The Pennsylvania State University

The Graduate School

Department of and Environmental Microbiology

CHARACTERIZATION OF 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

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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 . 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 , 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 iv 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 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

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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 #: 2010- 51181-21140): “Integrated management of zoosporic pathogens and irrigation water quality for a sustainable green industry” for making this research project possible. 1

Chapter 1

LITERATURE REVIEW

The Genera Pythium and Phytopythium

Pythium Pringsh. is a genus of organisms in the class Oomycota and kingdom , subgroup-Stramenopiles/Heterokontophyta (7). Pythium has a cosmopolitan distribution and there are over 130 described species in the genus (24; 49; 70). , belonging to the grouping Pseudofungi (55), are -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 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 , 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). 2

Pythium species utilize two life cycle stages that establish them as persistent pathogens. From , 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 from antagonistic microorganisms (33). Wet soils favor oospore 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 . The resulting oospore can germinate, produce a , and then directly infect the plant, or it can produce zoospores that can swim away and germinate near and infect a new plant. The 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 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 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 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 cavity spot, non- pathogenic 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 roots, (37) indicating that some species do act as commensals. Nonetheless, relatively non- pathogenic 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 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 zero- sum 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 zero- sum 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.

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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 co- inoculated.

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

Literature Cited

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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 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 ebb- and-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 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 (March- December, 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); 40- 48.5%, 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. OOMYA1702- 08. 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, non- inflated 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. OOMYA1646- 08, 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 () 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 12

10 Pythium sp. nov OOMYA1702-08 (B2) Clade E2-1 unknown 8 Pythium coloratum Phytopythium helicoides 6 Clade E2-2 unknown Pythium middletonii

Number of Isolates NumberIsolates of Baited 4 Clade B2 unknown Pythium sp. nov OOMYA1646-08 (E2) 2 Pythium rostratifingens

0 Clade A unknown

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

7

6

5 Clade E2-1 unknown 4 Pythium coloratum 3 Phytopythium litorale Clade E2-2 unknown

2 Number of Isolates NumberIsolates of Baited 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 Minimum Optimum Maximum Daily Colony (number of isolates) Growth Growth Growth Growth Morphology Temp (°C) Temp (°C) Temp (°C) (mm) Clade E2-1 unknown (3) 9,5 35,30 40,35 13.11 Ra Pythium coloratum (37) 5,9,15 30,35,25 35,40,30 11.74 Ra,R,N,R/Ra, Ra/C Phytopythium helicoides 15,5 35 40 20.00 Ra,N (9) Clade E2-2 unknown (6) 5,15 30 35,40 12.64 Ra Pythium middletonii (1) 5 25 30 9.76 Ra Clade B2 unknown (4) 9,5,15 35,30 40,35 14.39 Ra,R Pythium sp. nov. 5 25,30,35 30,35 10.48 Ra, Ra/C, OOMYA1646-08 (E2) (6) R/C Pythium rostratifingens 9,5 25 30 6.31 C (3) Phytopythium 9,15,5 30 35 15.13 Ra, Ra/R chamaehyphon (7) Clade A unknown (4) 9,5,15 30,35 35,40 0.08 C/Ra, R/Ra, Ra Pythium 9 35,30 40 10.55 Ra/N aphanidermatum (2) Pythium irregulare (1) 9 30 35 15.44 N Pythium cryptoirregulare 5 25 35 20.77 Ra/N (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 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).

B 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 B

C 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 Oogonia Oospore Hyphae Discharge Sporangia Antheridia Zoospore designation diameter wall width tube diameter Width diameter thickness length X X length width Clade E2-1 40.5 4.5±.5 4.3±1.3 4.4 x 2.7 25.9±4.3 9.8±1.5 unknown (1) (4) (3) (1) (7) (5) 34.1 x 48.3 Pythium 17.4±5.7 2.2±.8 0.4±.1 14.4±8.3 x 3.4±.5 coloratum (4) (6) (2) 41.7±13.2 (5) (9)

Pythium sp. nov. 18.7±1.1 2.7±.6 3.7±1.6 28.7±5.4 7.15±1.2 OOMYA1646- (7) (6) (3) (8) (5) 08 (E2) 15.9 x 23.6 Phytopythium 11.5 1.3 2.3±.2 3.3±1.3 x 25.3±3.2 x 2 (1) 7.7±.8 (4) helicoides (1) (1) (3) 4.6±1.8 26.2±4.1 (4) (5) 23.5±16.8 (2) Clade E2-2 20.35±1.3 2.3±.1 3.1 (1) 5.3±1.2 x 25.6±2.8 9.1±1.3 unknown (3) (3) 4.6±.7 (3) (5) (5)

Pythium 21.2±.6 2.65±.2 2.75±.75 28±1.3 7.95±.7 middletonii (3) (3) (3) (7) (4)

Clade B2 30.2±1.4 4.35±1.3 4 x 2.8 (1) 12 x 6.9 15.4 (1) 9.1 (1) unknown (3) (3) (1) 28±1.8 (4) Pythium 17±.3 (2) 2.4±.1 1.5 (1) 20.3±2.9 6.5±.5 rostratifingens (2) (2) (2)

Phytopythium 2.2±.1 3.6 x 3.8 24.6±2.9 9.5±.3 chamaehyphon (2) (1) (4) (4)

Clade A 15.5±1.1 1.4±.6 3.3±.4 8.8±7.2 3.9 (1) 9.7 (1) unknown (1) (2) (2) (6)

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.

