Effects of Rehydration on the Conidial Viability of Metarhizium flavoviride Mycopesticide Formulations

The rehydration of dried conidia of Metarhizium flavoviride was investigated in an attempt to increase speed of kill of locusts and grasshoppers by formulations of this fungus. Conidia were dried to 4-5% moisture content with no apparent adverse effects on viability, but rapid rehydration (by putting dried conidia directly in free water) reduced viability. Rehydration in an atmosphere of high humidity allowed dry conidia to absorb sufficient moisture to avoid imbibition damage. Rehydrating and pre-germinating conidia prior to spraying (in an oil-based formulation) on to the desert locust, Schistocerca gregaria, did not decrease the time to death, suggesting that moisture uptake by dry conidia on the desert locust cuticle is easily achieved.     

Susceptibility of the Hymenopteran Parasitoids Apoanagyrus ( = Epidinocarsis ) lopezi (Encyrtidae) and Phanerotoma sp. (Braconidae) to the Entomopathogenic Fungus Metarhizium anisopliae var. acridum (Deuteromycotina: Hyphomycetes)

Laboratory and so-called extended laboratory bioassays were conducted in Benin, West Africa, to investigate the pathogenicity and virulence of the entomopathogenic fungus Metarhizium anisopliae var. acridum , a biocontrol agent against locusts and grasshoppers, to two hymenopteran parasitoids, Apoanagyrus ( = Epidinocarsis ) lopezi and Phanerotoma sp. Treatments were carried out under simulated field conditions at standard field dose rates of 2.5 and 5.0 ×10 12 conidia ha -1 . Test organisms were continuously (3 weeks) exposed to spray residues in artificial or simulated natural environments. The standard strain IMI 330 189 of the mycopesticide Green Muscle caused a significant reduction of 24% in the longevity ( = average survival time, AST) of A. lopezi , relative to the untreated control. Mycosis was confirmed in 16% of all cadavers. AST was shorter under low relative humidity (RH) conditions, and these conditions seemed to enhance susceptibility to fungal infection. However, this effect was only marginally significant. In contrast, average longevity of untreated A. lopezi was slightly yet significantly shorter at low RH (50-60%) than at high RH (80-90%). In the extended laboratory assay, the same isolate had no significant effect on mortality, parasitoid emergence ( = beneficial capacity) and sex ratio. In a further screening test with isolates IIBC I91 609, IIBC I93 833 and IMI 330 189 (reference), no infection of A. lopezi was confirmed. Similarly, Phanerotoma sp. was not susceptible to IMI 330 189. It is concluded that mycopesticides based on the three strains tested pose a low risk to parasitic hymenopterans under field conditions.     

Long-term Storage of Metarhizium flavoviride Conidia in Oil Formulations for the Control of Locusts and Grasshoppers

Freshly harvested conidia of Metarhizium flavoviride (Gams & Rozsypal) were stored in two vegetable oils, groundnut or soya, or a mineral oil, Edelex. They were diluted with either Shellsol K or deodorized kerosene, and antioxidants were added to half of the vegetable oil formulations. Dried non-indicating silica gel was added to half of the formulations before storage at 8 or 17 C. Undried conidia, those without silica gel, lost viability rapidly, with germination dropping below 40% after 9 and 32 weeks at 17 and 8 C respectively. After 127 weeks (ca. 30 months) in storage, germination remained at over 60 and 80% for the dried formulations at 17 and 8 C respectively (after an unexplained drop in germination after 16-18 months in storage). Comparable figures for 160 weeks (ca. 37 months) were 47 and 68%. These figures represented germination after 24 h of incubation; after 48 h of incubation, germination was 79 and 89% from samples stored for 160 weeks at 17 and 8 C respectively. Representative formulations of the stored conidia were tested in bioassays against the desert locust Schistocerca gregaria (Forskal) up to 30 months into the experiment, and were found to have retained full virulence compared with freshly prepared formulations.     

