Application of Metarhizium anisopliae (Metsch.) Sor. conidia to control Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae) larvae on glasshouse pot plants

The efficacy of the entomogenous fungus Metarhizium anisopliae was assessed against vine weevil (Otiorhynchus sulcatus) larvae in the glasshouse. Prophylactic application of M. anisopliae conidia to begonia resulted in total larval control, but curative applications were less effective with only 65% control when conidial application was delayed until 8 weeks after egg infestation. Prophylactic applications also provided effective larval control on begonia plants which received multiple egg applications over a six week period. Larval mortality was monitored on cyclamen plants which had received a prophylactic drench of M. anisopliae conidia. The population was reduced by 78% within 5 weeks of egg application and control rose to 90% after 17 weeks, although the increase was not significant. Prophylactic conidial drenches were compared with a similar number of compost incorporated conidia on cyclamen, but there was no significant difference between the two spore application strategies. Application of M. anisopliae conidia to impatiens modules before potting-on resulted in over 89% larval control compared to over 97% control when a similar number of conidia were applied to the plants after potting. Larval control was further reduced to 79% when the module drenches were reduced to one quarter of the highest dose (5 × 10⁷ compared to 2 × 10⁸ conidia per module). The persistence of three M. anisopliae strains was examined over a 20 week period on impatiens. There was no overall decline in efficacy over this period, although there was variability in the performance of the different strains and it was suggested that this was linked to temperature. The results of these experiments suggest that M. anisopliae has considerable potential as a microbial control agent for O. sulcatus on glasshouse ornamentals.     

Synergism between Metarhizium anisopliae (Deuteromycota: Hyphomycetes) and Boric Acid against the German Cockroach (Dictyoptera: Blattellidae)

Mortality of German cockroaches, Blattella germanica (L.), caused by Metarhizium anisopliae (Metschnikoff) Sorokin strain AC-1 alone and in combination with different formulations of boric acid, was evaluated in laboratory bioassays. Topical application of M. anisopliae alone (8.96 × 10⁹ conidia/m²) required 28 days to cause >92% cockroach mortality (LT₅₀ = 10 days). In contrast, in combination with boric acid (topically applied as a dust or in drinking water), M. anisopliae killed cockroaches significantly faster than without boric acid. M. anisopliae conidial dust (8.96 × 10⁸ conidia/m²) with either 12.5% (w/w) boric acid dust or 0.1% (w/v) boric acid in drinking water killed 100% of the cockroaches in only 8 days (LT₅₀ = 5 days) and 10 days (LT₅₀ = 6 days), respectively, without compromising the fungus emergence from cadavers. Replacement of M. anisopliae with flour dust or heat-killed M. anisopliae conidia eliminated this effect, demonstrating that it was not the consequence of greater boric acid ingestion due to more extensive cockroach grooming upon exposure to M. anisopliae conidia. Moreover, injections of a low dose of M. anisopliae, which caused only 30% mortality, together with sublethal concentrations of boric acid into the cockroach hemocoel resulted in a doubling of mortality. Statistical analysis demonstrated a synergistic interaction between these two insecticides.     

Infection of Blissus antillus (Hemiptera: Lygaeidae) Eggs by the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana

This study determined the pathogenicity and virulence of Beauveria bassiana and Metarhizium anisopliae to eggs of the chinch bug Blissus antillus (Hemiptera: Lygaeidae). Eggs were inoculated under laboratory conditions by immersion in concentrations of 1 × 10⁴ and 5 × 10⁶ conidia/ml. Inoculated eggs were kept under controlled conditions. Evaluations were carried out daily for 20 days. M. anisopliae isolates were highly virulent to eggs, even at 1 × 10⁴ conidia/ml. All B. bassiana isolates tested were considered to be of low virulence or avirulent. The most virulent isolate tested was ESALQ 818 (M. anisopliae), which caused 96.7% infection, when eggs were immersed in suspensions of 1 × 10⁴ conidia/ml. Conidial production on infected eggs was observed to be highest for M. anisopliae isolate CG144, with a mean value of 11.6 × 10⁵ conidia/ml/egg. Infection of Blissus eggs oviposited on plant stems was greater when M. anisopliae isolate CG144 was formulated in mineral oil (63.5% mortality) than when formulated in Tween 80 (27.1% mortality).     

Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to Entomopathogenic Fungi and Their Extracts

The effectiveness of seven strains of entomopathogenic fungi against Ceratitis capitata adults was evaluated in the laboratory. Adults were susceptible to five of seven aqueous suspensions of conidia. Metarhizium anisopliae and strain CG-260 of Paecilomyces fumosoroseus were the most pathogenic fungi, with 10-day LD₅₀ values of 5.1 and 6.1 × 10³ conidia/fly, respectively, when applied topically. Sublethal effects on fecundity and fertility of the fungal-exposed females were also studied. The most effective fungus in reducing fecundity was P. fumosoroseus CECT 2705, with reductions on the order of 65% at 1 × 10⁶ conidia/fly. M. anisopliae and Aspergillus ochraceus also showed significant reductions of fecundity (40–50% for most of the assayed concentrations). Fertility was moderately affected by the fungi. M. anisopliae at 1 × 10⁶ conidia/fly was the most effective fungus, showing egg eclosion reduction of over 50% compared with the control. In addition, culture broth dichloromethane extracts from the entomopathogenic fungi were tested for insecticide activity against C. capitata, including effects on fecundity and fertility. The extract from M. anisopliae was the most toxic, resulting in about 90% mortality at a concentration of 25 mg/g of diet; under these conditions, fecundity and fertility of treated females were reduced by 94 and 53%, respectively, compared with untreated controls.     

Cellular and humoral immune defenses of Oxya japonica (Orthoptera: Acrididae) to entomopathogenic fungi Metarhizium anisopliae

One Indonesian isolate of the fungus Metarhizium anisopliae, named Majalengka strain, was evaluated not only for its virulence but also for the immune response of rice grasshopper Oxya japonica (Orthoptera: Acrididae) as a target organism. Five aqueous suspensions with different conidia concentrations in logarithmic series were prepared. The fungus showed high virulence as it caused 100% mortality at low conidia concentration (1.5 × 10² conidia/mL). Remarkable changes in the cellular and humoral responses were also observed when adult grasshoppers were infected with the fungus. The number of hemocytes decreased significantly within 12 h after infection. In addition, the total number of granulocytes increased rapidly in the first 12 h then gradually decreased 24 and 48 h after infections, while the number of coagulocytes fluctuated over time. The infection influenced the humoral response by increasing the phenoloxidase activity.     

Selection of promising fungal biological control agent of the western flower thrips Frankliniella occidentalis (Pergande)

Aims: Larval stages of Frankliniella occidentalis are known to be refractory to fungal infection compared with the adult stage. The objective of this study was to identify promising fungal isolate(s) for the control of larval stages of F. occidentalis. Methods and Results: Ten isolates of Metarhizium anisopliae and eight of Beauveria bassiana were screened for virulence against second‐instar larvae of F. occidentalis. Conidial production and genetic polymorphism were also investigated. Metarhizium anisopliae isolates ICIPE 7, ICIPE 20, ICIPE 69 and ICIPE 665 had the shortest LT₅₀ values of 8·0–8·9 days. ICIPE 69, ICIPE 7 and ICIPE 20 had the lowest LC₅₀ values of 1·1 × 10⁷, 2·0 × 10⁷ and 3·0 × 10⁷ conidia ml^?1, respectively. Metarhizium anisopliae isolate ICIPE 69 produced significantly more conidia than M. anisopliae isolates ICIPE 7 and ICIPE 20. Internally transcribed spacers sequences alignment showed differences in nucleotides composition, which can partly explain differences in virulence. Conclusion: These results coupled with the previous ones on virulence and field efficacy against other species of thrips make M. anisopliae isolate ICIPE 69 a good candidate. Significance and Impact of the Study: Metarhizium anisopliae isolate ICIPE 69 can be suggested for development as fungus‐based biopesticide for thrips management.     

