Microbial interference of zoosporogenesis of the plant pathogen aphanidermatum

Eric A. Carr and Eric B. Nelson Cornell University Department of and Plant-Microbe Biology

Background and Objectives Can direct via germ tubes cause plant infection?

• Pythium aphanidermatum is a soil-borne plant pathogen that infects over 50 different plant hosts (Farr • Direct germination in Pythium species is poorly understood and has been described only once for P. aphanidermatum (Stanghellini and Burr 1973) since the initial description of the species. Therefore, it is not known whether germ tubes et. al., 1989). are capable of infecting plants. This knowledge is essential to understand mechanisms underlying disease suppression.

• During plant pathogenesis, P. aphanidermatum progresses through a series of developmental stages • To answer this question we first exposed P. aphanidermatum sporangia to the additional treatment of concentrated that ultimately lead to the production of motile spores called zoospores. cucumber seed exudates (Cucumis sativum cv ‘Marketmore 76’). Seed exudates were shown previously to induce direct germination (Stanghellini and Burr 1973) and we verified this in our own experiment below. • Zoospores serve as the main infective stage of the pathogen (see Figure below). Results 4. Zoospores detect the presence of exudates • Seed exudates caused nearly 95% direct germination of sporangia (Fig. 2). No vesicles or zoospores were formed. released from seeds (blue zone surrounding seed) and swim chemotactically against the exudate gradient P. aphanidermatum sporangia before 125 germination. Bar = 50 μm indirect germination direct germination seed 100

5. Zoospores reach Figure 2. Observed 75 the seed surface, germination of sporangia 1. Sporangia are asexual pathogen 2. Sporangia produce vesicles where 3. Motile zoospores are released encyst, and then structures required for plant infection infective zoospores develop from vesicles into the soil. directly penetrate flooded with either water, and kill the seed vermicompost leachates, 50 Conclusions and or seed exudates. Significance 25 • The development of zoospores, or zoosporogenesis, begins when asexual pathogen structures percent germination called sporangia are flooded with water. Within 1-3 h, sporangial cytoplasm migrates into vesicles • Pathogen development and zoosporogenesis 0 where zoospores are subsequently differentiated and released. can be altered by microbial and non-microbial Water Vermicompost Sterile Seed exudate properties of vermicompost leachates. vermicompost

• Uncharacterized compounds in seed and root exudates serve as chemical cues to attract zoospores Interpretation to susceptible hosts (Mitchell & Deacon, 1986; Donaldson and Deacon 1993). Upon reaching the host • We can take advantage of this finding that seed exudates induce direct germination to understand whether • The suppression observed in vermicompost surface, zoospores encyst and penetrate the plant directly. vermicompost leachates can suppress germ tube-induced infections. leachates may have been a function of lower inoculum potential. Vermicompost leachates

• Although plant-associated microbes may block this homing response (Huengens & Park, 2003), other alter the germination mode of sporangia, complex microbial communities in soils are also known to reduce infection from Pythium species resulting in fewer infection foci (1-2 germ tubes (Martin & Loper, 1999). However, the mechanisms by which complex microbial communities impact If germ tubes cause infection, can infection be suppressed vs. 10-20 zoospores per vesicle). sporangial or zoospore development are not known. by vermicompost leachates? • Since germ tubes are the primary absorptive life cycle stage where Pythium species acquire • The goal of our study is to understand how soil and host-associated microbiota interfere with P. aphanidermatum sporangial germination and zoospore release and whether this may explain plant • To answer this question we exposed cucumber seeds to P. aphanidermatum sporangia flooded with the following carbon and energy, the suppression in disease protection. To achieve this goal we tested the hypothesis that complex microbial treatments in a transplant bioassay: vermicomposts may result in long-term communities associated with vermicomposted dairy manure suppress sporangial germination 1. Water, which induced indirect germination and zoospore release impacts on fitness and survival. and zoospore formation. 2. Vermicompost leachates, which induced direct germination and germ tube formation 3. Seed exudates, which induced direct germination and germ tube formation 4. Non-inoculated water control

