© 2003 Plant Management Network.
Evaluation of Soybean Plant Introductions from China for Resistance to Brown Stem Rot
Megan E. Patzoldt, Graduate Research Fellow, Department of Crop Sciences, 262 National Soybean Research Center, 1101 W. Peabody Drive, University of Illinois, Urbana 61801; Weidong Chen, Research Plant Pathologist, USDA-ARS, 303 Johnson Hall, Washington State University, Pullman 99614; and Brian W. Diers, Associate Professor, Department of Crop Sciences, 268 National Soybean Research Center, 1101 W. Peabody Drive, University of Illinois, Urbana 61801
Patzoldt, M. E., Chen, W., and Diers, B. W. 2003. Evaluation of soybean plant introductions from China for resistance to brown stem rot. Online. Plant Health Progress doi:10.1094/PHP-2003-0702-01-RS.
A new set of soybean accessions from south-central China were added to the USDA germplasm collection in 1996. Previous studies have shown that accessions with high levels of resistance to brown stem rot (BSR) can be found in germplasm collected from central and southern China. The objective of this study was to screen these accessions and identify those with resistance to BSR. In a preliminary study, 85 of 623 accessions tested were identified as resistant to BSR. In the second study, these 85 accessions were challenged with multiple biotypes of Phialophora gregata f. sp. sojae to identify those accessions with the strongest resistance. From these two studies, ten accessions were identified that had BSR resistance equal to or greater than the current resistant sources.
The soilborne fungus Phialophora gregata (Allington & Chamberlain) W. Gams f. sp. sojae Kobayashi, Yamamoto, Negishi, and Ogoshi, has been identified as the fungal pathogen that causes brown stem rot (BSR) in soybean [Glycine max (L.) Merr] (2,14). In early summer, P. gregata f. sp. sojae invades the root tissue of young soybean plants and then slowly spreads upwards in the plant vascular and pith tissues via mycelial and conidia production (20). Current control measures aimed at reducing yield losses from BSR infection rely on genetic resistance of the soybean and cultural practices. However, P. gregata f. sp. sojae has been shown to be highly variable in pathogenecity (7,10,19), and may overcome current plant resistance genes with time. Therefore, new sources of resistance are needed if genetic control of this disease is to continue.
There are two classification systems for describing isolates of P. gregata f. sp. sojae. The first system was described by Gray (10) and distinguishes between isolate types based on plant symptomology. Type I isolates cause distinct stem browning in the pith and vascular tissues. Foliar symptoms are caused by a toxin produced by Type I P. gregata f. sp. sojae isolates (11) and are characterized by interveinal chlorosis and necrosis leading to eventual defoliation. Type II isolates cause only browning in stem and petiole vascular and pith tissues with no leaf symptoms (10).
A newer classification system for P. gregata f. sp. sojae is based on fungal DNA polymorphisms (7). Specific primers were developed for the variable intergenic spacer (IGS) region in the nuclear DNA. Sequence data obtained from PCR products utilizing these primers reveal a 188 bp insertion/deletion of the IGS region between the two biotypes of P. gregata f. sp. sojae that infect soybean. Isolates that typically infect susceptible soybean cultivars are usually genotype A (the larger PCR product), whereas the isolates that typically infect resistant soybean cultivars are usually genotype B (the smaller PCR product). Hughes et al. (13) showed that genotype A isolates are the same as pathotype I isolates and that genotype B isolates are the same as pathotype II isolates described in the Gray classification system. Both pathotypes of P. gregata f. sp. sojae are routinely found in both resistant and susceptible germplasm.
Previous screenings of plant introductions (PIs) have been successful in identifying sources of BSR resistance and it is likely that there are other sources of BSR resistance present in the collection. In a screen of 2,060 accessions, Chamberlain and Bernard (6) identified the resistant accession PI 84946-2 from soybean lines collected in Korea. Germplasm from S. Korea, Japan, eastern USSR, and northeastern China was screened by Nelson et al. (17). Out of 3,400 accessions tested, 38 or 1.1% were identified as resistant to BSR. Recent disease screens of germplasm from China showed a higher rate of resistance. In germplasm from central China, Bachman et al. (3) identified 64 resistant accessions of 559 screened (11.4%). In germplasm from central and southern China, Bachman and Nickell (4) reported 29% (241 of 829) of the accessions they screened had resistance to BSR.
