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.
