A rapid and efficient inoculation method for Tomato spotted wilt tospovirus.

A rapid and efficient method of inoculation for Tomato spotted wilt tospovirus (TSWV) was achieved by applying the inoculum with a device consisting of a spray gun, an atomizer and a CO2-powered sprayer. The inoculum contained infected leaf sap prepared in 0.1M phosphate buffer, pH 7.0, 0.2% sodium sulfite and 0.01 M 2-mercaptoethanol (1g: 10 ml) and 1% each of Celite 545 and Carborundum 320 grit. The spray application of chilled inoculum at the rate of 1.1 ml/plant and at an air pressure of 4.1 bar resulted in systemic infection nearly to a 100% of the tobacco (Nicotiana tabacum) plants inoculated. The inoculation procedure was successfully applied to two other important host species of TSWV, peanut (Arachis hypogaea) and tomato (Lycopersicon esculentum), where 75.0-100% and 72.2-91.6% plants developed systemic infection, respectively. The approach facilitated a much faster inoculation of test plants with TSWV as it was estimated to be about 50 times quicker (depending on the plant species) than the hand inoculation. The procedure is suitable for rapid and simultaneous inoculation of a large number of test plants with TSWV and should facilitate screening of germplasm and breeding lines for virus resistance.

First Report of Pinaceae in Georgia Naturally Infected with Tomato spotted wilt virus.

In October 2004, three pine tree seedlings included in an ongoing survey of annual weeds elicited positive reactions for Tomato spotted wilt virus (TSWV [family Bunyaviridae, genus Tospovirus]) using double assay sandwich-enzyme linked immunosorbent assay (DAS-ELISA) (Agdia Inc. Elkhart, IN). All the seedlings appeared healthy with no visible adverse effects from the virus. Over the next 12 months, an additional 1,326 samples of various pine species representing different growth stages were screened for TSWV. Samples were comprised of local populations of Pinus elliottii Engelm., P. taeda L., and P. palustris P. Mill., with the majority (n = 886) of samples being seedlings collected from southern Georgia. Along with the seedlings, needles, stem sections, and roots from saplings, as well as needles from mature trees, were screened for the virus. Of the trees sampled, 5.35% (n = 71) tested positive for TSWV, and of the seedlings 6.77% (n = 60) tested positive. The DAS-ELISA positive threshold was obtained using a figure of three times the average plus two standard deviations of healthy negative pine tissue control absorbance readings at 405 nm. A number of saplings testing positive (n = 6) were marked for further evaluation, and the needles from these saplings consistently screened positive for TSWV in subsequent testing. Furthermore, several samples were processed in modified burlese funnels to detect the possible presence of thrips. No thrips were ever identified in any of the burlese funnel collections. Different tissue types (needles, roots, stem sections, and reproductive organs) were screened, but the virus was only detected in needles. This suggests that local infections are only at feeding sites of viruliferous thrips. The known thrips vectors for TSWV are not considered to be pine feeders, and there is no indication that pine trees are a reproductive reservoir for any local thrips species. However, pine-feeding thrips may also feed on known weed hosts, thus pines could be a perennial reservoir. Mechanical inoculations from surface-sterilized infected pine needles onto known TSWV indicator plants (Nicotiana glutinosa L., N. benthamiana, and Emilia sonchifolia L. (DC)) were inconsistent. Successful transmission occurred 24% of the time. To further verify serological data, total RNA extracts of pine sap were purified and subjected to immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) using primers specific to the nucleocapsid gene of TSWV (1). IC-RT-PCR was used due to the inability to obtain useful total RNA from the pine tissues. This may be due to a secondary metabolite interfering with the total RNA extraction protocol. The IC-RT-PCR products were analyzed with electrophoresis using 0.01% ethidium bromide stain in a 0.8% agarose gel. Amplicons produced at the expected size (bp = 774) were considered positive for TSWV. Several were sequenced and were consistent with known, local TSWV isolates. There is no indication that TSWV is detrimental to pine trees, but considering the widespread distribution of the genus Pinus and the potential of serving as a reservoir of TSWV, it may play a role in the overall epidemiology of TSWV in southern Georgia. Reference: (1) R. K. Jain et al. Plant Dis. 82:900, 1998.

First Report of Yellow Nutsedge (Cyperus esculentus) and Purple Nutsedge (C. rotundus) in Georgia Naturally Infected with Impatiens necrotic spot virus.

