|Wraight, Stephen - Steve|
Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/14/2010
Publication Date: 9/6/2010
Citation: Faria, M., Hotchkiss, J.H., Hajek, A.E., Wraight, S.P. 2010. Debilitation in conidia of the entomopathogenic fungi Beauveria and Metarhizium anisopliae and implications with respect to viability determinations and mycopesticide quality assessments. Journal of Invertebrate Pathology. 105(1):74-83. Interpretive Summary: The principal ingredients in most biopesticide products based on insect pathogenic fungi (mycopesticides) are dry spores called conidia, and viability of these conidia is one of the most commonly reported indicators of product quality (insecticidal activity). Nevertheless, there are no standardized protocols for determining viability. Viability (germinability) of mycopesticides is most commonly determined by mixing dry conidia from storage directly into water and then inoculating samples of the resulting spore suspension onto a germination substrate and incubating for 16–24 hours. Numerous researchers have reported, however, that in many cases, substantially higher viabilities can be obtained if conidia are incubated for longer periods of time or if the dry conidia are slowly rehydrated by exposure to high humidity before mixing in water. In a series of tests designed to identify the most useful/meaningful germination protocol, we observed that differences in the estimates of viability from different protocols increase markedly after conidia were exposed to various stress factors, such as high levels of temperature and moisture, during storage. Further research revealed that protocols using slow rehydration and/or prolonged incubation enabled germination of weakened conidia that would not otherwise have been counted as viable. Studies have shown that conidia in a weakened or debilitated state are less effective as pest control agents, and our research thus identifies protocols employing fast rehydration and short incubation times (24 hours or less) as providing the most reliable assessments of overall biopesticide quality. It is anticipated that these results will also lead to greater standardization of viability protocols among mycopesticide researchers and producers.
Technical Abstract: Viabilities of entomopathogenic fungal conidia comprising biopesticide products are most commonly determined by suspension of dry conidia from storage in a water/surfactant solution immediately before inoculation onto an agar-based germination substrate; conidia are then incubated at a moderate temperature (usually around 25 degrees C) for 24 hours. In this study, germination rates determined from this fast rehydration (FR) protocol were compared to those obtained when dry conidia were subjected to slow rehydration (SR) via exposure to high humidity for 24 hours prior to aqueous suspension. Differences in viability estimates obtained using the two protocols increased markedly after conidia were exposed to various stress factors during storage (high levels of moisture, temperature, and O2), with the SR protocol producing higher estimates of viability in all cases. For example, after Beauveria bassiana (Bb) conidia were stored under moist conditions for 21 days at 25 degrees C, the SR estimate of viability was 21 percent greater than the FR estimate. Germination of stressed Bb and Metarhizium anisopliae conidia increased substantially when incubation time on the germination substrate was increased from 24 to 72 hours, whereas germination of non-stressed conidia showed little change. Testing ultimately revealed that stress-induced changes in conidial quality were the underlying causes of the differences in viability estimates from the various protocols. Conidia debilitated by stress were characterized by hypersensitivity to lethal imbibitional damage, which is mitigated by slow rehydration, or by slow germination. These findings suggest that viability protocols incorporating short incubation times (24 hours) and fast rehydration may provide the most reliable assessments of overall mycopesticide quality.