Phytophthora stem and root rot (PSRR), caused by Phytophthora sojae, is a soilborne oomycete pathogen that ranks among the top five pathogens causing economic losses of soybean annually in the United States (Allen et al. 2017). In Iowa, it is estimated 7.1 million bu were lost to PSRR from 2010 to 2019 (Crop Production Network, 2020). Reducing losses to PSRR relies on single resistance (Rps) genes or partial resistance (governed by many genes) that are deployed in commercial soybean varieties.
Recently Matthiesen et al. (2021) reported the population of P. sojae in Iowa continues to gain virulence on varieties with Rps genes. Furthermore, pathotype complexity (the number of Rps genes an isolate was virulent on) of the isolates had increased since previous surveys. Traditionally it has been postulated that deploying Rps genes exerts selective pressure on the pathogen population so the number of pathotypes within a population increases and/or becomes more complex. In the past 10 years, however, there has been little change in the Rps genes that are deployed in commercial soybean (Matthiesen et al. 2021) and yet the population continues to evolve. Why?
Slusher and St Claire (1973) reported the roots of a cultivars with partial resistance to P. sojae had greater dry weight but as many oospores as the roots of a susceptible cultivar, while the roots of a cultivar with a Rps gene was considerably less oospores. Oospores are sexual thick-walled spores that overwinter in the soil. Thus, planting varieties with partial resistance may be contributing to a buildup of inoculum in the field.
We hypothesize that partial resistance exerts as much or more selection pressure on the population than Rps resistance and is responsible for the increased pathotype complexity in P. sojae reported across the Midwest. Our proposed research will test this hypothesis under controlled conditions. An improved understanding of how resistance in soybean affects P. sojae is crucial to breeders and pathologists to enable improved management of PSRR.

Objective 1: To compare the number of oospores produced on soybean varieties with different types of resistance to P. sojae.
Objective 2: To pathotype oospores of P. sojae recovered from soybean varieties with different types of resistance to P. sojae.

• Enumeration oospores in the roots of PSRR susceptible, resistant and partially resistant varieties
• Improved understanding of contribution of resistance to inoculum levels in the soil.
• Pathotype diversity of oospores recovered from the roots of PSRR susceptible, resistant and partially resistant varieties.
• Validation of molecular method for pathotyping pathotypes of P. sojae.
• Knowledge of the effect of resistance in soybean on pathotype diversity in P. sojae.
• Guidance for soybean breeders developing PSRR resistant cultivars.
• Presentations at various ISUEO extension events (Integrated Crop Management Conference, Crop Advantage Series, Research Farm Field Days) and Crops Team Publications (ICM Newsletter, ICM Blog), and the annual Soybean Breeders workshop.
• Research update reports posted to the National Soybean Checkoff Research Database.

Update:
Single zoospore isolates of four strains of P. sojae have been isolated and pathotyped. Single zoospore isolates with the same pathotype as the parent isolate have been identified and will be used as inoculum in our hydroponic trials.
We have been optimizing the Lebreton hydroponic assay for use in our lab. We had difficulties with soybean growth in the system but have made some modifications. Moreover, rather than using a zoospore suspension as inoculum in the system we have tried using a slurry of P. sojae-colonized media (unsuccessful) or P.sojae-colonized colonized discs. In the latter method susceptible soybean were stunted with more severe Phytophthora root and stem symptoms than resistant soybean.
We increased seed of Conrad in the greenhouse over the winter, so we now have seed of a partially resistant soybean variety, plus Sloan (susceptible to P.sojae) and Williams 82 (Rps1k) to inoculate in our modified hydroponic system.
Preliminary PCR analysis of isolates from P. sojae from our lab with primers developed by Lebreton et al. for pathotyping P. sojae suggest they may not be consistent. We hypothesize genotypes used to pathotype isolates may not result in consistent pathotypes, and consequently these primers may not be universal.

Update:
We have been struggling to modify the the Lebreton et al. (2018) hydroponic system (Fig. 1A). The system was developed to pathotype isolates of P. sojae. To determine pathotypes, soybean differentials are usually inoculated with P. sojae when the cotyledons emerge and open (growth stage VE). When we inoculated soybeans at VE in the system, Phytophthora root rot (PRR) developed on our susceptible variety Sloan (Fig. 1B) However, we are interested in how partial resistance affects oospore production, and consequently our soybean plants need to be at a later stage of development, when the unifoliate leaves are fully unfolded (VC) when they are inoculated. Partial resistance only becomes active after VC. Unfortunately we have struggled to get PRR to develop on our soybeans when they are inoculated at VC (Fig 1C). Although we are continuing to modify the system, we are also exploring other avenues of inoculating soybean roots with P. sojae after VC.
We have been optimizing the Dussault-Benoit et al. (2018) PCR method for pathotyping three isolates of P. sojae, P6497 (type strain), and Iowa isolates Rm 15 and S5.5a. Correct sized products have been amplified for 6 of the 9 primer combinations for P6497 (type strain) and two Iowa isolates (Fig 2A). Presence of the band indicates an AVR gene is present in the isolate, and consequently no PRR will develop on the soybean differntial with the corresponding Rps gene. An interesting result was different sized amplicons detected for the Iowa isolates with the Avr1a-indel primer set. This suggests that the particular mutation on which this primer set was developed, is not present in Iowa isolates. We are optimizing conditions for the remaining 2 primers (Avr 1c and Avr 1k) for which no product was amplified. We then plan to multiplex the reaction as Dussault-Benoit et al. did.
The three isolates we used in the PCR work were also pathotyped on soybean differentials in a Williams background, and corresponding differentials in a Harasoy background. There are some discrepancies in the pathotype data between differentials from the different genetic backgrounds (Fig. 2B), and the PCR data. These data suggest the Rps genes in the different genetic backgrounds may differ - either they represent different alleles of the Rps gene, or they are different Rps genes that are closely linked. Over the next year of funding, we plan to evaluate the PCR method on several more Iowan and regional isolates, as well as examine their pathotypes on Williams and Harasoy backgrounds.

View uploaded report

To study how partial resistance affects oospore production in soybean, and pathotype diversity in soybean, we needed methods. This year was spent modifying methods developed in Richard Belanger's lab at Laval University, Canada.
The first method in this project was to incorporate a hydroponic system for inoculating soybean with Phytophthora sojae reported by Lebreton et al. 2018 into the Robertson Lab. The system would be used to study soybean-P. sojae interactions such as production of oospores on varieties with partial resistance to the pathogen, and how selection pressure from partial resistance might influence the pathotype diversity of P. sojae. The system has been successfully used for inoculating soybean at growth stage VE and getting Phtophthora root rot (PRR) to develop. However, we have been unable to get PRR to develop when older plants are inoculated. Partial resistance only become active in soybeans after growth stage VC.
A second method in this project was to incorporate a PCR method developed by Dussault-Benoit et al. 2018 to pathotype isolates of P. sojae. Pathotyping is usually done by inoculating 7-day old soybean differentials with known Rps genes with P. sojae, and then recording the percent of plants that die seven days later. A PCR method would enable us to pathotype an isolate of P. sojae in an afternoon. We have had successful results with 6 of the 9 primers reported, and are currently modifying conditions for two primers. For one primer, we amplified a different sized DNA product to that reported by Dussault-Benoit et al., suggesting a different mutation occurs in the Iowa isolates we used to that reported in the isolates used in their study.

Improved resistant varieties for soybean farmers.
Reduced losses to PSRR and improved profitability for farmers. From 2010-2020, PSRR losses for Iowa were estimated at $0.73 per acre (Crop Protection Network).