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Variation in the Resistance of Japanese Soybean Cultivars to Phytophthora Root and Stem Rot during the Early Plant Growth Stages and the Effects of a Fungicide Seed Treatment
Plant Pathol. J. 2019;35:219-233
Published online June 1, 2019
© 2019 The Korean Society of Plant Pathology.

Hajime Akamatsu1,* , Masayasu Kato2, Sunao Ochi3,4, Genki Mimuro3,5, Jun-ichi Matsuoka1, and Mami Takahashi1

1Division of Lowland Farming, Hokuriku Research Center, Central Region Agricultural Research Center, National Agriculture and Food Research Organization, 1-2-1 Inada, Joetsu, Niigata 943-0193, Japan, 2Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan, 3Division of Plant Disease Management, Central Region Agricultural Research Center, National Agriculture and Food Research Organization, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8666, Japan, 4Research Center for Agricultural Information Technology, National Agriculture and Food Research Organization, 3-5-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-0013, Japan, 5Agricultural Research Institute, Toyama Prefectural Agricultural, Forestry and Fisheries Research Center, Toyama, Toyama 939-8153, Japan
Correspondence to: *Phone) +81-25-526-3243, FAX) +81-25-524-8578
E-mail) akamatho@affrc.go.jp
Received November 10, 2018; Revised March 28, 2019; Accepted April 7, 2019.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Soybean cultivars susceptible to Phytophthora root and stem rot are vulnerable to seed rot and damping-off of seedlings and young plants following an infection by Phytophthora sojae. In this study, the disease responses of Japanese soybean cultivars including currently grown main cultivars during the early growth stages were investigated following infections by multiple P. sojae isolates from Japanese fields. The extent of the resistance to 17 P. sojae isolates after inoculations at 14, 21, and 28 days after seeding varied significantly among 18 Japanese and two US soybean cultivars. Moreover, the disease responses of each cultivar differed significantly depending on the P. sojae isolate and the plant age at inoculation. Additionally, the treatment of ‘Nattosyo-ryu’ seeds with three fungicidal agrochemicals provided significant protection from P. sojae when plants were inoculated at 14–28 days after seeding. These results indicate that none of the Japanese soybean cultivars are completely resistant to all tested P. sojae isolates during the first month after sowing. However, the severity of the disease was limited when plants were inoculated during the later growth stages. Furthermore, the protective effects of the tested agrochemicals were maintained for at least 28 days after the seed treatment. Japanese soybean cultivars susceptible to Phytophthora root and stem rot that are grown under environmental conditions favorable for P. sojae infections require the implementation of certain practices, such as seed treatments with appropriate agrochemicals, to ensure they are protected from P. sojae during the early part of the soybean growing season.

Keywords : chemical control, Glycine max, partial resistance, Rps gene, zoospore
Materials and Methods

Phytophthora sojae cultures

The following five P. sojae isolates were obtained from symptomatic soybean plants collected in Ibaraki prefecture, Japan in 2006–2014: 13-B-RO-1, 13-B-RO-2, 13-7-B1-1, 13-8-11-2, and Ps1001 (Table 1). We isolated P. sojae as previously described by Sugimoto et al. (2006), but with some modifications to the contents of the V8 juice agar medium. Diseased soybean stems were washed with tap water or sterile distilled water, after which stem sections containing the edge of lesions and the adjacent healthy part were excised. The stem sections were immersed in 70% ethanol for 10–20 s and 0.5% sodium hypochlorite for 1–2 min and then rinsed twice with sterile distilled water. After air-drying on sterile filter paper, the stem sections were placed on BNPRA-V8 juice agar medium (200 ml V8 juice, 3 g CaCO3, 1.5% agar, 10 mg/l benomyl, 10 mg/l nystatin, 7.5 mg/l pentachloronitrobenzene, 5 mg/l rifampicin, and 500 mg/l ampicillin) supplemented with 50 mg/l 3-hydroxy-5-isoxazole. Samples were then maintained at 25°C in a Cool Incubator (Mitsubishi Electric Engineering Co., Ltd., Tokyo, Japan). Mycelial tips derived from the stem pieces were transferred to Petri plates containing fresh BNPRA-V8 juice agar medium supplemented with 50 mg/l 3-hydroxy-5-isoxazole. For each isolate, the elongating hyphal tips were transferred to fresh V8 juice agar medium and grown at 25°C in a Cool Incubator.

Four of the 17 isolates, Ps060726-2-1, Ps080806-3-5, Ps060626-4-1, and Ps060629-5-1, were collected from Ibaraki, Fukushima, Miyagi, and Toyama prefectures, respectively (Table 1), and were provided by the Hokuriku Research Center, Central Region Agricultural Research Center of the National Agriculture and Food Research Organization (CARC/NARO).

Eight P. sojae isolates were obtained from the soil collected from soybean fields in Akita, Nagano, Fukuoka, and Saga prefectures, Japan in 2014 (Table 1). The soil samples were maintained at room temperature in a plastic bag until used. ‘Nattosyoryu’ seeds were sown in plastic pots (1/10,000 a) filled with field soil. After 3–4 days under glasshouse conditions, the potted plants were flooded for 22 h. Two days later, plants with Phytophthora root and stem rot lesions were collected for a subsequent isolation of P. sojae according to the above-mentioned procedure.