46

Table 2-3. Results of the poison plate assay

Isolate designation (number of isolates) Number of isolates in each Fungicide Response Classification HS S MS I MR R HR Clade E2-1 unknown (4) 1 3 Pythium coloratum (39) 2 23 2 2 1 1 8 Phytopythium helicoides (9) 4 1 4 Clade E2-2 unknown (6) 1 4 1 Pythium middletonii (1) 1 Clade B2 unknown (5) 4 1 Pythium sp. nov. OOMYA1646-08 (E2) 1 5 (6) Pythium rostratifingens (3) 1 2 Phytopythium chamaehyphon (7) 3 2 2 Clade A unknown (5) 2 3 Pythium aphanidermatum (1) 1 Pythium cryptoirregulare (1) 1 Pythium irregulare (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.) OMYA- labeled 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 #: 2010- 51181-21140): “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 peat- based 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 re- isolating 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 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 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 , 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 (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 Pathogenic Co-inoc with Co-inoc with P. Co-inoc with P. (number of isolates) P. irregulare cryptoirregulare apahanidermatum Clade E2-1 unknown (-)(+) S P Pythium coloratum (-)(+) S, P S S, P Phytopythium helicoides (+) Clade E2-2 unknown (-)(+) Clade B2 unknown (-)(+) P Pythium rostratifingens (-) P Phytopythium (-)(+) P chamaehyphon Clade A unknown (+) S S

S- slowed disease progression, P-promoted disease progression. (+) = pathogenic, (-) = non- pathog enic. 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 % of times Were both pathogenic baited isolate species isolate recovered recovered? recovered Clade E2-1 unknown 95.6 4.4 yes Pythium coloratum 64.6 35.4 yes Phytopythium helicoides 100 0 no Clade E2-2 unknown 95.7 5.3 yes Pythium middletonii 100 0 yes Clade B2 unknown 100 0 yes Pythium sp. nov. OOMYA1646-08 86.7 13.3 yes (E2) Pythium rostratifingens 93 7 yes Phytopythium chamaehyphon 90.9 9.1 yes Clade A unknown 100 0 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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24. Raftoyannis, Y., and M. W. Dick. 2006. Zoospore encystment and pathogenicity of Phytophthora and Pythium species on plant roots. Microbiological Research 161(1):1-8 doi:10.1016/j.micres.2005.04.003. 25. Rodriguez, R. N. 2011. SAS. Wiley Interdisciplinary Reviews: Computational Statistics 3(1):1-11 doi:10.1002/wics.131. 26. Roncadori, R. W. 1974. Effect of Pythium irregulare on cotton growth and yield, and joint action with other soil-borne pathogens. Phytopathology 64(10):1303 doi:10.1094/Phyto-64-1303. 27. Rosindell, J., S. P. Hubbell, and R. S. Etienne. 2011. The unified neutral theory of biodiversity and biogeography at age ten. Trends in Ecology & Evolution 26(7):340-348 doi:http://dx.doi.org/10.1016/j.tree.2011.03.024. 28. Simon, A. L. 1970. Community equilibria and stability, and an extension of the competitive exclusion principle. The American Naturalist 104(939):413-423 doi:10.2307/2459310. 29. Suffert, F., and M. Guibert. 2007. The ecology of a Pythium community in relation to the epidemiology of carrot cavity spot. Applied Soil Ecology 35(3):488-501 doi:10.1016/j.apsoil.2006.10.003. 30. van der Plaats-Niterink, A. J. 1981. Monograph of the genus Pythium. Vol. no. 21. Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. 31. Weiland, J. E., B. R. Beck, and A. Davis. 2013. Pathogenicity and virulence of Pythium species obtained from forest nursery soils on douglas-fir seedlings. Plant Disease 97(6):744-748 doi:10.1094/PDIS-09-12-0895-RE.

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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 Inoculations 1 Ph. helicoides 2 P. aphanidermatum + Ph. helicoides 3 P. aphanidermatum + Ph. helicoides + P. coloratum 4 P. aphanidermatum + P. coloratum 5 P. aphanidermatum + Ph. helicoides + P. coloratum 6 No inoculum 7 P. coloratum 8 P. aphanidermatum + P. coloratum 9 P. aphanidermatum 10 P. aphanidermatum + Ph. helicoides + P. coloratum 11 P. aphanidermatum + Ph. helicoides + P. coloratum 12 P. aphanidermatum + Ph. helicoides 13 P. coloratum 14 No inoculum 15 Ph. helicoides 16 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 A- 10. 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 0- 471 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 Hours between 25° and 30° C Tank 1 Phytopyhtium helicoides 25.5 Tank 2 P. aphanidermatum + Ph. helicoides 0 Tank 4 P. aphanidermatum + P. coloratum 29.3 Tank 6 No inoculum 339.2 Tank 8 P. aphanidermatum + P. coloratum 325.8 Tank 9 P. aphanidermatum 340.8 Tank 10 P. aphanidermatum + Ph. helicoides + P. coloratum 471.2 Tank 12 P. aphanidermatum + Ph. helicoides 323.7 Tank 13 P. coloratum 131 Tank 14 No inoculum 176.7 Tank 16 P. aphanidermatum 290

Table 4-2. The number of hours the tanks had water temperatures between 25 and 30 degrees C.

