© 2009 Plant Management Network.
Within-field Pathogenic Diversity of Phytophthora sojae in Commercial Soybean Fields in Iowa
Alison E. Robertson, Assistant Professor, Silvia R. Cianzio, Professor, Sarah M. Cerra, Former Graduate Student, and Richard O. Pope, Extension Program Specialist, Department of Plant Pathology, Iowa State University, Ames, IA 50011
Robertson, A. E., Cianzio, S. R., Cerra, S. M., and Pope, R. O. 2009. Within-field pathogenic diversity of Phytophthora sojae in commercial soybean fields in Iowa. Online. Plant Health Progress doi:10.1094/PHP-2009-0908-01-RS.
Phytophthora root and stem rot (PRR), caused by the oomycete Phytophthora sojae, is an economically important soybean disease in the north central region of the United States, including Iowa. Previous surveys of the pathogenic diversity of P. sojae in Iowa did not investigate whether multiple pathotypes of the pathogen existed in individual fields. Considering the many pathotypes of P. sojae that have been reported in Iowa, we hypothesized multiple pathotypes could exist within single fields. In the research reported herein, several soil samples were collected systematically from each of two commercial fields with a history of PRR in Iowa, and each soil sample was baited separately for isolates of P. sojae. Numerous pathotypes of P. sojae were detected from both fields. As many as four pathotypes were detected in some soil samples (each consisting of six to eight soil cores), which suggests that a single soybean plant could be subjected to infection by more than one pathotype. This possibility presents important implications in breeding resistant cultivars and in the management of PRR.
Phytophthora root and stem rot (PRR), caused by Phytophthora sojae, is a soybean disease of economic importance in the USA. In 2005, yield loss due to PRR was estimated to reach over $250 million (20). This disease has been observed in up to 63% of Iowa soybean fields (14). Estimates of yield loss between 1996 and 1998 in Iowa, averaged 92,480 metric tons, with an approximate value of $13 million (21). Greater losses can occur during years with favorable environmental conditions for disease development, i.e., heavy rainfall and soil temperatures above 21°C (22).
Over 55 physiologic races or pathotypes of P. sojae have been identified throughout the north central United States (1,4,9,10,12,13,15,17,22). Pathotype designations of P. sojae isolates indicate the Rps genes possessed by soybean varieties that the isolates can infect. Originally, specific pathotypes of isolates of P. sojae were assigned race numbers; however, this system has become cumbersome due to increasing pathogen diversity. Therefore, it is now becoming more common for the pathotype designation to be used. Knowing the pathotype of an isolate is far more informative since it indicates which Rps genes can be infected by the tested pathotype.
The pathogenic diversity of P. sojae in Iowa has increased considerably since the pathogen was first identified in the state in 1966 (14,18,22). However, these data based on state-wide surveys of the pathogen did not provide information on pathogenic diversity of P. sojae within fields. Research in Ohio (5), Arkansas (9), and Australia (15), showed that more than one virulence phenotype of the pathogen can occur within a single field. In Arkansas (8) and Australia (15) isolates of several pathotypes were identified in fields that had been used as breeding nurseries for P. sojae-resistant soybeans. Dorrance et al. (3) reported the occurrence of numerous races within each of two commercial soybean fields in Ohio. In Iowa, knowledge about racial diversity of endemic populations of P. sojae within commercial fields is not available. These data are necessary to provide guidelines to both researchers and producers on deploying P. sojae-resistance genes in commercial varieties to protect soybean yields.
Use of resistant cultivars is the most effective and economic management tool for PRR. Fourteen resistance (Rps) genes have been identified in soybean germplasm (3,16); however, only Rps1c, Rps1k, Rps3a, and Rps6 have been deployed in commercial soybean varieties in the north central region of the United States (8). In Iowa, Rps1k is the most common resistance gene used at present, followed by Rps1c, but the increased prevalence of race 25 (pathotype 1a, 1b, 1c, 1k, 7) in Iowa (14) has prompted the incorporation of Rps6 into germplasm for Iowa.
