Association of Pythium and Phytophthora with Pre-emergence Seedling Damping-off of Soybean Grown in a FieldConverted from a Paddy Field in Japan

Abstract In Japan, soybean is usually cultivated in fields that have been converted from rice paddies, and poor seedling establishment due to pre-emergence seedling damping-off is often observed during the rainy season. In this study, the factors that cause the damping-off in flooded soil were investigated under high soil moisture conditions in a greenhouse and in agricultural fields. In sterilized soil sampled from a soybean field, seedlings emerged well under 48-hr flooded conditions. In unsterilized soil, soybean seeds treated with the fungicide, mancozeb+metalaxyl exhibited much higher emergence rates even under 48-hr flooded conditions than the seeds treated with oxytetracycline +streptomycin, benomyl, or flutolanil. Pythium, Phytophthora, Mucorales, Trichoderma, Geotrichum-like microorganisms, and some fungi producing conidia in a false head, were isolated from decayed seedlings. Of the isolated microorganisms, oomycete microorganisms, Pythium helicoides, other Pythium sp., and Phytophthora sp. were pathogenic to soybeans under flooded conditions. As the length of the flooding period increased, pre-emergence seedling rot also increased. However, the pathogenic oomycetes rarely caused pre-emergence seedling rot under conditions without flooding. Furthermore, under flooded conditions, the damage caused by these pathogens was reduced by treating the seeds with mancozeb+metalaxyl. These results indicate that oomycete microorganisms are involved in pre-emergence seedling damping-off under flooded soil conditions.

In Japan, approximately 80% of soybean [Glycine max (L.) Merr.] crops are grown in fields that have been converted from rice paddies. The soil drainage of these fields is generally poor, and consequently soil moisture levels are high. Soybean is usually sown during the rainy season, i.e., from mid-June to mid-July, in the central and western region in the country, and the fields are frequently flooded due to excessive rainfall. When soybean seeds are subject to flooding before emergence, pre-emergence seed rot occurs, as well as pre-emergence damping-off. This leads to a significant reduction in seedling stands, and hence potential yields. To improve seedling establishment, it is necessary to identify the factors that cause the failure of seedling emergence under flooded conditions.
Flooding causes physical and physiological damage to soybean. Leguminous seeds are known to be intolerant to water immersion (Woodstock and Taylorson, 1981). Rapid water uptake by dry seeds of soybean causes physical destruction to the cotyledons (Nakayama et al., 2004;Kokuryu et al., 2009), and injures the membranes. Anaerobic conditions force soybean seeds to change from respiration to fermentation (Woodstock and Taylorson, 1981), the latter reducing the level of energy acquisition.
There have been many reports on the microbial plant pathogens that are associated with pre-emergence seed rot and pre-emergence damping-off of soybean. For example, Phytophthora sojae is a common plant pathogen that causes root and stem rot worldwide, including pre-emergence damping-off (Schmitthenner, 1999). Several species of Pythium associated with damping-off have also been reported (Kageyama et al., 1982(Kageyama et al., , 2006Naito et al., 1993;Rizvi and Yang, 1996;Broders et al., 2007;Kirkpatrick et al., 2006;Kodama et al., 2010). Fusarium solani, Rhizoctonia solani, and Sclerotium rolfsii also cause damping-off (Killebrew et al., 1993;Naito et al., 1993). These soil-borne pathogens are opportunistic parasites, and their incidence is largely affected by soil moisture (Cook and Papendick, 1970). However, little information is available on the causal agents of pre-emergence seedling damping-off of soybean grown under moist conditions in the fields converted from rice paddies in Japan.
In this study, we examined some of the factors that might be responsible for the failure of seedling establishment. In the fields where seedling stands are poor, seeds often become soft and rotten in the soil, indicating microbial infection. Hence, here we hypothesized that microbial infection is probably the most important factor associated with the failure of seedling establishment. Therefore, we examined the effects of seed treatment with agrochemicals on pre-emergence seedling damping-off of soybean under the wet soil condition, subsequently identified the fungi and fungus-like pathogens associated with seedling damping-off, and finally determined appropriate control measures against these pathogenic microorganisms.

