Comparison between woody indexing and a rapid hybridisation assay for the diagnosis of little cherry disease in cherry trees*

Abstract

Sweet cherry (Prunus auium) and sour cherry (Prunus cerasus) trees from orchards in the Kootenay and Okanagan Valleys of British Columbia, Canada were assayed for the presence of little cherry disease by three different methods: Northern blot analysis of double-stranded RNA, woody indexing for fruit symptoms on sweet cherry cv. Lambert, and woody indexing for foliar symptoms on cv. Canindex 1. Results of the three methods were in agreement for 85% of the samples. Of the 78 orchard trees tested, double-stranded RNA isolated from 48 trees hybridised with a radiolabelled cloned probe specific for little cherry disease. When the 48 trees were tested by woody indexing, buds from 41 trees induced fruit symptoms on cv. Lambert, but only 32 yielded foliar symptoms on cv. Canindex 1 under the conditions of the experiment. Of the 30 orchard trees that did not yield a positive response to the Northern blot analysis, 26 samples were negative on cv. Lambert and 26 were negative on cv. Canindex 1. Northern blot analysis of the 78 cv. Lambert indicator trees revealed that there was an absolute correlation between the presence of little cherry disease-associated double-stranded R N A and the development of typical little cherry disease symptoms on the indicator trees. Reliability of woody indexing of orchard samples was impaired by poor transmission of the disease from the inoculating bud to the indicator tree. Woody indexing with cv. Canindex 1 was particularly prone to a large number of apparently erroneous negative results. Of the three protocols used, diagnosis of little cherry disease by Northern blot analysis was found to be the most reliable and offered a greatly accelerated means of diagnosis.

High-Throughput Sequencing Assists Studies in Genomic Variability and Epidemiology of Little Cherry Virus 1 and 2 infecting Prunus spp. in Belgium

“…The LChV-1 and -2 incidence and observed variability in all surveyed regions can mainly be attributed to the contribution of different plant material sources, their co-infectious status and their mode of propagation in long-established orchards. In a context of rapid and widespread disease worldwide, aspects like multiple infections in symptomless plant reservoirs are readily known to play a key epidemiological role [8,21,27,28,29,30,31,32,33,34,35]. Severe damage has occurred on sweet and sour cherry trees on which the LChD was observed, often leading, together with possible other biotic and abiotic factors, to full tree decline in devastated old orchards, as has been reported for most sensitive sweet cherry cultivars ([8], Supplementary Material).…”

“…Prior to RT-PCR, cDNA was synthesized from a representative selection of RNA samples with an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the supplier’s instructions. The generated cDNA was used as a template for different PCR reactions using specific primers [22,23,29,30] or primers designed for this study (see Table S1). Amplifications were carried out in a total volume of 25 μL of PCR mixture containing FastStart ™ Taq DNA Polymerase reaction mix (Roche, Mannheim, Germany) in an ABI9700 GeneAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA).…”

“…The wide intra- and inter-host genetic diversity of LChV-1 and LChV-2 has been characterized only in a few countries [2,3,4,27,28,29,30,31,32,33,34,35,36]. Both viruses are confirmed to be present in Belgium, however no comprehensive phylogenetic characterization of the LChD has been published so far.…”

Partial Nucleotide Sequence and Genome Organization of a Canadian Isolate of Little cherry virus and Development of an Enzyme-Linked Immunosorbent Assay-Based Diagnostic Test

“…LChV-3 p39 antiserum was tested for its ability to react to several LChV-3 variants collected from British Columbia (7) and from an isolate from the Netherlands and an isolate from Germany originally from flowering cherry. Several of the isolates gave positive reactions (J. Theilmann and D. Rochon, unpublished data), suggesting that the p39 antiserum may be useful in detecting a wide range of isolates.…”

“…Transmission of LChD can also occur by grafting or budding (13). Several studies suggest the partial or sole involvement of a virus in LChD (5,7). Moreover, the cloning and sequencing of an approximately 0.5-kb segment of the double-stranded (ds)RNA associated with LChD has been reported, and nucleotide sequence comparisons indicate that the dsRNA is derived from the single-stranded (ss)RNA genome of a closterovirus (previously designated Little cherry virus [LChV]-LC5 but identical to LChV-3 described here) that is phylogenetically related to other mealybug-transmitted closteroviruses (6).…”