Soil Sampling and Isolation of Phytophthora sojae
During the 2005 growing season, soil samples were collected from two commercial soybean fields with histories of losses due to PRR. One field was located near Albion, in Marshall Co., IA, and the second field was located near Albert City, Buena Vista Co., IA. Each 80-acre field was divided into 30 subsections on a 3 × 10 grid. A total of 30 soil samples, each consisting of six to eight soil cores (2.5 cm diameter by 15 cm deep), were systematically collected from the center of each subsection. The samples were stored at 4°C for 1 month before baiting for P. sojae.
Two methods were used to isolate P. sojae from each soil sample: seedling bioassay and leaf-disc baiting (17). No differences in isolate recovery were observed between the two methods, therefore data from the two detection methods were combined for presentation here. Each soil sample was mechanically mixed and then divided into two sub-samples; one was placed in a 4-inch plastic pot, and the other subsample into one 16-oz polystyrene cup. Each subsample was baited three consecutive times using either seedlings or leaf discs of the soybean variety Sloan, which is susceptible to infection by all pathotypes of P. sojae. To ensure zoospores did not survive from one baiting event to the next, soil subsamples were air-dried for approximately one week between each event. Plastic pots each filled with a soil subsample were placed in a growth chamber on a diurnal cycle of 25°C for 16 h/20°C for 8 h, with sequential 12 h light/dark cycles. Soil subsamples were moistened and allowed to warm 24 h in the growth chamber and then 10 seeds of Sloan were planted in each subsample. Three days after planting, the pots were flooded for 24 h. Thereafter, every three days, pots were watered with tap water by flooding the pot and letting the water drain from the bottom of the pot. Phytophthora sojae was isolated from symptomatic soybean stem tissue by plating small pieces of tissue removed from the leading edge of the stem lesions on half-strength V8 juice agar (100 ml V8 juice per liter) amended with neomycin sulphate (50 µg/ml), hymexazol (20 µg/ml), and chloramphenicol (10 µg/ml) (˝ V8JAA). In the leaf disc method, polystyrene cups half-filled with each soil sub-sample were flooded with deionized water. Leaf discs were floated on the water surface for up to 24 h before being placed on ˝ V8JAA. Putative isolates of the pathogen were confirmed to be P. sojae microscopically on the basis of morphological attributes and were subsequently purified by single zoospore isolation (17).
The pathotype of each isolate baited from the soil was determined by inoculating 10 seedlings of 14 standard soybean test varieties (often referred to as differentials), namely Parker (Rps1a), L77-1863 (Rps1b), Williams79 (Rps1c), PI 103091 (Rps1d), Williams82 (Rps1k), L76-1988 (Rps2), L83-570 (Rps3a), L92-7857 (Rps3c), L85-2352 (Rps4), L85-3059 (Rps5), L89-1581 (Rps6), Harosoy (Rps7), PI 399073 (Rps8), and Sloan (susceptible). Seedlings were inoculated with a slurry of isolate placed in a syringe and injected into the hypocotyl (7). The inoculated seedlings were grown at room temperature with 14 h light and 10 h darkness for 7 to 10 days. The pathotype of the inoculated isolate was determined by rating plant death of differentials as described by Dorrance and others (6). Seedlings that developed either stem lesions or 50% or greater root rot severity were classified as susceptible. Pathotype determination of each isolate was repeated at least once to confirm results.
Pathogenic Diversity of Phytophthora sojae
The 2005 growing season was characterized by abnormally dry weather not conducive to disease development. Disease pressure was consequently low at the Albion field, and no PRR was observed at the Albert City field. From the two fields, a total of 34 isolates of P. sojae were detected (Table 1) from 17 out of 60 soil samples collected (28.3% recovery). Pathotype or race designation (when available) of 31 isolates was determined. Pathotype testing of three isolates gave inconsistent results and therefore these isolates were not classified. Our results indicated that in both fields the endemic population of P. sojae was pathogenically diverse. Furthermore, 11 races and 12 pathotypes not previously reported in Iowa were identified in the study (Table 1). Of all the isolates detected, 25.8% were able to cause disease on Williams79, the soybean differential variety containing Rps1c, 51.6% were able to cause disease on Williams82 (Rps1k) and 35.5% caused disease on L89-1581 (Rps6). No data were available for resistance genes Rps8 and Rps2 due to poor germination of the particular soybean differential varieties.
