of temperatures within that
range
99
Table A-7. Average water temperature (°C) for 7 day periods ending on the sampling date in
two commercial greenhouses.
Sampling date
3/22/2011
3/28/2011
4/4/2011
4/11/2011
4/18/2011
4/25/2011
5/2/2011
5/9/2011
5/18/201
5/26/2011
6/1/2011
6/8/2011
6/16/2011
6/23/2011
7/1/2011
7/8/2011
7/14/2011
7/20/2011
7/27/2011
8/2/2011
8/9/2011
8/16/2011
8/22/2011
8/30/2011
9/13/2011
9/20/2011
9/27/2011
10/4/2011
10/12/2011
10/18/2011
10/24/2011
10/31/2011
11/7/2011
11/14/2011
11/21/2011
11/29/2011
12/6/2011
Tank L
16.14
15.96
15.55
15.84
15.98
16.38
16.02
16.21
Missing data
Missing data
16.33
18.95
18.28
18.50
21.18
23.17
20.52
20.22
22.25
22.75
18.02
17.66
18.62
19.35
19.25
18.36
18.03
18.29
18.83
18.73
18.67
18.44
Greenhouse E
Tank R
Tank G
19.85
19.54
19.25
20.28
20.43
19.73
21.29
20.06
22.16
23.31
26.49
25.57
25.61
25.33
26.12
26.29
27.22
26.82
22.64
26.25
22.47
26.27
25.91
26.49
25.85
26.80
26.77
26.85
26.65
26.65
20.33
22.07
22.91
21.95
20.77
19.34
18.55
17.86
17.42
17.22
17.75
17.40
22.20
23.17
20.90
20.41
Missing data
20.81
20.15
19.88
20.36
20.24
20.09
19.64
Tank SB
Greenhouse S
Sampling date
Tank C
3/22/2011
18.90
3/28/2011
20.47
4/5/2011
20.37
4/11/2011
20.25
4/18/2011
20.13
4/25/2011
20.20
5/2/2011
20.97
5/9/2011
20.38
5/16/11
22.42
5/ 25/11
22.45
5/31 /11
25.17
6/6 /11
25.12
25.95
29.31
29.83
31.44
27.20
25.59
20.82
21.19
20.77
19.55
18.37
18.41
18.53
17.65
16.37
9/ 16/11
9/22 /11
9/ 26/11
10/4 /11
10/11/11
10/18/11
10/25/11
11/1/11
11/8/11
11/15/11
11/22/11
11/29/11
12/6/11
12/13/11
12/20/11
24.03
22.56
21.82
21.82
21.20
20.21
20.09
20.18
19.84
19.28
19.21
19.22
19.20
18.14
17.84
Shaded cells indicate times when the tanks were not in use. Greenhouse S tank C was sampled
27 weeks, Greenhouse E tank L was sampled 32 weeks, G was sampled 21 weeks, R was
sampled 33 weeks, and tank SB was sampled 15 weeks.
100
Table A-8. Isolate pathogenicity on Pelargonium X hortorum geranium seedlings grown on
filter paper moistened with soluble fertilizer (300 ppm N; 15% N, 15% P2O5; 15% K2O) and coinoculation results.
Type
Isolate
Clade E2-1
unknown
E7.20.11GT
E6.16.11GB
E6.16.11LT
S5.2.11CT
S5.2.11CB
S9.26.11CB
S11.29.11CT
E3.22.11LT
E5.9.11LT
S11.29.11CT
E5.2.11LT
E6.16.11GT
S3.22.11CB
S4.18.11CB
S10.18.11CB
S3.22.11CT
E8.16.11LT
S4.18.11CT
E7.20.11LT
Pythium
coloratum
Phytopythium
helicoides
Clade E2-2
unknown
Clade B2
unknown
Pythium
rostratifingen
s
Phytopythium
chamaehypho
n
E10.4.11LT
E7.20.11LB
E6.23.11RT
E7.14.11SBB
E7.20.11SBB
E7.27.11SBT
E11.29.11SB
TP
E11.21.11SB
TP
S3.28.11CB
S11.22.11CB
P
S11.29.11CB
S11.22.11CT
P
Pathogenic
Coinoc
(+)=yes (-) = no with P.
irregula
re
+
S
+
P
+
S
S
+
S
+
+
+
S
+
+
+
S
+
+
P
+
+
+
+
+
-
P
S
S
S
S
S
S
P
P
P
+
+
+
Coinoc with
Coinoc with P.
P.
apahanidermat
cryptoirregula um
re
P
101
Clade A
unknown
Not
Sequenced
S9.22.11CB
S12.6.11CB
S10.4.11CT
E4.27.11LT
+
+
+
P
P
E8.9.11LB
-
S
S
S
S- slowed disease progression, P-promoted disease progression.
Table A-9. The representative isolates used in the greenhouse pathogenicity and co-inoculation
tests.