Field screening for BSR resistance can be difficult as infection and subsequent disease development are highly influenced by the environment (3,6,17). Factors such as soil fertility, inoculum levels (1,15), and isolate aggressiveness (8,10,19), along with air temperature (2,5,18), soil fertility (24), and soil moisture (16) all impact disease severity. Screening lines in the greenhouse for BSR resistance allows for control of these factors which can confound BSR progression, thereby improving the chance of discovering truly resistant lines. The objective of this study was to identify those accessions, in the new set from China, that have the highest level of BSR resistance.
Experiment 1: Single Isolate Screening
A new set of soybean accessions (PI 594392 - PI 594892) was obtained and added to the USDA soybean germplasm collection in 1996. These accessions originated in south-central China and there is no record that they have been previously tested for BSR resistance. During the winter seasons of 1999-2000 and 2000-2001, a preliminary screen for BSR resistance was conducted using a single isolate of P. gregata f. sp. sojae in a greenhouse as described in Sebastian et al. (21). The isolate used for the initial screening was OH2, classified as both a Type I (10) and a genotype A isolate (7). This isolate was selected because of its ability to remain pathogenic and produce both stem and foliar symptoms consistently in the greenhouse.
Liquid culture broth media, modified from Gray (10), was prepared using steamed, strained, and autoclaved seed from the BSR susceptible cultivar Century 84 (25). Broth solution was allowed to cool for 24 to 48 hours before inoculation with three agar plugs (approximately 3 mm2) from a P. gregata f. sp. sojae plate culture. Minimal agar for the plate culture was made from stem tissue of the susceptible cultivar Century 84, Bacto-agar (Difco Laboratories, Detroit, MI), and ddH20 (2). Liquid cultures were incubated in the dark at 24°C for four weeks and shaken once daily. Root dip inoculum was prepared by blending the seed broth cultures at high speed for 90 s. The initial concentration of conidial and mycelial fragments in the slurry was measured with a hemocytometer, and then adjusted to a final concentration of 1.2 × 106 propagules/ml. Carboxylmethylcellulose was added at a rate of 7.5 g/liter to help the fungus adhere to the roots. Inoculum was used within two hours of preparation.
The accessions were evaluated in the greenhouse for BSR resistance as described by Sebastian and Nickell (21). Briefly, ten seeds were planted in sand and when the plants reached the V2-3 growth stage (9), five uniform healthy plants were selected. Roots of each plant were rinsed, gently blotted dry, and collectively immersed in a beaker containing 50 ml of root dip inoculum for approximately 1 min. The five inoculated plants were then transplanted into 15 cm diameter steam-sterilized clay pots containing steam-disinfested 1:1 sand:top soil mixture. Remaining inoculum was poured over the roots and absorbed into the soil. The roots are then covered with the soil mixture to approximately 1 cm below the cotyledon. Each experimental unit was a single pot containing 5 plants. Previous experiments have shown that non-inoculated controls do not develop BSR symptoms in our greenhouse tests. Therefore, these controls were not included in this experiment so that all available bench space could be devoted the experiment. Plants in the greenhouse were grown under a 14-hour photoperiod with an average temperature ranging from 18°C at night to 24°C during the day. Plants were watered as needed and each pot received a weekly application of a 150 ml fertilizer solution containing 0.098 g N, 0.089 g P2O5, 0.085 g K2O, 0.12 mg chelated Cu, Mn, Zn, 0.5 mg B, and 0.24 mg chelated Fe.
When the plants reached R1-R2 growth stage (9) or about 6 to 8 weeks post inoculation, they were evaluated for BSR reaction. Foliar ratings were taken by counting the number of leaflets on each plant that exhibited foliar symptoms. These symptoms were reported as the proportion of nodes showing symptomatic leaves divided by the total number of nodes on the plant. Stem ratings were taken by splitting each stem longitudinally and then counting the number of nodes with brown pith tissue. The stem reactions were also reported as a proportion calculated by dividing the number of nodes showing browning by the total number of nodes in the plant. Disease data were collected and analyzed with a Proc GLM of SAS v.8.2.
