Entomopathogenic fungi as potential biocontrol agents for grapevine phylloxera

Entomopathogenic Beauveria spp. and Metarhizium spp. (Ascomycetes: Hypocreales) fungi occur in the soil worldwide. Research presented in this study provides information on the abundance, distribution and species identity of these fungi in vineyard soils from south eastern Australia, and their effect on the root galling (radicola) form of grapevine phylloxera (Daktulosphaira vitifoliae). In addition, this study explores the ability of B. bassiana to endophytically colonise the roots of Vitis vinifera, and examines the first 72 hours of the infection cycle by B. bassiana and M. pingshaense.
Soil samples from eight Australian vineyards, four in each of the states of New South Wales and Victoria, were surveyed. Two isolation methods, insect baiting using Tenebrio molitor and soil dilution were used to isolate Beauveria spp. and Metarhizium spp. from the soil samples. DNA sequencing of the B locus nuclear intergenic region (Bloc) for Beauveria spp. resulted in the identification of B. bassiana, B. australis and B. pseudobassiana, while sequencing of the elongation factor-1 alpha (EFT1) for Metarhizium spp. resulted in the identification of M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus.
The entomopathogenic fungus B. bassiana has been shown to endophytically colonise a wide range of plants, including the leaf tissue of the European grapevine V. vinifera. This study demonstrated the ability of B. bassiana to endophytically colonise the roots of V. vinifera. This was achieved by either drenching the roots with conidial suspension or by mixing conidia grown on solid rice culture into the potting media.
Selected isolates from the vineyard soil surveys were tested for their virulence and pathogenicity in vitro. Initially, the efficacy of eight Beauveria spp. and 15 Metarhizium spp. isolates was tested on green peach aphid (Myzus persicae). M. robertsii, M. brunneum, M. guizhouense, M. pingshaense, B. bassiana and B. australis resulted in 100 % morality of M. persicae in 9-14 days post inoculation with a conidial suspension. The efficacy of five Beauveria spp. and six Metarhizium spp. isolates were also tested on three radicola grapevine phylloxera endemic to Australia and Europe. None of the isolates were effective in achieving 100 % mortality of the phylloxera tested.
Scanning Electron Microscopy (SEM) was used to assess, within the first 72 hours, the infection of black bean aphid (Aphis fabae) or radicola grapevine phylloxera with a single strain of B. bassiana and M. pingshaense. Both fungi adhered to the cuticle of both insect species and geminated 48 hours post-inoculation (hpi). However, at 72 hpi A. fabae cadavers treated with either fungus had an abundance of fungal mycelia, while at the same time point, phylloxera were still alive without conidia or fungal hyphae on their surface. The expression of adhesin-like protein 1 genes were observed in M. anisopliae 24, 48 and 72 hpi in A. fabae and phylloxera while for B. bassiana it was observed at all three time points only in A. fabae.
The findings of this research have laid the groundwork for further investigations into the use of fungal entomopathogens against phylloxera and other vineyard pests. These could encompass the testing of other entomopathogenic fungal genera to test against phylloxera, in depth investigations of the potential benefits of endophytically established entomopathogenic fungi in V. vinifera roots and further investigations on the mechanisms employed by phylloxera to defend itself from entomopathogenic fungi.