3.3.23
A MOLECULAR METHOD FOR THE SPECIFIC DETECTION OF PEA SEEDBORNE MOSAIC VIRUS

VA TOROK and JW RANDLES

Department of Crop Protection, University of Adelaide (Waite Campus), PMB 1, Glen Osmond SA 5064, Australia

Background and objectives
Pea seedborne mosaic potyvirus (PSbMV) infects pea and other legumes. It is both seed and aphid borne so that a low level of seed infection can lead to a high incidence of disease resulting in decreased crop yield and quality. Three PSbMV pathotypes (PSbMV pathotype P-1, P-4 and L-1) have been recognised by their ability to infect a number of pea differential genotypes [1]. The virus is believed to have spread world-wide through the exchange of infected germplasm. It was first introduced into Australia through quarantine in 1978 from seed imported from Sweden. Over the past 3 years 16 PSbMV isolates have been collected from 3 states in Australia from breeders' seed lines, field trials, and commercially available pea cultivars.

PSbMV is generally identified by symptomatology or ELISA. We are developing a sensitive and specific detection system for PSbMV based on the reverse transcription polymerase chain reaction (RT-PCR). This test targets a part of the PSbMV genome that is specific to PSbMV and not other legume infecting potyviruses.

Materials and methods
Twenty-three full length potyviral genome sequences and two PSbMV types isolate sequences were obtained from the genome database "ANGIS" and compared using the multiple sequence alignment program "clustalw". Regions specific to PSbMV and not the other potyviral sequences were selected and PCR primers designed. Total nucleic acids were extracted from infected leaf tissue using a modified CTAB method. Reverse transcription (RT) was primed with primer VT04, targeted to a region of the P3 gene, to generate cDNA and polymerase chain reaction (PCR) was done with nested primers VT02 and VT03, targeted to regions in the HC-Pro gene. RT-PCR was carried out on 16 PSbMV isolates collected in Australia, 14 PSbMV isolates collected in Pakistan [2] and 2 PSbMV type isolates, as well as with the other legume infecting potyviruses: clover yellow vein virus (ClYVV), bean yellow mosaic virus (BYMV), pea mosaic virus (PMV), lettuce mosaic virus (LMV), bean common mosaic virus (BCMV), peanut mottle virus (PeMV) and turnip mosaic virus (TuMV). Published general potyviral primers were used as controls [3].

Results and conclusions
RT-PCR with VT04 and nested primers VT02/VT03 generated a single 1.1kb fragment from PSbMV infected tissue but not with nucleic acids from healthy pea or ClYVV, BYMV, PMV, LMV, TuMV, BCMV, PeMV or TMV infected samples. RT-PCR on the same samples with the general potyvirus primers generated a fragment of 1.6-2kb from those samples infected with a potyvirus but not from healthy pea or the unrelated tobamovirus, TMV. Sensitivity was limited by the RT step with reliable detection of PSbMV RNA at 100pg in a 10ul RT reaction mixture. RT-PCR for PSbMV is 103-105 times more sensitive than the serological test DIBA.

RT-PCR with VT04 and the nested primers VT02/VT03 is sensitive and specific for PSbMV and not other legume infecting potyvirus, but is not PSbMV isolate specific because it detected all of the 32 PSbMV isolates which included representatives of several pathotypes.

This RT-PCR is to be developed further for virus diagnosis in pea germplasm.

References
1. Alconero R, Provvidenti R, Gonsalves D, 1986. Plant Disease 70, 783-786
2. Ali A, Randles JW, 1997. Plant Disease 81, 343-347
3. Gibbs A, Mackenzie A, 1997. Journal of Virological Methods 63, 9-16.