1.11.37
CHARACTERIZATION AND CONTROL OF SOYBEAN SEVERE STUNT, A NEW SOILBORNE VIRUS DISEASE AFFECTING SOYBEAN IN THE USA

TA EVANS, RP MULROONEY and RB CARROLL

Department of Plant and Soil Science, University of Delaware, Newark, USA

Background and objectives
Soybean severe stunt (SSS) is a soilborne disease caused by the soybean severe stunt virus (SSSV) affecting soybeans (Glycine max) in Delaware, USA [1]. At present, ca 60 fields are known to be affected by SSS. Symptoms occur on the first true leaves, and infected plants have shortened internodes resulting in severe stunting, thickened, dark-green mottled leaves, and a reduced number of flowers, pods and seeds. The virus can kill plants and is associated with a reduction in germination and seedling emergence. In the field, the disease occurs in localized, circular areas. SSSV is transmitted through soil, and dagger nematodes, Xiphinema americanum, are consistently associated with infected plants in the field. Greenhouse studies using field soils infested with the dagger nematode and planted to susceptible soybean cultivars consistently resulted in transmission of SSS and recovery of SSSV from symptomatic plants. To date, all attempts to transmit SSSV using single nematodes in vitro have failed. SSSV has been partially characterized and determined to have 29-30 nm diameter, labile virions that are difficult to purify intact [1]. SSSV has tested negatively with antisera for all nepoviruses known to affect soybean in the USA and a number of other isometric labile viruses, including ilarviruses [1], and no SSSV-specific antiserum has yet been produced. A main objective of this research is to identify resistant soybean cultivars and to develop specific cDNA probes for the detection of SSSV and its vector in infected plants.

Materials and methods
30 soybean cultivars in 1995, 24 in 1996, and 34 in 1997 (maturity groups (V and V) were evaluated for resistance to SSSV. Field trials were in loamy-sand soils that had a history of SSS. Beginning in 1996, all soybean cultivars tested in the field were also screened for resistance to SSSV in the greenhouse using a newly developed soybean root inoculation bioassay. Characterization of SSSV continues and we are developing CDNA probes specific for it. Purification of intact virions of SSSV has been problematic. Instead, total RNA isolations were carried out directly using systemically infected soybean or N. benthamiana. Both total RNA and poly(A)-RNA from healthy and SSSV-infected plants were treated with RNase and DNase following standard protocols, and bioassayed. Work in progress includes double stranded (ds) RNA isolation and differential display using random primers.

Results and conclusions
The cultivars Stine S4650, Defiance, Cisne, Delsoy 4710 and Chesapeake were very resistant to SSSV in field and greenhouse tests, and produced acceptable yields [2]. Untreated poly(A)-RNA from SSSV-infected soybean plants was infective and was inactivated by RNase but not by DNase treatment. Results of dsRNA isolation and analyses were inconclusive. All four bands of CMV dsRNA (doublet and two other bands) were detected but no dsRNA was detected from healthy soybean or SSSV-infected soybean leaf tissue 5 weeks after infection. Differential display using random primers with total RNA from healthy and SSSV-infected tissue has produced probably unique bands which we are attempting to PCR.

References
1. Weldekidan T, Evans TA, Carroll RB, Mulrooney RP, 1992. Plant Disease 76, 747-750.
2. Mulrooney RP, Evans TA, Taylor RW, 1997. Biological and Cultural Control Tests 12, 85.