Species
Clade E2-1 unknown
P. coloratum
Ph. helicoides
Clade E2-2 unknown
Isolate
E6.16.11LT
E7.20.11GT
E6.16.11GB
S3.22.11CT
E5.2.11LT
S3.22.11CB
S5.2.11CT
E5.9.11LT
S11.29.11CT
S4.18.11CT
E8.16.11LT
E6.23.11LB
E4.14.11RT
E3.28.11RB
S4.11.11CT
E7.8.11LT
E3.28.11RT
E4.4.11RT
E4.18.11RT
S5.2.11CB
E6.16.11GT
S4.18.11CB
S10.18.11CB
E7.20.11LT
S9.16.11 10
E10.4.11LT
E7.20.11LB
102
Clade B2 unknown
P. sp. nov. OOMYA1646-08 (E2)
Pythium rostratifingens
Ph. chamaehyphon
Clade A unknown
P. cryptoirregulare
P. aphanidermatum
P. irregulare
Other
E7.14.11SBB
E7.20.11SBB
E7.27.11SBT
E6.23.11RT
E3.22.11LT
E4.27.11RT
E.11.21.11SBTP
E11.29.11SBTP
S9.22.11CB
S12.6.11CB
S3.28.11CB
11.29.11CB
S11.22.11CBP
S11.22.11CTP
S10.4.11CT
E4.27.11LT
S9.26.11CB
P123
P128
P84
E8.9.11LB
S9.29.11CT
103
Table A-10. A list of the isolates used for the lab soil pathogenicity tests.
Species
Clade E2-1 unknown
P. coloratum
P. sp. nov. OOMYA1646-08
(E2)
Ph. helicoides
Clade E2-2 unknown
P. rostratifingens
Ph. chamaehyphon
Clade A unknown
P. irregulare
P. cryptoirregulare
P. aphanidermatum
Isolate
E6.16.11LT
F2:4
S3.22.11CT
E4.11.11RT
E8.16.11LT
S12.13.11CB
E10.31.11RT
E7.1.11GB
E7.14.11SBT
S9.16.11 10
S1
E10.4.11LT
E11.21.11SBTP
E12.16.11RB
S3.28.11CB
S10.4.11CT
S9.26.11CB
C13
S2
P84
P123
P128
P220
P223
104
Table A-11. Average weekly tank temperatures for the tank simulation experiment
7/237/29
7/308/5
8/68/12
8/138/19
8/208/26
8/279/2
9/3-9/9
9/109/16
9/179/23
9/249/30
10/110/7
10/810/14
10/1510/21
10/2210/28
10/2911/4
11/511/11
11/1211/18
11/1911/25
11/2612/2
12/312/9
12/1012/14
Tank Tank Tank Tank Tank Tank Tank Tank Tank Tank Tank
1
2
4
6
8
9
10
12
13
14
16
22.2 na
22
24.1 23.7 23.9 24.8 24.3 23.8
24.1 24.6
22.1
na
22.1
23.9
23.6
23.8
24.2
23.8
23.1
23.3
23.6
21.8
na
22.3
24
23.9
24.2
24.2
23.8
23
22.9
23.6
21.1
na
21.3
22.6
22.3
22.7
23.4
22.9
22.4
22.6
22.8
22.7
22.5
22.2
23.2
22.9
23.4
24.3
24
23.6
23.7
23.7
23
22.7
22.5
24.3
23.7
24.4
24.9
24.5
24
24.2
24.4
22.5
20.4
22.3
20.1
22.2
20.2
24
21.2
23.2
20.7
24
21.2
24.5
22
24.1
21.8
23.7
21.5
23.8
21.6
24.1
21.7
20.1
19.7
19.8
21.5
20.3
21.4
22.2
21.9
21.6
21.5
21.8
21.2
20.9
20.8
22.3
22.1
22.2
22.8
22.7
22.5
22.1
22.5
19.3
19
19.1
19.7
18.5
19.5
20
20.1
20.1
19.8
20.1
19
18.8
18.9
19.5
18.8
19
19.7
19.8
19.6
20
19.7
18.1
17.9
18.2
18.4
17.3
18.2
18.9
19.2
19.1
19.1
18.9
19.3
18.8
19.5
19.8
19.5
19.4
19.8
19.9
19.9
19.4
18.9
18
17.7
18.1
18.1
18
17.8
18
18
18
17.7
16.9
18.1
17.9
18.3
18.6
18.5
18.2
18.4
18.5
18.6
17.7
17.5
15.6
15.7
15.7
16.3
16
16
15.5
15.7
15.7
14.6
14.6
16.4
16.3
16.6
16.5
16.8
16.4
16
16.2
16.3
15.3
15.0
15.1
15.3
15.5
15
15.7
15.6
15.2
15.3
15.3
14.7
13.9
14.8
14.8
15
15.3
