Development of a Prunus necrotic ringspot virus -based viral vector for characterization of gene functions and control of viral diseases in stone fruits. H. CUI AND A. WANG. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada
Virus has ability to induce gene silencing that degrades RNA molecules sharing similar nucleotide sequence, termed as virus-induced gene silencing (VIGS). VIGS may be used to silence a target gene to characterize its function in planta. VIGS also has potential to be used to control viral diseases by directly silencing the target virus or silencing a host factor gene that is required by the virus to fulfill its life cycle. To develop a viral vector suitable for studying gene functions as well as against viral pathogens in stone fruits, Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus in the world, was selected for this study. As the first step to develop a PNRSV-based vector, the incidence and induced symptoms of PNRSV were investigated. A total of 69 samples including 32 from cherry and 37 from peach were randomly collected from an experimental orchard in the Niagara Fruit Belt, Ontario, Canada. Enzyme-linked immunosorbent assay (ELISA) showed that 18 out of 32 cherry trees and 12 out of 37 peach samples were infected by PNRSV with an average incidence of 43.5%. The presence of PNRSV in those ELISA-positive samples was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). No obvious phenotypic differences were observed among PNRSV-positive or negative trees. Phylogenetic analysis of the coat protein gene clustered these 30 isolates into two groups, PV96 and PV32, with the former as the predominant group. The entire genome, including RNAs1, 2 and 3 of two PNRSV isolates, Chr3 from cherry (the PV96 group) and Pch12 from peach (the PV32 group), were cloned and sequenced. Sequence analyses revealed that RNAs 1 and 2 are monocistronic, encoding proteins P1 and P2, respectively, and RNA 3 is bicistronic, encoding the 5'-proximal movement protein (MP) and the 3'-proximal coat protein (CP). The full-length cDNAs of PNRSV isolates Chr3 and Chr12 were further cloned into a binary vector under the control of 35S promoter. Agrobacterium-mediated transfection was used to deliver this vector into two herbaceous model plant species, Nicotiana benthamiana and Cucumis sativus, and its natural host, peach (Prunus persicae). ELISA and RT-PCR results indicated that those constructs were infectious in all the three plant species tested. Although mosaic, necrotic and ringspot symptoms were found in C. sativus, no symptoms were evident in P. persicae or N. benthamiana. Taken together these data suggest that PNRSV is highly endemic in the Niagara region and suitable for being developed to study gene functions and develop VIGS-based resistance to viral diseases in stone fruits.
Fungicides for control of stemphylium leaf blight in onions. M. T. TESFAENDRIAS AND M. R. MCDONALD. Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
Stemphylium leaf blight of onion (Allium cepa L.), caused by Stemphylium vesicarium (Wallr.) Simmons, has been observed in the Holland/Bradford Marsh, Ontario since 2008. In 2010, yield losses were associated with this disease. There are no registered fungicides for this disease on onions. In 2011, a field trial was conducted to determine the efficacy of several fungicides on stemphylium leaf blight severity. A randomized complete block design with four replicates per treatment was used. Treatments were: Pristine (pyraclostrobin 25.2%, boscalid 12.8%), Bravo 500 (chlorothalonil 50%), Manzate 750F (mancozeb 75%), Switch 62.5WG (cyprodinil 37.5%, fluodioxinil 25.0%), Fontelis 20SC (penthiopyrad 20%), Inspire (difenoconzole 23.2%) and Luna Tranquility (fluopyram 11.3%, pyrimethanil 33.8%). An untreated control was also included. Plots were sprayed six times at weekly intervals starting 12 July and assessed weekly. Stemphylium leaf blight was rated using a scale 0–9, where 9 = >85% foliar area diseased per plot. After the final fungicide application, ten plants from each replicate were pulled and assessed for percent of foliage infected. All of the fungicides reduced disease severity compared to the untreated control. The new products, Fontelis, Luna Tranquility and Inspire were most effective in reducing stemphylium leaf blight with 19.8, 20.7 and 24.5% foliage with symptoms respectively, as compared to 67% in the untreated control. No differences in yield or size distribution were found. However, marketable yield decreased in relation (r = −0.45) to increasing leaf length with disease symptoms. The percent of cull onions also increased (r = 0.43) with an increase in disease severity.