Seed agrochemical treatment
Four agrochemicals, oxytetracycline (1.5%)+streptomycin sulfate (18.8%), benomyl (50.0%), flutolanil (25%), and mancozeb (55.0%) +metalaxyl (10.0%), were used in this study. Each of these treatments is effective against different types of microbial pathogens. Soybean seeds were covered with a film containing one of the four chemicals at a seed weight of 1%, or with a film containing a mixture of the four agrochemicals, 1% each, 4% in total of seed weight. A diluted film coat material [film coat material : water=2 : 3 (v/v)], named "Agro-red" (Incotec Japan Co. Ltd., Niiza, Japan), was used to improve chemical attachment to the seed coat, at 0.7% of seed weight. Seeds were treated 1 d before sowing. Seeds treated with only the film coat material were included as an untreated control.

Effects of soil sterilization and seed chemical treatments
on seedling stand under greenhouse and field conditions Soil (muck) from a soybean farm that had been converted from a paddy field in Tsukuba (140º8΄E, 36º6΄N, Field E in Table 1), Ibaraki, Japan, was sampled in November 2008, after the soybean harvest. The soil passed through a 4-mm grid sieve (250 mL) was placed in pots (113 mm in diameter) with a hole in the bottom, and 12 soybean seeds (cv. Tachinagaha, a major cultivar in Ibaraki) each were subjected to one of the six treatments: one of the four agrochemicals, a mixture of all four agrochemicals, and an untreated control were placed on the soil, and then covered with another 150 mL soil. For the soil-sterilization treatment, the soil was autoclaved (121ºC for 30 min), and the seeds were treated with the film coat material only. The pots were placed in a greenhouse at 19 to 25ºC. Twenty to 24 hr after sowing, the soil was flooded from the bottom hole just above the soil surface for 45 to 48 hr. A little water was applied to the pots to keep soil moisture high during the following 8 to 10 d. Emerging seedlings were counted, and non-emerging seedlings were dug up and examined for the presence of rot. Seedlings with the cotyledons completely above the soil surface were designated as emerging seedlings. Five pots were used for each treatment, and the experiment was conducted twice.
In the field experiments, one of the four agrochemicals, a mixture of all four agrochemicals, and untreated control-were applied to the seeds of the cultivars 'Tachinagaha' and 'Natto-shoryu', the latter of which is another major cultivar in Ibaraki. The proportions of emergence of these seeds were confirmed more than 95% in the different greenhouse experiments under the unflooded conditions. The seeds were sown in the experimental fields of the National Agricultural Research Center and one farmer's field in Tsukuba, Ibaraki, Japan. If irrigation was available, the fields were flooded for 24 or 48 hr. If irrigation was not available, the seeds were sown after a heavy rain or when the forecast was rain. Thus, the fields received either a flooding treatment or 44 to 60 mm of precipitation before or within 3 d after sowing. The field size and the number of replicates differed with the experiments owing to the availability of fields (Table 1). The experimental design consisted of a randomized complete block design with two replicates. In three experiments (field experiments 1, 4, and 6), the fields contained rotary-cultivated (conventional tillage) or nontilled plots. Since no statistical difference was found between tilled and non-tilled plots, the tillage factors were omitted from further statistical analysis. Emerging seedlings were counted for all plants from 8 to 14 d after sowing. Volumetric soil water content was monitored in the field experiment 7 by inserting time domain reflectrometer sensors (EC-5, Decagon Devices Inc., Pullman, WA, USA) at 5 cm depth. Soil temperature at about 3 cm depth was monitored in the field experiments 1, 2, 4, 5 and 6 using a data logger with a thermistor sensor (TR-52i, T&D Corporation, Matsumoto, Japan).

Isolation of fungi and fungus-like organisms from diseased seedlings
Soil collected from the converted rice paddy described above were placed in deep vats (390 mm × 280 mm × 135 mm, with a hole on both sides), and placed in a greenhouse. Soybean seeds (cv. Tachinagaha) were treated with each of the four agrochemicals as described above. Thirty seeds from each treatment and untreated seeds were sown in the soil of the vat. At 21 to 24 hr post-sowing, the holes were plugged and the soil was flooded for 48 hr. The soils were then watered daily. Non-germinating seeds and pre-emerging seedlings were dug up 10 d after sowing to isolate the potential causal agent of disease. The seeds and seedlings were washed with running water. Two small segments from decayed seedlings were cut with a flamesterilized blade. One segment was surface-sterilized and placed on a 1.5% agar plate with lactate. The other segment was placed on V8 juice agar (V8A) medium containing benomyl, nystatin, pentachloronitrobenzene, rifampicin, and sodium ampicillin (BNPRA V8A) at 10, 25, 25, 10, and 500 mg L -1 , respectively (Masago et al., 1977).

