PCR-RFLP analysis of chitinase genes enables efficient genotyping of Metarhizium anisopliae var. anisopliae.

A new genotyping tool has been developed and evaluated for Metarhizium anisopliae var. anisopliae. The tool is based on Restriction Fragment Length Polymorphism (RFLP) analysis of three chitinase genes that are functionally linked to insect-pathogenicity of this fungus. It allowed for discrimination of 14 genotypes among 22 M. anisopliae var. anisopliae strains of a world wide collection. Analyses revealed that the approach may also be applicable to other Metarhizium varieties. The new tool will be useful for genetic characterization of M. anisopliae var. anisopliae strains, and it is applicable for laboratories with limited access to molecular diagnostic equipment.

An optimized microsatellite marker set for detection of Metarhizium anisopliae genotype diversity on field and regional scales.

Thirty-three Metarhizium anisopliae isolates sampled across Switzerland as well as 35 and 36 M. anisopliae isolates sampled from two field sites were assembled in three isolate collections. All isolates were analyzed using 27 newly developed and 14 previously published microsatellite markers. The 41 markers allowed for detection of 25 genotypes in the Swiss collection while 30 and 11 genotypes were detected in the two field collections. This indicated high genetic diversity on a regional as well as on a field scale. In order to improve genotyping efficiency, an optimized marker set, which allows discrimination of a large number of genotypes with as few markers as possible was developed. The optimized marker set consisted of 16 common markers, which provided resolution close to maximal resolution in all three collections (91-93 %). The results demonstrated that optimized marker sets have to be validated before large scale application to previously unassessed collections in order to avoid suboptimal resolution. This genetic tool will be valuable for analyses of genetic population structure of M. anisopliae in different habitats on a regional as well as on a field scale.

The expression of the t-SNARE AtSNAP33 is induced by pathogens and mechanical stimulation.

The fusion of vesicles in the secretory pathway involves the interaction of t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) on the target membrane and v-SNAREs on the vesicle membrane. AtSNAP33 is an Arabidopsis homolog of the neuronal t-SNARE SNAP-25 involved in exocytosis and is localized at the cell plate and at the plasma membrane. In this paper, the expression of AtSNAP33 was analyzed after different biotic and abiotic stresses. The expression of AtSNAP33 increased after inoculation with the pathogens Plectosporium tabacinum and virulent and avirulent forms of Peronospora parasitica and Pseudomonas syringae pv tomato. The expression of PR1 transcripts encoding the secreted pathogenesis-related protein 1 also increased after inoculation with these pathogens and the expression of AtSNAP33 preceded or occurred at the same time as the expression of PR1. AtSNAP33 was also expressed in npr1 plants that do not express PR1 after pathogen inoculation as well as in cpr1 plants that overexpress PR1 in the absence of a pathogen. The level of AtSNAP33 decreased slightly in leaves inoculated with P. parasitica in the NahG plants, and eds5 and sid2 mutants that are unable to accumulate salicylic acid (SA) after pathogen inoculation, indicating a partial dependence on SA. AtSNAP33 was also expressed in systemic noninoculated leaves of plants inoculated with P. syringae. In contrast to the situation in infected leaves, the expression of AtSNAP33 in systemic leaves was fully SA dependent. Thus, the expression of AtSNAP33 after pathogen attack is regulated by SA-dependent and SA-independent pathways. Mechanical stimulation also led to an increase of AtSNAP33 transcripts.