A novel mite-transmitted virus with a divided RNA genome closely associated with pigeonpea sterility mosaic disease.

ABSTRACT The agent of sterility mosaic, a disease that is a major constraint on pigeonpea (Cajanus cajan) production in the Indian subcontinent, is transmitted by the eriophyid mite, Aceria cajani. This agent has remained elusive for decades despite intensive efforts but we report the isolation of highly flexuous filamentous virus-like particles (VLPs) of 3 to 10 nm in width and of undefined lengths from sterility mosaic disease (SMD)-affected pigeonpea plants. Purified VLP preparations from virus-infected pigeonpea and Nicotiana benthamiana had a buoyant density in cesium chloride of 1.22 to 1.23 g cm(-3) and contained a major virus-specific protein species of approximately 32 kDa and 5 to 7 RNA species of approximately 6.8 to 1.1 kb. The sequence of some complementary DNA clones to RNA from purified VLP preparations had no significant matches in database searches. Two oligonucleotide primers derived from one such sequence, when used in reverse transcriptase-polymerase chain reaction assays, amplified a product of 321 bp specifically from SMD-affected pigeonpea plants. Purified VLP preparations were used to produce polyclonal antibodies that, in infected plants, detected the virus using enzyme-linked immuno-sorbent assay (ELISA) and the virus-specific 32-kDa protein in western immunoblotting (WIB). In such assays, the virus was detected consistently in all SMD-affected pigeonpea plant samples from several different locations in India, but not in samples from symptom-free pigeonpea plants from the same locations. In experimental studies, all pigeonpea plants inoculated with viruliferous A. cajani and those plants graft-inoculated with SMD-affected tissue were infected with the virus as assessed by ELISA and WIB, but not any uninfected pigeonpea plants. This virus, tentatively named Pigeonpea sterility mosaic virus (PPSMV), has some properties similar to virus species in the genera Tospovirus and Tenuivirus and with the eriophyid mite-transmitted High plains virus (HPV) but is distinct from these and from all other characterized viruses. The combination of novel properties shown by PPSMV and HPV suggest that they may constitute species in a new genus of plant viruses.

Transmission of Pigeon pea sterility mosaic virus by the Eriophyid Mite, Aceria cajani (Acari: Arthropoda).

The transmission characteristics of Pigeon pea sterility mosaic virus (PPSMV) to pigeon pea (Cajanus cajan) by its eriophyid mite vector, Aceria cajani, were studied. Nonviruliferous A. cajani colonies were established on detached healthy leaflets of a PPSMV-immune pigeon pea cultivar floating on water. The transmission efficiency of single A. cajani was up to 53% but was 100% when >5 mites per plant were used. A. cajani acquired PPSMV after a minimum acquisition access period (AAP) of 15 min and inoculated virus after a minimum inoculation access period (IAP) of 90 min. No latent period was observed. Starvation of A. cajani prior to, or following, PPSMV acquisition reduced the minimum AAP and IAP periods to 10 min and 60 min, respectively, and mites retained virus for up to 13 h. None of the mites that developed from eggs taken from PPSMV-infected leaves transmitted the virus, indicating that it is not transmitted transovarially. Taken together, these data suggest a semipersistent mode of transmission of PPSMV by A. cajani.

Improved PCR Detection of Blackcurrant reversion virus in Ribes and Further Evidence that It Is the Causal Agent of Reversion Disease.

Within 5 years of mechanically inoculating blackcurrant cultivars with partially purified preparations of particles of Blackcurrant reversion virus (BRV), infected plants developed leaf and flower bud symptoms typical of reversion disease, demonstrating that BRV is the causal agent of this disease. To improve the erratic immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) detection of BRV in Ribes plants, various stepwise changes were made to the original protocol. Significant improvement in the reliability and sensitivity of BRV detection was made by extracting RNA from trapped BRV particles using Triton-X 100, the design of new primers with higher annealing temperatures, and the use of 'Ready-to-go' RT-PCR beads. These features, combined with other minor changes to the protocol, improved BRV detection in reverted blackcurrant plants from <50% to >90% but the reliability of BRV detection in red currant was always very much less and was possible only using nested PCR that was developed for this purpose.

A New Badnavirus in Ribes Species, its Detection by PCR, and its Close Association with Gooseberry Vein Banding Disease.

Gooseberry vein banding disease (GVBD) affects Ribes species and cultivars worldwide. It is the second most important virus-like disease in these crops after black currant reversion disease. In this paper, we describe a bacilliform virus, Gooseberry vein banding associated virus (GVBAV), which is associated closely with GVBD, and provide evidence that GVBAV is a distinct species within the genus Badnavirus. Purified GVBAV particles were ca. 120 × 30 nm in size and contained dsDNA. The sequence of a 1.5-kb DNA fragment amplified from viral genomic DNA was similar to those of a wide range of badnaviruses and contained motifs characteristic of the RNase H domain of the badnavirus open reading frame (ORF) III polyprotein. Phylogenetic analyses suggest that GVBAV is most closely related to Spiraea yellow leaf spot virus. Using sequence derived from the polymerase chain reaction (PCR)-amplified DNA fragment, virus-specific primers were designed. These primers were used in PCR to assay for GVBAV in a range of Ribes germplasm affected with GVBD, with other unrelated virus-like diseases and viruses found in Ribes, and in healthy plants. GVBAV was detected in all of 58 GVBD-affected plants from diverse sources, but not from healthy Ribes plants nor from plants infected with other viruses.

Incidence and Distribution of Raspberry bushy dwarf virus in Commercial Red Raspberry (Rubus idaeus) Crops in Scotland.

A survey was done in 1998 to determine whether Raspberry bushy dwarf virus (RBDV) was established in raspberry fruiting plantations in Scotland. Raspberry-producing holdings were selected according to geographical area and size. Samples (201), each comprising 60 shoots per stock, were obtained from 77 holdings and tested by enzyme-linked immunosorbent assay (ELISA). ELISA-positive shoots from each infected stock were grafted onto cultivar Glen Clova, which is resistant to the Scottish-type isolate of RBDV (RBDV-S), to establish whether the virus is a resistance-breaking (RB) isolate. RBDV was detected in 22% of the stocks sampled, with 2 to 80% incidence of infection. No RBDV was in any of the 40 plantations containing cultivars resistant to RBDV-S or in Glen Clova plants, which were grafted successfully with samples from 15 infected plantations, indicating that no RB isolates were detected. The percentage of infected plantations increased with time from the planting date. In order to investigate possible sources of infection, ELISA for RBDV was made in 1999 on samples of stocks of raspberry cultivars entered for the lowest certified grade (Standard Grade) in Scotland and, in 1994 to 1997, on certified stocks planted with material originating from outside Scotland. No RBDV was detected in any of the samples. RBDV was found only rarely in samples of wild raspberry in Angus and Perthshire.