Richard A. Daoust

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of temperature and relative humidity on conidial viability and virulence against mosquitoes

Abstract Unformulated conidia of three strains of Metarhizium anisopliae were stored at four temperatures (viz., 37°, 26°, 19°, and 4°C) at each of six relative humidities (RH) (viz., 0, 12, 33, 53, 75, and 98%) and at −20°C at 0% RH. Conidial viabilities and virulence against two species of mosquitoes were determined at various intervals over a 2-year period. Conidia were found to survive longest when moderate temperature-high RH (26°C–97% RH or 19°C–97% RH) or low temperature-low RH (4°C–0% RH) were used, while intermediate RH values between 33 and 75%, depending upon the strain, were most lethal. As the temperature declined from 37° to 4°C at nearly all RH values the longevity of spores increased. Conidia remained highly virulent under favorable storage conditions for at least 18 months against Culex pipiens pipiens or 12 months against Anopheles stephensi larvae.

Virulence of Natural and Insect Passaged Strains of Metarhizium Anisopliae to Mosquito Larvae

Abstract Forty-seven isolates of Metarhizium anisopliae var. anisopliae (small-spored form) and five isolates of M. anisopliae var. major (large-spored form) obtained from widely separated geographical regions from various insect hosts were screened for virulence against Culex pipiens pipiens larvae. Pathogenesis was variable with mortalities ranging from 0 to 100%. However, much of the variation in mortality among small-spored isolates was due to lowered natural viabilities. The most virulent isolates were from Austria, Australia, and Brazil from insect species in three different orders. Isolates from the major strain were generally avirulent. There was no correlation of strain morphology, geographical region of isolation, or original host species with strain virulence. The strains most virulent to C. pipiens larvae were also highly infective to Aedes aegypti and Anopheles stephensi larvae. Virulence of two strains (E6 and E9) to C. pipiens larvae was significantly enhanced by one passage through a C. pipiens larval siphon. Relative potencies increased approximately 1.63 to 2.45 times. A smaller increase in virulence, depending upon the isolate, was also shown when these same strains were tested against A. aegypti and A. stephensi. Virulence of strain E9 was also increased significantly by passage through an alternate host, Nilaparvata lugens.

Effect of formulation on the viability of Metarhizium anisopliae conidia

Abstract Fourteen oils, including mineral oil, cod liver oil, and 12 vegetable oils, were highly detrimental to Metarhizium anisopliae conidial viability after 2 months storage at 26° and 19°C. Most oilconidial suspensions (8 of 14) also lost conidial viability more rapidly than unformulated conidia at 4°C. The viscosity of oils was not related to the rapid loss of conidial viability in these formations. Other liquid vehicles, including petroleum-based oils, organic acids, and water, with or without a surfactant, were also highly lethal to conidia after 4 months of storage. In contrast to liquid formulations, high conidial viabilities were retained on most dry formulations, including four different granular carriers and 12 different dust diluents, over a 1-year period at 4°C. At 20°C, however, conidial viabilities declined to below 50% with most dry diluents. Dust diluents were generally more compatable with conidia than granular carriers. Unformulated conidia mixed with two desiccants lost germination capacity sooner than conidia exposed to, but separated from, anhydrous CaCl2. On the other hand, conidia in a dust formulation mixed with kaolinite clay and powdered silica gel survived longer than unformulated conidia either mixed with or separated from the desiccant. This study confirms the need for careful assessment of the compatibility of formulation components with conidia prior to their use in formulations.

Studies on the Prolonged Storage of Metarhizium Anisopliae Conidia. Effect of Growth Substrate on Conidial Survival and Virulence Against Mosquitoes

Abstract Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.

Effect of formulation on the virulence of Metarhizium anisopliae conidia against mosquito larvae

Abstract Metarhizium anisopliae conidia were formulated with three granular carriers and nine dust diluents and stored over an 8- to 12-month period at 4° or 20°C. The virulence of formulations, with the exception of two dust preparations, was reduced significantly compared to unformulated conidia against Culex pipiens pipiens larvae. The formulation components most detrimental to conidial virulence were corn cob granules, diatomaceous earth, and two Kaolinite diluents. This was exampled by a decline in virulence from ca. 100% for unformulated conidia to 36% or below for these formulations. LT 50 values also increased from 2.4–2.6 days for unformulated conidia to above 6 days. In contrast, a diluent derived from dried castor oil (Thixcin R) significantly enhanced conidial virulence at several doses above that of unformulated conidia against C. pipiens larvae. Enhancement occurred whether conidia were formulated prior to storage or stored separate from the diluent and mixed prior to application. The Thixcin R formulation was more effective against Anopheles stephensi larvae, but virulence was reduced against Aedes aegypti larvae. A bentonite formulation (Bentone-38) also maintained conidial virulence effectively, but Thixcin R was a superior diluent. It was shown that conidial virulence of formulations was not correlated with differences in conidial viability. The preparations that were applied dry by a surface method were more virulent than when an aqueous suspension containing a surfactant was used. The results demonstrate the need to assess efficacy of mycoinsecticidal formulations in a virulence bioassay prior to field testing.