Pathogenicity trials
There were significant differences in Pp stage in mortality levels caused by the 3 fungal strains tested (H = 5.49, p = 0.03) (Fig. 1). However, no differences were observed against L2 (H = 2.49, p = 0.32), L3 (H = 1.16, p = 0.61), L4 (H = 3.62, p = 0.16), or L5 (H = 0.82, p = 0.75) immature stages. Among the larval instars, the CM ranged between 21.65 ± 14.43% (CEP591-L4) and 81.6 ± 2.89% (CEP591-L5). The highest CM (99.98 ± 0.0%) was registered for the Pp stage (CEP591). Mortality percentages among control treatments (not shown) ranged between 2% (L3) and 7% (L2, L5). To our records, this is the first time that the susceptibility of larval stages of L. botrana to M. anisopliae has been demonstrated. Probit analysis for LT50 measured in hours showed that the highest larval instar (L5) had lower lethal times for CEP413 (110), CEP589 (112), and CEP591 (113), when compared to L3 instar (138, 139, 150), respectively.
All the three Metarhizium isolates infected and killed the larvae and pupae of the grapevine species. Additional studies to further define their potential role in integrated pest management programs for this pest seem warranted. The study presents a new evidence of demonstrating that some native strains of M. anisopliae derived from arid zones within Argentina were active against different stages of the vine moth, especially the older larval instars. It is widely accepted that among immature stages, eggs are more difficult to infect than larval stage (Skinner et al. 2014) and that pupae are typically very resistant to succumb to infection (Vestergaard et al. 1995). However, this is not always the case, and some biocontrol strategies effectively targeted pupal stage (Ansari et al. 2008).
The majority of biological control studies on the vine moth have focused on the use of Bacillus thuringiensis (Roditakis 1986 and De Escudero et al. 2007). However, Cozzi et al. (2013) tested 11 fungal strains belonging to Fusarium (3 strains), Beauveria (6 strains), Paecilomyces (1 strain), and Verticillium (1 strain) genera. They obtained a maximum mortality of 55% on L. botrana larvae with Beauveria bassiana under field conditions. Although the obtained results cannot be compared directly to those of Cozzi et al. (2013), additional fungal entomopathogens were identified that may be useful in the biocontrol of L. botrana. In addition, it is the first report to demonstrate the susceptibility of immature stages of grapevine moth to Metarhizium.
Botrytis cinerea growth inhibition
The ANOVA test showed that inhibition of strains B11 and B15 were first detected at 72 h post inoculation. No inhibition was detected prior to 48 h post inoculation (Figs. 2 and 3). There was no difference in the percent inhibition of strain B11 between 72 and 168 h post inoculation (F = 0.67, p = 0.619). However, differences were significant for B15 (F = 5.28, p = 0.002). There were no differences in the levels of B. cinerea inhibitions among the 3 EPF strains during 72–168 h post inoculation for B11 (F = 0.14, p = 0.873) and B15 (F = 1.93, p = 0.163). The level of inhibition ranged between 48 and 64% (B11) and 47–62% (B15).
With respect to the B. cinerea trials, the level of growth inhibition observed suggests that the same isolates provide additional benefits through their ability to inhibit growth of the pathogen in situ. The capacity of different entomopathogenic strains to suppress B. cinerea growth could improve the overall level of control obtained with selective fungicides. The tested strains had a similar inhibitory effect that reached 64%. Although there is no evidence from previous studies about using EPF to decrease the growth rate of the gray rot fungi, the study by Molina et al. (2006) proved that inhibitory effects on B. cinerea, using Clonostachys spp. provided similar results. According to Campbell (1989), Botrytis is highly vulnerable to competition for nutrients and substrate, which may in part explain the growth inhibition observed in the Petri dish assays. Recent studies (Hwi-Geon et al. 2017) found that B. bassiana and M. anisopliae can inhibit B. cinerea and control Myzus persicae. Therefore, there is a pre-existing antecedent, using Metarhizium as a potential fungicide/fungistatic agent.
Fungicide susceptibility
In general terms, the ANOVA test detected statistical differences among treatments (Table 1). On the one side, the 3 EPF strains were equally inhibited by dicarboximide (87–91%, F = 0.78, p = 0.464), copper oxychloride (73–80%, F = 2.02, p = 0.143), cyprodinile-fludioxonile (94–96%, F = 1.68, p = 0.197), and miclobutanil (97–98%, F = 0.98, p = 0.384). Nevertheless, some fungicides affected the strains differently. Carbendazim for example, completely inhibited strains CEP589 and CEP591. However, growth of CEP413 was inhibited by 56%. Similarly, the inhibitory effect caused by iprodione was higher for CEP413 (89%) than for CEP591 (74%) and CEP589 (61%). In the case of fenhexamide, growth inhibition was higher for CEP413 (79%) than for CEP589 (58%) and CEP591 (41%).
All of the EPF strains were highly sensitive to dicarboximide, copper oxychloride, miclobutanil, and cyprodinile—fludioxonile with inhibition percentages ranging from 73 to 98%. Therefore, the use of these fungicides together with the tested EPF strains is probably unadvisable. However, CEP413 in contrast to CEP589 and CEP591, was moderately resistant to carbendazim (56%). Equally, fenhexamide caused only a moderate inhibition on CEP589 and CEP591 (58 and 41%, respectively).