Because of limited greenhouse space, the accessions were tested in seven sets that each included approximately 150 accessions and multiple pots of each control genotype. The accessions were not replicated in this first screen and pots were arranged in a completely randomized design for each set. Controls were replicated five times in each set and included the three resistance sources; Rbs1 in germplasm line L78-4094 (derived from PI 84946-2) (12), Rbs2 in PI 437833 (12), and Rbs3 in PI 437970 (26), and Century 84 as the susceptible control (25). The one hundred forty three accessions that showed resistance in the first round of testing were re-evaluated with isolate OH2 in a single replicate test.
Despite multiple inoculation sets used to complete the screen, there was no significant (P < 0.05) interaction between genotype and screening set, based on the response of control genotypes over all inoculation groups. Of the 623 lines available for testing, 85 accessions (13.6%) showed a lack of visible BSR disease symptoms (data not shown) over the two replications. Because of the large number of accessions identified as resistant, more sophisticated testing was needed to determine which accessions have the strongest resistance to P. gregata f. sp. sojae.
Experiment 2: Multiple Isolate Screening
Accessions that gave a resistant response after both inoculations with OH2 were inoculated with multiple isolates of P. gregata f. sp. sojae. Inoculum preparation and inoculation procedures were the same as described above. For this experiment, the inoculum contained equal parts of four highly pathogenic genotype A isolates and four highly pathogenic genotype B isolates (8) (Table 1). This inoculum mixture was used to identify accessions with a high level of resistance to multiple biotypes of P. gregata f. sp. sojae. The experiment was replicated twice with replicates spaced over time. Within each replicate, the genotypes were randomized, each accession was included once, and the control genotypes listed in Experiment 1 were included three times.
After inoculation with the eight isolates, Century 84 exhibited significantly (P < 0.001) greater BSR stem and foliar symptoms than the resistant controls, L78-7094 (Rbs1), PI 437833 (Rbs2), and PI 437970 (Rbs3) (Table 2). The level of visible infection for the resistant controls was varied, ranging from 4% stem infection for L78-4094 (Rbs1) to 20% stem infection for PI 437970 (Rbs3). This indicates that these genes did not provide an equal level of resistance to the mixture of eight P. gregata f. sp. sojae isolates.
Table 1. Collection history and categorization of highly pathogenic P. gregata f. sp. sojae isolates used in this study. Information was adapted from Chen (8).
a Available from the American Type Culture Collection as isolate ATCC MYA - 735.
b Available from the American Type Culture Collection as isolate ATCC MYA - 734.
Table 2. List of brown stem rot (BSR) resistant accessions from southeastern China, their maturity groups, and BSR stem and foliar ratings from the greenhouse root dip inoculation experiment.
a Percent of nodes that showed browning symptoms in stem pith tissues.
b Percent of nodes that showed foliar chlorosis and necrosis.
Ten of the 85 accessions tested in this study had less stem pith browning than the most resistant control genotype, L78-4094 (Rbs1) (Table 2). These accessions originated in the provinces Sichuan (1), Fujian (2), Hunan (2), Guizhou (3), and Yunnan (2) in south-central China. Of these ten, nine accessions had less foliar symptoms than PI 437970 (Rbs3), the control genotype with the least amount of foliar symptoms. Two accessions (PI 594536 and PI 594638B) had no visible pith browning in the stem in either replication, indicating a very high level of resistance to infection by P. gregata f. sp. sojae. It is possible that these lines may have been colonized by P. gregata f. sp. sojae but did not develop the characteristic browning of the pith tissues. Studies have demonstrated that pith browning may not always indicate the extent of colonization by P. gregata f. sp. sojae (22,23). The accessions PI 594500D, PI 594598A, PI 594637, PI 594650A, PI 594827, and PI 594858B had no detectible foliar BSR symptoms even though they showed slight stem browning. The results indicate that these accessions may be resistant to the toxin produced by P. gregata f. sp. sojae and may also be useful for further tests.
All ten selected Chinese accessions demonstrated high levels of resistance to even highly virulent strains of P. gregata f. sp. sojae and any of them could potentially be useful sources of resistance. The maturity group (MG) IV accessions will likely be the most useful in the short term to soybean breeders in the Midwest U.S.A., as they will be easiest to cross with northern soybean germplasm. However, the most important step in utilizing these accessions will be to determine if they have unique BSR resistance genes.
This research was supported by the United Soybean Board.
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