A PCR analysis using a species-specific primer pair (PSOJF1 and PSOJR1) (Bienapfl et al., 2011) confirmed that the expected amplicon was produced for all isolates except for four isolates provided by the Hokuriku Research Center, CARC/NARO. The four isolates from the Hokuriku Research Center have been already identified as P. sojae by means of morphological observation, PCR-based molecular characterization, and inoculation of soybean seedlings (Moriwaki, 2010).

Plant materials and seed treatment with pesticides

Eighteen Japanese soybean cultivars were used in this study (Table 2). Nine of the 18 cultivars (‘Tachinagaha’, ‘Enrei’, ‘Fukuyutaka’, ‘Toyomusume’, ‘Ryuhou’, ‘Miyagishirome’, ‘Yukihomare’, ‘Satonohohoemi’, and ‘Osuzu’) were among the 10 most planted soybean cultivars in Japan in 2015 (i.e., in terms of cultivated area). The nine other cultivars were as follows: ‘Himeshirazu’, ‘Nattosyoryu’, ‘Suzumaru’, ‘Suzuyutaka’, ‘Tamahomare’, ‘Sachiyutaka’, ‘Toyokomachi’, ‘Tanbakuro’, and ‘Hatayutaka’. ‘Suzumaru’ and ‘Sachiyutaka’ were among the 10 most planted soybean cultivars in 2012 and 2012–2014, respectively. The statistical data was obtained from the governmental official website for Ministry of Agriculture, Forestry and Fisheries of Japan. The Rps genes have not been detected in these 18 Japanese cultivars, although some of the cultivars are reportedly resistant to certain P. sojae races (Jiang et al., 2017; Yamashita et al., 2012). Two US cultivars, ‘Williams’ and ‘Harosoy’, which lacks Rps genes (rps) and expresses low levels of partial resistance and possesses Rps7 (Anderson and Buzzell, 1992; Burnham et al., 2003; Wu et al., 2011), respectively, were also inoculated with Japanese P. sojae isolates.

Soybean seeds (approximately 25 seeds per cultivar) were sown in plastic pots (1/10,000 a) containing 700 ml vermiculite (Burnpiece, S size; Hakugen Earth, Tokyo, Japan), and covered with 200 ml vermiculite. The vermiculite in pots was completely moistened with water, after which the pots were placed on a plastic tray (Sanko vat No. 1, 7 cm deep; Sanko Co., Ltd., Gifu, Japan). Water was then supplied to the plants through a hole at the bottom of the pots starting from 2–3 days after seeding. The pots were incubated in a glasshouse at CARC/NARO as described by Kato et al. (2013) to prepare the soybean plants for the inoculation test. To investigate the effects of pesticides on P. sojae, ‘Nattosyoryu’ seeds were treated with three commercially available pesticides (Table 3) at the recommended rates. The plants resulting from the pesticide-treated seeds were prepared for the inoculation test according to the above-mentioned procedure.

Inoculation and evaluation

Soybean plants were inoculated with P. sojae cultured on V8 juice agar medium as described by Mukobata and Sekihara (2006), with some modifications (i.e., P. sojae–V8 culture flooding method). Specifically, seven small pieces (approximately 5 × 5 mm) of a P. sojae culture grown on V8 juice agar medium were used to inoculate fresh V8 juice agar medium in 9 cm plastic Petri plates (Ina-Optika Co., Ltd., Osaka, Japan). The plates were incubated for 14 days at 25°C in an incubator. The P. sojae–V8 culture was roughly cut into small pieces (approximately 2 × 2 cm) using a spatula and then added to the vermiculite in each pot. The pots were flooded to approximately 2 cm above the vermiculite surface and then incubated for 5 days in the glasshouse. The inoculated plants were grown for an additional 5 days, during which plants were watered through a hole at the bottom of the pots. The inoculated soybean plants were evaluated in terms of the number of dead plants per emerged plants. Example of the disease symptoms caused by the P. sojae inoculation is shown in Fig. 1. None of ‘Nattosyoryu’ plants treated with only V8 juice agar pieces died, while all ‘Nattosyoryu’ plants were killed by inoculation with the Ps060626-4-1-cultured V8 juice agar pieces (Fig. 1). The extent of the disease resistance among inoculation tests was compared based on the proportion of dead plants.

Data analysis

Data were analyzed using R software (version 3.4.4) (R Core Team, 2018). In the time course experiment, the rates of dead plants due to the P. sojae infection underwent an analysis of variance (ANOVA) for each soybean cultivar grown for a different number of days before being inoculated. To determine significant differences among the mean rates of dead plants, Tukey’s honestly significant difference (HSD) test was completed by inputting the ANOVA results into the glht function of an R package, multcomp. The rates of dead plants due to the P. sojae infection were also compared among the 20 soybean cultivars using the above-mentioned method. In the time course evaluation of the efficacy of pesticide treatments of ‘Nattosyoryu’ seeds, the rates of dead plants due to the P. sojae infection underwent an ANOVA for the non-treated control plants and the plants derived from seeds treated with one of three pesticides. Significant differences between the mean rates of dead plants for pesticide-treated and non-treated plants were determined by analyzing the ANOVA results with the glht function of multcomp according to Dunnett’s multiple comparison test.

