© 2008 Plant Management Network.
Isolation, Storage, Pathotype Characterization, and Evaluation of Resistance for Phytophthora sojae in Soybean
Anne E. Dorrance and Sue Ann Berry, Department of Plant Pathology, The Ohio State University-OARDC, Wooster 44691; Terry R. Anderson and Chuck Meharg, Agriculture and Agri-Food Canada, Harrow, Ontario, Canada N0R 1G0
Dorrance, A. E., Berry, S. A., Anderson, T. R. and Meharg, C. 2008. Isolation, storage, pathotype characterization, and evaluation of resistance for Phytophthora sojae in soybean. Online. Plant Health Progress doi:10.1094/PHP-2008-0118-01-DG.
Phytophthora sojae is one of the predominant soybean pathogens in production regions with poorly drained soils (8). It can infect soybeans at all growth stages and causes seed rot, pre- and post-emergence damping-off, root and stem rot. The severity of disease is directly related to the length of time soils are saturated as well as the susceptibility of the cultivar. This pathogen is effectively managed through the development and deployment of cultivars with single resistance genes (Rps) as well as partial resistance which is inherited quantitatively (8). Soybean breeders face the challenge to assess which Rps genes are present in a given cultivar as well as an assessment of the level of partial resistance. The presence of an effective Rps gene can mask the level of partial resistance and other Rps genes.
There are over 50 described races of Phytophthora sojae, but many more pathotypes have been reported throughout the US and Canadian soybean production regions (8). Many of the isolates that have been collected in the past ten years are more complex in that they can cause disease on plants with three or more Rps genes (6). As a result of this increasing complexity in P. sojae populations, soybean seed companies not only stack or combine Rps genes but also increase the levels of partial resistance. This diagnostic guide provides some standard assays to assist in the identification of virulence types of P. sojae and genetic resistance in soybean.
Soybean [Glycine max (L.) Merrill] is the primary host. Cranesbill (Geranium carolinianum L.), Lupinus spp., Lima bean (Phaseolus lunatus L.), and string bean (P. vulgaris L.) were described as hosts, but these were only in greenhouse inoculations. None have been reported as hosts in the field [reviewed by Erwin and Ribeiro (7)].
Phytophthora root and stem rot, pre and post-emergence damping-off.
Phytophthora sojae Kaufmann & Gerdemann. Synonyms include: Phytophthora megasperma var. sojae Hildebrand, P. megasperma f. sp. glycinea Kuan and Erwin, and P. sojae f. sp. glycines (7).
Erwin and Ribeiro (7) provide a thorough overview of the taxonomic changes that have occurred since P. sojae was first described by Herr in 1957.
Symptoms and Signs
P. sojae causes damping-off, root rot and stem rot of soybean. Seedlings colonized by P. sojae turn brown, wilt, and die. Lesions can begin at any place on the root, hypocotyl, or cotyledon. The symptoms are indistinguishable from damping-off caused by Pythium spp. The most characteristic symptom is in the stem rot phase after development of the primary leaves, in which a chocolate-brown colored lesion develops from the base of the plant up the stem.
In the US and Canada, P. sojae has been reported from all soybean production regions. It has also been reported from Australia, Argentina, Hungary (9,12), and more recently from Brazil, China, Japan, and Republic of Korea (4,14,20,21).
Seedlings. Plant tissue is washed with hand soap to gently remove all of the soil associated with the plants then rinsed thoroughly with tap water. The tissue is then rinsed for 10 sec in 0.5% sodium hypochlorite solution, rinsed with sterile distilled water, and blotted dry on sterile paper towels (Fig. 1a and 1b). The sections from the edge of the lesion are placed on PBNIC agar (see Appendix 1).
