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.
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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.