First Report of Kudzu (Pueraria montana) Infections by Tobacco ringspot virus in Mississippi

Virus-like symptoms were observed in several kudzu patches in Mississippi during a survey of viruses infecting soybean carried out in late summer/fall of 2013 as a part of a project funded by the Mississippi Soybean Promotion Board. Symptomatology consisted of chlorotic mottle and ringspots, vein-associated feathering, necrosis, and leaf deformation, which were often observed in combination on the same plant. In order to identify the virus(es) involved in the disease, young leaves from a symptomatic kudzu sample collected in Kemper County were crushed in 10 volumes of 0.1 M phosphate buffer (pH 7.2) and mechanically inoculated onto celite-dusted leaves of two soybean varieties (Asgrow AG4605 and AG4730), each represented by 10 plants. Sap from an asymptomatic kudzu sample from Oktibbeha County was used as a control. Both varieties reacted by systemic mottle, stunting, and apical leaf necrosis approximately 2 weeks after inoculation, while no symptoms could be observed in controls. Partially purified preparations from both symptomatic soybean cultivars exhibited the presence of putative intact and empty spherical virus particles ~30 nm in diameter. ELISA tests with antisera to several soybean viruses were performed on the original kudzu sample and inoculated AG4605 and AG4730 soybean plants. These tests revealed the presence of Tobacco ringspot virus (TRSV) in all symptomatic samples. In order to better understand the incidence of this virus in kudzu in Mississippi, a total of 127 samples from 28 counties were collected during October 2013 and tested using ELISA. A total of 11 samples collected in 8 different counties were positive for TRSV. To further confirm these results, one step RT-PCR test was performed on total nucleic extracts from all ELISA-positive and four negative kudzu samples using TRSV-specific primers (3). A specific PCR product of 766 bp was present in all ELISA-positive samples and positive controls, whereas no visible bands were present in negative samples. PCR products generated from samples, collected in Kemper, Tippah, and Jefferson Davis counties, were cloned and custom sequenced. Pair-wise comparisons indicated conserved nucleotide (95 to 98%) and amino acid (98 to 99%) contents among sequenced products. Kudzu isolates from Mississippi shared 91 to 96% and 98 to 99% conserved nucleotides and amino acids, with TRSV sequences currently available in the NCBI/GenBank database. This is the first report of TRSV infection of kudzu in Mississippi. The possible implications to the soybean industry are yet to be determined since kudzu occupies approximately 202,000 ha in Mississippi and TRSV has historically been reported associated with bud blight in soybean (1). Nonetheless, results of our study, along with the recent report from Louisiana (2), strongly suggest that kudzu, due to its widespread distribution in the region, may represent a major reservoir of TRSV in the southeastern United States. References: (1) G. L. Hartman et al. Compendium of Soybean Diseases. American Phytopathological Society, St. Paul, MN, 1999. (2) Khankhum et al. Plant Dis. 97:561, 2013. (3) S. Sabanadzovic et al. Plant Dis. 94:126, 2010.