Characterization, identification and virulence of Metarhizium species from Cuba to control the sweet potato weevil, Cylas formicarius Fabricius (Coleoptera: Brentidae).

AIMS: Entomopathogenic Metarhizium fungi are widely recognized for their biological control potential. In Cuba, several fungus-based bio-insecticides have been developed and are produced as part of integrated pest management (IPM) programmes for economically relevant agricultural pests. Screening of fungal isolates from the INISAV strain collection was used for the development of bio-insecticides against important pest insects as, for example the sweet potato weevil, Cylas formicarius. METHODS AND RESULTS: Six fungal isolates from Cuba were microscopically, morphologically and molecular-taxonomically characterized using marker sequences ef1a, rpb1 and rpb2, and the 5TEF region of the ef1a gene. Five isolates were assigned to the species Metarhizium anisopliae sensu stricto and one isolate to Metarhizium robertsii. The pathogenic potential was evaluated against adults of C. formicarius, and growth and conidial production on different nutritional media were determined. Metarhizium anisopliae strain LBM-267 displayed pronounced virulence against the sweet potato weevil and abundant conidia production on several culture media. CONCLUSIONS: Entomopathogenic fungal isolates from Cuba were assigned to the taxonomic species M. anisopliae sensu stricto and M. robertsii. Virulence assessment with respect to C. formicarius led to the identification of two M. anisopliae isolates holding biocontrol potential. Isolate LBM-11 has previously been developed into the bio-insecticide METASAVE-11 that is widely used to control several species of plant pathogenic weevils, Lepidoptera and thrips in Cuba. Isolate LBM-267 has not been employed previously but is as virulent against C. formicarius as LBM-11; its growth and conidial production capacities on different nutritional media will likely facilitate economically feasible bio-insecticide development. SIGNIFICANCE AND IMPACT OF THE STUDY: Metarhizium anisopliae isolate LBM-267 has been selected as a promising candidate for biocontrol of the sweet potato weevil, an economically important agricultural pest in Cuba, and for further R&D activities within the framework of the Biological Control Program of Cuba.

History of the International Symposium on Fungal Stress - ISFUS, a dream come true!

The International Symposium on Fungal Stress (ISFUS) was born in a dream that Drauzio Eduardo Naretto Rangel had in 2013. This article reviews the first three ISFUSs and prospects for the future meetings. Although ISFUS was born as a small family organized meeting, since the first meeting, ISFUS has achieved great success, receiving very important research grants from FAPESP, FAPEG, and CAPES to bring international scientists to Brazil. Moreover, three special issues in leading journals have been published with articles relating to the talks presented at each ISFUS. For the first meeting, most speakers published in a special issue in Current Genetics. From the second and third meeting, articles from the speakers were published in special issues of the top mycology journal, Fungal Biology, published by Elsevier on behalf of the British Mycological Society. Here we show that following the dreams with a full heart and adding lots of love, passion, and hard work can achieve success.

Occurrence, Identification, and Virulence of Native Fungal Pathogens Isolated From Mexican Fruit Fly (Diptera: Tephritidae) Larvae From Soils of Three Cropping Systems.

The Mexican fruit fly, Anastrepha ludens Loew, is a significant pest in mango and citrus production areas of Mexico. In this study, we evaluated the effects of some geographic characteristics, rainfall period, soil micro-environmental, and soil coverage variables on the occurrence of entomopathogenic fungi (EPF) associated with A. ludens larvae in soils of mango, grapefruit and mixed crops in central Veracruz state, Mexico. EPF isolates were characterized morphologically and identified by sequence analysis of elongation factor (EF1-1018F, EF1-1620R). We recorded four species of EPF (Metarhizium robertsii J.F. Bisch, S.A. Rehner & Humber [Hypocreales: Cordycipitaceae], M. brunneum Petch [Hypocreales: Cordycipitaceae], M. pinghaense Q.T. Chen & H.L. Guo [Hypocreales: Cordycipitaceae], and Beauveria bassiana (Balsamo) Vuillemin [Hypocreales: Cordycipitaceae]), of which Metarhizium robertsii was the most abundant and the most virulent. Also, we found that rainfall period, organic matter, coverage of herbs and forbs, and calcium levels modulated EPF occurrence. We estimated lethal concentrations for A. ludens larvae of the four most promising isolates, V3-123, V3-160, V1-332, and V3-369. Our results suggest that M. robertsii obtained from agricultural soils holds potential as a biological control agent for A. ludens.

Species of the Metarhizium anisopliae complex with diverse ecological niches display different susceptibilities to antifungal agents.

Species of the Metarhizium anisopliae complex are globally ubiquitous soil-inhabiting and predominantly insect-pathogenic fungi. The Metarhizium genus contains species ranging from specialists, such as Metarhizium acridum that only infects acridids, to generalists, such as M. anisopliae, Metarhizium brunneum, and Metarhizium robertsii that infect a broad range of insects and can also colonize plant roots. There is little information available about the susceptibility of Metarhizium species to clinical and non-clinical antifungal agents. We determined the susceptibility of 16 isolates comprising four Metarhizium species with different ecological niches to seven clinical (amphotericin B, ciclopirox olamine, fluconazole, griseofulvin, itraconazole, tebinafine, and voriconazole) and one non-clinical (benomyl) antifungal agents. All isolates of the specialist M. acridum were clearly more susceptible to most antifungals than the isolates of the generalists M. anisopliae sensu lato, M. brunneum, and M. robertsii. All isolates of M. anisopliae, M. brunneum, and M. robertsii were resistant to fluconazole and some were also resistant to amphotericin B. The marked differences in susceptibility between the specialist M. acridum and the generalist Metarhizium species suggest that this characteristic is associated with their different ecological niches, and may assist in devising rational antifungal treatments for the rare cases of mycoses caused by Metarhizium species.