14.4
15
14.9
15
15.1
14.6
13.9
14.4
14.5
14.6
14.6
13.9
14.4
14.1
14.2
14.3
13.5
13.1
105
Figure A-1. ITS and cox sequences from a representative isolate of each species baited
Clade E2-1 unknown
E6.16.11LT
ITS:
GCGTGCTGCCTGGTATGATTTTTAATTAGATTGTATCGGCGTGTGCGCGGGCTCGGCTGATCGAAGGCT
TTGCTTTCTGCTGCGAGTGTGTGCGTGCTTTTCGGAGCGCGCGTGTGTCTTGCGGCGGGGCGGGCTGAC
TTACTCTTTCAAACCCCTTCCTTTATTACTGATGTATACTGTGAGGACGAAAGTCTTTGCTTTTAAACTA
GATAACAACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCGATACG
TAATGCGAATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCATATTGCACTTTCGGGTTATGCCT
GGAAGTATGTCTGTATCAGTGTCCGTACATCAACCTTGCCTCTCTTAGTCGGTGTAGTCCGGTTTGGAG
ACGAGCAGATCTGAAGCGTCTCGCGTCGTTGCCTCTGCAATGGTGCGAGTCCTTTTGAAACGACACGA
TCTCTTCTATTTGCCTTTAGCAACTCGCTTTGGTTTGAACGCATCGGTCTTGTACTCGTTTGCAGTCTCC
GGCGACCTTGGCTTTGGACATTATGGAGGGCACCTCACTTCGCGGTATGTTAGGCTCTTTGTGGCTGAA
CAATGTTGCGTTTGTGGGCGTGTGTATTTCCGTCTTTGGCTTTGAGGTGTACTGTGGGGTTGTGGGCTT
GAGTGCTTGTGCTGTGTGTTAGTAGCTCGGAGGCGGTGTGTTTGCTATTGGATTCTGCGCGTTGCGTGG
GTAGAGGGGTTTCCATTTGGGAAATACTGTACTGCGGCTCGTTTTC
cox:
CGGTGCTTTTTCAGGTGTAGTAGGTACTACTTTATCTGTTTTAATTAGAATGGAATTAGCACAACCTGG
TAATCAAATTTTTGAAGGTAATCATCATTTATATAATGTAGTAGTTACTGCACATGCTTTTATTATGATT
TTTTTTATGGTTATGCCTGTTTTAATTGGCGGTTTTGGTAATTGGTTTGTACCTTTAATGATAGGAGCAC
CTGATATGGCTTTTCCTCGTATGAATAATATTAGTTTTTGGTTATTACCTCCATCATTATTATTGTTAGT
ATCATCAGCTATTGTTGAATCAGGTGCTGGTACAGGTTGGACTGTATATCCACCCTTATCAAGTGTTCA
AGCTCATTCAGGCCCTTCAGTAGATTTAGCTATTTTTAGTTTACATTTATCAGGTATTTCTTCATTATTA
GGTGCAATAAATTTTTTATCAACAATTTATAATATGAGAGCTCCAGGTTTAAGCTTTCATAGATTACCT
TTATTTGTTTGGGCTATATTTATTACAGCTTTCTTATTATTATTAACATTACCTGTTTTAGCTGGTGCAAT
CACTATGTTATTAACAGATAGGAATTTAAATACTTCATTTTATGATCCATCAGGTGGAGGTGATCCTGT
ATTATATCAACATTTATTTTGGTT
P. sp. nov. OOMYA1646-08
E10.31.11RT
ITS:
CTGTTTGTATCCGATTCGCGCCGGGTTTCGAGCGTGTTTGTATTCGTTACTGTGTAATGCAGTGATAGT
GCAAGCAATGCGAGGAGCTTTGGCTGATCGAAGGTCGTTGCGCAAGTATTTATATGCGCGCTTCGGCT
GACTTATACTTTCAAACCCCTTACTTTAAAAACTGATCAATACTGTGAGGACGAAAGTCTTTGCTTTAA
AACTAGATAACAACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCG
ATACGTAATGCGAATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCATATTGCACTTTCGGGTTA
TACCTGGAAGTATGTCTGTATCAGTGTCCGTACATCAACCTTGCCTCTCTTTGTCGGTGTAGTCCGGCTT
GGAGCATGTGCAGATGTGAGGTGTCTCGCGGCGTGTGTGTGTGTTGTAAAATGCATACGCTTGCTGCG
AGTCCCTTTAAAACGACACGATCTTTCTATTTGCTTTCTACGGAGCGCGTATTTCGAACGCGGCGGTCC
TCGGATCGCTCGCAGTCGACAGCGACTTCAGCGGAGACATATGGAAGAAACCACTATTCGCGGTACGT
TAGGCTTCGGCTCGACAATGTTGCGTTTCAGTGTGTGGATTCCGTTTTCGCTTTGAGGTGTACTGTTCG
GTTGTGGGCTTGAACCTTGTGTCTCGCTTTGTTAGTAGAGGTGTGTCGATTTCTGTGGTTTGATTCCGCA
CTTTATGTGTGGGTAGAGAGACTCCATTTGGGAAACATTGTACTGCGCGTACGCTTTCGGGCGTGTGCG
TGTGT
cox:
TTTATATTTAATTTTTGGTGCTTTTTCAGGTGTAGTTGGTACTACATTATCTGTTTTAATTAGAATGGAA
