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). Reducing losses to PSRR relies on single resistance (Rps) genes that are deployed in commercial soybean varieties. In Iowa, the most commonly deployed Rps genes are Rps 1c and Rps 1k; while less than 10% of commercial varieties have Rps 1a and less than 5% have Rps 3a (Matthiesen et al. 2021). However, P. sojae can naturally change overtime to cause disease on Rps genes (Dorrance et al. 2003). Consequently, incorporating Rps genes together with partial resistance has been encouraged (Dorrance et al. 2016). Partial resistance has no Rps genes, is multigenic and is effective against all pathotypes of the pathogen (Schmitthenner 1985). 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 cultivars with partial resistance to P. sojae had more root mass but as many oospores as the roots of a susceptible cultivar, while the roots of a cultivar with a Rps gene had 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. More importantly however, sexual reproduction increases genetic diversity within a population.
Stewart et al. (2016) recovered more pathotypes of P. sojae from rotations of soybean that included a cultivar with partial resistance compared to rotations in which cultivars with only Rps genes were grown. Thus, planting varieties with partial resistance may be contributing to increased pathotype diversity of P. sojae in Iowa. 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.

• Improved understanding of contribution of resistance to inoculum levels in the soil.
• Knowledge of the effect of resistance in soybean on pathotype diversity in P. sojae.
• Guidance for soybean breeders developing PSRR resistant cultivars.

Update:
We are continuing to work on optimizing the hydroponic method to inoculate soybean with P. sojae. We are still struggling to get infection after V1. If we want to evaluate the role of partial resistance in shaping the diversity of P. sojae, we need to consistently get infection after V1.

We have also been evaluating a molecular assay based on discriminant mutations leading to genetic variations associated with seven Avr genes that was recently published. The assay accurately predicted virulence of Canadian isolates of P. sojae on Harosoy differentials based on the presence or absence of each Avr gene amplicon. We'd like to use this assay to pathotype oospores we recover from our infected soybeans. We evaluated the assay with P. sojae isolates from the United States (US) pathotyped on Harasoy and Williams differentials. Strain P6497 was used as a positive control to compare our results with the published assay. While virulence was predicted correctly for some Avr genes of some isolates, inconsistencies were also observed. For some isolates virulence varied between backgrounds for the same Rps gene, suggesting a different Rps allele or a linked Rps gene may have been incorporated on each background. Virulence on a differential did not always correspond with absence of the associated amplicon, suggesting Avr mutations present in the US population of P. sojae differ from those occurring in the Canadian population.

An abstract for a poster describing our work on the molecular assay was submitted to the annual APS meeting that will be help in Denver in August 2023.

Update:
We continue to work on developing a modified hydroponic method to inoculate soybean with zoospores of P. sojae which simulates natural infection better than inoculating the hypocotyl with a slurry of mycelium. We have successfully caused infection of soybean seedlings and will use this method to study the effect of DNA methylation on the soybean-P. sojae interaction (see leveraged funding). We plan to test this method on V1 soybean, which is the growth stage at which partial resistance is activated.

We are finishing up our work comparing a molecular assay for pathotyping with the standard hypocotyl assay. We have evaluated approximately 30 isolates of P. sojae, some of which were used to develop the molecular assay. As our initial work suggest, there were inconsistencies between the two methods. Moreover, there were inconsistencies among the differentials used in the hypocotyl test. We plan to follow up with some sequencing work to identify if different mutations are present in the Avr loci of the isolates we have been evaluating compared to the isolates used to develop the molecular pathotyping test.

Updated May 9, 2024:
We evaluated a hydroponic method (Lebreton et al. 2018) to inoculate soybean with P. sojae. This method was developed to infect soybean seedlings that were approximately 12-days old, at growth stage VE-VC. While we were able to get infection of seedlings 7-14 days old, we were unable to get infection after V1. This was surprising because, according to published literature, soybean is susceptible to infection by P. sojae at all growth stages. Since partial resistance (also known as field tolerance) is only activated after the first true leaves form (V1) we were unable to evaluate the role of partial resistance in shaping the diversity of P. sojae. We are continuing to try different methods of inoculating soybean at different growth stages with the pathogen.