Results

In a preliminary inoculation test involving 20 soybean cultivars, ‘Himeshirazu’ and ‘Nattosyoryu’ appeared to be susceptible to P. sojae isolate 13-B-RO-1. The average rates of dead plants for ‘Himeshirazu’ and ‘Nattosyoryu’ were greater than 90% when these cultivars were inoculated at 14 days after seeding or earlier. None of the control soybean plants treated with only V8 juice agar or water developed disease symptoms (Fig. 1). To investigate the temporal changes in the disease resistance levels of the two cultivars, plants grown for 16–40 days after sowing were inoculated with P. sojae isolate 13-B-RO-1 (Fig. 2). On average, more than 75% of ‘Himeshirazu’ and ‘Nattosyoryu’ plants died following the inoculations at 16–23 days and 16–18 days after sowing, respectively. Compared with the data for the plants inoculated at highly susceptible ages, the rates of dead plants for ‘Himeshirazu’ and ‘Nattosyoryu’ significantly decreased when plants were inoculated at 25–40 days and 26–40 days after sowing, respectively (Tukey’s HSD test, p < 0.05). The average rates of dead plants were less than 16% in ‘Himeshirazu’ plants grown for 29–40 days, and less than 5% in ‘Nattosyoryu’ plants grown for 29–40 days. To investigate whether the rates of dead plants decreased in other Japanese cultivars, the following 18 cultivars were challenged with 17 P. sojae isolates from Japanese soybean fields (Table 1) at 14, 21, and 28 days after seeding (Fig. 3): ‘Himeshirazu’, ‘Nattosyoryu’, ‘Tachinagaha’, ‘Enrei’, ‘Fukuyutaka’, ‘Toyomusume’, ‘Suzumaru’, ‘Ryuhou’, ‘Miyagishirome’, ‘Suzuyutaka’, ‘Tamahomare’, ‘Sachiyutaka’, ‘Yukihomare’, ‘Toyokomachi’, ‘Tanbakuro’, ‘Hatayutaka’, ‘Satonohohoemi’, and ‘Osuzu’ (Table 2). Two US cultivars, ‘Williams’ and ‘Harosoy’, were also inoculated with the same isolates to investigate their responses to Japanese P. sojae isolates (Fig. 3). Inoculations at 14 days after seeding resulted in an average rate of dead plants that exceeded 75% for the following six Japanese cultivars: ‘Himeshirazu’, ‘Nattosyoryu’, ‘Enrei’, ‘Suzumaru’, ‘Hatayutaka’, and ‘Osuzu’. The average rate of dead plants ranged from 27.7% to 74.8% for the 12 other Japanese cultivars. The extent of the resistance to the 17 P. sojae isolates varied significantly among the 20 cultivars (ANOVA, p < 0.001).

The average rate of dead plants decreased as the age of the plants when inoculated increased to 21 and 28 days after seeding. Moreover, there were significant differences in the extent of the resistance among cultivars for each age selected for inoculations (ANOVA, p < 0.001). During the inoculation of plants at 21 days after seeding, ‘Himeshirazu’ was the only cultivar with an average rate of dead plants that was greater than 75%. In contrast, ‘Nattosyoryu’, ‘Enrei’, and ‘Suzumaru’, which were also highly susceptible to disease when plants were inoculated at 14 days after seeding, had average rates of dead plants between 45.6% and 64.5%. ‘Hatayutaka’ and ‘Osuzu’ had even lower average rates of dead plants, at 26.4% and 7.8%, respectively. Of the plants inoculated at 14 days after seeding, only ‘Miyagishirome’ had an average rate of dead plants less than 30%. Meanwhile, during the inoculations at 21 days after seeding, the following 10 cultivars had an average rate of dead plants of 4.7%-26.4%: ‘Fukuyutaka’, ‘Toyomusume’, ‘Ryuhou’, ‘Miyagishirome’, ‘Tamahomare’, ‘Sachiyutaka’, ‘Yukihomare’, ‘Tanbakuro’, ‘Satonohohoemi’, and ‘Osuzu’. There were no significant differences in the rates of dead plants following the inoculations at 14 and 21 days after seeding for ‘Himeshirazu’, ‘Nattosyoryu’, ‘Tachinagaha’, ‘Toyomusume’, ‘Suzumaru’, ‘Ryuhou’, ‘Tamahomare’, ‘Yukihomare’, ‘Toyokomachi’, and ‘Satonohohoemi’. For the other cultivars, the rates of dead plants were significantly lower for the inoculations at 21 days after seeding (Tukey’s HSD test, p < 0.01). Of the plants grown for 28 days before being inoculated, only ‘Himeshirazu’ exhibited a significantly decreased degree of resistance against the P. sojae isolates (Tukey’s HSD test, p < 0.01). The average rate of dead plants for ‘Himeshirazu’ was 32.3%, which was considerably higher than the corresponding rate for the 17 other cultivars (0.2–2.5%). Moreover, for all cultivars, the average rates of dead plants were lower for the inoculations at 28 days after seeding than for the inoculations at 14 days after seeding (Tukey’s HSD test, p < 0.01). Thus, the inoculation test involving 18 Japanese cultivars and 17 P. sojae isolates revealed that the disease responses of the cultivars differed depending on the isolate used and the plant ages when inoculated.