Tank Date 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 8/19 c c c c c c 8/20 c c c c c c 8/21 h c c c c c h 8/28 c c c h c c c c h c 9/4 c c c c P9/8 c h c c h h h 9/11 h c h c c 9/18 c h c h c 9/25 c c c h c 10/2 c c c 10/9 c c h 10/16 c c h 10/23 c c c 10/30 c c c c h P11/1 11/7 c c c c 11/13 c c c c 11/20 c c c c 12/4 c c c c c c 12/11 c c c c c c c 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 #: 2010- 51181-21140): “Integrated management of zoosporic pathogens and irrigation water quality for a sustainable green industry”.

78

Literature Cited

1. Agrios, G. N. 2005. Plant Pathology. 5th ed. Elsevier. 2. Al-Sadi, A. M., Al-Ghaithi, A. G.,Al-Balushi, Z. M., and Al-Jabri, A. H. 2012. Analysis of diversity in Pythium aphanidermatum populations from a single greenhouse reveals phenotypic and genotypic changes over 2006 to 2011. Plant Disease 96(6):852-858 doi:10.1094/PDIS-07-11-0624. 3. Al-Sheikh, H. 2010. Two pathogenic species of Pythium: P. aphanidermatum and P. diclinum from a wheat field. Saudi Journal of Biological Sciences 17(4):347-352 doi:10.1016/j.sjbs.2010.05.001. 4. Al-Sheikh, H., and Hani, M. A. A. 2012. Occurrence, identification and pathogenicity of Pythium aphanidermatum, P. diclinum, P. dissotocum and Pythium "Group P" isolated from Dawmat Al-Jandal Lake, Saudi Arabia. Research Journal of Environmental Sciences 6(6):196-209 doi:DOI: 10.3923/rjes.2012.196.209. 5. 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. 6. Burgos-Garay, M. L. 2013. Effect of heterotrophic bacerial communities on Pythium spp. in recycled irrigation water, Plant Pathology Department, University Park, PA, The Pennsylvania State University. Ph. D. 7. Bush, E. A., C. Hong, and E. L. Stromberg. 2003. Fluctuations of Phytophthora and Pythium spp. in components of a recycling irrigation system. Plant Disease 87(12):1500- 1506. 8. Ghimire, S. R., P. A. Richardson, P. Kong, J. Hu, J. D. Lea‐Cox, D. S. Ross, G. W. Moorman, and C. Hong. 2011. Distribution and diversity of Phytophthora species in nursery irrigation reservoir adopting water recycling system during winter months. Journal of Phytopathology 159(11‐12):713-719 doi:10.1111/j.1439-0434.2011.01831.x. 9. Gull, C., Labuschagne, N., Botha, W.J. 2004. Pythium species associated with wilt and root rot of hydroponically grown crops in South America. African Plant Protection 10(2):109-116. 79

10. Hong, C. X., and G. W. Moorman. 2005. Plant pathogens in irrigation water: Challenges and opportunities. Critical Reviews in Plant Sciences 24(3):189-208 doi:10.1080/07352680591005838. 11. Ivors, K. L., and G. W. Moorman. 2014. Oomycete Plant Pathogens in Irrigation Water. Pages 57-64. in: Biology, detection, and management of plant pathogens in irrigation water C. Hong, G. Moorman, W. Wohanka, and C. Büttner, eds. APS Press, The American Phytopathological Society, St. Paul, Minnesota. 12. Lévesque, C., A. , and A. W. A. M. De Cock. 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycological Research 108(12):1363-1383 doi:10.1017/s0953756204001431. 13. 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. 14. Nechwatal, J., A. Wielgoss, and K. Mendgen. 2008. Diversity, host, and habitat specificity of oomycete communities in declining reed stands (Phragmites australis) of a large freshwater lake. Mycological Research 112(6):689-696 doi:10.1016/j.mycres.2007.11.015. 15. Shrestha, S. K., Y. Zhou, and K. Lamour. 2013. Oomycetes baited from streams in Tennessee 2010-2012. Mycologia 105(6):1516-1523 doi:10.3852/13-010. 16. Stanghellini, M. E. 1973. Effect of soil water potential on disease incidence and oospore germination of Pythium aphanidermatum. Phytopathology 63(12):1496 doi:10.1094/Phyto-63-1496. 17. Thines, M. 2009. Bridging the gulf: Phytophthora and downy mildews are connected by rare grass parasites. Plos One 4(3):e4790 doi:10.1371/journal.pone.0004790. 18. van der Plaats-Niterink, A. J. 1981. Monograph of the genus Pythium. Vol. no. 21. Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. 19. Yang, X., P. A. Richardson, H. A. Olson, and C. X. Hong. 2013. Root and stem rot of begonia caused by Phytopythium helicoides in Virginia. Plant Disease 97(10):1385-1385 doi:10.1094/PDIS-05-13-0472-PDN.