Table 1. Characteristics of Phytophthora sojae isolates
w Virulence formulae based on standard differentials (5).
x Identified in the present study and also reported in the 1996
y Identified in the present study and also reported in the 2004
z Results of pathotype screening inconsistent.
Our study detected multiple pathotypes of P. sojae within individual fields in Iowa for the first time. This is likely because multiple soil samples were taken from both fields surveyed, thus allowing isolation and pathogenic characterization of the pathogen from different sites within the same field. The sampling technique used in previous studies (14,22), in which several soil samples collected from a single field were bulked before being baited, allowed determination of pathogen variability at the county and state levels but precluded determination of pathogen variability at the field level. The difference in soil sampling method between our study and previous research could lead to the speculation that within-field variation may have been present in the past, but not observed due to the sampling method used.
At the Albert City field, 21 isolates of P. sojae were collected (Table 1), primarily in the lower lying areas along the northern one-third of the field (Fig. 1). The predominant soils mapped in this field include Canisteo clay loam (40.6%), Nicollet loam (27.2%), and Wacousta mucky silty clay loam (11.8%). Canisteo and Wacousta soils are poorly drained, while Nicollet is somewhat poorly drained (19). Survival of the pathogen and subsequent disease development is likely favored under poor drainage conditions. Canisteo clay loam was formed in recent glacial till (Fig. 1) and is calcareous, consequently exhibiting high soil pH (19). Wacousta mucky silty clay loam has the highest organic-carbon content recorded in the upper soil profile (19). Nine documented races (2, 3,1 0, 14, 17, 22, 29, 33, and 54) were identified in addition to nine pathotypes not corresponding to any described races of P. sojae. The virulence phenotype of two isolates could not be consistently determined. The population of P. sojae in this field was very diverse and infected plants with any of the following Rps genes: 1a, 1b, 1c, 1d, 1k, 3a, 3c, 4, 5, 6, and 7. More importantly, at five of the 30 sampling locations within the field, between two and four pathotypes of P. sojae were detected per location (Fig. 1).
In the Albion field, 13 isolates of P. sojae (Table 1) were collected, primarily from the lower lying, northern half of the field (Fig. 1). The predominant soils in this field were Muscatine silty clay loam (65.5% of the field area) and Garwin silty clay loam (29.3% of the field area), which were both formed in loess (19). Garwin soils are poorly drained and occur on nearly level (0 to 2% slopes) upland areas with limited surface drainage while the Muscatine soils are somewhat poorly drained, on slightly sloping (2 to 5%) uplands (Fig. 1) (19). Survival of P. sojae and PRR development should be favored by poor soil drainage. The isolates of P. sojae detected in this field belonged to eight documented races and an additional three pathotypes of the pathogen. Thus the population of P. sojae in this field was equally diverse as that of the Albert City field, and one or more isolates were able to infect plants with genes Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps3a, Rps3c, Rps4, Rps6, and Rps7 (Table 1). Likewise, at one location within the Albion field, three isolates belonging to races 11, 20, and 25, (M44, M12, and M4, respectively) were detected (Table 1, Fig. 1) and at each of another three locations in the same field, two isolates were detected that could similarly overcome multiple Rps genes.
That some soil samples in both fields yielded up to four pathotypes of P. sojae suggests that soybeans could be subjected to infection by more than one pathotype of the pathogen. Moreover, the potential for further diversity within the endemic population of P. sojae increases Phytophthora sojae is homothallic (self-fertile); however, outcrossing has been shown to occur in the laboratory between isolates when they are grown in mixed culture (2). In addition, Layton and Khun (11) showed soybean plants could be simultaneously colonized by more than one pathotype of P. sojae in a greenhouse assay and demonstrated the potential for crosses to occur between these races in planta and possibly lead to the formation of new pathotypes.
