Mysore R. Sudarshana

Fate of Escherichia coli O157:H7 in field-inoculated lettuce.

Impact of drip and overhead sprinkler irrigation on the persistence of attenuated Escherichia coli O157:H7 in the lettuce phyllosphere was investigated using a split-plot design in four field trials conducted in the Salinas Valley, California, between summer 2007 and fall 2009. Rifampicin-resistant attenuated E. coli O157:H7 ATCC 700728 (BLS1) was inoculated onto the soil beds after seeding with a backpack sprayer or onto 2- or 4-week-old lettuce plant foliage with a spray bottle at a level of 7 log CFU ml⁻¹. When E. coli O157:H7 was inoculated onto 2-week-old plants, the organism was recovered by enrichment in 1 of 120 or 0 of 240 plants at 21 or 28 days post-inoculation, respectively. For the four trials where inoculum was applied to 4-week-old plants, the population size of E. coli O157:H7 declined rapidly and by day 7, counts were near or below the limit of detection (10 cells per plant) for 82% or more of the samples. However, in 3 out 4 field trials E. coli O157:H7 was still detected in lettuce plants by enrichment 4-weeks post-inoculation. Neither drip nor overhead sprinkler irrigation consistently influenced the survival of E. coli O157:H7 on lettuce.

Limitations on Geminivirus Genome Size Imposed by Plasmodesmata and Virus-Encoded Movement Protein: Insights into DNA Trafficking

Animals and plants evolved systems to permit non-cell-autonomous trafficking of RNA, whereas DNA plays a cell-autonomous role. In plants, plasmodesmata serve as the conduit for this phenomenon, and viruses have evolved to use this pathway for the spread of infectious nucleic acids. In this study, a plant DNA virus was used to explore the constraints imposed on the movement of DNA through this endogenous RNA trafficking pathway. The combined properties of the geminivirus-encoded movement protein and plasmodesmata were shown to impose a strict limitation on the size of the viral genome at the level of cell-to-cell movement. Size-increased viral genome components underwent homologous and nonhomologous recombination to overcome this strict limitation. Our results provide insights into the genetic mechanisms that underlie viral evolution and provide a likely explanation for why relatively few types of plant DNA viruses have evolved: they would have had to overcome the constraints imposed by an endogenous system operating to ensure that DNA acts in a cell-autonomous manner.

Complete Genome Sequence of a Novel Vitivirus Isolated from Grapevine

ABSTRACT A novel virus-like sequence from grapevine was identified by Illumina sequencing. The complete genome is 7,551 nucleotides in length, with polyadenylation at the 3′ end. Translation of the sequence revealed five open reading frames (ORFs). The genomic organization was most similar to those of vitiviruses. The polymerase (ORF1) and coat protein (ORF4) genes shared 31 to 49% nucleotide and 40 to 70% amino acid sequence identities, respectively, with other grapevine vitiviruses. The virus was tentatively named grapevine virus F (GVF).

Biology and Molecular Characterization of Cucurbit leaf crumple virus, an Emergent Cucurbit-Infecting Begomovirus in the Imperial Valley of California

Cucurbit leaf crumple virus (CuLCrV) is an emergent and potentially economically important bipartite begomovirus first identified in volunteer watermelon plants in the Imperial Valley of southern California in 1998. Field surveys indicated that CuLCrV has become established in the Imperial Valley; and field plot studies revealed that CuLCrV primarily infects cucurbits, including cantaloupe, squash, and watermelon. Full-length DNA-A and DNA-B clones of an Imperial Valley isolate of CuLCrV were obtained by polymerase chain reaction (PCR) with overlapping primers. These clones were infectious in various cucurbits and common bean (cv. Topcrop); symptoms included stunted growth and leaf crumple, curl, and chlorosis. CuLCrV was not sap-transmissible, and immunolocalization and DNA in situ hybridization studies revealed that it is phloem-limited. A CuLCrV agroinoculation system was generated, and host range studies revealed differential susceptibility in cucurbits, with squash, watermelon, cantaloupe, and honeydew melon being most to least susceptible, respectively. Germplasm screening studies identified a number of resistant cantaloupe and honeydew melon cultivars. The genome organization of this CuLCrV isolate (CuLCrV-CA) is similar to other bipartite begomoviruses, and phylogenetic analysis placed CuLCrV in the Squash leaf curl virus (SLCV) cluster of New World bipartite begomoviruses. A CuLCrV-specific PCR test was developed which allows for differentiation from other begomoviruses, including SLCV.

Phylogeny of Geminivirus Coat Protein Sequences and Digital PCR Aid in Identifying Spissistilus festinus as a Vector of Grapevine red blotch-associated virus

Grapevine red blotch-associated virus (GRBaV) is a single-stranded DNA virus, proposed to be a member of the family Geminiviridae, and is associated with grapevines showing red blotch symptoms in North America. The existence of the virus was reported in 2012, and subsequently detected in grapevines in major grape production regions. We investigated if a vector exists that can transmit GRBaV in vineyards. Phylogenetic analysis of the predicted amino acid sequence of coat protein (CP) of GRBaV with the CP of 23 geminiviruses representing all seven genera of the family Geminiviridae revealed that GRBaV-CP was most similar to that of Tomato pseudo-curly top virus, a geminivirus known to be transmitted by a treehopper (Membracidae), a family that is closely related to leafhoppers (Cicadellidae). To identify vectors of GRBaV, hemipteran species within and nearby wine grape vineyards where virus spread was suspected were collected and transmission assays were conducted. Among the species tested, the three-cornered alfalfa hopper Spissistilus festinus (Hemiptera: Membracidae) was able to both acquire the virus from a grapevine infected with GRBaV and transmit the virus to healthy grapevines in the laboratory. In commercial vineyards, lateral shoots of grapevines girdled due to feeding injury by the adult three-cornered alfalfa hopper also tested positive for the virus using digital PCR. These findings represent an important step in understanding the biology of GRBaV and develop management guidelines.