Stem lesions. Leaves, petioles, and unwanted roots from the main stem containing the lesion are removed from the plant with hand clippers. The stems are washed with dry powdered laundry soap (Tide, Proctor and Gamble, Cincinnati, OH), then rinsed thoroughly with water. The stems are placed in 0.5% sodium hypochlorite solution in a laminar flow hood to surface-sterilize the tissue. Thinner tissues are placed in solution for 10 sec, while thicker tissues may be in solution for 30 sec for large tissues up to 1 min. Another method of surface-sterilizing is to rinse the tissue with 70% ethanol for 5 to 10 sec, then rinse with sterile distilled water. Both methods require blotting the excess moisture with sterile paper towels. The outer layer, or epidermis, of the stem is removed aseptically and small wedges are cut from the advancing margin of the lesion or slightly higher. Wedges are placed on PBNIC agar (Fig. 1C).
After placing symptomatic tissue on agar plates, lift the agar from the plate with a 5-cm spatula (Fig. 1D). Flip the agar, to relocate the pieces between the bottom of the plate and the agar, cut agar layer close to plate edge and press gently around each piece with the spatula so the agar is sealed to the bottom of the plate (Fig. 1E). In Ontario, the isolation procedure is modified slightly. Stem sections, 1 to 2 cm in length, are surface sterilized in 1.25% sodium hypochlorite solution for 3 min, blotted, and sectioned into 1-mm sections that are placed in LBA (Appendix 1). Plates are incubated in the laboratory and monitored for growth of Phytophthora with a dissecting microscope at a regular schedule. After 3 days incubation, hyphal tips of Phytophthora are removed aseptically and transferred to fresh LBA to monitor for bacterial contamination. On LBA, bacteria can sometimes be viewed through the plate under a compound scope at 20×, colonies will grow along the mycelium. Another check is to transfer a 2-mm P. sojae colonized agar plug to a vial containing 5 ml of nutrient broth. If bacteria are present, the broth will appear cloudy in less than 24 h.
Soil (bioassays). P. sojae survives in soil primarily as oospores which can readily be baited to obtain isolates for identification and pathotype screening (16). Seed planted immediately into soil may not detect Phytophthora since oospores require some time to break dormancy and seedlings may be large enough to escape damage by the time enough active inoculum is present for infection. Soil is treated to induce oospore germination before planting. To prepare soil, air dry (Fig. 2A), then grind to fine particles (IER Improved Soil Grinder, The Fen Machine Co., Cleveland, OH or other suitable piece of equipment) to make potting easier as well as to mix the soil (17). Flood the soil for 24 h (Fig. 2B), then drain and air dry until the moisture content approaches approximately –300 mb matric potential [soil cracks or pulls away from the side of container although it is still damp (17)]. Place containers of soil in polyethylene bags and incubate at room temperature for a total of 2 weeks (Fig. 2D). Oospores will germinate and form sporangia during this period. Following the incubation period, seeds of a moderately susceptible cultivar are placed on top of the soil in the pots and covered with wet coarse vermiculite. The pots are placed into plastic bags to prevent drying out during seed germination. Three days after planting, when the seedling roots are 5 cm long, the containers are flooded again for 24 h (Fig. 2B). Containers are placed on benches to drain. Phytophthora sojae can readily be isolated from collapsed hypocotyls of emerging seedlings over the next 10 days (Fig. 2D).
P. sojae has a distinct growth pattern on PBNIC agar which is slow, the mycelium will not appear until after 2 or 3 days. After a week, the colony may only be 5 to 10 mm in size (Fig. 1F). If the colony is thick and covers the plate in the same time period, it is most likely a Pythium spp. The mycelium is coenocytic and on PBNIC there are many branches and hyphal tips are curved.
P. sojae is homothallic and oospores will form within three days on LBA. Oospores are predominately paragynous, but amphigynous can also be found (Fig. 3). Sporangia and hyphal swellings are rarely formed in culture.
Two methods for storage are recommended, in a 15°C incubator or in liquid nitrogen. The advantage of liquid nitrogen storage is the maintenance of both virulence pathotype and aggressiveness for at least 4 years (Dorrance et al., unpublished data).