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 co- inoculation 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 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 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 Isolate number Zoospore Morphological Possible designation release Characteristics Characteristics date Pythium sp. nov. E3.28.11LB OOMYA1702- 08 (B2) Clade E2-1 E6.16.11LT 4 12 unknown E6.16.11GB 6,14 E7.14.11GT 4,25 E7.20.11GT 6,16 E7.27.11GT 4 E8.2.11GT 4 E8.9.11GT 10 E8.16.11GT 10 Pythium S3.22.11CB 4 coloratum S3.22.11CT 2, 9 E3.22.11RB 19-20, 24,30,36 E3.28.11RB E3.28.11RT 4 E4.4.11RB E4.4.11RT S4.5.11CB 7 S4.11.11CT 21,42 S4.11.11CB 7 21,42 E4.11.11RB 8 E4.11.11RT 8 S4.18.11CB S4.18.11CT E4.18.11RT 8 S4.27.11CB 7 S4.27.11CT 7 S5.2.11CB 7 S5.2.11CT 7 12 E5.2.11LT 4 E5.9.11LT 8 S5.9.11CT 7 8 S5.16.11CB S5.16.11CT S5.25.11CT S6.6.11CB 8 87

S6.6.11CT E6.16.11GT 8 E6.23.11LB 8 E6.23.11LT 4,12 E6.23.11GB 4 E6.23.11GT 3-4,8,13,20,24,26,28-30,33,37,41-42 E7.8.11LB 8 E7.8.11LT 8 E8.9.11LT 8 E8.16.11LT 10,14 S10.11.11CT 24, 26 8 S10.18.11CB 9,21,22,24,26 S10.18.11CT 9,24,26 S10.25.11CT 8 S11.1.11CB 4 S11.8.11CB 4 S11.15.11CB 4,8 S11.15.11CT S11.22.11CT 8 S11.29.11CT 8 S12.6.11CT 4 S12.13.11CBP 8 S12.13.11CT S12.13.11CTP 8 Phytopythium S3.28.11CT 6 20, 37 litorale Phytopythium E6.16.11RB 4 helicoides E7.1.11RT 4,21,22,24,42 E7.1.11RB 4,21-22,24,26 E7.1.11GB 4,17,21,24,26,37,39 E7.1.11GT 4,17,21,22,24,26,30,37,39,42 E7.8.11RB 4 15 E7.8.11RT 3,4,12 15 E7.14.11RBa 20-22,24,26,37,42 E7.14.11RT 4,15,21,22,24,26 E7.14.11SBT 21,22,24,25,42 E7.20.11LT 3,4,15,22,24,42 E7.20.11SBT 4,15 E7.20.11RB 4,24,42 E8.2.11SBT 4,23,24,26,42 E8.9.11SBB 8 88

Clade E2-2 E7.20.11LB 4 unknown E7.27.11LT 4,24,25 E7.27.11SBB 4 S9.16.11 screen 4, 13-14 S9.16.11 10 4, 13-14 S9.16.11 60 4 S9.16.11 100 4, 13-14 S9.16.11 270 4, 13-14 E10.4.11LT 4,24,26,42 S10.4.11CB 4 Pythium E8.22.11LB 4 middletonii E8.30.11LT 3,4,16,24,26,37 E9.13.11LB 4,14,24,26,31 E9.13.11LT 4,24,26,31 Clade B2 E5.9.11RB 8 unknown E5.9.11RT 8 E5.18.11RT 8 E5.26.11RB 8 E5.26.11RT 8 E6.1.11RB 8 E6.1.11RT 8,12 E6.8.11RB 8 E6.8.11RT 8 E6.16.11RT 8 E6.23.11RB 8 E6.23.11RT 4,8,15 E7.8.11SBB 8 E7.14.11SBB 4,20-22,24,26,27 E7.20.11SBB 4 E7.27.11SBT 6 E8.2.11SBB 4 E8.9.11SBT 8 E8.9.11RT 8,24,26,37,39,42 Pythium sp. nov. E3.22.11LT 4, 36 OOMYA1646- E4.27.11RT 4,17-18,21-22,24,26,30 08 (E2) E10.31.11RT 4,24,26,41 E11.7.11RT 4,24,26,37,39,41,42 E11.29.11RT 4,26,42 E11.29.11RT poinsettia 4,22,26,37,42 E12.6.11RT 4,21,24,26,30,36-39,42 E12.6.11RT poinsettia 4,24,26,30,36,37,39,42 38 89

Pythium E11.21.11SBT 4,24,26,37,42 rostratifingens poinsettia E12.6.11RB 24,42 4 E12.6.11RB poinsettia 26,26,42 4 Phytopythium S3.28.11CB 4 chamaehyphon S4.5.11CT 6 S9.22.11CB 24,26,36,42 14 S9.22.11CT 4 S9.26.11CT 24,26 S10.4.11CT 4 S10.11.11CBb 24, 26 8, 43 S11.1.11CT 4,24,42 S11.8.11CT 4 S11.22.11CB 4 14 poinsettia S11.22.11CT 4 poinsettia S11.29.11CT 4 poinsettia S11.29.11CB 4 S12.6.11CB 4 S12.6.11CB 4, 24, 26 poinsettia S12.13.11CB 8 Clade A E4.27.11LT unknown S5.31.11CB 8 S9.26.11CB P. S9.16.11 Bay 15 7, 22, 24, 26, 29,30,34,40-42 aphanidermatum Other E3.22.11RT 4, 19-21, 36 E4.18.11RB 8 E4.22.11RB 17-18,21- 4,36 22,24,26,30,37 E5.2.11RB 7 E5.2.11RT 7 S5.9.11CB 8 E5.18.11RB S5.25.11CB 45 8 E7.14.11RBb E7.14.11LT 4,25,42 E7.20.11RT 4 E7.27.11RB E7.27.11RT 8 90