Impatiens necrotic spot virus (INSV), family Bunyaviridae, genus Tospovirus, is an emerging virus found mostly in ornamentals under greenhouse production. INSV has been detected in peanut (Arachis hypogaea L.) in Georgia and Texas (3) and recently in tobacco (Nicotiana tabacum L.) in the southeastern United States (2) but little is known about INSV distribution and impact on these crops. Noncrop plant hosts are likely to contribute to disease spread by serving as reservoirs for the virus and reproductive hosts for thrips (Frankliniella occidentalis Pergande), which transmit the virus. Yellow nutsedge, a native of North America, and purple nutsedge introduced from Eurasia, are considered serious weed problems in the southeastern United States. To date, there are no reports of natural INSV infections in these weeds. A survey was conducted at two research farms in Tift County, Georgia to determine if yellow and purple nutsedge plants were naturally infected with Tomato spotted wilt virus (TSWV) and INSV. The first field at the Black Shank Farm had been planted with flue-cured tobacco K-326 earlier in the year and fallow at the time of sampling. The second field at the Ponder Farm was planted at the time of sampling with yellow squash (Cucurbita pepo L.) and cabbage (Brassica oleracea L.). In early October 2002, 90 nutsedge plants were taken at random from each site. Leaf and root tissues of each of the nutsedge plants were tested for TSWV and INSV using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) alkaline phosphatase antisera kits (Agdia Inc., Elkhart, IN). No visible symptoms of INSV or TSWV were observed. Samples from the field at the Black Shank Farm resulted in 2 of 26 positive for INSV in purple nutsedge plants and 6 of 64 in yellow nutsedge plants. At the Ponder Farm, 3 of 12 were positive for INSV in purple nutsedge plants and 14 of 78 in yellow nutsedge plants. None of the samples in either site tested positive for TSWV. The DAS-ELISA positive samples were verified for INSV using reverse transcription-polymerase chain reaction (RT-PCR) as previously described by Dewey et al. (1). Total RNA extracts were obtained from the DAS-ELISA positive nutsedge samples using RNeasy extraction kits (Qiagen Inc., Valencia, CA). The RT-PCR was carried out with primer 1F: 5'-TCAAG(C/T) CTTC(G/T)GAA(A/G)GTGAT 3' (1) and primer 2R: 5'-ATGAACAAAGCAAAGATTACC 3' specific to the 3' end of the INSV N gene open reading frame (GenBank Accession No. NC003624). DAS-ELISA negative tissues of Cyperus esculentus L. and Emilia sonchifolia (L.) DC and an E. sonchifolia DAS-ELISA positive for INSV were included in the reactions as controls. All of the DAS-ELISA positive nutsedge samples yielded an amplification product with the expected size of 298 bp when PCR products were resolved by agarose (0.7%) gel electrophoresis. The relatively high occurrence of INSV found in the sampled fields may explain the recent increase in incidence of INSV in susceptible field crops. Although yellow nutsedge is more common than purple nutsedge in North America, the potential for dispersal of INSV in both species could be significant because of the nature of nutsedge tuber survival and spreading capabilities. References: (1) R. A. Dewey et al. J. Virol. Methods 56:19, 1996. (2) N. Martínez-Ochoa et al. On-line publication. doi:10.1094/PHP-2003-0417-01-HN. Plant Health Progress, 2003. (3) S. S. Pappu et al. Plant Dis. 83:966,1999.

Development of Multi-Component Transplant Mixes for Suppression of Meloidogyne incognita on Tomato (Lycopersicon esculentum).

The effects of combinations of organic amendments, phytochemicals, and plant-growth promoting rhizobacteria on tomato (Lycopersicon esculentum) germination, transplant growth, and infectivity of Meloidogyne incognita were evaluated. Two phytochemicals (citral and benzaldehyde), three organic amendments (pine bark, chitin, and hemicellulose), and three bacteria (Serratia marcescens, Brevibacterium iodinum, and Pseudomonas fluorescens) were assessed. Increasing rates of benzaldehyde and citral reduced nematode egg viability in vitro. Benzaldehyde was 100% efficacious as a nematicide against juveniles, whereas citral reduced juvenile viability to less than 20% at all rates tested. Benzaldehyde increased tomato seed germination and root weight, whereas citral decreased both. High rates of pine bark or chitin reduced plant growth but not seed germination, whereas low rates of chitin increased shoot length, shoot weight, and root weight; improved root condition; and reduced galling. The combination of chitin and benzaldehyde significantly improved tomato transplant growth and reduced galling. While each of the bacterial isolates contributed to increased plant growth in combination treatments, only Brevibacterium iodinum applied alone significantly improved plant growth.