Effects of the Sunscreen Oxybenzone on Field Efficacy and Persistence of Metarhizium flavoviride Conidia Against Kraussella amabile (Orthoptera: Acrididae) in Mali,West Africa

A field trial was performed at Mourdiah, northwest Mali, to evaluate whether the performance of Metarhizium flavoviride conidia would improve if 2% oxybenzone, an oil-soluble ultraviolet light protectant, was used. Conidia were applied in an ultra-low volume formulation against nymphs of the grasshopper Kraussella amabile . Mortality was assessed by collecting grasshoppers directly from field plots (sweep net samples) or by exposing untreated nymphs to sprayed vegetation for 48 h (field cage samples). For sweep net samples, the mortalities in M. flavoviride treatments varied between 94.5 and 99.9% up to 7 days after treatment, while control mortalities were significantly lower (20.7-55.0%). There were no significant differences between M. flavoviride treatments with or without oxybenzone. The average survival times were found to be significantly lower for fungal treatments (4.9 days) compared with the control treatment (11.8 days). W ith field cage samples, the mortality in M. flavoviride treatments declined from > 95.0% for nymphs exposed just after application to < 30.0% for nymphs exposed to treated vegetation on the 10th day after application. The control mortalities varied between 5.1 and 37.2%, and were significantly lower than fungal treatments for samples placed just after application and 10 days after application. The overall average survival times were 10.9 and 10.7 days for M. flavoviride with and without oxybenzone respectively, and 13.9 days with the control treatment. An exponential regression was fitted using cage mortality data which indicated that the half-life for conidial infectivity was 4.3 days on treated vegetation.     

Field Trial with the Entomopathogenic Fungus Metarhizium anisopliae var. acridum against Bands of the Grasshopper Rhammatocerus schistocercoides in Brazil

The efficacy of a mycoinsecticide formulated in vegetable oil was tested in Brazil against the grasshopper Rhammatocerus schistocercoides . A set of experiments was conducted in the Chapada dos Parecis region (Mato Grosso state), a permanent zone of outbreaks for this pest. Experiments were performed in zones of natural vegetation, against grasshopper bands in the third nymphal instar. Three nymphal bands were treated with a mycoinsecticide formulation based on conidia of the entomopathogenic fungus Metarhizium anisopliae var. acridum ( =M. flavoviride ), strain CG 423. Three non-treated bands were used as control. The application was made with the aid of a hand-held ULV sprayer adjusted to deliver 2 l of the formulation ha -1 , each containing 1 ×10 13 conidia. Treatments were limited to the surface of the grasshopper bands and their immediate borders (5-10 m). The efficacy of the mycoinsecticide was evaluated through band survival after treatment (grasshopper numbers, surface, density, behaviour and daily movement of the band), allowing the insects to move freely in their natural environment. Insects were regularly surveyed and maintained in the laboratory, allowing estimates of the infection rate. Field and laboratory studies showed a clear effect of the product 10 days after treatment. At 14 days post-spraying, mortality caused by the mycoinsecticide in the field was approximately 88%.     

Viability of Stored Conidia of Metarhizium flavoviride Gams and Rozsypal, Produced under Differing Culture Regimes and Stored with Clays

Conidia of Metarhizium flavoviride were cultured under a range of conditions and stored , with or without clays and silica gel , as powder or in oil . Fungal biomass was produced in shake flasks in liquid media containing nutrients before being added to sterilized rice for conidial production . Conidia pre - cultured under a low C:N ratio , or dried over a 9 - day period compared with 2 days before being placed in storage , showed greatest loss of viability . Conidia stored at 10 o C survived better than those stored at + 30 o C . A range of clays had no significant effect on storage of dried conidia but montmorillonite K10 clay was often harmful in terms of conidial viability . Conidia survived better when stored as dry powder than when stored in a mixture of mineral and vegetable oils . The addition of silica gel proved beneficial even when dried conidia were being stored .     

Development of a Spray-drying Technique for Submerged Spores of Entomopathogenic Fungi

The stability of blastospores or submerged conidia of entomopathogenic fungi is one of the key problems associated with the practical use of fungal biopreparations. A spray-drying technique was developed which allows the drying of blastospores and/or submerged conidia, here called submerged spores, without a significant loss of viability and with only a slight delay in eY cacy. The method was tested successfully using the fungal species Metarhizium anisopliae , M. flavoviride, Beauveria bassiana and Paecilomyces fumosoroseus . For all experiments, a laboratory spray dryer was used. The optimum inlet and outlet temperatures were found to be 64 2oC and 48 2oC respectively. The best protective agent for submerged spores was skimmed milk powder at concentrations of 10 or 20%. The addition of sugars, especially 2.5% sugar-beet syrup, slightly improved the viability after spray-drying. Submerged spores of eight isolates of M. anisopliae, M. flavoviride, B. bassiana and P. fumosoroseus were suspended in 20% skimmed milk powder and 2.5% sugar-beet syrup, and spray-dried. Germination rates of about 90% were achieved, which were comparable to those of freshly produced submerged spores. Finally, the virulences of spray-dried submerged spores of M. anisopliae (Ma 97) and M. flavoviride (Mfl 5) were tested in bioassays using third- and fourth-instar nymphs of the African locust, Locusta migratoria . In contrast to M. flavoviride , there was no significant diVerence in the median lethal time between spray-dried and fresh submerged spores of M. anisopliae . The investigations demonstrated that spray-drying is possible to preserve sensitive submerged spores of entomopathogenic fungi.     