E8.2.11RB 4,15,24,26,44 E8.2.11LT 8,17,23,24,26,32 E8.9.11LB 10 E8.22.11GT 10 S10.11.11CBa 35 4 E10.31.11RB 4,24,26,41 S11.22.11CB 8 S11.29.11CB 4 poinsettia S12.6.11CTP 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 Isolate/s used Clad Number Months Isolated Months Isolated Designation in Phylogenetic e of Isolates from Greenhouse from Greenhouse E Analysis S Pythium sp. nov. E3.28.11LB B2 1 none March OOMYA1702-08 (B2) Clade E2-1 E7.14.11GT E2 8 September 2013 June- August unknown Pythium E6.23.11LB B2 57 March-June March-August coloratum S3.22.11CT October-December Phytopythium S3.28.11CT K 1 March none litorale Phytopythium E7.20.11RB K 16 none June- August helicoides Clade E2-2 E10.04.11LT E2 12 September 2011 & July and October unknown 2013 Pythium E9.13.11LB E2 4 none August & September middletonii Clade B2 E6.01.11RB B2 19 none May-August unknown Pythium sp. nov. E11.07.11RT E2 9 March- April December OOMYA1646-08 October-December (E2) Pythium E12.06.11RBP E1 4 none November & rostratifingens December Phytopythium S9.26.11CT K 10 September- none chamaehyphon December March-April Clade A unknown S5.31.11CB A 6 May & September, April September 2013 All isolates are from 2011 unless specified as September 2013.

92

Table A-3. The cardinal temperatures of the isolates, mean daily growth rates at 25C on PCA, and colony morphology on PCA.

Isolate Isolate number Minimu Optimu Maximu Daily Colony designation m m m Growt Morpholog Growth Growth Growth h (mm) y Temp (C) Temp Temp (C) (C) Clade E2-1 E6.16.11LT 9 30-35 35 13.42 Ra unknown E7.20.11GT 9 35 40 13.74 Ra F2:4 5 35 40 12.16 Ra Pythium S4.27.11CB 5 30-35 35 13.92 Ra coloratum E3.28.11RB 5 30 35 13.75 Ra/SR E3.28.11RT 5 30 35 12.62 Ra/SR E4.11.11RB 5 30 35 10.66 N/R E4.11.11RT 5 30 35 9.36 N E4.18.11RT 9 35 40 5.57 R S10.11.11CT 5 30-35 35 13.63 Ra S11.1.11CB 5 30 35 13.71 Ra S11.1.11CT 5 30 35 14.31 R S12.6.11CT 5 30 35 13.33 Ra S4.11.11CB 5 30 35 14.87 Ra/R S4.11.11CT 5 30 35 14.97 Ra S4.27.11CT 9 30 40 10.93 N S11.22.11CTP 5 30 35 13.02 R/Ra S3.22.11CT 5 30 35 14.31 R S11.29.11CT 5 30 35 10.18 Ra/SR S3.22.11CB 9 30 35 12.20 N S10.18.11CB 5 30 35 11.48 Ra/R S4.18.11CB 9 30 35 9.65 Ra/R S4.5.11CT 15 30 35 13.99 Ra S5.16.11CB 5 30 35 11.48 Ra/N S12.13.11CB 9 25 30 10.31 Ra/C E6.23.11LB 9 30 35 12.53 R/Ra E6.23.11LT 9 30 35 8.97 Ra/R E6.16.11GT 9 35 35 8.95 R E5.9.11LT 9 30 35 8.26 Ra/R E4.14.11RT 5 30 35 12.79 Ra/R E4.4.11RB 9 30 35 10.35 N E5.9.11RB 9 30-35 40 5.21 R E7.8.11LT 5 30 35 11.74 Ra/R S12.13.11CBP 5 30 35 9.79 Ra/C S12.13.11CT 5 30 35 11.61 R 93

S12.13.11CTP 5 30 35 14.34 Ra/SR S4.18.11CT 5 30 35 14.51 R/Ra E8.16.11LT 5 30 35 12.84 R/Ra S5.2.11CT 5 30 35 13.32 R/Ra S5.2.11CB 5 30 35 10.95 R Phytopythium E7.14.11SBT 5-15 35 40 19.06 Ra helicoides E7.20.11LT 15 35 40 23.72 Ra E7.14.11RT 15 35 40 20.99 Ra E7.1.11RT 15 35 40 21.01 Ra E7.20.11RB 5 35 40 21.00 Ra E7.8.11RT 15 35 40 20.80 Ra E7.1.11GB 15 35 40 15.64 N E7.1.11RB 5-15 35 40 20.01 Ra E7.20.11SBT 5-15 35 40 17.75 Ra Clade E2-2 E7.20.11LB 5 30 35 12.65 Ra unknown E10.4.11LT 5 30 40 12.57 Ra S9.16.11 270 15 30 35 11.13 Ra S9.16.11 10 5 30 35 13.29 Ra F1:5:1 5 30 35 13.55 Ra S1 5 30 35 12.66 Ra Pythium E8.30.11LT 5 25 30 9.76 Ra middletonii Clade B2 E6.23.11RT 9 35 40 1.27 R unknown E7.20.11SBB 5-15 35 40 22.31 Ra E7.27.11SBT 9 30 35 13.45 Ra E7.14.11SBB 15 35 40 20.54 Ra Pythium sp. E12.6.11RTP 5 30-35 35 13.06 Ra nov. E10.31.11RT 5 25 30 8.70 R/C OOMYA1646 E11.29.11RT 5 25 30 9.88 Ra -08 (E2) E11.7.11RT 5 25 30 10.14 Ra/C E3.22.11LT 5 25 30 10.62 Ra E4.27.11RT 5 25 30 9.44 Ra/C Pythium E11.29.11SBTP 9 25 30 6.73 C rostratifingens E11.21.11SBTP 9 25 30 6.32 C E12.6.11RB 5 25 30 5.87 C Phytopythium S9.26.11CT 15 30 35 16.65 Ra chamaehypho S10.4.11CT 9 30 35 12.60 Ra n S12.6.11CB 9 30 35 15.64 Ra S9.22.11CB 15 30 35 16.94 Ra S11.29.11CB 15 30 35 14.96 Ra S3.28.11CB 5 30 35 13.00 Ra S9.22.11CT 9 30 35 16.10 Ra/R Clade A C13 9 30 35 0.00 C/Ra 94