Pathogenicity of fungi and fungus-like organisms
Various types of fungi and fungus-like microorganisms were isolated from soybean seedlings that exhibited preemergence damping-off. After classifying the isolates by colony morphology and microscopic examination, 12 representative isolates (Table 2) were tested for pathogenicity. Twelve soybean seeds (cv. Tachinagaha) were sown in pots containing sterilized pot soil (light-colored andosol). A small agar segment (ca. 3 mm × 3 mm), containing mycelia of isolates grown on potato dextrose agar (PDA) or V8A plates at 25ºC for 7 to 8 d, was attached to the surface of a seed without wounding it, before covering with soil. The pots were maintained in a greenhouse between 18ºC and 30ºC. Flooding treatment for 48 hr was started 24 hr after sowing. The number of emerged seedlings was counted 10 d after sowing. Two pots were used per isolate. Soybean seeds (8 of cv. Natto-shoryu and 10 of cv. Tachinagaha) were sown in a tray (150 mm × 55 mm × 100 mm), and placed on a laboratory bench at 25ºC with natural light for 6 to 7 d. Then an approximately 1 cmlong slit was made with a surgical blade longitudinally in a hypocotyl at a distance of 1 cm below a cotyledonary node. Small culture segments of the isolates that caused preemergence seedling rot, which had been grown on V8A for 6 to 7 d, were inserted into the slit. The seedlings were then covered with polyethylene bags for 2 d, and examined for the presence of disease 4 d after the inoculation.

Identification of Pythium and Phytophthora isolates
Potential isolates of Pythium and Phytophthora were cultured on V8A agar, and single hyphal tips were cut and placed onto new V8A media. The colonies were observed under a light microscope for sexual organ production, until 4 wk after inoculation. An agar plug (approx. 5 mm square) from the middle parts of the colonies of 5-to 7-d-old cultures was cut out with a sterile blade, and placed in a sterile polystyrene petri dish. Sterile water was added to immerse the agar plug. The petri dishes were maintained in an incubator at 25ºC in the dark. The morphology of sexual and asexual fungal structures was examined daily under a light microscope until 7 d after immersion. Morphological identification of these oomycetes was based on van der Plaats-Niterink (1981) and Erwin and Ribeiro (1996). To confirm morphological identification further, we determined the rDNA ITS sequences of the isolates by a method described by Uzuhashi et al. (2008). The species identity of isolates was estimated by conducting a BLAST analysis for each sequence.

Effects of flooding periods on the pathogenicity of
Pythium and Phytophthora Soil preparation, sowing, and inoculation were conducted by the same method as used for the pathogenicity test. Three flooding treatments (0, 24, and 48 hr) were started 24 hr after sowing. After the flooding treatment, pots were watered daily. Emerged seedlings were counted 10 d after sowing. Each treatment for the combination of isolates and flooding periods consisted of two pots. In another experiment including the Ps1001 isolate of Phytophthora sojae, two flooding treatments (0 and 48 hr) were conducted with four pots. Each experiment was conducted twice.

Control of Pythium and Phytophthora with seed chemical treatment
Twelve soybean seeds (cv. Tachinagaha), treated with oxytetracycline +streptomycin, benomyl, flutolanil, and mancozeb +metalaxyl were sown in a pot containing sterilized soil in a greenhouse. Small segments with Pythium sp. V1, Py. helicoides V80, and Phytophthora sp. V24 grown on V8A were attached to each seed. Flooding treatments for 48 hr were started 1 d after sowing, and seedling diseases examined 10 d after sowing. Each treatment consisted of three pots and the experiments were conducted twice.

