Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7.

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified. One of those species, GLRaV-7, was first reported from a symptomless white-fruited wine grape cultivar from Albania. Since its discovery, GLRaV-7 has been reported from 14 countries. Although serological assays have been developed to detect GLRaV-7, commercially available antibodies produce high background signals making them unsuitable for regulatory testing. Furthermore, while molecular detection assays have been shown to be more sensitive when compared to the serological assays, published molecular assays, except the one Reverse Transcription-quantitaive Polymerase Chain Reaction (RT-qPCR) assay based on heat shock protein 70 homologue (HSP70h) gene, have been reported to be inadequate in detecting all reported isolates of GLRaV-7. Availability of multiple assays provides flexibility to diagnostic laboratories in cases where the chosen assay fails to detect a strain or an isolate of a pathogen due to variation in its targeted region or where additional confirmation of the results is required. In this study, we developed a sensitive and specific RT-qPCR assay, based on a region of p61 gene of GLRaV-7, which detected all available isolates.

The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains.

We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed at low but detectable levels. Posttranscriptional gene silencing using VIGS resulted in a compatible reaction with a normally incompatible stem rust pathogen. Allele sequencing also validated the candidate Rpg5 gene. Allele and recombinant sequencing suggested that the probable rpg4 gene encoded an actin depolymerizing factor-like protein. Involvement of actin depolymerizing factor genes in nonhost resistance has been documented, but discovery of their role in gene-for-gene interaction would be novel and needs to be further substantiated.

First Report of Hemp Canker Caused by Sclerotinia sclerotiorum in Alberta, Canada.

Hemp (Cannabis sativa L.) is a herbaceous annual grown mainly for its blast fiber and seed oil. In 1999, Health Canada issued licenses to plant 12,145 ha of hemp in Canada. Of these, 730 ha were in Alberta. During the last week of August, hemp plants (cv. Fasamo) in a central Alberta field showed the following symptoms and signs: wilting foliage turning light brown; dry tan to gray lesions on stems; shredding and breaking of stems at the lesion; presence of white mycelium in the lesion; and black round, irregular, or oblong sclerotia (up to 5 mm diameter and 2 to 11 mm long) present externally at the lesion on the stem and inside the pith cavity. Lesions were found at the crown, near the inflorescence, and along the entire stem length. Disease incidence in a survey of six commercial fields (40 ha) ranged from 1 to 8%. The organism isolated from lesions on potato dextrose agar produced white aerial mycelia and large numbers of sclerotia characteristic of Sclerotinia sclerotiorum. Pathogenicity was confirmed by inoculating 23-day-old greenhouse-grown hemp plants (cv. Fasamo) with autoclaved wheat grains colonized for 14 days with a S. sclerotiorum culture previously isolated from an infected hemp plant. The grains were placed on soilless growing medium near the plant and covered very lightly. One week after inoculation, grayish lesions appeared on the stems, white mycelia appeared on lesions, and plants wilted. The pathogen was reisolated from the lesions. This is the first report of S. sclerotiorum on hemp in Alberta, Canada. The disease known as hemp canker has been reported to cause severe losses under cool wet conditions in the Netherlands (1). Reference: (1) J. M. McPartland. J. Int. Hemp Assoc. 3:19, 1996.

First Report of Powdery Mildew of Greenhouse-grown Tomatoes in Alberta, Canada.

In Alberta, powdery mildew disease of greenhouse-grown tomatoes (Lycopersicon esculentum Mill.) appeared for the first time in 1995 as circular white colonies on leaves, petioles, and stems. Since then it has been found every year and is becoming an economically important disease of tomatoes. This is the first report of the disease from Alberta, Canada. In Canada, powdery mildew on greenhouse tomatoes was previously reported in 1994 from the province of Quebec (1). The pathogen had unbranched conidiophores with average length and width of 62.3 µm and 8.5 µm, respectively. Conidia were clear or hyaline, elliptical to oval in shape, and were borne singly or in short chains. Average length and width of conidia were 36.0 µm and 17.6 µm, respectively. The conidia contained numerous vacuoles but fibrosin bodies were not observed. Germ tubes were straight and formed at the ends or very close to the ends of conidia. Rarely, a conidium produced two germ tubes. Cleistothecia were not found. Six-week-old, greenhouse-grown, healthy tomato cv. Trust plants were inoculated by shaking conidia from powdery mildew-infected plants onto the leaves of the healthy plants. The plants developed powdery mildew symptoms within 9 days after the inoculation. The symptoms on inoculated plants and morphological characteristics of the pathogen were similar to those for naturally infected plants. Based on the characteristics of the asexual stage, the pathogen was identified as an Erysiphe sp. until its identity is confirmed by the characteristics of its sexual stage. An Erysiphe sp. has also been reported to cause powdery mildew of greenhouse-grown tomatoes in Canada (1), the U.S. (3), and Spain (2). Optimal temperature and relative humidity for germination of conidia of the pathogen were 20 to 25°C and >90%, respectively. Myclobutanil, fenarimol, sulfur, triademefon, and triforine showed promise for effective management of this disease. Myclobutanil and sulfur are now registered for control of this disease in Canada. Since cleistothecium has not been found, there is a need to identify the sources of primary inoculum to understand the disease cycle and effective management of the disease. References: (1) R. R. Bélanger and W. R. Jarvis. Plant Dis. 78:640, 1994. (2) L. Olalla and J. A. Torés. Plant Dis. 82:592, 1998. (3) J. F. White, Jr., et al. Plant Dis. 81:227, 1997.