unknown C3 5 30 35 0.00 Ra/C S2 15 35 40 0.04 R/Ra S9.26.11CB 9 30-35 35 0.27 Ra Not Yet E5.16.11RB 9 30 35 9.60 Ra Sequenced C5 15 30 35 0.00 Ra/SC C6 5 35 40 13.90 Ra C1 15 30-35 35 0.31 Ra/C C14 15 25 35 0.10 C/Ra C2 9 35 40 13.98 C/Ra C12 9 25 35 0.22 C/Ra Other P. 9 35 40 10.55 Ra/N aphanidermatu m P. irregulare 9 30 35 15.44 N P. 5 25 35 20.77 Ra/N cryptoirregular e S9.16.11 Bay 9 30-35 40 20.02 N 15 Ra -radiate, C-chrysanthmum, R-rosette, N-no pattern, S-slight pattern; if more than one pattern is observed,s the pattern that most strongly visible is listed first in the classification.

Table A-4. Full results of the poison plate assay.

Isolate Isolate number Growth with Fungicide designation mefenoxam/ growth Sensitivity without mefenoxam (%) Classification Clade E2-1 E6.16.11GB 22 S unknown E6.16.11LT 19.5 S E7.20.11GT 20 S F2:4 5.5 HS Pythium S4.27.11CB 7.5 HS coloratum E3.28.11RB 30 S E3.28.11RT 32.5 S E4.11.11RB 35 S E4.11.11RT 14.5 S E4.18.11RT 23.5 S S10.11.11CT 36 S S11.1.11CB 40 S S11.1.11CT 34.5 S S12.6.11CT 23.5 S S4.11.11CB 39.5 S S4.11.11CT 23 S 95

S4.27.11CT 37 S S11.22.11CTP 25 S S3.22.11CT 34 S S11.29.11CT 17.3 S S11.29.11CTP 18.5 S S3.22.11CB 30.5 S S10.18.11CB 43.5 MS S4.18.11CB 40.5 MS S11.22.11CBP 49.7 I S4.5.11CT 48.5 I S5.16.11CB 64 R S12.13.11CB 99.5 HR E6.23.11LB 98 HR E6.23.11LT 91.5 HR E5.2.11LT 101 HR E6.16.11GT 92 HR E5.9.11LT 102 HR E7.8.11LT 99 HR S12.13.11CBP 7.5 HS S12.13.11CT 36 S S12.13.11CTP 35.5 S S4.18.11CT 52.5 MR E8.16.11LT 98 HR S5.2.11CT 34.5 S S5.2.11CB 35 S E4.14.11RT 26 S E4.4.11RB 19.5 S Phytopythium E7.14.11SBT 44 MS helicoides E7.20.11LT 42.1 MS E7.14.11RT 51 I E7.1.11RT 54.5 MR E7.20.11RB 54.5 MR E7.8.11RT 56 MR E7.1.11GB 45.5 MS E7.1.11RB 59 MR E7.20.11SBT 47.5 MS Clade E2-2 E7.20.11LB 32.8 S unknown E10.4.11LT 35 S S9.16.11 270 5.5 HS S9.16.11 10 12 S S1 53.5 MR F1:5:1 11 S Pythium E8.30.11LT 88 R 96