Statistical analysis
The proportion of emerged seedlings was arcsine transformed and analyzed by ANOVA using JMP statistical software (ver. 8.0.1; SAS Institute Japan, Tokyo, Japan). The differences of means between treatments were analyzed by Tukey's HSD (honestly significant difference) test.

Effects of seed chemical treatment on seedling stand under greenhouse conditions
When the seeds without any agrochemicals were planted benomyl established only a few seedlings comparable to the untreated seeds (Table 3).

Pathogenicity of fungi and fungus-like organisms isolated from diseased seedlings
From rotten soybean seeds, microorganisms belonging to Mucorales, Trichoderma spp., Geotrichum-like fungi, and fungi that produce conidia in a false head, were commonly isolated on acidified water agar. The Mucorales fungi, Pythium, and Phytophthora were commonly isolated on BNPRA V8A ( Table 2). All of the isolates of Pythium and Phytophthora, except for Pythium isolate V61, caused preemergence rot that was similar to the original symptoms and reduced seedling emergence rates (Fig. 2). Isolates belonging to Mucorales, Geotrichum-like fungi, Trichoderma spp., and fungi producing conidia in a false head did not cause pre-emergence rot (Fig. 2). Seedlings from the seeds inoculated only with PDA or V8A plugs emerged well, even with flooding treatment (Fig. 2). Pythium isolates V1, V12, and V80 and Phytophthora isolates V24, V53, and W6 usually caused the cotyledons to rot before root elongation (Fig. 3  top). Soil became firmly adhered to the rotten seeds, and was not washed off easily. Phytophthora sojae isolate Ps1001 produced brown water-soaked lesions on the hypocotyls, roots, and occasionally on cotyledons, particularly on the basal part of cotyledons; however, the cotyledons usually remained firm (Fig. 3 bottom), and soil was easily washed off. When the hypocotyls of soybean were woundinoculated with agar pieces containing mycelia of Pythium isolates V1, V12, V80, and Phytophthora isolates V24 and V53, at the cotyledon unfolding stage, no susceptible reactions were observed (data not shown). When isolates of Ph. sojae (Ps060726-5-1 and Ps1001) were woundinoculated as pathogenic controls, the seedlings shriveled, and ultimately died.
in sterilized soil, almost all seedlings emerged, even with the flooding treatment (Fig. 1). When soybean seeds not treated with any agrochemical were planted in unsterilized soil and flooded for 48 hr, seedlings emerged from only approximately half of the seeds sown (Fig. 1). Most of the non-germinating seeds produced a soil ball that was firmly adhered to soil with rotten cotyledons. Among the soybean seeds flooded after the treatment with an agrochemical, the seeds treated with mancozeb+metalaxyl exhibited the highest emergence rate, which was comparable to the emergence of untreated seeds planted in sterilized soil (Fig. 1). When seeds treated with oxytetracycline + streptomycin sulfate were flooded, the proportion of emerged seedlings was less than that of untreated seeds, and the proportion of pre-emerging seedlings with rotten cotyledons was the highest (Fig. 1). Seeds treated with benomyl and flutolanil showed intermediate proportions of emergence (Fig. 1).

Effects of seed chemical treatment on seedling stand under field conditions
Soil water content was monitored only in the field experiment 7. This field received 24.0 mm precipitation before sowing and 36.5 mm within 3 d after sowing (Table 1). During this period, volumetric soil water content increased from 22% to 42%.
There was no statistical difference in seedling emergence among seeds treated with each agrochemical in cv. Natto-shoryu (Table 3). However, the seeds of cv. Tachinagaha treated with mancozeb +metalaxyl exhibited higher seedling emergence rates than those treated with oxytetracycline +streptomycin sulfate, flutolanil, benomyl, and the untreated controls (Table 3). Seeds treated with a mixture of the four agrochemicals (mixed treatment) exhibited emergence rates similar to the seeds treated with mancozeb +metalaxyl (Table 3). The seeds treated with oxytetracycline +streptomycin sulfate, flutolanil, and a The proportion of seedling emergence was arcsine transformed and analyzed by one-way ANOVA. Values followed by the same letter within the same row are not significantly different between the agrochemical treatments using Tukey's HSD test (P < 0.05). b "Mixed" indicates that the soybean seeds were treated with all four agrochemicals.