TTAGCACAACCTGGTAATCAAATTTTTGAAGGTAATCATCATTTATATAATGTTGTTGTTACTGCTCAC
GCATTTATTATGATTTTTTTTATGGTTATGCCTGTTTTAATTGGTGGTTTTGGTAACTGGTTTGTACCTTT
AATGATTGGTGCTCCAGATATGGCTTTTCCTCGTATGAATAATATTAGTTTTTGGTTATTACCCCCATCT
TTATTATTATTAGTATCATCAGCTATTGTTGAATCAGGTGCTGGTACAGGTTGGACAGTATATCCTCCA
TTATCTAGTGTACAAGCTCACTCAGGTCCTTCAGTAGATTTAGCTATTTTTAGTTTACATTTATCAGGTA
106
TATCATCATTATTAGGTGCTATTAATTTTTTATCAACTATTTATAATATGAGAGCTCCTGGTTTAAGTTT
TCATAGATTACCTTTATTTGTTTGGGCTATATTTATTACAGCTTTTTTATTATTATTAACATTACCAGTAT
TAGCAGGTGCAATTACTATGTTATTAACTGATAGAAATTTAAATACATCTTTTTATGATCCTTCTGGTG
GAGGTGATCCAGTATTATATCAACATTTATTTTGGTTC
Clade E2-2 uknown
E10.4.11LT
ITS:
TGTCTTACGAGATTCGCGCCGTGACGTGTGTTGTCGCTGTGTGTGCTGTACATATGTATGGTGCGCATG
GTGGCGACTGCGTGGGTCGGCTGATCGAAGGTCGCATTGTGCTGTATTGCGCAGTGTGGCTGACTTATT
CTTTCAAACCCATTCCTTAATGACTGATTCATACTGTGAGGACGAAAGTCTTTGCTTTTACTAGATAAC
AACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCGATACGTAATGC
GAATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCATATTGCACTTTCGGGTTATACCTGGAAGT
ATGTCTGTATCAGTGTCCGTAAATCAAACTTGCCTCTCTTTGTCGGTGTAGTCCGGCTTGGAGTGCGCA
GATGTGAAGTGTCTCGCGCTACGTCAGTCTATTTACGATAGACTAGGCGCGCGAGTCCTTTTAAATGG
ACACGATCTTTCTATTGCTTTCTGCGGAGCGCATCATTTGAACGCGGCGGTCTTGGGATCGCCTGCAGT
CGATAGCGACTTTGGTAGAGACATATGGAATGACCCTCATTTCGCGGTACGTTAGGCTTCGGCTCGAC
AATGTTGCGTCGTGAGTGTGTTGTTTCGTCTTTGCTTTGAGGTGTACTGTCGGTTGTGGGCTTGAACCG
AAGTATTGTGTGTTAGTAGAGTGTGTCGTTTTCTGTGGTTAGTGTCTGTGTGTGGCCTTGTGTCGCGCAT
AGGTAGAAGGGTATCATTTGGGAAACATTGTACTGCGCGCTGCAAAGCGTGTGTGT
cox:
TACTACATTATCTGTTTTAATTAGAATGGAATTAGCACAACCTGGTAATCAAATTTTTGAAGGTAATCA
TCATTTATATAATGTTGTTGTTACTGCTCATGCATTTATTATGATTTTTTTTATGGTTATGCCTGTTTTAA
TTGGTGGTTTTGGTAATTGGTTTGTACCTTTAATGATTGGTGCACCAGATATGGCATTTCCTCGTATGA
ATAATATTAGTTTTTGGTTATTACCTCCATCTTTATTACTATTAGTATCTTCAGCTATTGTTGAATCAGG
TGCTGGTACAGGTTGGACTGTATATCCACCTTTATCAAGTGTACAAGCTCACTCTGGTCCTTCAGTAGA
TTTAGCTATTTTTAGTTTACATTTATCAGGTATATCATCTTTATTAGGTGCAATTAATTTTTTATCAACT
ATTTAYAATATGAGAGCTCCTGGTTTAAGTTTTCATAGATTACCTTTATTTGTTTGGGCTATATTTATTA
CAGCTTTTTTATTATTATTAACTTTACCTGTATTAGCTGGTGCAATTACAATGTTATTAACAGATAGAA
ATTTAAATACATCTTTTTATGATCCATCAGGTGGAGGTGATCCAGT
Clade B2 unknown
E7.14.11SBB
ITS:
CTCTCTCTCGGGAGAGCTGAACGAAGGTGGGCTGCTGTTATGGTAGTCTGCCGATGTACTTTTAAACCC
ATTACACTAATACTGAACTATACTCCAAAAACGAAAGTATTTGGTTTTAATCAATAACAACTTTCAGCA
GTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCGATACGTAATGCGAATTGCAGAA
TTCAGTGAGACATCGAAATTTTGAACGCACATTGCACTTTCGGGTTATGCCTGGAAGTATGCCTGTATC
AGTGTCCGTACATCAAACTTGCCTTTCTTTTTTTGTGTAGTCAAGAAGAGAGATGGCAGACTGTGAGGT
GTCTCGCTGACTCCCTCCTCGGAGGAGAAGACGCGAGTCCCTTTAAATGTACGTTCGCTCTTTCTTGTG
TTTAAGATGAAGTGTGACTTTCGAACGCAGTGATCTGTTTGGATCGCTTTGCTCGAGTGGGCGACTTCG