Effects of a Mycoinsecticide on Feeding and Fecundity of the Brown Locust Locustana pardalina

The entomopathogenic fungus Metarhizium anisopliae var. acridum is being developed for use as a mycoinsecticide against locusts and grasshoppers in Africa. In addition to causing significant mortality, studies have shown the pathogen to cause reductions in feeding of a number of target species during the disease incubation period. The present study extends this work by demonstrating significant reductions in feeding following infection in another important pest species, the brown locust Locustana pardalina (Walker). Insects collected from the field following a spray application and subsequently maintained in a field laboratory showed a significant reduction in per capita feeding, as indicated by faecal production, as early as 2 days after treatment; this was before any pathogen-induced mortality. This per capita reduction in feeding by infected individuals contributed to a total reduction in feeding of 65% relative to controls. In addition to this laboratory based assessment, experiments were conducted to examine the effects of infection on mortality, feeding and fecundity in the field. Mortality rates of infected individuals in the field were significantly slower than those in the laboratory (average survival time of approximately 39 days compared with 6 days), although total mortality and percentage mycosis were equivalent. Treated individuals showed a significant reduction in body fat accumulation at sexual maturity compared with controls. This may reduce dispersal ability and total reproductive fitness. However, treated individuals exhibited a reduced pre-oviposition period resulting in them laying more eggs than untreated individuals within the first weeks following fledging. Thus, no significant reductions in fecundity were recorded by the end of the assessment period when mortality of treated individuals exceeded 90%. Given that many untreated locusts were still alive and had the potential to continue oviposition for several more weeks, however, it is likely that differences in fecundity would have become apparent if the assessment had continued. These results provide important insights for evaluating overall impacts of spray applications of the mycoinsecticide.     

Effects of Moisture Content and Temperature on Storage of Metarhizium flavoviride Conidia

The effects of moisture content and temperature on the medium-term (3-4 months) storage of conidia of Metarhizium flavoviride were investigated. Conidia harvested after 24 days of culturing on rice showed greater tolerance to long storage than conidia from 12-day cultures. The moisture content of the conidia was of greatest importance; at harvest from the culture, conidial moisture contents could be 40%, while the optimal moisture content for storage was found to be 4-5%. Dried conidia stored in oil benefited from the addition of dried silica gel, as did conidia stored as powder. A range of mineral oils proved satisfactory for storage, and when dried silica gel was added to suspensions, germination levels were 79.8% after 105 days at 28-32 C. Dried conidia stored in oil maintained germination levels of up to 96 and 85% after 80 days at 10-14 C and 28-32 C respectively. Dried conidia stored as powder retained germination levels of 95% at 10-14 C, but only up to 27% at 28-32 C. In another experiment, dried conidia maintained greater than 90% germination over 128 days, with or without silica gel at 10 - 14 C or -15 - -18 C.     

Influence of Moisture Content on Temperature Tolerance and Storage of Metarhizium flavoviride Conidia in an Oil Formulation

Investigations of the temperature tolerances of Metarhizium flavoviride conidia, of different moisture contents, in an oil formulation were carried out. The conidia were dried to 5, 9, 15 or 19% moisture contents and exposed to a range of temperatures, -15, 12, 25, 38, 48 and 55 C. Viability declined due to high temperatures and high moisture contents. After 4 months of storage, the conidia were at over 80% viability in all but the 19% moisture content group of the 25 C treatment and all the samples stored at lower temperatures. In a second experiment, conidia dried to 4-5% moisture content showed greater temperature tolerance than conidia with higher moisture contents. Short-term storage under adverse conditions could be feasible with conidia dried to a suitable degree.