Detection of Grapevine leafroll-associated virus 7 using real time qRT-PCR and conventional RT-PCR

Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.

Lettuce infectious yellows virus-encoded P26 induces plasmalemma deposit cytopathology

Lettuce infectious yellows virus (LIYV) encodes a 26 kDa protein (P26) previously shown to associate with plasmalemma deposits (PLDs), unique LIYV-induced cytopathologies located at the plasmalemma over plasmodesmata pit fields in companion cells and phloem parenchyma. To further characterize the relationship of P26 and PLDs, we assessed localization and cytopathology induction of P26 expressed from either LIYV or a heterologous Tobacco mosaic virus (TMV) vector using green fluorescent protein (GFP) fusions, immunofluorescence microscopy, biochemical fractionation, and transmission electron microscopy (TEM). TEM analyses demonstrated that P26 not only associated with, but induced formation of PLDs in the absence of other LIYV proteins. Interestingly, PLDs induced by P26-expressing TMV were no longer confined to phloem cells. Putative P26 orthologs from two other members of the genus Crinivirus which do not induce conspicuous PLDs exhibited fractionation properties similar to LIYV P26 but were not associated with any PLD-like cytopathology.

Detection and genetic diversity of Grapevine red blotch-associated virus isolates in table grape accessions in the National Clonal Germplasm Repository in California

Abstract Grapevine red blotch-associated virus (GRBaV) is a recently discovered ssDNA virus that is widespread in wine grapes in California. We investigated whether GRBaV infection was present in 156 table grape accessions of Vitis vinifera that included 53 accessions exhibiting leafroll-like symptoms and 81 accessions from diverse geographic origins. Cane samples were collected during the dormant season in 2012 and analysed for GRBaV infection by PCR. A total of 73 accessions showed presence of GRBaV and these included raisin and table grape accessions with black, green and red berries. A 557 bp amplicon obtained by PCR was purified and sequenced, and the phylogenetic relationship among GRBaV isolates was examined by the maximum likelihood method. The maximum genetic variability among the isolates was only 8% and they belonged to two clades. Although it is not yet known if GRBaV is present outside of North America, 54 accessions from sources originating outside of North America tested positive for the virus.

An Evaluation of the Flora Adjacent to Wine Grape Vineyards for the Presence of Alternative Host Plants of Grapevine red blotch-associated virus

Grapevine red blotch-associated virus (GRBaV) is a recently discovered virus of concern to wine grape production in North America. While the vector of this virus is unknown, other elements of virus epidemiology are essential to develop guidelines for the management of the virus as well as to assist in the search for its vector. The objective of this study was to evaluate vegetation within and surrounding GRBaV-infected vineyards to identify potential virus reservoirs that may serve as sources of inoculum. In this study, 13 plant species were sampled throughout the year and were tested for the presence of GRBaV. Of the 13 species tested, two species, Rubus armeniacus and wild grapes (Vitis californica × V. vinifera), tested positive by quantitative PCR. Of these two species, only wild grapes were determined to be a true host. This study documents the first time GRBaV has been confirmed in an alternative host or in a species outside of a commercial vineyard and suggests that a mechanism exists by which GRBaV moves between plant species that is not human-mediated. The precise role that wild grapes play in the epidemiology of GRBaV remains unknown.

Effects of Grapevine red blotch-associated virus (GRBaV) infection on foliar metabolism of grapevines

Abstract Grapevine red blotch-associated virus (GRBaV), which causes grapevine red blotch disease, is an emerging problem for grapevine production in the United States. However, little is known about how viruses, such as GRBaV, affect host physiology even though it is crucial to understanding host-pathogen interactions, symptom development, and potential effects on other pathogens and insect pests including potential vectors. Thus, foliar levels of amino acids, sugars, phenolics and terpenoids were examined in healthy or GRBaV-infected ‘Cabernet Franc’ (CF) or ‘Cabernet Sauvignon’ (CS) grapevines both before and after development of red blotch symptoms, in July and September of 2014, respectively. Particular amino acids were increased both before and after symptom development in both cultivars, with some of these amino acids having previously defined associations with host defences. Fructose and glucose were increased in GRBaV-infected CF at both sampling times. However, for CS only, glucose was increased in infected grapevines and only when pre-symptomatic. Phenolic levels were greater in GRBaV-infected CF and CS after symptom expression. Terpenoids were greater in infected CF in July, with no other apparent differences. Taken together, these results demonstrate the effects of GRBaV infection on host physiology, with shifts potentially associated with symptom development and changes in resistance to other organisms.