Three Methods Assessing the Association of the Endophytic Entomopathogenic Fungus Metarhizium robertsii with Non-Grafted Grapevine Vitis vinifera

Abstract

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.

Keywords: ddPCR; endophytes; fungal entomopathogens; rhizosphere.

Figure 1

(a) Time-course of quantification of rhizospheric V. vinifera association of two M. robertsii strains (EF3.5(2), red boxes; GFP transformed strain ARSEF-2575-GFP, white boxes) and a control treatment (blue boxes) using ddPCR. The boxplots represent the logarithm of the number (nb) of DNA copies (of non-null values) of M. robertsii per microliter of DNA extracted of mixed non-disinfected grapevine roots with adhering soil (rhizosphere) 14-, 35-, 63- and 96–98 days post inoculation (dpi). Significant differences are indicated by small letters above to boxes (Kruskal–Wallis test, p < 0.05); (b) Time-course of quantification of endophytic V. vinifera root association of two M. robertsii strains using ddPCR. The boxplots represent the logarithm of the number (nb) of DNA copies (of non-null values) of M. robertsii per microliter of DNA extracted of mixed disinfected grapevine roots (root endosphere) 14-, 35-, 63- and 96–98 days post inoculation (dpi).

Figure 2

Time-course of detection of two M. robertsii strains (EF3.5(2); GFP-transformed strain ARSEF-2575-GFP) and a control treatment on V. vinifera roots. The bars represent the percentage of M. robertsii colonized grapevines at (a) the rhizosphere and (b) the root endosphere evaluated with two methods: culture-based method (EF3.5(2): red bars, ARSEF-2575-GFP: grey bars, controls: dark blue bars) and ddPCR (EF3.5(2): pink bars, ARSEF-2575-GFP: white bars, controls: light blue bars). Evaluation was made 14-, 35-, 63- and 96–98 days post inoculation (dpi).

Figure 3

Confocal images of grapevine root association with M. robertsii ARSEF-2575-GFP expressing green fluorescent protein (GFP) observed at (a) 14 dpi, and at (b–d) 31 dpi. All scale bars are 5 μm and total magnification was ×63 using an oil-immersion objective.