Occurrence of two distinct Tomato spotted wilt virus subgroups in southern Italy

A fragment of the NSM gene of naturally occurring Tomato spotted wilt virus (TSWV) isolates recovered in Apulia (southern Italy) from different cultivated and wild plant species was amplified by RT-PCR. The resulting 670 bp amplicon was cloned, sequenced and compared with available NSM sequences of other tospoviruses. The primary nucleotide sequence was 94.3 to 100% identical in all the Apulian TSWV isolates and had a 94-99% identity with TSWV-BR01, TSWV-L3, TSWV-JP, TSWV-A and TSWV-D. With other tospoviruses, sequence identity was in the range of 81-83% with Tomato chlorotic spot virus and Chrysanthemum stem necrosis virus and, 74-79% with Groundnut ring spot virus, Zucchini lethal chlorosis virus and Impatiens necrotic spot virus, 53-55% with Iris yellow spot virus, Peanut bud necrosis virus and Watermelon silver mottle virus. The phylogenetic tree showed three primary branches departing from the bulk of all NSM sequences; all the TSWV sequences clustered in two subgroups, denoted A-like and D-like, clearly distinct from the other tospoviruses. The inclusion in a group of any of the Apulian isolates of TSWV was apparently not determined by the year, the place or the host from which they were recovered. The A-like isolates clustered with TSWV-A, an isolate that was able to overcome host and pathogen-mediated resistance in tomato whereas the remaining six isolates clustered with TSWV-D, an isolate that did not overcome these forms of resistance. A simple and rapid procedure based on RT-PCR followed by enzymatic digestion was developed to identify isolates of the two subgroups