The influence of soil moisture and Rhizoctonia solani anastomosis and intraspecific group on the incidence of damping-off and the incidence and severity of rhizoctonia crown and root rot in sugar beet. C. L. TRUEMAN AND L. E. HANSON. Ridgetown Campus, University of Guelph, 120 Main Street East, Ridgetown, ON N0P 2C0, Canada; and (L.E.H.) USDA-ARS, East Lansing, MI 48824, USA
Rhizoctonia crown and root rot [Rhizoctonia solani Kühn] reduces plant stand, sugar quality and yield in sugar beet. To evaluate the influence of R. solani anastomosis (AG) and intraspecific groups and soil moisture on disease development, a trial was established in Ridgetown, Ontario using the susceptible variety ‘Crystal 824’. Four moisture treatments (rain-fed and drip irrigation once, twice or three times per week) and three R. solani isolates identified as AG-2-2 IIB, AG-2-2 IV, and AG-4 were included. Plant stand declined 30 percent more in soil inoculated with AG2-2 IIIB than AG-2-2 IV or AG-4. There were no differences among moisture treatments and no interaction among R. solani isolate and moisture treatments. Disease incidence was higher in beets irrigated once per week than in beets not irrigated or irrigated two times per week 64 days after seeding (DAS) but not 96 and 137 DAS, which may indicate that seedling infection is more responsive to moisture than mature plant infection. Disease incidence and severity for AG-2-2 IIIB were higher than AG-2-2 IV and AG-4 on all dates. Beet weight was lower in plots inoculated with AG-2-2 IIIB than AG-2-2 IV or AG-4 at 137 DAS and there were no differences among moisture treatments. Preliminary results agree with previous reports that AG-2-2 IIIB is more aggressive at the seedling and mature beet stages than AG-4 and AG-2-2 IV, and this is true regardless of the frequency of irrigation events. Further analysis of actual soil moisture and temperature data and disease severity is required.
Understanding and control of plant viruses. A. WANG. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada
Plant viruses cause multi-billion US dollars of yearly loss in crop yields in the world. Genetic resistance is considered the most effective means to control these viruses. Unfortunately, resistant resources in nature are very rare. The development of novel antiviral strategies relies on a better understanding of the virus infection process such as replication and movement. In the past several years, we used three potyviruses, i.e., Turnip mosaic virus, Soybean mosaic virus, and Tobacco etch virus as model virus to study virus-plant interactions. We have shown that the potyviral 6K membrane protein is responsible for inducing the formation of ER-derived membranous vesicles. The vesicular pathway and actomysin motility system are required for the trafficking of the 6K vesicles from the ER to chloroplasts. We also have shown that the chloroplast-associated vesicles contain viral RNA, double-stranded RNA, and viral replicase components in infected cells. Thus, potyviruses sequentially recruit the endoplasmic reticulum and chloroplasts for their genome replication. These studies support the assumption that plant viruses encode only a very limited set of proteins, and, therefore, largely depend on host gene products to fulfill their life cycle. We have also applied four genomics approaches, i.e., expression sequence tags, microarray (both oligoarray and cDNA array), cDNA-AFLP, and the yeast-two hybrid system to study gene expression networks in peach (Prunus persica), soybean (Glycine max), Arabidopsis thaliana, and Nicotiana benthamiana in response to potyviral infection. Through these studies, we have constructed a database containing over 4000 genes associated with potyviral infection. Moreover, we have evaluated approximately 200 genes and identified eight genes that may be manipulated against these viruses. Moreover, we engineered genetic resistance into the natural host of some potyviruses such as Plum pox virus through the hairpin-mediated RNA silencing (RNAi) approach. The resistant transgenic plants generated provide an alternative resistant source for the control of potyviruses.