Identification of Pythium and Phytophthora isolates
On the basis of morphological characteristics and rDNA-ITS sequences, the seven isolates of Pythium and Phytophthora isolated from the rice paddy were identified as Pythium mercuriale (V61), Py. helicoides (V80), an unknown Pythium sp. (V1 and V12), and an unknown Phytophthora sp. (V24, V53, and W6). Pythium isolates V61 and V80 only produced asexual structures that corresponded with those of Py. mercuriale (Belbahri et al., 2008) and Py. helicoides (van der Plaats-Niterink, 1981), respectively. The ITS sequences of the isolate V61 showed high sequence identity (99.4%) with the ex-type isolate STE-U 6204 of Py. mercuriale (Belbahri et al., 2008). The sequence of the isolate V80 also showed high sequence identity (96.3%) with the extype isolate CBS286.31 of Py. helicoides (Lévesque and de Cock, 2004). The unknown Pythium sp. isolates V1 and V12 produced zoospores in vesicles formed from globose zoosporangia, but did not form sexual organs until 4 wk. Their ITS sequences were identical to each other. BLAST analysis of the sequences suggests that the isolates showed highest similarity with Py. grandisporangium isolate CBS 211.85 (72.0%). The isolates require further investigation for species identification. The unknown Phytophthora sp. isolates V24, V53, and W6 differentiated zoospores in ovoid zoosporangia. They did not form sexual organs. They had identical ITS sequences that did not match any of the known Phytophthora species based on the BLAST analysis.

Effects of flooding period on the pathogenicity of
Pythium and Phytophthora isolates Under the unflooded condition, seeds inoculated with isolates of Pythium helicoides (V80), unknown Pythium sp. (V1 Fig. 2. Emergence of soybean seedlings inoculated with potential pathogens isolated from rotten pre-emergence seedlings under a 48-hr flooded condition. The taxonomic groups of the isolates are shown in Table 2. Bar graph shows the average proportion of emerged seedlings with the standard errors from two replications. and V12) and Phytophthora spp. (V24, V53, and W6) emerged at high percentages (Fig. 4). The proportion of seedling emergence decreased as the length of the flooding period increased. Seeds inoculated with only V8A plugs emerged well, irrespective of flooding periods of up to 48 hr (Fig. 4). In comparison, seeds inoculated with Ph. sojae isolate Ps1001 produced the lowest number of healthy seedlings, even under the unflooded condition, along with isolate V80, in this experiment (Fig. 5).

Effects of agrochemical seed treatment on the pathogenicity of Pythium and Phytophthora in an inoculation test
Treatment with mancozeb +metalaxyl effectively controlled pre-emergence seedling rot in seeds inoculated with Pythium helicoides isolate V80, unknown Pythium sp. isolate V1, and Phytophthora sp. isolate V24 (Fig. 6). Although mancozeb+metalaxyl treatment did not improve seedling emergence from the seeds inoculated with isolate V1, it reduced pre-emergence seedling rot (Fig. 6). The other three chemical treatments did not reduce the level of seedling rot (Fig. 6).