GTTAGAACATTAAAGGAAGCAACCTCTATTGGCGGTATGTTAGGCTTCGGCCCGACTTTGCAGCTGAC
GGTGTGTTGTTTTCTGTTCTTTCCTTGAGGTGTACCTGTCTTGTGTGAGGCAATGGTCTGGGCAAATGGT
TGTTGTGTAGTAGGAGGTTGCTGCTCTTAGACGCTCTTCGGAGTAAAGAAGACAACACCAATTTGGGA
TAGTCAATCTATGATTGGCGCTCTTTC
cox:
ACTCTATATTTAATTTTCGGTGCTTTTTCAGGTGTTGTTGGTACAACTTTATCAGTTTTAATCAGAATGG
AATTAGCACAACCTGGTAATCAAATTTTTATGGGAAATCATCAACTATATAATGTAGTTGTAACTGCTC
ATGCTTTCATTATGATTTTCTTCATGGTTATGCCTGTTTTAATAGGTGGTTTTGGTAATTGGTTTATTCCT
TTAATGATAGGTGCTCCAGATATGGCTTTCCCTAGAATGAATAATATTAGTTTTTGGTTATTACCACCA
TCTTTATTATTATTAGTATCTTCTGCTATTGTAGAATCTGGTGCTGGTACAGGTTGGACAGTATATCCAC
CTTTATCAAGTGTTCAAGCTCACTCAGGACCTTCTGTAGATTTAGCTATTTTTAGTTTACACTTATCAGG
107
TATTTCATCTTTATTAGGTGCTATTAATTTCTTATCAACTATTTATAATATGAGAGCTCCAGGTTTAAGC
TTCCATAGATTACCATTATTCGTTTGGTCTGTATTTATTACAGCTTTCTTATTATTATTAACATTACCTGT
ATTAGCTGGTGCTATTACAATGTTATTAACAGATAGAAACTTAAATACATCTTTCTATGATCCATCAGG
TGGAGGTGATCCAGTATTATATCAACATTTATTTTGGTTCTTTGGTCACCCAGAA
Clade A unknown
S5.31.11CB
ITS:
GTTCTGTGCTCCTCTCGGGGAGCTGAACGAAGGTGAGCTGCTGTTATGGTGGCTTGCCGATGTACATTT
CAAACCCATTACTTTAATACTGAACTATACTCCAAAAACGAAAGTCTTTGGTTTTAATCAATAACAACT
TTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAGCGCTGCGAACTGCGATACGTAATGCGAAT
TGCAGAATTCAGTGAGTCATCGAAATCTTGAACGCACATTGCACTTTCGGGTTATGCCTGGAAGTATG
CCTGTATCAGTGTCCGTACAACAAACTTGCCTCTTTTTTTCTGTGTAGTCAGGGAGAGAGATGGCAGAA
GGTGAGATGTCTCGTTGACTCCCTCTTCGGAGGAGAAGACGCGAGTCTCTTTAAACGTACGTTCGCTCT
TTCTTGTGTTCGATGTAGAAGTGTGGCTTGCGAACGCGGTGATCTGTTTGGATCGCTTTGCGCTTTCGG
GCGACTTCGGTTAGGACATTAAAGGAAGCAACCTCTATTGGCGGTATGTTAGGCTTCGGCCCGACTTT
GCAGCTGACGGAGTGTGGTTTTCTGTTCTTTCCTTGAGGTGTACCTGAAAATAGTGTGAGGCAATGGTC
TGGGCAAATGGTTGCTGTGTAGTAGTGGGTCGCTGCTCTCGGACGCTCTTGCTTCGGTGAGAGTAAAG
GAGGCAACACCAATTTGGGACCGTGGCGCTTTT
cox:
TTCTTTATATTTAATTTTTGGTGCTTTTTCAGGTGTAGTTGGTACAACTTTATCTGTTTTAATTAGAATG
GAATTAGCACAACCAGGTAATCAAATTTTTATGGGAAATCATCATTTATATAATGTTGTAGTTACAGCT
CATGCTTTTATTATGATTTTTTTCATGGTTATGCCTGTATTAATAGGTGGTTTTGGTAATTGGTTTGTAC
CTTTAATGATTGGTGCTCCAGATATGGCTTTCCCAAGAATGAATAATATTAGTTTTTGGTTATTACCTCC
TTCTTTATTATTATTAGTATCTTCAGCTATTGTAGAATCGGGTGCTGGTACAGGTTGGACAGTTTATCCA
CCATTATCAAGTGTTCAAGCACACTCAGGTCCTTCTGTTGATTTAGCTATTTTTAGTTTACACTTATCAG
GTATTTCATCATTATTAGGTGCTATTAATTTCTTATCTACTATTTATAATATGAGAGCTCCTGGTTTAAG
TTTTCATAGATTACCTTTATTTGTATGGGCTATTTTTATTACAGCTTTCTTATTATTATTAACTTTACCTG
TGTTAGCTGGTGCAATTACAATGCTTTTAACAGATAGGAATTTAAATACTTCATTTTATGATCCATCAG
GTGGTGGAGATCCTGTATTATATCAACATTTATTCTGGTTTTTGGACACCC
Pythium (middletonii)
E8.30.11LT
ITS:
TGTCTTACGAGATTCGCGCCGTGACGTGTGTTGTCGCTGTGTGTGCTGTATTTATATCGTGCGCATGGT
GTCGACTGCGTGGGTCGGCTGATCGAAGGTCGCGTTGTGCTTTATTGCGCATTGTGGCTGACTTATTCT
TTCAAACCCATTTCTTTATTACTGATTCATACTGTGAGGACGAAAGTCTTTGCTTTTACTAGATAACAA
CTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCGATACGTAATGCGA
ATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCATATTGCACTTTCGGGTTATACCTGGAAGTAT
GTCTGTATCAGTGTCCGTAAATCAAACTTGCCTCTCTTTGTCGGTGTAGTCCGGCTTGGAGTGCGCAGA
TGTGAAGTGTCTCGCGCTACGTCAGTCTTTATTGTACTAGGCGCGCGAGTCCTTTTAAATGGACACGAT
CTTTCTATTGCTTTCTGCGGAGCGCATCATTTGAACGCGGCGGTCTTGGGATCGCCTGCAGTCGATAGC
GACTTTGGTAGAGACATATGGAAGAACCCTCATTTCGCGGTACGTTAGGCTTCGGCTCGACAATGTTG
CGTAGTGAGTGTGTTGTTTCGTCTTTGCTTTGAGGTGTACTGTCGGTTGTGGGCTTGAACCCAAGTATT
GTGTGTTAGTAGAGTGTGTCGATTTCTGTGGTTAGCGTCTATGTGTGGCTTTATGTCGTACGTAGGTAG
AAGGGTATCATTTGGGAAACATTGTACTGCGCGCTGCAAGGCGTGTGTGT
cox:
108
TTAATTTTCGGTGCTTTTTCAGGTGTAGTTGGTACTACACTATCTGTTTTAATTAGAATGGAATTAGCAC
AACCTGGTAATCAAATTTTTGAAGGTAATCATCATTTATATAATGTTGTTGTTACTGCTCATGCATTTAT
TATGATTTTTTTTATGGTTATGCCTGTTTTAATCGGTGGTTTTGGTAATTGGTTTGTACCTTTAATGATTG
GTGCACCAGATATGGCTTTTCCTCGTATGAATAATATTAGTTTTTGGTTATTACCACCATCTTTATTATT
ATTAGTATCTTCAGCTATCGTTGAATCAGGTGCTGGTACAGGTTGGACTGTATATCCACCTTTATCAAG
TGTACAAGCACACTCGGGTCCTTCAGTAGATTTAGCGATTTTCAGTTTACATTTATCAGGTATATCATC
TTTATTAGGTGCAATTAATTTTTTATCTACTATTTATAATATGAGAGCTCCTGGTTTAAGTTTTCATAGA
TTACCTTTATTTGTTTGGGCTATATTTATTACAGCTTTTTTATTATTACTAACTTTACCTGTATTAGCTGG
TGCAATTACAATGTTATTAACAGATAGAAATTTAAATACATCTTTTTATGATCCATCAGGTGGAGGTGA
TCCTGTATTATATCAACATTTATTTTGGTTTTTCGGTCATCC
P. coloratum
E6.16.11GT
ITS:
CGTTGTAACTATGTTCTGTGCTCTCTTCTCGGAGAGAGCTGAACGAAGGTGGGCTGCTTAATTGTAGTC
TGCCGATGTACTTTTAAACCCATTAAACTAATACTGAACTATACTCCGAAAACGAAAGTCTTTGGTTTT
AATCAATAACAACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCGA
TACGTAATGCGAATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCACATTGCACTTTCGGGTTAT
GCCTGGAAGTATGCCTGTATCAGTGTCCGTACATCAAACTTGCCTTTCTTTTTTTGTGTAGTCAAGAAG
AGAGATGGCAGACTGTGAGGTGTCTCGCTGACTCCCTCTTCGGAGGAGAAGACGCGAGTCCCTTTAAA
TGTACGTTCGCTCTTTCTTGTGTTTAAGATGAAGTGTGACTTTCGAACGCAGTGATCTGTTTGGATCGCT
TTGCTCGAGTGGGCGACTTCGGTTAGGACATTAAAGGAAGCAACCTCTATTGGCGGTATGTTAGGCTT
CGGCCCGACTTTGCAGCTGACTGGAGTTGTTTTCTGTTCTTTCCTTGAGGTGTACCTGTCTTGTGTGAGG
CAATGGTCTGGGCAAATGGTTATTGTGTAGTAGGAAGTTGCTGCTCTTAAACGCTCTAGCTTCGGTTAG
AGTAAAGGAGGCAACACCAATTTGGGATAGTCGTTGATTTATCAATG
cox:
GTACTCTATATTTAATTTTTCGGTGCTTTTTCAGGTGTTGTAGGTACAACTTTATCCGTTTTAATCAGAA
TGGAATTAGCACAACCTGGTAATCAAATTTTTATGGGAAATCATCAACTATATAACGTAGTTGTAACT
GCTCACGCTTTTATTATGATTTTCTTCATGGTTATGCCTGTTTTAATAGGTGGTTTTGGTAATTGGTTTA
TTCCTTTAATGATAGGTGCTCCAGATATGGCTTTCCCTAGAATGAATAATATTAGTTTTTGGTTATTACC
ACCATCATTATTATTATTAGTATCTTCAGCTATTGTAGAATCAGGTGCTGGTACTGGTTGGACTGTTTAT
CCACCTTTATCAAGTGTACAAGCTCACTCAGGACCTTCAGTAGATTTAGCTATTTTTAGTTTACACTTAT
CAGGTATCTCATCTTTATTAGGTGCTATTAATTTCTTATCAACTATTTATAACATGAGAGCTCCTGGTTT
AAGCTTCCACAGATTACCATTATTCGTTTGGTCTGTATTCATTACAGCTTTCTTATTATTATTAACATTA