The average rates of dead plants for the US cultivars, ‘Williams’ and ‘Harosoy’, were 91.2% and 69.1%, respectively, following the inoculation at 14 days after seeding. These averages for ‘Williams’ and ‘Harosoy’ decreased to 43.4% and 36.6%, respectively, following the inoculation at 21 days after seeding, and decreased further to 5.7% and 5.3%, respectively, following the inoculation at 28 days after seeding (Tukey’s HSD test, p < 0.01).

The results described herein indicated that the resistance levels against P. sojae isolates varied among the tested Japanese soybean cultivars. Additionally, none of the cultivars were completely resistant to all P. sojae isolates during the seedling and young plant stages. Moreover, for all cultivars, greater resistance (i.e., fewer dead plants) was observed with increasing plant age prior to inoculation. However, the plant ages at which the number of dead plants significantly decreased varied among the cultivars.

The effects of pesticide treatments of soybean seeds on the development of Phytophthora root and stem rot during the early soybean growth stages were investigated using three registered agrochemicals (Table 3, Fig. 4). The active ingredients against P. sojae in the three pesticides are amisulbrom, metalaxyl-M, and cyazofamid (Table 3). The rates of ‘Nattosyoryu’ plants killed by a P. sojae infection were significantly lower for the seed-treated samples than for the non-treated controls following the inoculations at 14, 16, 20, 24, and 28 days after seeding (Dunnett’s test, p < 0.001). For the inoculations at 32, 36, and 40 days after seeding, an average of ≤ 5.8% of ‘Nattosyoryu’ plants were killed regardless of whether seeds were treated with the pesticides. This result suggested that the three pesticides can control the pathogen during the growth period important for stand establishment. Additionally, the pesticide-induced decrease in the number of dead plants due to the pathogen was maintained for at least 28 days after treated seeds were sown.

To investigate the efficacy of the pesticides against different P. sojae isolates, ‘Nattosyoryu’ plants were grown for 14 days after the seeds treated with the pesticides containing metalaxyl-M and cyazofamid were sown. The plants resulting from the seeds treated with metalaxyl-M were then challenged with the following seven P. sojae isolates: 13-B-RO-1, 13-B-RO-2, 13-7-B1-1, 13-8-11-2, Ps1001, Ps080806-3-5, and Ps060626-4-1. Meanwhile, the plants that emerged from seeds treated with cyazofamid were inoculated with the same P. sojae isolates, except for 13-B-RO-2. The number of dead plants significantly decreased in response to both pesticides (Fig. 5, Dunnett’s test, p < 0.05). The average rate of dead plants for the nontreated controls was 72.7%-100%, while the corresponding rate for the samples treated with metalaxyl-M was 0%-12.7%. Regarding the cyazofamid-treated samples, under the conditions in which an average of 63.7%–100% non-treated control plants were killed by the six P. sojae isolates, all of the pesticide-treated samples survived the inoculations.

Discussion

Phytophthora root and stem rot of soybean has threatened Japanese soybean production, especially when P. sojae infections occur during the early soybean growth stages. Soybean cultivation in Japan mainly involves the use of converted rice paddy fields. However, such a crop rotation system is favorable for P. sojae infections and disease development because of the soil type and poor drainage of the converted fields (Kato et al., 2013; Sugimoto et al., 2012). Vertical resistance via the expression of Rps genes provides complete protection from the corresponding pathogen races/populations (Dorrance, 2018), but Rps genes have not been exploited in Japan. Thus, the onset of Phytophthora root and stem rot during the early part of the soybean growing season leads to seed rot and seedling damping-off, with adverse consequences for stand establishment (Kato et al., 2013; Schmitthenner and Dorrance, 2015). In this study, we assessed the disease resistance of Japanese soybean cultivars, including the most widely grown cultivars in terms of planted area, by inoculating the aboveground part of the lower hypocotyl. Although none of the Japanese soybean cultivars were totally resistant to all tested P. sojae isolates during the first month after seeding, all cultivars exhibited increased resistance when plants were grown for more than 14 days after seeding before being inoculated. Additionally, a seed treatment with one of three pesticides protected soybean plants from P. sojae during the early vegetative growth phase, which is an important period for stand establishment. The protective effects of the pesticides were observed for at least 28 days after the treated seeds were sown. In the absence of strong resistance, like that mediated by Rps gene expression, in soybean seeds or seedlings immediately after germination, certain practices (e.g., seed treatments with agrochemicals) are needed to protect soybean plants from P. sojae infections during the early growth stages.

