Quantification of fungal growth and destruxin A during infection of Galleria mellonella larvae by Metarhizium brunneum.

Destruxin A is among the major secondary metabolites produced by the entomopathogenic ascomycete Metarhizium sp., and the lack of studies concerning production of destruxin A by the fungus is most likely the biggest obstacle for the registration of new fungal strains. Although several studies focus on the production of destruxin A in culture media, few studies examine destruxin A in vivo during host infection. In the current work, Galleria mellonella was used as an insect model to develop for the first time in vivo real-time PCR- and HPLC-MS-based quantification of fungal growth and metabolite production, respectively, during infection by two strains of M. brunneum. Total mortality of sixth instar G. mellonella larvae that were immersed in a suspension of 1.0×108conidiamL-1 of M. brunneum EAMa 01/58-Su or BIPESCO5 strains reached 85.5% and 78.8%, respectively, and the percentage of cadavers with fungal outgrowth was low at 12.2% and 4.4%, respectively. The average survival time of treated larvae was 5.5days for both fungal strains. Using EAMa 01/58-Su and BIPESCO5 specific primer set, real-time PCR showed that the patterns of fungal growth were different for the two strains, whereas no significant differences were detected in the number of fungal sequence copies recovered from the infected larvae. EAMa 01/58-Su and BIPESCO5 strains secreted destruxin A from days 2 to 6 and from days 2 to 5 post treatment, respectively. For EAMa 01/58-Su and BIPESCO5, the maximum titer of destruxin A in the host was on day 4 at 0.369 and 0.06µg/larva, respectively, and throughout the pathogenic process, the total production was 0.6 and 0.09µg/larva, respectively. These results demonstrated that the strains pose a low hazard, if any, to humans and the environment. The methods used in this study to quantify fungal growth and metabolite production provided valuable data to better understand the role of destruxin A during the growth of M. brunneum in the host larvae and to monitor the fate of destruxin A in food chains.

Selection and evaluation of micro-organisms for biocontrol of Verticillium dahliae in olive.

AIMS: To identify potential biological control agents against Verticillium wilt in olive through a mass screening approach. METHOD AND RESULTS: A total of 47 strains and nine mixtures of micro-organisms were evaluated against Verticillium dahliae in a three stage screening: (i) in vitro, by the effect on the mycelial growth and spore germination of the pathogen; (ii) in natural infested soil, by the effect on the reduction of microsclerotia of the pathogen; (iii) in planta, by the effect on the infection of olive plants under controlled conditions. Various fungal and bacterial strains and mixtures inhibited the pathogen and showed consistent biocontrol activity against Verticillium wilt of olive. CONCLUSION: The screening has resulted in promising fungi and bacteria strains with antagonistic activity against Verticillium, such as two non-pathogenic Fusarium oxysporum, one Phoma sp., one Pseudomonas fluorescens and two mixtures of micro-organisms that may possess multiple modes of action. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a practical basis for the potential use of selected strains as biocontrol agents for the protection of olive plants against V. dahliae infection. In addition, our study presented an effective method to evaluate antagonistic micro-organisms of V. dahliae in olive.

Enhanced production of microsclerotia in recalcitrant Verticillium dahliae isolates and its use for inoculation of olive plants.

AIMS: The optimization of a simple protocol for the mass production of viable microsclerotia (MS) of Verticillium spp., even for recalcitrant isolates, to the inoculation of olive cuttings. METHOD AND RESULTS: Four Verticillium spp. isolates were characterized by growth rate and morphology. Then, the production ability and the viability of MS over time were assessed in seven solid culture media and five aqueous media. The best culture medium, according to the quantity and the quality (size) of the MS produced, was the alkaline-modified sodium polipectate (AMSP) aqueous medium. The MS viability was higher in peat moss substrates. Finally, the MS obtained in this work were infective causing 100% incidence of Verticillium wilt (VW) disease in inoculated olive plants. CONCLUSION: This study demonstrates that the modified sodium polipectate medium amended with 0·1% agar is the most suitable for the production of MS of Verticillium dahliae isolates that have lost the ability to produce MS in standard culture media. SIGNIFICANCE AND IMPACT OF THE STUDY: Mass production of MS for artificial infestation of soil is critical to the study of epidemiological and control aspects of the VW. To overcome the failure in the production of MS in recalcitrant isolates, a culture media was optimized and a successful plant inoculation experiment was carried out with artificial MS.