Changes in pathotype (race) populations have been documented in previous surveys in Iowa, and the shift has considerably expanded the number of races identified in the state since 1994 (14,22). The second survey conducted between 1992 and 1994 reported that 1% of isolates of P. sojae detected could infect plants with the Rps1k gene (22). A survey between 2002 and 2004 found that 45% of the isolates collected could infect plants with the Rps1k gene (14). Our study identified over 50% of the isolates collected could infect plants with the Rps1k gene. Moreover, races previously reported at high prevalence, such as race 1, 3, 4, 25, and 28 (14) were collected at lower frequencies, if collected at all, in our study. We collected races 3 and 25 at less than 4% frequency, while races 1, 4, and 28 were not detected. Furthermore, the occurrence of isolates that could overcome Rps6 is disturbing since this gene has yet to be commercially released in Iowa.
Previous surveys in Arkansas and Australia studying within-field diversity of P. sojae sampled trial sites used for screening soybean breeding materials (9,15). In Arkansas, 8 races and 7 pathotypes were found in a single field (9). Jackson et al. (9) concluded the diverse population found in the field could be attributed to the selection pressure placed on the P. sojae population by the large number Rps genes in the soybean varieties that had been planted there. Ryley et al. (15) found eight races within a single disease nursery in Australia. In our study, we sampled two growers’ fields and found a similar degree of diversity to that found in the Arkansas study. Therefore, complex pathogenic diversity of P. sojae in soybean production fields may be more common in commercial soybean fields than has been previously reported. Indeed, in the Ohio study, up to 56 different pathotypes were detected from one intensively sampled soybean production field (4).
Our data suggest that stacked gene combinations, high partial resistance (tolerance), or Rps genes in combination with high partial resistance are necessary in order to protect soybean yields in Iowa. Although partial resistance to P. sojae is active across all races, it does not express in the plant until the VC stage of growth (7,9), and therefore additional management practices including the use of seed treatments may be necessary to protect germinating seedlings.
In Iowa, PRR as a seedling disease is becoming less important but more so as a mid- to late-season disease problem, primarily because soybeans are being planted earlier in the spring when soil temperatures are below favorable temperatures for P. sojae development. Thus, in Iowa, seed treatments for the control of damping off caused by P. sojae may not be necessary. Varieties with partial resistance would be a useful and practical management tool for a mid- to late-season disease problem, considering pathogen variability within fields and the possible appearance of new pathotypes. During the 2008 growing season, incidence of PRR in some fields in southeast Iowa was as high as 30% (A. Robertson, personal observation) as a result of field flooding that occurred in mid to late June, which indicated that genetic resistance to P. sojae is an important trait in soybean production in Iowa.
Phytophthora root and stem rot (PRR), caused by the oomycete Phytophthora sojae, is an economically important soybean disease in the north central region of the United States, including Iowa. Data regarding the pathogenic diversity of P. sojae within commercial fields is necessary to provide guidelines to both researchers and producers on deploying P. sojae-resistance genes in commercial varieties to protect soybean yields. In Iowa, Rps1k is the most common resistance gene used at present, followed by Rps1c, but the increased prevalence of race 25 (pathotype 1a, 1b, 1c, 1k, 7) in Iowa has prompted the incorporation of Rps6 into germplasm for Iowa. Two commercial fields with a history of PRR in Iowa were intensively sampled and the pathogenic diversity of isolates of P. sojae detected from each field determined. Our results indicated that in both fields the endemic population of P. sojae was pathogenically diverse. Of all the isolates detected, 25.8% were able to infect plants with the P. sojae resistance gene Rps1c, 51.6% were able to infect plants with Rps1k, and 35.5% could infect plants with Rps6. Our data suggest that stacked gene combinations or Rps genes in combination with high partial resistance (tolerance) might be necessary in order to protect soybean yields in Iowa.
We thank Greg Gephardt for assistance with soil sampling, and undergraduates who assisted with baiting. Funding for this work was provided by the Iowa Soybean Association and the North Central Soybean Research Program.
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