In this study, we evaluated the resistance of Japanese soybean cultivars to Phytophthora root and stem rot using a P. sojae–V8 culture flooding method modified from the procedure described by Mukobata and Sekihara (2005). Diverse inoculation methods have been developed, including the hypocotyl inoculation, greenhouse inoculum-layer test, and slant-board test as well as the application of a zoospore suspension and a mixture of agar culture homogenate and soil (Dorrance et al., 2004b, 2008; Dorrance and Mc-Clure, 2001; Guérin et al., 2014; Jiang et al., 2017; Stewart and Robertson, 2012; Sugimoto et al., 2006; Tooley and Grau, 1982). Some of these methods are used to evaluate complete resistance (e.g., Rps-mediated resistance), while others have been designed to assess incomplete resistance. In our method, the P. sojae agar culture is directly added to the water flooding the vermiculite in pots. Zoospores from the sporangia in the water initiate the infection, likely reproducing the soybean–pathogen interactions in the aboveground part of the lower hypocotyl when soybean plants are waterlogged during the early part of the growing season. This inoculation method enables P. sojae zoospores to mainly infect the aboveground part of the lower hypocotyl because negative geotaxis causes the zoospores to accumulate near the water surface (Cameron and Carlile, 1977). The damage to the lower hypocotyl caused by P. sojae severely inhibits soybean growth because of poor transport of water and/or nutrients from the roots to the upper plant parts, which leads to the damping-off of soybean seedlings and young plants. Our simple inoculation method does not require special techniques or expertise and results in the consistent induction of disease symptoms in soybean plants during the early vegetative growth phase. Moreover, this inoculation method revealed that the tested Japanese cultivars are differentially resistant to P. sojae. Although there is relatively little available information regarding the presence/absence of Rps genes in Japanese soybean cultivars (Athow et al., 1980; Rennie et al., 1992; Sugimoto et al., 2011, 2012), some of the tested cultivars were highly resistant to certain P. sojae isolates, suggesting that such strong resistance responses may be attributed to a race-specific resistance via the expression of Rps genes. A published report recommended the use of the hypocotyl inoculation test to assess Rps-mediated resistance to ensure P. sojae isolates compatible with the tested Japanese soybean cultivars prior to an evaluation of incomplete resistance (Dorrance et al., 2008). Because Rps-mediated resistance was not the focus of our study, compatible/incompatible interactions between the 18 Japanese cultivars and the 17 P. sojae isolates were not assessed before applying the P. sojae–V8 culture flooding method to evaluate the resistance of Japanese cultivars to P. sojae at different plant ages.

Partial resistance cannot completely prevent pathogen multiplication, while tolerance refers to the ability of the plant host to endure the presence of the pathogen (Parlevliet, 1979). In soybean, both resistance types are equivalently applied along with quantitative resistance, field resistance, and rate-reducing resistance (Dorrance, 2018; Schmitthenner, 1985). Tolerance to Phytophthora root and stem rot is defined as a relative characteristic based on the degree of plant loss or the decrease in growth or yield in the presence of the pathogen races for which a soybean cultivar lacks specific resistance genes (Anderson and Buzzell, 1982). Although it is unclear whether Japanese soybean cultivars carry certain Rps genes, we observed that the resistance of the tested soybean cultivars increased as the age of the plants when inoculated increased (i.e., decrease in the extent of plant death caused by the P. sojae infection). The inoculation of plants at 14 days after seeding revealed that six Japanese cultivars (‘Himeshirazu’, ‘Nattosyoryu’, ‘Enrei’, ‘Suzumaru’, ‘Hatayutaka’, and ‘Osuzu’) had an average rate of dead plants that exceeded 75%. With the exception of ‘Miyagishirome’, the other cultivars were also highly susceptible to certain P. sojae isolates. Thus, the resistance of the Japanese cultivars to P. sojae infections is not fully expressed in 14-day-old plants, in which the first trifoliate leaves are usually expanding or have expanded. Depending on the cultivars, the average rates of dead plants following a P. sojae infection significantly decreased when plants were inoculated at 21 or 28 days after seeding. Considering the partial resistance of soybean to Phytophthora root and stem rot is effective during or after the growth stage when the first trifoliate leaf forms (Dorrance and McClure, 2001; Schmitthenner and Dorrance, 2015), the plant age-dependent resistance observed in this study may be an example of partial resistance to the Japanese P. sojae isolates. Several plant species exhibit a disease resistance at various developmental stages, with varying levels depending on plant age or tissue maturity (Develey-Rivière and Galiana, 2007). The relationship between plant age/maturity and disease resistance has been studied in a soybean–P. sojae pathosystem (Dorrance and McClure, 2001; Jimenez and Lockwood, 1980; Lazarovits et al., 1981; Paxton and Chamberlain, 1969). Consistent with the results described herein, soybean plants reportedly become more resistant to P. sojae as they mature. For example, a previous study revealed that nine susceptible cultivars grown for 7 days before being inoculated were killed by P. sojae at a rate of 90% or higher, while the same cultivars grown for 21 days before being inoculated died at a rate of 30% or lower (Paxton and Chamberlain, 1969). In the hypocotyl, newer tissues are more susceptible to P. sojae than older tissues (Lazarovits et al., 1981). Additionally, an inoculation of ‘Altona’ plants grown for 6 days with zoospores of an incompatible race 4 isolate reportedly induces a resistance reaction in all hypocotyl parts, implying that the Rps-mediated incompatible interaction has been established in 6-day-old hypocotyls. The observed diseased or dead plants resulting from the inoculation of Japanese soybean cultivars at 14 days after seeding, suggests there is a lack of strong Rps-mediated resistance in most cultivar–isolate interactions at this plant growth stage. Soybean cultivar ‘Conrad’, which does not carry Rps genes and expresses a high level of partial resistance (Fehr et al., 1989), is highly susceptible to P. sojae infections at 0–5 days after seeding, with a seedling emergence rate significantly lower than that of plants inoculated later (Dorrance and McClure, 2001). Jimenez and Lockwood (1980) observed that even ‘Hark’, which is a susceptible cultivar with little field tolerance, exhibits increased resistance to P. sojae when plants are inoculated at 16 days after seeding or later. In the current study, the rates of dead plants due to a P. sojae infection for two susceptible cultivars, ‘Himeshirazu’ and ‘Nattosyoryu’, were significantly lower for plants inoculated at 25 and 26 days after seeding or later than for plants inoculated at 16–21 days and 16–18 days after seeding, respectively. The resistance of these two cultivars may not be expressed as quickly as that of ‘Conrad’. Although different inoculation and evaluation protocols were used, disease resistance increased as the age of plants at inoculation increased for all of the tested Japanese soybean cultivars, which is consistent with the results of previous studies (Dorrance and McClure, 2001; Jimenez and Lockwood, 1980; Lazarovits et al., 1981; Paxton and Chamberlain, 1969). The plant ages associated with increases in resistance varied among the cultivars, but the timing of the expression of resistance in the Japanese cultivars is likely consistent with that of partial resistance (Schmitthenner and Dorrance, 2015). Varying levels of partial resistance to P. sojae were shown in ‘Conrad’ and ‘Sloan’ hypocotyls, while a lack of partial resistance was observed in the roots of these two cultivars (Yamamoto et al., unpublished). However, partial resistance to P. sojae is reportedly expressed in the roots of cultivars with high levels of resistance (i.e., limited root rot and the absence of stem rot) (Dorrance et al., 2007; Mideros et al., 2007; Schmitthenner, 1985; Tooley and Grau, 1984). The partial resistance expression site in the current study might be inconsistent with that of several other studies that assessed root damage (Abeysekara et al., 2016; Burnham et al., 2003; Jia and Kurle, 2008; Lee et al., 2013a, 2013b; McBlain et al., 1991; Schneider et al., 2016; Stewart and Robertson, 2012; Tucker et al., 2010; Wu et al., 2011). This discrepancy was likely because our evaluation was based on the damping-off elicited by the severe infection around the lower hypocotyl and not root damage.