Discussion
In the first stage of our study, we evaluated whether microbiological organisms were responsible for the reduction of seedling emergence. The seeds used in the greenhouse and field experiments were confirmed to have a high ability to emerge under the unflooded conditions. The reduction in the proportion of emergence may be caused by some factors other than the reduction of emergence ability. Comparison of emergence of soybean sown in sterilized and natural soil under flooded conditions showed that microbiological organisms are responsible for pre-emergence seedling rot.
We then investigated which fungi or fungus-like organisms cause pre-emergence seedling damping-off. Oomycete pathogens, such as Pythium and Phytophthora, are known to be causal agents of damping-off of soybean, particularly in poorly drained soils (Schmitthenner, 1999;Yang, 1999). Phytophthora sojae is a well-known causal agent of dampingoff of soybean in Japan. Some species of Pythium (Kageyama and Ui, 1983;Kodama et al., 2010) have also been reported as damping-off causal agents. Other microorganisms, including F. solani, Calonectria ilicicola, Sclerotium rolfsii, and R. solani, also occasionally attack pre-emerging seedlings of soybean, and kill such seedlings. In our study, the Pythium and Phytophthora isolates. Bar graph shows the average proportion of emerged seedlings to 12 seedlings, each with two replicates in three separate experiments. Bars with the same letters within each isolate indicate no significant difference between flooding periods by Tukey's HSD test (P < 0.05). Fig. 5. Pathogenicity of Pythium and Phytophthora isolates to soybean seedlings under unflooded and 48-hr flooded conditions. Isolates V12, V80, V24, and Ps1001 belong to Pythium sp., Py. helicoides, Phytophthora sp., and Ph. sojae, respectively. V8A indicates inoculation of V8 juice agar pieces only. Bars show average proportion of healthy seedlings to 12 seedlings, with four replicates per treatment in a representative experiment. Bars with the same letters within each flooding period indicate no significant difference by Tukey's HSD test (P < 0.05) using arcsine-transformed data. The two replicates of the experiment showed similar results. ab agrochemical seed treatment experiments in the greenhouse and the field indicated that several species of Pythium and Phytophthora are associated with pre-emergence seedling rot of soybean. Although soilborne pathogens other than oomycetes may have been present in the tested soil, chemicals specifically effective against bacteria, ascomycetes, and basidiomycetes did not improve seedling emergence in the current study. Therefore, microorganisms other than oomycetes were considered to contribute less to preemergence seedling damping-off of soybean under flooded conditions. In Iowa, the United States, Pythium and Phytophthora were most important agents of seedling disease of soybean, followed by R. solani; Fusarium spp. were isolated at a relatively low frequency (Rizvi and Yang, 1996). In their report, Fusarium spp. were considered as secondary colonizers. Although wet soils are also favorable for F. solani, C. ilicicola, and R. solani, flooded conditions may be much more conducive for Pythium and Phytophthora. This is because oomycetes produce zoospores in saturated soil, and the motile spores allow them to reach the root and crown tissue of their host more easily. Therefore, oomycetes probably contribute to the reduction of seedling emergence that is frequently observed in soybean sown during the rainy season in Japan. Some Pythium spp., such as Py. ultimum, Py. myriotylum, (Kageyama et al., 1982;Kageyama and Ui, 1983), and Py. spinosum , in addition to Ph. sojae, have been reported as causal agents of the damping-off of soybean in Japan. The present study found that preemergence seedling damping-off was caused by Py. helicoides and unidentified Pythium and Phytophthora species. In Ohio, USA, 12 Pythium spp., including Py. helicoides, have been isolated from diseased soybean seedlings using a soybean bait (Broders et al., 2007). In Arkansas, a state that is part of the southern soybean cropping area of the United States, Pythium is the only genus of filamentous microorganisms whose isolation frequency increases with flooding (Kirkpatrick et al., 2006). In the present study, Ph. sojae was not isolated from soybean commercial agricultural farming soils. In fact, Phytophthora root and stem rot did not occur on cv. Tachinagaha, which was used as bait in the experiments, but did occur on cv. Natto-Shoryu in the farmer's field. It is possible that cv. Tachinagaha has a resistance gene to the Ph. sojae races infesting the field. If cv. Natto-shoryu had been used as bait, Ph. sojae also would have been isolated from decayed seedlings. Hence, several species of Pythium and Phytophthora are probably responsible for seedling damping-off in flooded soybean fields. The optimum temperatures for the mycelial growth of Pythium (Hendrix and Campbell, 1973) and the Fig. 6. Improvement in the emergence of soybean inoculated with three Pythium and Phytophthora isolates, with agrochemical treatment under flooded conditions. Isolates V12, V80, and V24 belong to Pythium sp., Py. helicoides, and Phytophthora sp., respectively. V8A indicates inoculation of V8 juice agar pieces only. The bar graph shows the average proportion