Entomopathogenic fungi in Portuguese vineyards soils: suggesting a ‘Galleria-Tenebrio-bait method’ as bait-insects Galleria and Tenebrio significantly underestimate the respective recoveries of Metarhizium (robertsii) and Beauveria (bassiana)

Abstract. Entomopathogenic fungi (EPF) are the natural enemies of insect-pests. However, EPF recoveries can be influenced by the soil habitat-type(s) incorporated and/or the bait-insect(s) used. Galleriamellonella (GM) as bait-insect, i.e. ‘Galleria-bait’, is arguably the most common methodology, which is sometimes used solely, to isolate EPF from soils. Insect baiting using Tenebriomolitor (TM) has also been employed occasionally. Here 183 soils were used to estimate the functional diversity of EPF in Portuguese Douro vineyards (cultivated habitat) and adjacent hedgerows (semi-natural habitat), using the TM bait method. Moreover, to study the effect of insect baiting on EPF recovery, 81 of these 183 soil samples were also tested for EPF occurrences using the GM bait method. Twelve species were found in 44.26% ± 3.67% of the total of 183 soils. Clonostachysroseaf.rosea was found in maximum soils (30.05% ± 3.38%), followed by Beauveriabassiana (12.57% ± 2.37%), Purpureocilliumlilacinum (9.29% ± 2.14%) and Metarhiziumrobertsii (6.01% ± 1.75%). Beauveriapseudobassiana (P < 0.001), C.roseaf.rosea (P = 0.006) and Cordycepscicadae (P=0.023) were isolated significantly more from hedgerows, highlighting their sensitivities towards agricultural disturbances. Beauveriabassiana (P = 0.038) and M.robertsii (P = 0.003) were isolated significantly more using GM and TM, respectively. Principal component analysis revealed that M.robertsii was associated both with TM baiting and cultivated habitats, however, B.bassiana was slightly linked with GM baiting only. Ecological profiles of B.bassiana and P.lilacinum were quite similar while M.robertsii and C.roseaf.rosea were relatively distant and distinct. To us, this is the first report on (a) C.cicadae isolation from Mediterranean soils, (b) Purpureocilliumlavendulum as an EPF worldwide; and (c) significant recoveries of M.robertsii using TM over GM. Overall, a ‘Galleria-Tenebrio-bait method’ is advocated to study the functional diversity of EPF in agroecosystems.