This study also determined the effective period for the treatment of seeds with three pesticides to provide protection against P. sojae. The active ingredients against Phytophthora root and stem rot in these pesticides are amisulbrom, metalaxyl-M, and cyazofamid. Metalaxyl-M, which is also known as mefenoxam, is the active isomer of metalaxyl, and is grouped with the phenylamide fungicides (Hirooka and Ishii, 2013), which are among the most commonly applied agrochemicals worldwide to control diverse oomycete-induced diseases (Dorrance, 2018; Dorrance et al., 2004a, 2007; Matheron and Porchas, 2014; Schmitthenner and Dorrance, 2015; Taylor et al., 2002). Meanwhile, amisulbrom and cyazofamid are quinone inside inhibitors that have similar protective effects against foliar and tuber blight caused by Phytophthora infestans (Hirooka and Ishii, 2013; Honda et al., 2007). Cyazofamid strongly inhibits the mycelial growth of oomycete species, with 0.2 μg/ml representing an effective concentration for inhibiting P. sojae growth by 50% (Mitani et al., 2001). At least 0.5 μg/ml is needed for an inhibition exceeding 90%, similar to metalaxyl. Additionally, mefenoxam is more effective than cyazofamid against Phytophthora capsici, with lesion development inhibited for at least 3 weeks when applied to roots (Matheron and Porchas, 2014). Although the three active ingredients are highly specific for oomycetes, previous studies focused primarily on the protective effects of metalaxyl and metalaxyl-M against Phytophthora root and stem rot of soybean (Anderson and Buzzell, 1982; Dorrance and McClure, 2001; Dorrance et al., 2009; Guy et al., 1989; Vaartaja et al., 1979). There have been relatively few studies examining the effects of amisulbrom and cyazofamid on soybean–P. sojae interactions (Mimuro, 2011). The current study revealed that the rates of dead plants due to P. sojae infections decreased significantly after seeds were treated with the pesticides. The protection provided by the pesticides continued for 28 days after the treatment. Because few plants were killed in the non-treated control at 29 days or later, the effective periods for these pesticides were not determined in this study. Mimuro (2011) reported that these three pesticides can protect soybean plants from Phytophthora root and stem rot for approximately 1 month after treated seeds are sown, which is consistent with our findings as well as the effective period indicated by the manufacturer of the pesticide containing metalaxyl-M.