It is important to characterize the pathotype of P. sojae causing Phytophthora stem and root rot (PSRR) in a field to identify what Rps genes should be used in future soybean plantings to reduce losses to PSRR. It is also important for soybean breeders to know what pathotypes are prevalent to guide breeding efforts and ensure the correct Rps genes are incorporated into commercial soybean varieties.

Dussault-Benoit et al. (2020) developed a molecular assay based on discriminant mutations that lead to genetic variations associated with seven Avr genes in P. sojae. Their assay accurately predicted the virulence of isolates of P. sojae on Harosoy differentials based on the presence or absence of each Avr gene amplicon. Traditionally, pathotypes of the pathogen are distinguished using a hypocotyl assay. The hypocotyl assay is laborious, requires considerable space and it may take up to 18 days for results. The molecular assay provides pathotype results within a few hours.

Thus, we evaluated the molecular assay to predict the virulence and thus pathotypes of isolates of P. sojae recovered from soil samples or diseased soybean plants from Iowa, Nebraska and Ohio. Four isolates that were used in the development of the molecular assay were used as checks. The objectives of the study were to:
1. Determine the pathotype of the isolates on Harosoy and Williams sets of differentials using the hypocotyl inoculation assay.
2. Compare the virulence of each isolate on differential lines in a Harosoy background compared to a Williams background.
3. Use the molecular assay to predict the pathotype of each isolate.
4. Compare the predicted pathotype with the pathotype of each isolate determined using the hypocotyl inoculation assay.

View uploaded report

For some isolates of P. sojae that we characterized for pathotype (formally known as race) using the hypocotyl inoculation method and a molecular assay, the ability to cause disease (virulence) varied among the differential lines of Harosoy and Williams carrying the same Rps gene (see Table 1). In fact, the pathotype determined by hypocotyl inoculation assay of the Harosoy and Williams differential lines was identical for only 5 isolates. These data suggest that differentials of a specific Rps gene in distinct genetic backgrounds may reflect the activity of distinct Rps alleles or even a different Rps gene that may be tightly linked. While virulence was predicted correctly for some Avr genes of some isolates using the molecular assay, it was not correctly predicted for many others. Absence or presence of the amplicon in the molecular assay was not associated with the actual virulence or avirulence (no disease) observed on the differentials. These results may reflect that Avr mutations present in the P. sojae isolates tested here differ from those occurring in the isolates used in the development of the molecular assay. Our study suggests additional evaluation of the molecular assay is recommended before its widespread use for determining the pathotypes of P. sojae present in a population.

Characterizing the pathotypes of P. sojae that are present in Iowa, and throughout the North Central Region informs soybean breeders of which Rps genes to incorporate into soybean varieties. This research demonstrated pathotype may vary depending on the soybean differential used. This research also suggests the molecular assay needs further evaluation before it can be used to characterize P. sojae. Some suggestions for future work include (i) evaluating discriminate mutations in Avr genes in a broader range of isolates of P. sojae to optimize primers that could be used in a molecular assay, (ii) assembling a standard set of isolates of P. sojae to be used as controls for comparing soybean differentials that each contain the same Rps gene and characterizing pathotypes of P. sojae, (iii) an improved understanding of the epidemiology of PSRR, specifically the susceptibility of different growth stages of soybean to P. sojae, and (iv) improved mapping, or cloning, of Rps genes in soybean to confirm their presence in a soybean differential set.

Publications
Watson, G. and Robertson, A.E. 2023. Validation of a molecular assay to identify Phytophthora sojae pathotypes (Abstr.). Phytopathology 113:S3.154. https://doi.org/10.1094/PHYTO-113-11-S3.1

Lopez-Nicora, H., Mangel, D., McCoy, A., Webster, R.W., Robertson A., Chilvers, M., Tenuta, A., Mueller, D. and Wise, K. 2024. An overview of Phytophthora root and stem rot. Crop Protection Network. (doi: to be determined)

• 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). Preventing losses to PSRR on half of the soybean acres on Iowa would equate to approximately $3.6 million return on the soybean checkoff investment of $65,673.