Interaction of Host Genotype, Medium of Inoculum, and Pathogen Species on Pathogenicity of Pythium Disease on Soybean

Soybean (Glycine max (L.) Merr) is one of the most important crops for edible oil and proteins in the United States. However, about 11% of soybean production is suppressed by soybean diseases including seedling disease [1,2]. In the U.S. and Ontario, Canada, seedling diseases caused a total yield loss of 241 million bushels from 2010 to 2014, being the second most destructive soybean diseases [1]. One of the most important pathogens causing soybean seedling diseases is Pythium, a genus of soil-borne, oomycete pathogens typically favored by cool, wet conditions and early planting, causing seed decay, root rot, and damping off [3]. More than 20 Pythium species have been identified with pathogenicity on soybean so far, such as P. ultimum, P. aphanidermatum, P. debaryanum, P. irregulare, P. myriotylum, P. torulosum and P. vexans, in Ohio, Iowa, Florida, Arkansas, Illinois, and North Dakota [4-12]. All the Pythium species cause similar symptoms, including soft and rotted seeds before germination, preor post-emergence damping-off, and hypocotyl or root discoloration [13,14]. Crop rotation is a traditional way to manage Pythium diseases [15], however, because Pythium has multiple hosts such as corn and wheat, crop rotation is considered less effective [13]. Another widely used method is the treatment of seeds with fungicide, such as metalaxyl, mefenoxam and some newer fungicides [16-18].


Copyright © Dechun Wang
MCDA.000625. 5 (5).2020 medium for inoculum [20][21][22][23][24][25][26][27][28][29]. However, it has not been clear yet about which medium is more suitable to evaluate the pathogenicity of Pythium on soybean, and moreover, it is still unknown whether there's any interactions between soybean genotypes, the inoculum medium, and different Pythium species, which would strongly impact our strategy of evaluating Pythium diseases. Therefore, this case study was designed to 1) explore the interactions of soybean genotype, inoculum medium, and Pythium species; 2) identify a more suitable medium for Pythium disease evaluation; and 3) Compare the pathogenicity of different Pythium species

Case Presentation
A total of 9 Michigan State University improved soybean lines were used in this study. For the pathogen, three Pythium isolates were used including CMISO2 5-14, MSIO 8-23, and CMISO2 2-30, which were classified as P. irregulare, P. oopapillum, and P. sylvaticum, respectively [3]. All isolates were maintained on slants of Potato Carrot Agar (PCA) at 15 °C in glass tube. A modified protocol from Howell [30] was used to prepare inoculum ( Figure 1A). Two cultural media, millet and white rice, were used. 625g millet seeds and 425g white rice (parboiled) grains were each mixed with 400mL ddH 2 O in an autoclave-safe plastic bag and autoclaved at 121 °C for 4 hours.
When bags cooled down to room temperature, one full plate of a 4-day-old isolate cultured at CMA medium was transferred to the millet or rice bag aseptically. After incubation at room temperature for 12 days, the inoculum was ready to use ( Figure 1B). The disease was evaluated at the Michigan State University greenhouse, with temperature at 20-22 °C and humidity at 20%. All soybean varieties were evaluated in a randomized incomplete block design with 12 seeds evaluated for each replicate. Three replicates were performed for inoculation group, and one replicate was used as control. To start the assay, 6-cell seed starting trays were first fulfilled with medium size vermiculite and soaked in water until the vermiculite was fully saturated. Then two 2cm-depth holes were made in each cell and approximately 2g inoculum and un-inoculated medium (or 6-8 rice grains or 15-20 millet grains) was placed at the bottom of each hole for inoculation. A single seed was then planted on the top of the inoculum in each hole and covered with vermiculite ( Figure 1C). After planting, seed starting trays were transferred to the greenhouse benches which had been covered with waterproof plastic. Benches were watered until the water height reached the height of inoculum. After that, benches were watered every other day to maintain the water saturation environment until two days before measurement. Data were collected when the control group reached V2 stage, or 14 days after inoculation ( Figure 1D). Root score and whole plant weight were measured. To rate root score, roots were removed from the pots, gently washed and cleaned with running tap water, and wrapped in a towel to absorb excess water.