middletonii Clade B2 E6.23.11RT 16 S unknown E7.20.11SBB 33.1 S E7.27.11SBT 28.5 S E7.14.11SBB 42.4 MS E5.9.11RB 9.5 S Pythium sp. nov. E12.6.11RTP 7 S OOMYA1646-08 E10.31.11RT 84.5 R (E2) E11.29.11RT 89.5 R E11.7.11RT 89.5 R E3.22.11LT 89.5 R E4.27.11RT 77 R Pythium E11.29.11SBTP 91 HR rostratifingens E11.21.11SBTP 97 HR E12.6.11RB 83.5 R Phytopythium S9.26.11CT 41 S chamaehyphon S10.4.11CT 22.8 S S12.6.11CB 24 S S9.22.11CB 43 MS S11.29.11CB 43.5 MS S3.28.11CB 50.7 I S9.22.11CT 50.5 I Clade A E4.27.11LT 69.3 R unknown S9.26.11CB 77.5 R S2 105.5 HR C13 96 HR C3 152.5 HR Not Yet E5.16.11RB 66.5 R Sequenced C5 85 R C6 3.5 HS C1 109.5 HR C14 111 HR C2 6 HS C12 82.5 R E8.9.11LB 14.5 S Other P128 2012 97 HR P. 98.6 HR aphanidermatum P. irregulare 8 HS P. 13 S cryptoirregulare 13-151A 37.5 S S9.16.11 Bay 15 1 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 Isolate Clade E2-1 unknown E6.16.11LT E7.20.11GT F2:4 Pythium coloratum E4.11.11RT E6.16.11GT S4.27.11CT S10.11.11CT S12.13.11CTP Pythium sp. nov. OOMYA1646-08 E4.27.11RT (E2) E11.29.11RT E12.6.11RTP S12.13.11CB Phytopythium helicoides E7.1.11RT E7.8.11RT E7.14.11SBT E7.20.11LT E7.27.11SBT Clade E2-2 unknown E10.4.11LT S9.16.11 10 S9.16.11 270 E10.4.11LT E7.20.11LB Pythium middletonii E8.30.11LT Clade B2 unknown E5.9.11RB E7.14.11SBB E7.20.11SBB E7.27.11SBT Pythium rostratifingens E11.21.11SBTP E11.29.11SBTP E12.16.11RB Phytopythium chamaehyphon S10.11.11CB S11.29.11CB Clade A unknown 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 Minimum Optimum Maximu during the week of initial baiting Growth Growth m Growth isolation Temp Temp Temp (°C) (°C) (°C) Pythium sp. 15.96 NA NA NA nov. OOMYA1702- 08 (B2) Clade E2-1 26.3-26.8 9,5 35,30 40,35 unknown Pythium 16-16.3, 18.1, 18.9-21.2, 22.4-26.1 5,9,15 30,35,25 35,40,30 coloratum Phytopythium 20.5 NA NA NA litorale Phytopythium 16.3, 18.3, 22.6, 25.6, 26.3, 26.8, 27.2, 15,5 35 40 helicoides 29.8 Clade E2-2 18.3-18.6, 24, 31.4 5,15 30 35,40 unknown Pythium 20.2, 22.3-22.8 5 25 30 middletonii Clade B2 20.6, 22.2, 23.3, 25.3-25.9, 26.5-26.7, 9,5,15 35,30 40,35 unknown 27.2, 29.3-29.8, 31.4 Pythium sp. 16.4, 17.4-17.8, 18.1-18.5, 19.7 5 25,30,35 30,35 nov. OOMYA1646- 08 (E2) Pythium 17.4-17.6, 18.5 9,5 25 30 rostratifingens Phytopythium 19.2, 20.4-20.5, 21.2-21.8, 22.6 9,15,5 30 35 chamaehyphon Clade A 16.4, 21.8, 25.2 9,5,15 30,35 35,40 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.

Greenhouse E Greenhouse S Sampling date Tank L Tank R Tank G Tank SB Sampling date Tank C 3/22/2011 16.14 19.85 3/22/2011 18.90 3/28/2011 15.96 19.54 3/28/2011 20.47 4/4/2011 15.55 19.25 4/5/2011 20.37 4/11/2011 15.84 20.28 4/11/2011 20.25 4/18/2011 15.98 20.43 4/18/2011 20.13 4/25/2011 16.38 19.73 4/25/2011 20.20 5/2/2011 16.02 21.29 5/2/2011 20.97 5/9/2011 16.21 20.06 5/9/2011 20.38 5/18/201 22.16 5/16/11 22.42 5/26/2011 23.31 5/ 25/11 22.45 6/1/2011 26.49 5/31 /11 25.17 6/8/2011 25.57 6/6 /11 25.12 6/16/2011 Missing data 25.61 6/23/2011 Missing data 25.33 26.12 7/1/2011 26.29 27.22 7/8/2011 16.33 26.82 25.95 7/14/2011 18.95 22.64 26.25 29.31 7/20/2011 18.28 22.47 26.27 29.83 7/27/2011 18.50 25.91 26.49 31.44 8/2/2011 21.18 25.85 26.80 27.20 8/9/2011 23.17 26.77 26.85 25.59 8/16/2011 20.52 26.65 8/22/2011 20.22 26.65 8/30/2011 22.25 9/13/2011 22.75 9/ 16/11 24.03 9/20/2011 18.02 20.33 22.20 9/22 /11 22.56 9/27/2011 17.66 22.07 23.17 9/ 26/11 21.82 10/4/2011 18.62 22.91 20.90 10/4 /11 21.82 10/12/2011 19.35 21.95 20.41 20.82 10/11/11 21.20 10/18/2011 19.25 20.77 Missing data 21.19 10/18/11 20.21 10/24/2011 18.36 19.34 20.81 20.77 10/25/11 20.09 10/31/2011 18.03 18.55 20.15 19.55 11/1/11 20.18 11/7/2011 18.29 17.86 19.88 18.37 11/8/11 19.84 11/14/2011 18.83 17.42 20.36 18.41 11/15/11 19.28 11/21/2011 18.73 17.22 20.24 18.53 11/22/11 19.21 11/29/2011 18.67 17.75 20.09 17.65 11/29/11 19.22 12/6/2011 18.44 17.40 19.64 16.37 12/6/11 19.20 12/13/11 18.14 12/20/11 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 co- inoculation results.