1. Storage at 15°C on dilute V8 juice agar slants (Appendix 1). Cultures are grown on 9 ml of agar in Wheaton scintillation vials. When cultures are 10 to 12-day old, cover with 2 ml of sterile de-ionized water and store at 15°C for up to 3 years. The virulence pathotype of isolates may change under this storage system.
2. Storage in liquid nitrogen in cryovials. A culture at least 2 weeks old is used to ensure oospore development and maturation. Five to six 5-mm colonized plugs containing oospores are transferred to a cryovial (Corning, Corning, New York) containing a sterilized solution of 10% glycerol and sterile distilled water. These are placed in -80°C overnight before placing into liquid nitrogen (Depending on how often an isolate will be needed, we routinely store 3 to 4 cryovials for isolates that we rarely use and 20 to 30 for those used more frequently). This procedure was first described by Tooley et al. (15). In the recovery of isolates, it is critical that they be brought to room temperature quickly to avoid the formation of ice crystals by placing them in a warm water bath (30°C). Stocks of 10% glycerol can be stored by autoclaving 10 ml aliquots of 10% glycerol and storing at 4°C until required. Dispense 1.5 ml into each cryogenic vial just prior to storage.
Race characterization and Rps genes. Soybeans have a race-specific interaction with P. sojae. Resistance has been identified at 8 loci with 14 dominant resistance genes (Rps genes). Several sets of differentials have been developed in the past including collections of the original source of the Rps gene, and lines developed from backcrosses into Williams, Harosoy, or Bedford background. Recently, PIs, Williams and Harosoy differentials were compared for their reaction to P. sojae isolates (5). The most current recommended set of differentials was recently described (5). During a meeting in Wisconsin in the early 1980s, soybean pathologists designated the following eight differentials for inclusion in race characterization of P. sojae isolates: Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps3, Rps6, and Rps7 (1). Rps3b, Rps3c, Rps4, and Rps5 were excluded because they were considered to be unstable. In fact, efforts to map Rps5 have been unsuccessful to date. Rps8 was only recently identified in PI399073 (3). The race or pathotype characterization of an isolate is primarily assessed through inoculations on the soybean hypocotyl. Both laboratory and several greenhouse assays can be used to characterize the pathotype of a given isolate but also to identify the Rps gene(s) present in a given cultivar.
Hypocotyl inoculation technique. There are many variations of this assay including placing seeds in germination towels or planting in soil or vermiculite. The greenhouse protocol is described here. Coarse vermiculite is fairly inexpensive, light in weight, and easily handled. Plant seeds in coarse vermiculite until the cotyledons have expanded (about 6 to 7 days). Emergence of Phomopsis infested or poor-quality soybean seed can be improved when sprayed with a solution of 0.3 g benomyl (0.6 g Benlate 50WP) in 500 ml of sterile distilled water until run-off. Inoculum is produced on soft (12 to 14 g agar/liter) dilute lima bean (see Appendix 1) or V-8 juice agar until the mycelium covers the plate at 21 to 25°C. The colony is cut in strips and placed in a 10-ml syringe. The agar culture is then forced through the syringe. The macerated culture is placed in the syringe a second time and a #18 needle placed on the end. Make a slit with the needle tip about 1 cm long in the hypocotyls of the seedling 1 cm below the cotyledonary node. Place 0.2 to 0.4 ml (approximately 200 to 400 cfu/ml) of the culture slurry into the slit with the syringe. Cover the plants with plastic for about 12 h to prevent the agar from drying and for the infection to take place. Incubate at 25°C in diurnal light for 1 week. Susceptible plants will die or develop distinct lesions 3 to 5 days after inoculation. Resistant plants or non-hosts will develop a hypersensitive reaction. Reactions are scored for pure lines (non-segregating lines) as follows: 0 to 2 seedlings dead is resistant; 3 to 5 seedlings intermediate; 6 to 8 susceptible or < 25% plants killed is resistant; 26 to 75% plants killed is intermediate; and > 75% plants killed is susceptible. An intermediate reaction can indicate contamination of the differential soybean line or a mixed culture of more than one pathotype.