CCAGTATTAGCTGGTGCGATTACAATGTTATTAACAGATAGAAACTTAAATACATCTTTCTATGATCCA
TCAGGTGGAGGTGATCCAGTATTATATCAACATTTATTTTGGTTTTTCGGTC
P. rostratifingens
E11.29.11SBTP
ITS:
ACTATCCACGTGAACCGTTAAGCAAACAAGTTAAGCAGGCGCGATTGGTGGTGCGTCTGGGAAGCGCT
TGTCGATATTCGATCGGATCGGATATTGTCGGGCGTCGTCCGGACACGCTGTCGATCGGAGTCGGCTA
AACGAAGGTCGGGCGTTCGCTATCGGAGCGATGTGCGCGTTGTCGCACGTTGCTGCAATGGCTCGAGC
AAGCGGCTGATTTATGTCTTTCAAACCATACGTGACGTACTGATTATACTGTGAGGACGAAAGTCCTTG
CTTTTACTAGATAACAACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAAC
TGCGATACGTAATGCGAATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCATATTGCACTTCCG
GGTTATGCCTGGAAGTATGTCTGTATCAGTGTCCGTACATTAAACTTGCCTCTCTTCGTCGGTGTAGTC
CGGCTTGGAGAAGGAGCAGAGGTGAAGTGTCTCGCGCCATGCTGGTGATCTATCTTCGGGTAGATGAC
GAGAGTGCACGAGTCCTTTGAAATGGACTCCGGTTTTTCTATTGCGTTGCTCAAGGGCGTGTATTTTGA
109
ACGCGGCAATCTCGTCGATTGCCTGCAGATGTTTACGACCTTGGCGAGAACATGTGGAAGCAACCTCC
TTTTCGCGGTACGTTAGGCTTCGGCTGGACAATGTTGTGAGAGGGTGTGTGTCTTTCGTTTTCGCTTGG
AGGTGTGTTTTGTACTGTGGGTGGTTAGCGTGTCTTTTGTCGGTAGTAGAGGTATGCGTTTGTCGGTGC
GCACTTGTTGTGTGGTTGATCGCGCTTGCGTGGTCGATTGCGCAGATAGAGAGACTGATTTGGGTAATT
CTGTGCTCCAGAGCACGCTACCGAGGTCGCTGTCTTTTGCGAGCTCGTGTGTGTGTGTGTGTTGGTAGT
ATCTCAATTGGACCTGATATCAGACA
cox:
GGTACTTTATATTTAATTTTTGGTGCTTTTTCTGGTGTAGTAGGTACTACATTATCTGTTTTAATTAGAA
TGGAATTAGCACAACCTGGTAATCAAATTTTTGAAGGTAACCATCATTTATATAATGTTGTTGTTACTG
CTCATGCATTTATTATGATTTTTTTTATGGTTATGCCAGTTTTAATTGGAGGTTTTGGTAACTGGTTTGT
ACCTTTAATGATTGGTGCTCCAGATATGGCTTTTCCTCGTATGAATAATATTAGTTTTTGGTTATTACCT
CCATCTTTATTATTATTAGTATCATCAGCTATTGTTGAATCAGGTGCTGGTACAGGTTGGACAGTATAC
CCACCTTTATCAAGTGTTCAAGCCCATTCAGGACCATCAGTAGATTTAGCTATTTTTAGTTTACATTTAT
CAGGTATATCATCATTATTAGGTGCTATTAATTTCTTATCAACTATTTATAATATGAGAGCTCCTGGTTT
AAGTTTTCATAGATTACCTTTATTTGTTTGGTCTATATTTATTACAGCATTTTTATTATTATTAACTTTAC
CAGTATTAGCAGGTGCTATTACTATGTTATTAACTGATAGAAATTTAAATACATCTTTTTATGATCCAT
CAGGTGGAGGTGATCCAGTATTATATCAACATTTATTTTGGT
Ph. chamaehyphon
S11.29.11CB
ITS:
TGAAACATACTGTGGGGACGAAAGTCTCTGCTTTAAACTAGATAGCAACTTTCAGCAGTGGATGTCTA
GGCTCGCACATCGATGAAGAACGCTGCGAACTGCGATACGTAATGCGAATTGCAGGATTCAGTGAGTC
ATCGAAATTTTGAACGCATATTGCACTTTCGGGTTATGCCTGGAAGTATGTCTGTATCAGTGTCCGTAC
ACTAAACTTGCCTCCTTTCGCGTCGTGTAGTCGGCGCGTGTGGGAATTGCAGCAGATGTGAGGTGTCTT
GTGGTCCTTCGCGGACAGCAAGTCCCTTGAAAGTCGGACGCGTATCTTTGCGTGCGTTGGGTGCTGGT
GGGCTGTGGGACGCGTCTGTTGACGAGTCTGGCGACCTTTGGCGCGTGCATGCTTGGGCACTGTGTATT
GGCGGTATGTTAGGCTGCGTTTCGTGCGCGGCTTTGGCAATGCAGCTGATGCGTGTGTTTGGGCGGCGT
GTGTTGTATGGGTGAACCGGATGGTCGACGGGTTTGACTCGTGTTTCGTTAGTCTGTAGCCGGTGTTCT
GTATCGCGCGCGGAGTGTGTCACCATTTGGGAATCTGTGTGGTCTTTCGTAGTATC
cox:
TTATATTTAATTTTTGGTGCTTTTTCAGGTATAGTTGCAACAACTATGTCTGTTTTAATTAGAATAGAAT