Riboflavin induces Metarhizium spp. to produce conidia with elevated tolerance to UV-B, and upregulates photolyases, laccases and polyketide synthases genes.

AIMS: The effect of nutritional supplementation of two Metarhizium species with riboflavin (Rb) during production of conidia was evaluated on (i) conidial tolerance (based on germination) to UV-B radiation and on (ii) conidial expression following UV-B irradiation, of enzymes known to be active in photoreactivation, viz., photolyase (Phr), laccase (Lac) and polyketide synthase (Pks). METHODS AND RESULTS: Metarhizium acridum (ARSEF 324) and Metarhizium robertsii (ARSEF 2575) were grown either on (i) potato dextrose agar medium (PDA), (ii) PDA supplemented with 1% yeast extract (PDAY), (iii) PDA supplemented with Rb (PDA+Rb), or (iv) PDAY supplemented with Rb (PDAY+Rb). Resulting conidia were exposed to 866·7 mW m-2 of UV-B Quaite-weighted irradiance to total doses of 3·9 or 6·24 kJ m-2 . Some conidia also were exposed to 16 klux of white light (WL) after being irradiated, or not, with UV-B to investigate the role of possible photoreactivation. Relative germination of conidia produced on PDA+Rb (regardless Rb concentration) or on PDAY and exposed to UV-B was higher compared to conidia cultivated on PDA without Rb supplement, or to conidia suspended in Rb solution immediately prior to UV-B exposure. The expression of MaLac3 and MaPks2 for M. acridum, as well as MrPhr2, MrLac1, MrLac2 and MrLac3 for M. robertsii was higher when the isolates were cultivated on PDA+Rb and exposed to UV-B followed by exposure to WL, or exposed to WL only. CONCLUSIONS: Rb in culture medium increases the UV-B tolerance of M. robertsii and M. acridum conidia, and which may be related to increased expression of Phr, Lac and Pks genes in these conidia. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhanced UV-B tolerance of Metarhizium spp. conidia produced on Rb-enriched media may improve the effectiveness of these fungi in biological control programs.

Occurrence of entomopathogenic fungi in tejocote (Crataegus mexicana) orchard soils and their pathogenicity against Rhagoletis pomonella.

AIMS: To determine the abundance and diversity of entomopathogenic fungi in tejocote orchard soils and evaluate their ability to infect Rhagoletis pomonella Walsh., the main pest of tejocote. METHODS AND RESULTS: Surveys were made in two locations in Mexico state and two in Puebla state. Soil from selected locations was baited for entomopathogenic fungi with Galleria mellonella (L.). All isolates were identified morphologically to genus level and to species level using Bloc and elongation factor 1-α gene sequence information, respectively; Beauveria bassiana ((Bals.-Criv.) Vuill.), B. pseudobassiana (S.A. Rehner & Humber) and Metarhizium robertsii (J.F. Bisch., Rehner & Humber) were found, with B. bassiana being the most abundant and widely distributed. Pathogenicity of five selected B. bassiana isolates and three M. robertsii isolates was evaluated against larvae and pupae of R. pomonella. All isolates infected larvae resulting in an average mortality of 35%. Pupae were not susceptible; however, adults emerging from inoculated pupae did die due to infection. CONCLUSIONS: At least three species of entomopathogenic fungi are present in the soil from tejocote orchards, with B. bassiana being the most abundant and widely distributed. Rhagoletis pomonella larvae were more susceptible to infection than pupae. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study has produced new information about the distribution of entomopathogenic fungi in cultivated soils from this region of North America, contributing to a better understanding of their natural occurrence and underpinning the development of biological control approaches.

Specific diversity of the entomopathogenic fungi Beauveria and Metarhizium in Mexican agricultural soils.

Prior knowledge of the local population structure of entomopathogenic fungi is considered an important requisite when developing microbial control strategies against major pests of crops such as white grubs. An extensive survey in the estate of Guanajuato, one of the main agricultural regions of Mexico, was carried out to determine the abundance and diversity of entomopathogenic fungi in soil. Soil collected from 11 locations was baited for entomopathogenic fungi using Galleria mellonella. In addition, all isolates were morphologically identified and selected isolates of Beauveria and Metarhizium isolates identified using Bloc and ITS or Elongation Factor 1-α and ITS sequence information respectively. Genotypic diversity was then studied using microsatellite genotyping. The proportion of isolates belonging to each genus varied amongst all locations. The species Beauveria bassiana, B. pseudobassiana and Metarhizium robertsii were found, with B. bassiana being the most abundant and widely distributed. Microsatellite genotyping showed that the 36 B. bassiana isolates were grouped in 29 unique haplotypes, but with no separation according to geographical origin.