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The fungus Neotyphodium sinicum forms an asymptomatic symbiosis with Roegneria grass species which are widely distributed in China. We collected 1,809 asymptomatic specimens of Roegneria, belonging to six species, from around 14 cities in nine provinces. 45.7% of the 1,809 asymptomatic Roegneria plants were endophyteinfected. Of 100 Neotyphodium isolates subsequently obtained, 31 were tested for cultural characteristics. These isolates showed morphological diversity and varying growth rates as well as differences in colony appearance and in conidial lengths. While conidial length of most of the isolates was typical of interspecific hybrid Neotyphodium species, conidial length of some isolates was smaller, similar to that of haploid species. A phylogenetic study was conducted of the tubB and tefA genes in 21 Neotyphodium isolates. The isolates were phylogenetically clustered into three types: 15/ 21 isolates had two divergent alleles of both tubB and tefA; 3/21 had two alleles of only tubB and 3/21 had two alleles of only tefA. In the tubB NJ tree, alleles-1 formed two distinct sub-clades in the Epichloë bromicola/E. yangzii clade (EBY); allele-2 was located in three sub-clades widely distributed within the E. typhina clade (ETC). The allele-1 of the tefA gene was also located within the EBY clade and formed two sub-clades; allele-2 was located in 3 sub-clades within the ETC clade. No relationship was observed between phylogenetic relationships and morphology in the N. sinicum isolates. The phylogenetic characteristics had little relationship to the host species or site of origin of the isolates. An explanation consistent with the phylogenetic findings is that N. sinicum is a complex of species that has resulted from multiple independent hybridization events.

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122–139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.

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