Precautions. Bacterial contamination and high temperatures (> 28°C) can easily influence the resistance response following inoculation of the soybean hypocotyls (2,11,19). Two simple procedures to assess for identification and contamination are to examine growth on potato dextrose agar (PDA) or nutrient broth. P. sojae does not grow on full strength PDA (10) and nutrient broth will become cloudy from the bacterial growth. On LBA, the bacteria that often contaminate cultures do not grow well. High temperatures induce susceptibility to P. sojae hypocotyl inoculation (2,11,19), thus it is necessary to monitor temperatures following inoculation.
There is one Rps gene, Rps2, in which the use of the hypocotyl technique was debated. In earlier studies, the expression of Rps2 in the hypocotyl ranged from resistant to intermediate. The expression of this resistance in roots is thought to be more consistent (18). An aeroponic system was developed and used to inoculate roots to evaluate the expression of Rps2 in a segregating population (13,18). In our experience, using the Williams differential (L76-1988), we have found a consistent response when the hypocotyl test is compared to other root inoculation assays.
Rps gene discovery. The Rps gene that is present in a soybean can be identified through a series of inoculations with specific P. sojae pathotypes (Appendix 2, Fig. 5, and Table 1). Any isolate can be used as long as the pathotype (virulence pattern) matches the virulence pattern on the described differentials in the isolates described here. Most P. sojae isolates have virulence to Rps7, thus this is not considered in the scheme, but should be kept in mind if a particular isolate does not have this virulence. The simplest gene to identify is Rps1a. Soybeans with this gene express resistance to isolates that are resistant to Race 1 and susceptible to Race 3, but can also be confirmed with Races 4, and 7. Several isolates are needed to identify Rps1k. Soybeans with Rps1k express resistance following inoculation to Races 1, 3, 4, and 7, but are susceptible to 25. Race 25 has virulence towards Rps1a, Rps1b, Rps1c, and Rps1k. Therefore if a soybean cultivar expresses susceptibility to Race 25 but resistance to Race 1, then Rps1a, Rps1b, Rps1c, or Rps1k may be present. All of these genes are believed to occur at the same locus in the soybean genome, thus a combination of these four genes is not possible. P. sojae races 2, 3, and 4 can then separate the remaining genes from Rps1k (Appendix 2, Fig. 5).
Table 1. Response of soybean differentials with Rps genes to P. sojae.
Partial resistance. In addition to Rps genes, soybeans have a race-non specific type of resistance to P. sojae that is quantitatively inherited and known as partial resistance as well as tolerance in older literature; this type of resistance reduces the overall rate of colonization of soybean and has different levels. Choice of isolate is critical to evaluate this trait correctly. Before completing a partial resistance assay, a hypocotyl test should be completed to ensure that the P. sojae isolate used will have a compatible interaction (susceptible response) with the soybean line(s) to be tested. The presence of effective Rps genes (resistant interaction) will mask this trait, thus the lesion growth in resistant lines will typically be 0 to 1 mm or no root rot will be present.
Layer test. Inoculum for the layer test consists of a 14-day-old P. sojae culture grown on lima bean agar in glass Petri dishes (9 cm). The agar layer is then placed 5 cm below the seed in cups (1.2-liter polystyrene containers with bottom drainage) containing coarse vermiculite. The diameter of agar disc must fit the cup diameter to prevent roots from growing around the inoculum. The amount of root rot and seedling death is rated three weeks after planting. The rating system for the layer test uses a scale of 1 to 9, in which 1 = no root rot, 2 = trace of root rot, 3 = bottom third of root mass rotted, 4 = bottom two thirds of root mass rotted, 5 = all roots rotted + 10% seedling kill, 6 = 50% seedling kill + moderate stunting on tops, 7 = 75% seedling kill + severe stunting of tops, 8 = 90% seedling kill, 9 = all seedlings dead.