Type Isolate Pathogenic Coinoc Coinoc with Coinoc with P. (+)=yes (-) = no with P. P. apahanidermat irregula cryptoirregula um re re Clade E2-1 E7.20.11GT - unknown E6.16.11GB + P E6.16.11LT - S Pythium S5.2.11CT + P coloratum S5.2.11CB + S9.26.11CB - S S S11.29.11CT - S E3.22.11LT - S E5.9.11LT + S S11.29.11CT + S E5.2.11LT - S S E6.16.11GT + S3.22.11CB + S S4.18.11CB + S10.18.11CB + S3.22.11CT + S S E8.16.11LT + S4.18.11CT + P P Phytopythium E7.20.11LT + helicoides Clade E2-2 E10.4.11LT - unknown E7.20.11LB + Clade B2 E6.23.11RT + unknown E7.14.11SBB + E7.20.11SBB + P E7.27.11SBT - Pythium E11.29.11SB - rostratifingen TP s E11.21.11SB - P TP Phytopythium S3.28.11CB - chamaehypho S11.22.11CB + n P S11.29.11CB + P S11.22.11CT + P 101

S9.22.11CB + P S12.6.11CB + P S10.4.11CT - Clade A E4.27.11LT + S S unknown Not E8.9.11LB - S Sequenced

S- slowed disease progression, P-promoted disease progression.

Table A-9. The representative isolates used in the greenhouse pathogenicity and co-inoculation tests.

Species Isolate Clade E2-1 unknown E6.16.11LT E7.20.11GT E6.16.11GB P. coloratum 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 Ph. helicoides E7.20.11LT Clade E2-2 unknown S9.16.11 10 E10.4.11LT E7.20.11LB 102

Clade B2 unknown E7.14.11SBB E7.20.11SBB E7.27.11SBT E6.23.11RT P. sp. nov. OOMYA1646-08 (E2) E3.22.11LT E4.27.11RT Pythium rostratifingens E.11.21.11SBTP E11.29.11SBTP Ph. chamaehyphon S9.22.11CB S12.6.11CB S3.28.11CB 11.29.11CB S11.22.11CBP S11.22.11CTP S10.4.11CT Clade A unknown E4.27.11LT S9.26.11CB P. cryptoirregulare P123 P. aphanidermatum P128 P. irregulare P84 Other E8.9.11LB S9.29.11CT

103

Table A-10. A list of the isolates used for the lab soil pathogenicity tests.

Species Isolate Clade E2-1 unknown E6.16.11LT F2:4 P. coloratum S3.22.11CT E4.11.11RT E8.16.11LT P. sp. nov. OOMYA1646-08 S12.13.11CB (E2) E10.31.11RT Ph. helicoides E7.1.11GB E7.14.11SBT Clade E2-2 unknown S9.16.11 10 S1 E10.4.11LT P. rostratifingens E11.21.11SBTP E12.16.11RB Ph. chamaehyphon S3.28.11CB S10.4.11CT Clade A unknown S9.26.11CB C13 S2 P. irregulare P84 P. cryptoirregulare P123 P. aphanidermatum P128 P220 P223

104

Table A-11. Average weekly tank temperatures for the tank simulation experiment

Tank Tank Tank Tank Tank Tank Tank Tank Tank Tank Tank 1 2 4 6 8 9 10 12 13 14 16 7/23- 22.2 na 22 24.1 23.7 23.9 24.8 24.3 23.8 24.1 24.6 7/29 7/30- 22.1 na 22.1 23.9 23.6 23.8 24.2 23.8 23.1 23.3 23.6 8/5 8/6- 21.8 na 22.3 24 23.9 24.2 24.2 23.8 23 22.9 23.6 8/12 8/13- 21.1 na 21.3 22.6 22.3 22.7 23.4 22.9 22.4 22.6 22.8 8/19 8/20- 22.7 22.5 22.2 23.2 22.9 23.4 24.3 24 23.6 23.7 23.7 8/26 8/27- 23 22.7 22.5 24.3 23.7 24.4 24.9 24.5 24 24.2 24.4 9/2 9/3-9/9 22.5 22.3 22.2 24 23.2 24 24.5 24.1 23.7 23.8 24.1 9/10- 20.4 20.1 20.2 21.2 20.7 21.2 22 21.8 21.5 21.6 21.7 9/16 9/17- 20.1 19.7 19.8 21.5 20.3 21.4 22.2 21.9 21.6 21.5 21.8 9/23 9/24- 21.2 20.9 20.8 22.3 22.1 22.2 22.8 22.7 22.5 22.1 22.5 9/30 10/1- 19.3 19 19.1 19.7 18.5 19.5 20 20.1 20.1 19.8 20.1 10/7 10/8- 19 18.8 18.9 19.5 18.8 19 19.7 19.8 19.6 20 19.7 10/14 10/15- 18.1 17.9 18.2 18.4 17.3 18.2 18.9 19.2 19.1 19.1 18.9 10/21 10/22- 19.3 18.8 19.5 19.8 19.5 19.4 19.8 19.9 19.9 19.4 18.9 10/28 10/29- 18 17.7 18.1 18.1 18 17.8 18 18 18 17.7 16.9 11/4 11/5- 18.1 17.9 18.3 18.6 18.5 18.2 18.4 18.5 18.6 17.7 17.5 11/11 11/12- 15.6 15.7 15.7 16.3 16 16 15.5 15.7 15.7 14.6 14.6 11/18 11/19- 16.4 16.3 16.6 16.5 16.8 16.4 16 16.2 16.3 15.3 15.0 11/25 11/26- 15.1 15.3 15.5 15 15.7 15.6 15.2 15.3 15.3 14.7 13.9 12/2 12/3- 14.8 14.8 15 15.3 14.4 15 14.9 15 15.1 14.6 13.9 12/9 12/10- 14.4 14.5 14.6 14.6 13.9 14.4 14.1 14.2 14.3 13.5 13.1 12/14

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