Characterizing the association of endophytic insect pathogenic fungi (EIPF) with plants is an important step in order to understand their ecology before using them in biological control programs. Since several methods are available, it is challenging to identify the most appropriate for such investigations. Here, we used two strains of Metarhizium robertsii: EF3.5(2) native to the French vineyard environment and ARSEF-2575-GFP a laboratory strain expressing a green fluorescent protein, to compare their potential of association with non-grafted grapevine Vitis vinifera. Three methods were used to evaluate the kinetics of rhizosphere and grapevine endospheric colonization: (i) Droplet Digital (ddPCR), a sensitive molecular method of M. robertsii DNA quantification in different plant parts, (ii) culture-based method to detect the live fungal propagules from plant tissues that grew on the medium, (iii) confocal imaging to observe roots segments. Both strains showed evidence of establishment in the rhizosphere of grapevines according to the culture-based and ddPCR methods, with a significantly higher establishment of strain EF3.5(2) (40% positive plants and quantified median of exp(4.61) c/μL) compared to strain ARSEF-2575-GFP (13% positive plants and quantified median of exp(2.25) c/μL) at 96–98 days post-inoculation. A low incidence of association of both strains in the grapevine root endosphere was found with no significant differences between strains and evaluation methods (15% positive plants inoculated with strain EF3.5(2) and 5% with strain ARSEF-2575-GFP according to culture-based method). ddPCR should be used more extensively to investigate the association between plants and EIPF but always accompanied with at least one method such as culture-based method or confocal microscopy.

Entomopathogenic fungi (EPF) are substantial biocontrol agents reducing the populations of economically important pests in numerous crops. Recent findings indicate that their role in agroecosystems is more complex and extends to affecting plant physiology and growth. This study examined the effects of Beauveria bassiana and Isaria fumosorosea, as well as Salicylic acid (SA), on physiological parameters of grapevine (Vitis vinifera cv. Sauvignon Blanc). Additionally, the impact of SA on spore germination and pathogenicity of EPF against larvae of the European grapevine moth (Lobesia botrana) was tested. Foliar application of EPF was found to increase the electron transport rate (ETR) from PSII to PSI, indicating higher photosynthetic activity compared to control plants. EPF also elevated the transpiration rate (E) and stomatal conductance (gs). In contrast, SA treatments decreased E and gs, while the high dose (10 mM) exhibited reduced Fv/Fm value, accompanied by phytotoxic spots on leaves. Spore germination of both fungi was significantly reduced only by the SA concentration of 2 mM, while 0.5 and 1 mM did not affect germination. Combination EPF and SA treatments presented the highest larval mortality of L. botrana (87.5% at 28 °C and 77.5% at 24 °C for B. bassiana and I. fumosorosea, respectively). However, SA reduced larval mycosis in most cases. Overall, the results suggest that EPF and SA can be co-applied and included in vineyard integrated strategies to support grapevine health.