Tray test. Lesion length is one of three components that contribute to the expression of partial resistance in soybean. Seed are planted in cups (1.2-liter polystyrene containers with bottom drainage) with coarse vermiculite on the bottom 2.54 cm of the cup then with fine vermiculite. After 7 days, when unifoliolate leaves are first visible, plants are gently removed from cups and the vermiculite gently washed from the roots under running tap water. Seedlings are placed on a food service tray with one of the raised sides removed (Fig. 4A). The plants are aligned on a polyester cloth that is placed on top of a wicking pad. A wound (~5 mm) is made on the root of each seedling approximately 20 mm below the stem/root interface. A mycelial slurry (approximately 0.5 mL) from a 7-day-old culture of P. sojae is placed over the wound (Fig. 4B). Lesion lengths on each seedling can be evaluated as early as three days, but seven days is more common, after inoculation from the inoculation point to the edge of the symptomatic tissue (Fig. 4C and 4D).
Many of the techniques in this Diagnostic Guide first appeared in Schmitthenner and Bhat (17). The descriptions in this diagnostic guide focus primarily on P. sojae and many have been updated. Funding for this project was primarily through a joint project from United States Department of Agriculture, United Soybean Board, American Seed Trade Association, USDA-IFAFS for a project entitled A National Soybean Pathogen Germplasm Conservation System. We would like to thank D. Mills, K. Broders, and S. Mideros Mora for pictures. Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.
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Appendix 1: Ingredients of dilute V-8 juice, PBNIC, and lima bean agar
Dilute V-8 Juice Agar. Autoclave the V-8 juice (40 ml), CaCO3 (0.6 g/liter), and 300 ml distilled water for 15 min at 121°C; filter through Whatman #1 filter paper with a 1-cm pad of diatomaceous earth; increase volume to 1000 ml with distilled water; add sucrose (1.0 g), Bacto yeast extract (0.2 g); and cholesterol (2 ml of a 0.01 g N,N, dimformamide solution); and disperse by shaking; add Bacto agar (20 g) and autoclave. A V-8 juice concentrate can be prepared by adding 0.6 g CaCO3 to 40 ml of V-8 juice, autoclaving, storing frozen, and unfiltered until needed. When preparing media, thaw and filter the concentrate, then proceed with the directions.
PBNIC agar. For 1 liter, add the following to the Dilute V-8 juice agar prior to autoclaving: Benlate (50% benomyl) (0.01 g); PCNB (pentachloronitrobenzene, 0.054 g); Neomycin sulfate (0.1 g); Chloramphenicol (0.01 g). After autoclaving, add Rovral (50% Iprodione, 0.04 g). Pythium spp. growth can be limited by adding hymexazole (20 mg/liter after autoclaving).
LBA, lima bean agar. For each liter, autoclave frozen lima beans (150 g) in 500 ml of distilled water for 30 min. Pass through a household sieve to remove seed coats. Add Bacto Agar (20 g) to extract and pulp and increase volume to 1 liter and autoclave.
Dilute frozen lima bean agar. For each liter, autoclave frozen lima beans (50 g) in 500 ml of distilled water for 30 min. Filter through sieve and mash cooked lima beans, add to water. Filter through a 2.54-cm pad of diatomaceous earth (Sigma D5509) on Whatman #1 filter paper. Increase volume to 1 liter. Add Bacto agar (20 g) and autoclave. For hypocotyl inoculations, ½ strength lima bean agar, add 12 to 14 g of Bacto agar.
Appendix 2; Inoculation scheme for detecting Rps genes in soybean cultivars