Production of thermotolerant entomopathogenic fungal conidia on millet grain.

Thermotolerance of entomopathogenic (insect-killing) fungi should be seriously considered before industrialization. This work describes the feasibility of millet grain as a substrate for production of thermotolerant Beauveria bassiana (Bb) GHA and ERL1170 and Metarhizium anisopliae (Ma) ERL1171 and ERL1540 conidia. First, conidial suspensions of the Bb isolates, produced on millet grain in polyethylene bags, were exposed to five temperatures (43-47 °C) at 15-min intervals for up to 120 min (experiment I). Agar-based quarter-strength (») Sabouraud dextrose agar supplemented with yeast extract (SDAY) and whey permeate media served as controls. Millet-grain-based culture was superior in producing the most thermotolerant Bb conidia, followed by whey permeate agar and »SDAY-based cultures. Secondly, to compare the thermotolerance of conidia produced at the same conditions, the Bb isolates were then produced on agar-based millet powder medium, with »SDAY and whey permeate agar media as controls, and the two Ma isolates were added (experiment II). They were then exposed to the same temperatures as above. More thermotolerant Bb and Ma conidia were produced on millet powder agar than on whey permeate agar and »SDAY overall. These results suggest that millet grain can be used as a substrate to produce thermotolerant conidia in a mass production system.

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay.

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and »SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45°C for 45min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14d were more hydrophobic and exhibited greater thermotolerance than those produced on »SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.

Whey for mass production of Beauveria bassiana and Metarhizium anisopliae.

Spore production of Beauveria bassiana and Metarhizium anisopliae was studied in a novel whey-based culture media. Spore yield and viability were determined for two B. bassiana (GHA-726 and CA-603) and two M. anisopliae (CA-1 and IMI 330189) isolates following production in three whey-based systems: solid, liquid, and a diphasic production system. Our study indicated that whey permeate can be used effectively for production of spores of entomopathogenic fungi. However, spore yield and viability were significantly influenced by fungal isolate, whey concentration, and the type of production process used. Under the conditions defined in the present study, spore yields ranging from 1.3x10(9)-10x10(11) spores l(-1) of whey medium could be obtained depending on the strain and production process used. Our study revealed that spores produced by all strains in whey-based solid and liquid media showed between 73-99% viability; germination rates were comparable with those obtained using the standard SDA medium. In the two-stage production process, the viabilities of conidia produced by GHA-726, CA-603, and CA-1 were 35-86, 32-98, and 6-29%, respectively; viability was correlated with whey concentration and isolates. Whey permeate can be used as a growth substrate for mass production of biocontrol fungi. We hypothesize that spore yield and viability could be improved by careful selection of whey content in the medium, incorporation of critical additives and optimization of culture conditions.