MCDA.000625. 5(5).2020
Root rot score for each plant was rated on a 0-to-5 scale ( Figure  1E), wherein, 0 represents a healthy root system with no symptoms of lesions or rot on the roots; 1 represents small lesions and discoloration on the roots, with approximately 1% to 20% of roots showing visible symptoms; 2 represents some lesions on roots and lateral roots with approximately 20 to 40% of roots rotted with visible symptoms of lateral roots reduction; 3 represents rot on lateral roots and visible symptoms of rot, with approximately 40 to 60% of the roots exhibiting visible symptoms; 4 represents both lateral roots and main tap roots having visible symptoms on roots and approximately 60 to 90% of the roots infected, roots and lateral roots reduced drastically, and 5 represents roots severely rotted or no germination with complete colonization on the seed.

Results and Discussion
The three-way ANOVA results indicated that there were significant interactions among soybean genotypes, inoculum medium, and Pythium species for both RR and RW with p=0.006 and p=0.026, respectively (Table 1). Among the three factors, soybean genotype did not have significant interaction with inoculum medium for either RR or RW (p=0.071, and p=0.126, respectively). The interaction between soybean genotype and Pythium species was significant for RR, but not significant for RW (p=0.046, and p=0.611, respectively). However, the interaction between inoculum medium and Pythium species was very significant with p<0.001 for both RR and RW. These results indicated that inoculum medium is important for evaluation of soybean diseases caused by different Pythium species. To further evaluate the effect of inoculum medium on soybean diseases, we conducted the marginal analysis as shown in Figure 2. For each soybean genotype, the RR values obtained from rice was consistently larger than that from millet ( Figure  2A). Averagely, the mean of RR for rice was 0.817, compared to that of millet which was 0.672. Because the higher value of the RR indicates higher disease pathogenicity, the inoculation method using rice as inoculum medium can therefore induce higher disease pressure to soybean plants. The results from RW was consistent with that from RR ( Figure 2B). For RW, the lower value indicates higher disease pressure. Obviously, all the nine soybean genotypes were consistently more susceptible using rice than using millet as inoculum medium, with RW averagely 0.394 and 0.645 for rice and millet, respectively. Our results indicated that for future studies on Pythium diseases, rice may be a better choice than millet as inoculum medium to obtain higher disease pressures.   Additionally, we also conducted the marginal analysis to compare the pathogenicity among the three isolates from the three Pythium species (Figure 3). The pathogenicity of three isolates was generally consistent on soybean genotypes using either RR or RW as evaluation method. For example, for soybean genotype1, CMISO2 2-30 (P. sylvaticum) was the most pathogenic as indicated by both RR and RW, followed by CMISO2 5-14 (P. irregulare) and MISO 8-23 (P. oopapillum), while for soybean genotype 9, the most aggressive isolate was the P. oopapillum. The P. oopapillum isolate was most aggressive on six soybean genotypes evaluated by RR (11,12,6,7,8 and 9) and on four soybean genotypes by RW (11,12,8 and 9). The P. irregulare isolate was most aggressive on two soybean genotypes (2 and 5) by RR, and on four genotypes (2,5,6, and 7) by RW. The P. sylvaticum isolate was the most aggressive only on soybean genotype 1.
The different response pattern of soybean genotypes to different species of Pythium suggested that soybean does not deploy a single genetic resistance mechanism against different Pythium species. This can be supported by the identification of different quantitative disease resistance loci (QDRL) for different types of Pythium species. For example, our previous study identified two QDRL on soybean chromosomes 11 and 20, respectively, for partial resistance to P. irregulare [23], while in our recent study for soybean resistance to P. sylvaticum, novel QDRLs were identified on chromosomes 10 and 18 (unpublished data). Nevertheless, some common genes may play critical roles in regulating soybean resistance to different Pythium species. For example, the QDRL on chromosome 20 was identified for both P. irregulare and P. sylvaticum (unpublished data).

Summary
This case study illustrated that soybean genotypes, inoculum media, and types of Pythium isolates, along with the interactions between inoculum media and types of Pythium isolates, and the interactions among the three factors, can significantly affect the soybean response to Pythium pathogen which was evaluated by RR and RW. Compared to millet medium, rice medium can induce heavier disease severity and may serve as a better choice for soybean disease resistance studies for Pythium root rot. In addition, this case study indicated that P. oopapillum was the most aggressive on 6 soybean genotypes evaluated by RR and on 4 genotypes by RW; P. irregulare was the most aggressive on 2 soybean genotypes evaluated by RR, and on 4 genotypes by RW; and P. sylvaticum was the most aggressive on one soybean genotype. Moreover, our results suggested that different mechanisms of resistance may be deployed by soybean plants although some common genes may play important roles for resistance to different Pythium species.