• Transplant bioassays were designed to expose seeds to either germ tubes (direct germination) or zoospores (indirect References Do complex microbial communities alter sporangial germination) to ensure only one germination mode in the bioassay. Mode of sporangial germination was induced using

the treatments stated previously and then cucumber seeds were placed in the flooded P. aphanidermatum culture (as Donaldson SP, Deacon JW (1993). Effects of Amino- germination and zoospore formation? shown below). The seeds and flooded culture were incubated for 7 h at 27ºC then the seed was sown in sterile sand. Acids and Sugars on Zoospore Taxis, Encystment and Seeds were kept moist and incubated for an additional 7 d. Cyst Germination in Pythium-Aphanidermatum (Edson) •To answer this question we exposed P. aphanidermatum sporangia to the following treatments: Fitzp, P-Catenulatum Matthews and P-Dissotocum 1. Pure water, which consistently induces zoospore formation Drechs. New Phytologist 123: 289-295. 2. Aqueous leachates of vermicomposted dairy manure as a source of Pythium-suppressive microbial communities

3. Sterile vermicompost leachates in which microbes were eliminated treatment Farr, D. F., Bills, G. F., Chamuris, G. P., and Rossman, each seed transplanted A. Y. (1989). Fungi on plants and plant products in the •Germination was recorded at 2 min intervals for up to 8 h using time-lapse photomicroscopy. Data were expressed as seed to sterile sand in 12-well United States. APS Press, St. Paul, Minnesota. the percent of sporangia that germinated over an 8 h period. plates culture

A B medium Heungens K, Parke JL (2000). Zoospore homing and 125 seeds and flooded culture incubated Figure 1. Observed infection events: Effects of the biocontrol bacterium indirect germination direct germination together for 7 h at 27ºC 100 germination of sporangia Burkholderia cepacia AMMDR1 on two oomycete flooded with either water or pathogens of pea (Pisum sativum L.). Applied and vermicompost leachates. (A) • Disease was measured based on a 0 to 5 disease rating scale (5 = healthy). 75 Environmental Microbiology 66: 5192-5200. Indirect germination resulted in development of viable vesicles 50 Results Germ tubes and release of zoospores, Martin FN, Loper JE (1999). Soilborne plant diseases • Water and seed exudate treatments caused significant disease compared to vermicompost and non-inoculated control direct germination resulted in caused by Pythium spp.: ecology, epidemiology, and 25 the development of germ tubes percent germination percent treatments (Fig. 3). prospects for biological control. Critical Reviews in (B). 0 B Plant Science 18: 111-181. 5 Water Vermicompost Sterile B

vermicompost Figure 3. – Measured Mitchell RT, Deacon JW (1986). Differential (host- Results 4 disease from transplant specific) accumulation of zoospores of Pythium on •When exposed to pure water, sporangia developed vesicles and released zoospores as predicted (Fig. 1A). This mode bioassay. Each roots of graminaceous and non-graminaceous plants. of germination is known as indirect germination. 3 seed/seedling was rated A New Phytologist 102: 113-122.

for disease (5=healthy). A •However, in the presence of vermicompost leachates, sporangia germinated almost exclusively by the formation of germ 2 Seeds not placed in Stanghellini ME, Burr TJ (1973). Germination in vivo of tubes and few or no vesicles and zoospores were formed (Fig. 1A). Removal of microbes from the vermicompost culture medium served as Pythium aphanidermatum in oospores and sporangia. leachate did not alter the germination behavior from that of non-sterilzed leachate. This mode of germination via germ a control. P = 0.05. 1 Phytopathology 63: 1493-1496. tubes is known as direct germination.

(5=healthy) rating disease 0 Interpretation Water Vermicompost Seed exudate Non-inoculated •Vermicompost leachates completely suppress the normal course of indirect germination in P. aphanidermatum sporangia and shift the germination mode to direct germination. This shift in germination behavior does not require the control presence of microbes and may therefore be due to the chemical properties of the vermicompost leachate. Interpretation Acknowledgements • Germ tubes induced by seed exudates caused similar levels of disease as zoospores. This suggests that the germ •These results lead directly to new questions concerning the mechanism associated with disease suppression: tubes produced in the presence of vermicompost leachates are susceptible to disease suppression by microbial or non- Funding: USDA Small Business Innovation Research 1. Can direct germination via germ tubes cause plant infection? microbial factors of vermicomposts. Phase II, NYSTAR Center for Advanced Technology 2. If germ tubes cause infection, can infection be suppressed by vermicompost leachates?