of emerged seedlings, pre-emergence seedlings without rot, and pre-emergence seedling with rot to 12 seedlings, each with three replicates in a representative experiment. sporangium formation of Phytophthora (Ribeiro, 1983) vary among species. Soil temperatures during the sowing period differ with the region because soybeans are sown in late May to early June in northern Japan and mid-June to mid-July in central to western Japan. In Hokkaido, the northernmost region of Japan, low temperatures at the pre-emergence stage promote damping-off of soybean by Py. spinosum and Py. ultimum var. ultimum . Therefore, different species of Pythium and Phytophthora are probably involved in damping-off and seedling rot among regions. Flooding conditions cause physical injury to cotyledons through rapid water uptake by soybean seeds (Kokuryu et al., 2009;Nakayama et al., 2004). In this study, the influence of physical injury was minimized by delaying flooding treatment to 1 d after sowing. Flooding also changes the physiological metabolism of seeds via fermentation under anaerobic conditions, and such changes can reduce the defense reactions of plants (Drew and Lynch, 1980). This study showed that almost all seedlings emerged in sterilized soil, even under the 48-hr flooding condition. This result indicates that the physiological changes due to 48-hr anaerobic conditions do not always result in a low seedling emergence rate. The oomycetes isolated from rotten seeds rarely caused preemergence seedling damping-off in the absence of flooding treatment. As the flooding period is prolonged, the occurrence of seedling damping-off was increased. These results indicate that isolated oomycetes, with the exception of Py. helicoides, are weak pathogens, and require flooding to cause pre-emergence seedling damping-off. Flooding activates oomycetes and makes soybean seeds less active, including their defense response. The isolates of Pythium and Phytophthora isolated from decayed seeds and seedlings may cause pre-emergence seedling rot only when the defense mechanisms of the host plant does not operate well. Py. helicoides and Ph. sojae do not necessarily require flooded conditions to cause pre-emergence seedling damping-off. Ph. sojae, moreover, can cause also postemergence seedling damping-off in the absence of flooding. The oomycetes isolated in this study are considered opportunistic pathogens, and are more dependent on flooding to infect soybean than Ph. sojae. Differences in the symptoms caused by the species of Pythium and Ph. sojae might be applied to develop a useful key in the diagnosis of pre-emergence seedling damping-off.
The effect of flooding treatment on the proportion of emergence differed with the cultivar. This may be due to the soil temperature. In experiment 1 and 3, where cv. Natto-shoryu was sown, the average soil temperature was 21.1 and 24.5ºC, respectively. In experiment 4, 5 and 6, where cv. Tachinagaha was sown, the average soil temperature was 25.9, 26.7 and 26.8ºC, respectively. Soil microorganisms grow vigorously under high soil temperatures. On the other hand, soybean seedlings become exhausted rapidly under high temperature and low oxygen conditions. The hundred seed weight was approximately 10 and 35 g in cv. Natto-shoryu and cv. Tachinagaha, respectively. A consistent correlation has not been observed between seed size and emergence (Edwards and Hartwig, 1971;Burris et al., 1973;Johnson and Luedders, 1974;Hopper et al., 1979;Longer et al., 1986). A difference in the level of susceptibility to weak oomycete pathogens between cultivars may explain the difference in emergence rate.
When seeds are physically injured, microbial infection may occur more easily. Injured tissues release exudates that serve as nutrients for microorganisms, and attract zoospores (Morris and Ward, 1992). A reduction in defense reaction, the formation of physical injury, and favorable conditions for oomycete development provide conditions that are conducive to the infection of soybean by Pythium and Phytophthora. Although oomycetes are considered the main cause of failure of seedling emergence, physical injury or physiological changes also are responsible for some of this failure, whether alone or by facilitating colonization and infection by plant pathogenic oomycetes.
In this study, treatment of soybean seeds with agrochemicals was effective against oomycetes, probably by suppressing or killing target organisms around the seed, thus improving seedling emergence and establishment under flooded or high soil moisture conditions. Culture techniques, such as improving field drainage and sowing on hills, reduce the frequency and duration of flooding, and hence prevent Pythium and Phytophthora from infecting soybean seeds. Fungicide treatments are effective against microbial infection, but not against physical injury due to rapid water uptake. Sowing of moisture-adjusted soybean seeds where moisture is elevated to ca. 15% of seed weight is recommended to avoid physical injury to the seed (Nakayama et al., 2004;Kokuryu et al., 2010). A combination of agrochemical treatment, culture techniques, and the use of moisture-adjusted soybean seeds might also contribute toward reducing pre-emergence seedling damping-off and improving seedling establishment in sowing during the rainy season in wet soils, including flooded conditions.