TATCACAACCAGGTAATCAAATATTTATGGGGAATCATCAATTATATAATGTTATGGTTACAGCACAT
GGATTATTAATGTTATTCTTTGTTGTTATGCCTATATTAGTTGGTGGTTTTGGTAATTGGTTTGTACCTTT
AATGTTAGGTGCACCTGATATGGCTTTTCCACGTTTAAATAATATTAGTTTTTGGTTATTACCTCCATCA
TTATTATTATTAGTATCTTCTGCATTAGTAGAATCTGGTGCGGGTACAGGTTGGACTGCTTATCCACCTT
TATCTAGTGTAGCTGCGCATTCAGGACCTTCAGTAGATTTAGCAATTTTTAGTTTACATTTATCTGGTAT
TTCTTCATTATTAGGTGCTATTAATTTTATTGCAACTATTTTTAATATGAGAGCTCCAGGTTTAAGTATG
CATAGAATGCCTTTATTTGTTTGGTCAATATTAATTACAGCTTTTCTTTTAGTATTAACTTTACCTGTAT
TTTCAGGTGCTATTACTATGTTATTAACAGATAGAAACTTTAATACATCATTTTATGATCCAGCAGGTG
GTGGTGATCCAGTATTATTCCAACATTTATTTTGGT
Ph. litorale
S3.28.11CT
110
ITS: NA
cox:
TTATCACAACCAGGTAATCAAATTTTTATGGGAAATCATCAATTATATAATGTTATGGTTACAGCACAT
GGATTATTAATGTTATTCTTTGTTGTTATGCCTATATTAGTTGGTGGTTTTGGTAATTGGTTTGTTCCTTT
AATGTTAGGTGCTCCTGATATGGCTTTTCCACGTTTAAATAATATTAGTTTTTGGTTATTACCTCCATCA
TTATTATTATTAGTATCTTCTGCTTTAGTAGAATCTGGTGCTGGTACAGGTTGGACAGCATATCCACCTT
TATCAAGCGTAGCTGCTCACTCGGGACCTTCAGTAGATTTAGCTATTTTTAGTTTACATTTATCTGGTAT
TTCATCTTTATTAGGTGCTATTAATTTTATTGCAACTATTTTTAATATGAGAGCTCCTGGATTAAGTATG
CATAGAATGCCTTTATTTGTTTGGTCTATATTAATTACAGCTTTTCTTTTAGTAATTACTTTACCAGTAT
TTTCTGGTGCTATTACAATGTTATTAACTGATAGAAATTTTAATACTTCTTTT
Ph. helicoides
E7.20.11LT
ITS:
TCTTTCCACGTGAACCGTTTGTGACATGGTTGGGCTTGTGCGTGTTCTCTCTGTTTTGGGGGGAGGCGT
GCGAGCTATCTGTAAACTTGTCAAACCCATTCTCTTTGATAACTGAAACATACTGTGGGGACGAAAGT
CTCTGCTTTGAACTAGATAGCAACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCT
GCGAACTGCGATACGTAATGCGAATTGCAGGATTCAGTGAGTCATCGAAATTTTGAACGCATATTGCA
CTTTCGGGTTATGCCTGGAAGTATGTCTGTATCAGTGTCCGTACACTAAACTTGCCTCCTTTGCGTCGT
GTAGTCGGCGCGTTGGAAATTGTGGCAGATGTGAGGTGTCTTGATTGTTGTGTCTTTTTTGATGCGTCG
GTCAAGTCCCTTGAAAGTCGGACGCGTATCTTTGCGTGCGTTGGGTGCCGGTGGGCTGTGGGACGCGT
CTGTTGACGAGTCTGGCGACCTTTGGCGCGTGCATGCTTGGGCACTGTGTATTGGCGGTATGTTAGGCT
GCGTTCGCGCCGCTTTGACAATGCAGCTGATGCGTGTGTTTGGGCTGTGGTGCTGTATGGGTGAACCG
GATGGTCGATGGGTTTTATATGCGTTTCTCGTGTCTGTTTTTATCCGGTGTTCTGTATCGTGCGTGGAGT
GTGTCATCATTTGGGAATTTGTACGTCTTCTTGTTTTGAGGGCGTATCTCATTTGGACCTGATATCA
cox:
GGTACTTTTATATTTAATTTTTGGTGCTTTTTCAGGTATAGTTGCAACAACTATGTCTGTTTTAATTAGA
ATAGAATTATCACAACCTGGTAATCAAATATTTATGGGAAATCATCAATTATATAATGTTATGGTTACA
GCACATGGATTATTAATGTTATTCTTTGTTGTTATGCCTATATTAGTTGGTGGTTTTGGTAATTGGTTTG
TACCTTTAATGTTAGGTGCACCTGATATGGCTTTTCCTCGTTTAAATAATATTAGTTTTTGGTTATTACC
TCCATCATTATTATTATTAGTATCTTCTGCATTAGTAGAATCTGGTGCTGGTACAGGTTGGACAGCTTA
TCCACCATTATCTAGTGTAGCAGCACACTCTGGACCTTCAGTAGATTTAGCTATTTTTAGTTTACATTTA
TCAGGTATTTCTTCATTATTAGGTGCTATTAATTTTATTGCAACTATATTTAACATGAGAGCTCCTGGTT
TAAGTATGCATAGAATGCCTTTATTTGTTTGGTCAATATTAATTACAGCTTTTCTTTTAGTATTAACTTT
ACCTGTATTTTCTGGTGCTATTACAATGTTATTAACTGATAGAAACTTTAATACATCTTTTTATGATCCA
GCAGGAGGGGGTGATCCAGTTTTATTCCAACATTTATTTTGGTTTTTTT