According to industry estimates, the treatment of soybean seeds with pesticides has dramatically increased from < 8% in 1996 to > 30% in 2008, which has improved disease management during the early part of the soybean growing season (Esker and Conley, 2012; Munkvold, 2009). Soybean cultivation in Japan has recently become increasingly dependent on seed treatments, mainly with metalaxyl-M-containing pesticides. This is because soybean plants grown in fields converted from rice paddies are at high risk of being infected by P. sojae. Although seed treatments with pesticides are important for protecting young soybean plants from P. sojae and enhancing stand establishment, the pesticides are costly and repeated applications of the same pesticide may decrease the sensitivity of P. sojae to the active ingredient (i.e., pesticide resistance) (Han et al., 2011; Hunger et al., 1982). Planting cultivars exhibiting complete and/or incomplete disease resistance may be a more economically viable option for combating Phytophthora root and stem rot of soybean. Consequently, a comprehensive characterization of the disease resistance in Japanese cultivars via an analysis of Rps genes may be important for elucidating the Japanese soybean–P. sojae pathosystem and may be useful for the development of new lines/cultivars that are resistant to Phytophthora root and stem rot. The fact that none of the 18 tested Japanese cultivars were completely resistant to all of the P. sojae isolates following inoculations at 14 days after seeding suggests these cultivars likely do not express effective resistance during the early growth stages. Thus, additional practices, such as seed treatments with agrochemicals, will need to be implemented, especially if these soybean cultivars lack Rps-mediated resistance and are grown in disease-conducive environments, including fields previously infected by P. sojae and fields that have been waterlogged for prolonged periods.

Acknowledgements

We gratefully acknowledge technical staffs of Central Region Agricultural Research Center of National Agriculture and Food Research Organization for their technical assistance. We also thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. This research was supported by the Commissioned Project Study entitled “Development of Soybean and Buckwheat Cultivars with Processability and Wide Area Adaptability According to the Users’ Needs” from the Ministry of Agriculture, Forestry and Fisheries of Japan.

Figures
Fig. 1. Soybean plants inoculated with Phytophthora sojae cultured on V8 juice agar medium (P. sojae–V8 culture flooding method). The photo shows ‘Nattosyoryu’ plants inoculated with Ps060626-4-1 isolate (the three pots in the front row) and treated with V8 agar as a control (the three pots in the back row).
Fig. 2. Temporal changes in the rates of dead soybean plants (%) following Phytophthora sojae inoculations of plants at different ages. Plants were inoculated with P. sojae isolate 13-B-RO-1 at 16–40 days after seeding. Mean values ± standard errors for three inoculated pots are plotted in two graphs (top: ‘Himeshirazu’; bottom: ‘Nattosyoryu’). Mean values with the same letters in each graph are not significantly different according to Tukey’s honestly significant difference test (p < 0.05).
Fig. 3. Comparison of the resistance to Phytophthora root and stem rot among 20 soybean cultivars at different plant ages. Soybean plants were grown for 14, 21, or 28 days and then inoculated with one of 17 Phytophthora sojae isolates. The boxplots were drawn based on the rates of dead plants (%) for the inoculation of plants grown for 14 days (A), 21 days (B), or 28 days (C). The open diamond in the plots indicates the average rate of dead plants for each cultivar. The mean values for each growth period were analyzed with Tukey’s honestly significant difference test. The same letters in boxplots indicate the mean values are not significantly different among the three growth periods (p < 0.01).
Fig. 4. Effects of a seed treatment with pesticides on the development of Phytophthora root and stem rot. ‘Nattosyoryu’ seeds treated with one of three pesticides containing fungicidal ingredients were sown, and plants grown for specific periods (14–40 days) were inoculated with Phytophthora sojae isolate 13-B-RO-1. The graph represents a plot of the mean rates of dead plants ± standard errors (%) for plants derived from pesticide-treated seeds (white square: amisulbrom; black circle: thiamethoxam, fludioxonil, and metalaxyl-M; grey triangle: cyazofamid) and non-treated controls (white circle). The mean values were significantly different between pesticide-treated samples and non-treated controls following inoculations at 14, 16, 20, 24, and 28 days after seeding according to Dunnett’s multiple comparisons test (p < 0.001).
Fig. 5. Effect of a seed treatment with pesticides on the inoculation with different Phytophthora sojae isolates. ‘Nattosyoryu’ seeds were treated with thiamethoxam, fludioxonil, and metalaxyl-M (A), or cyazofamid (B), and plants grown for 14 days after sown were inoculated with P. sojae isolates. The boxplots were drawn based on the rates of dead plants (%) caused by the inoculation. The open diamond in the plots indicates the average rate of dead plants for each treatment.
Tables

Phytophthora sojae isolates used in this study

Code Location Isolation date Isolation origin
Ps060626-4-1 Furukawa, Osaki, Miyagi June 26, 2006 Tanrei
Ps060629-5-1 Ogou, Imizu, Toyama June 29, 2006 Enrei
Ps060726-2-1 Higashiyonou, Chikusei, Ibaraki July 26, 2006 Nattosyoryu
Ps080806-3-5 Minatomachi-Shizukata, Aizu-Wakamatsu, Fukushima August 6, 2008
13-B-RO-1 Kannondai, Tsukuba, Ibaraki September 20, 2013 Himeshirazu
13-B-RO-2 Kannondai, Tsukuba, Ibaraki September 20, 2013 Tanbakuro
13-7-B1-1 Hikawa, Tsukuba-Mirai, Ibaraki 2013 Nattosyoryu
13-8-11-2 Tamura, Tsukuba-Mirai, Ibaraki 2013 Tachinagaha
Ps1001 Kihara, Miura, Inashiki, Ibaraki August 23, 2010 Tachinagaha
59-3-2 Iitagawa-Iizuka, Katagami, Akita February 6, 2014 Ryuhoua
66-1 Kanbayashi, Matsumoto, Nagano February 21, 2014 Nakasennaria
69-2 Kanbayashi, Matsumoto, Nagano February 21, 2014 Nakasennaria
90-1-3 Nijomasue, Itoshima, Fukuoka March 7, 2014 Fukuyutakaa
97-1-2 Nakasokoino, Nakama, Fukuoka March 7, 2014 Fukuyutakaa
103-3 Nanatsue, Yanagawa, Fukuoka March 10, 2014 Fukuyutakaa
117-3-2 Kawasoe, Saga, Saga March 21, 2014 Fukuyutakaa
129-2-3 Shiota, Ureshino, Saga March 27, 2014 Fukuyutakaa

Soybean cultivars grown in the fields from which the soil samples for isolation of Phytophthora sojae were collected.


Soybean cultivars used in this study and their planted area in Japan in 2012–2015a

Ranking 2012 2013 2014 2015




for Cultivar Cultivation area Cultivar Cultivation area Cultivar Cultivation area Cultivar Cultivation area




planted ares ha % ha % ha % ha %
1 Fukuyutaka 33,488 24.50 Fukuyutaka 33,467 25.98 Fukuyutaka 34,507 26.22 Fukuyutaka 35,57125.05
2 Enrei 14,756 10.80 Enrei 13,143 10.20 Yukihomare 12,118 9.21 Yukihomare 14,069 9.91
3 Yukihomare 10,764 7.87 Yukihomare 11,745 9.12 Enrei 11,831 8.99 Enrei 11,595 8.17
4 Ryuhou 10,135 7.41 Ryuhou 10,013 7.77 Ryuhou 9,600 7.29 Ryuhou 10,295 7.25
5 Tachinagaha 8,724 6.38 Tachinagaha 8,039 6.24 Tachinagaha 7,485 5.69 Tachinagaha 7,065 4.98
6 Osuzu 4,160 3.04 Miyagishirome 4,760 3.70 Miyagishirome 4,439 3.37 Satonohohoemi 6,635 4.67
7 Miyagishirome 4,137 3.03 Osuzu 4,147 3.22 Satonohohoemi 4,176 3.17 Yukishizuka 5,202 3.66
8 Sachiyutaka 3,335 2.44 Sachiyutaka 3,462 2.69 Osuzu 4,064 3.09 Miyagishirome 4,701 3.31
9 Iwaikuro 3,182 2.33 Toyomusume 3,034 2.36 Yukishizuka 3,791 2.88 Osuzu 4,578 3.22
10 Suzumaru 3,008 2.20 Iwaikuro 3,006 2.33 Sachiyutaka 3,334 2.53 Toyomusume 4,066 2.86
Toyomusume 2,817 2.06 Suzumaru 2,048 1.59 Suzumaru 1,940 1.47 Sachiyutaka 3,374 2.38
Tanbakuro 2,781 2.03 Tanbakuro 2,861 2.22 Toyomusume 3,325 2.53 Suzumaru 1,906 1.34
Nattosyoryu 1,741 1.27 Nattosyoryu 1,643 1.28 Tanbakuro 2,837 2.16 Tanbakuro 2,986 2.10
Tamahomare 1,153 0.84 Tamahomare 1,099 0.85 Nattosyoryu 1,410 1.07 Nattosyoryu 1,183 0.83
Satonohohoemi 1,102 0.81 Satonohohoemi 2,224 1.73 Tamahomare 1,033 0.78 Tamahomare 951 0.67
Suzuyutaka 268 0.20 Hatayutaka 236 0.18 Hatayutaka 274 0.21 Hatayutaka 244 0.17
Hatayutaka 238 0.17 Suzuyutaka 207 0.16 Suzuyutaka 120 0.09 Suzuyutaka 11 0.01
Others 31,417 22.98 Others 24,109 18.72 Others 25,710 19.54 Others 27,82319.59
Total 136,700 100 Total 128,800 100 Total 131,600 100 Total 142,000 100

Cultivars highlighted in dark and light grey were used in this study. The dark grey-highlighting cultivars were those ranked in top 10 of planted area in Japan. The statistical data was obtained from the governmental official website for Ministry of Agriculture, Forestry and Fisheries of Japan. The cultivation area (ha) represents the sum of the statistics for each cultivar in the data, whereas those for two cultivars: Himeshirazu and Toyokomachi were not determined due to no data available at the website.


Pesticides used in this study

Pesticidea Active ingredient (a.i.) Mode of actionb Application volume

Target site (Code) FRAC code
Vortex FSc Amisulbrom (50%) Complex III: cytochrome bc1 at Qi site (C4) 21 8 ml/kg seed
Cruiser MAXXd Metalaxyl-M (1.7%) RNA polymerase I (A1) 4 8 ml/kg seed
Ranman flowable Cyazofamid (10%) Complex III: cytochrome bc1 at Qi site (C4) 21 20 ml/kg seed

The three pesticides are manufactured by Nissan Chemical Industries, Ltd., Syngenta Japan K.K., and Ishihara Sangyo Kaisya, Ltd, respectively.

Mode of action of the pesticides is based on Fungicide Resistance Action Committee (2018).

The manufacturing of the product has been discontinued, and it has been excluded from agricultural chemical registration in 2016.

The product contains two additional ingredients: 1.1% fludioxonil and 22.6% thiamethoxam against fungal and insect pest, respectively.

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