Soil properties affect the availability, movement, and virulence of entomopathogenic fungi conidia against puparia of Ceratitis capitata (Diptera: Tephritidae)

The Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae), is the major tephritid pest in the Mediterranean region. This insect may overwinter as pupae inside fruits or in soil. Therefore, infection with entomopathogenic fungi is a potentially useful control technique during the insect’s soil-dwelling stage. Entomopathogenic fungi have an important role in Integrated Pest Management programs as an alternative to conventional chemical control, but they have been usually selected on the basis of laboratory results with little regard to fungal ecology. In this work, we designed several experiments to study the availability and movement of the EF Beauveria bassiana (Balsamo) Vuill. and Metarhizium anisopliae (Metsch.) conidia in 16 soils differing widely in pH, texture, organic matter, and carbonate contents. Experiments of adsorption and drag of conidia by soil particles suspended in CaCl₂ solutions of different ionic strength showed B. bassiana conidia to be retained by clay particles, and this effect disappeared with increasing ionic strength. The availability of M. anisopliae conidia in the suspension tended to be lower for sandy than for clayey soils and was not influenced by ionic strength. Regardless of soil properties, over 90% of the added fungal propagules were recovered from the surface layer of columns of packed soils representing model combinations of texture (sandy or clayey) and pH values (acid or alkaline). However, retention of B. bassiana conidia in the surface layer was higher in clayey than in sandy soils, and the retention of M. anisopliae conidia in the surface layer was higher in sandy than in clayey soils. Finally, neither soil texture nor ionic strength affected the infectivity of conidia of both fungal strains to C. capitata puparia.     

Cover crop and conidia delivery system impacts on soil persistence of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) in sugarbeet

The sugarbeet root maggot, Tetanops myopaeformis (Röder), is a major North American pest of sugarbeet, Beta vulgaris L. Previous research suggests that moderate T. myopaeformis control is possible with the entomopathogen Metarhizium anisopliae (Metch.) Sorok. We conducted a three-year (2002–2004) experiment to assess impacts of oat, Avena sativa L. and rye, Secale cereale L., cover crops on persistence of corn grit-based granular or spray formulations of M. anisopliae isolate ATCC 62176 (i.e. MA 1200) applied at 8×10¹² viable conidia/ha in sugarbeet. More colony forming units (CFUs) were detected immediately after application [0 days after treatment (DAT)] in spray plots than granule-treated plots. However, 76–92% declines in CFUs per gram of soil occurred in spray plots within 30 DAT. Substantially (i.e. 83–560%) more rainfall occurred in June 2002 than during June of any other year. Subsequently, 71–670% increases in CFU concentrations occurred by 60 DAT in M. anisopliae granule-treated plots with oat or rye cover crops that year. CFU density increases were higher in cover crops in 2002, but no significant cover crop effects were detected. Conidia persisted for up to 30 DAT in M. anisopliae spray plots and 60 DAT in granule-treated plots in 2002; however, no increases occurred in the years with less June rainfall. Trends suggest that M. anisopliae aqueous sprays result in greater conidia concentrations than granules at sugarbeet plant bases in June during T. myopaeformis oviposition and larval establishment on host plants. Increases are possible when delivering conidia via granules, but high post-application rainfall could be necessary for conidia production.     

Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat

To evaluate the pathogenicity of Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes) a bioassay was designed under laboratory conditions against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat. The fungus was applied at the dose rates of 8 × 10³, 8 × 10⁵, 8 × 10⁷ and 8 × 10⁹ conidia/kg of wheat and the bioassay was conducted at 25°C with 60% relative humidity. The data regarding the mortality was recorded after 7 and 14 days exposure intervals. All the treatments gave the significant mortality of R. dominica and M. anisopliae of 8 × 10⁹ conidia/kg was found to be the most effective after a 14-day exposure interval. There was greater production of progeny when the low rate of M. anisopliae was applied to wheat. Overall, our study showed that M. anisopliae is vigorous when applied at a high dose rate which revealed an effective control of R. dominica and also played a pivotal role in the integrated pest management program (IPM) of stored wheat insect pests.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

Application of hydrophilic–lipophilic balance (HLB) number to optimize a compatible non-ionic surfactant for dried aerial conidia of Beauveria bassiana

The hydrophilic–lipophilic balance (HLB) number system was used to optimize a compatible non-ionic surfactant, TDA (polyoxyethylene tridecyl ether) in formulations for two Beauveria bassiana strains, NI8 and GHA. The optimal HLB number for TDA was determined on the basis of wetting times for conidial powders. The results indicated that optimal HLB number of TDA for B. bassiana strain NI8 was 8, while the optimum HLB number for strain GHA was 10. The optimized TDA surfactants required significantly less wetting times than the commonly used laboratory surfactants, Triton X-100, Span 80, and Tween 80. These optimized TDA surfactants were further characterized on their ability to produce conidial suspensions of the two strains after 5 min of mixing, TDA HLB 8 and TDA HLB 10 produced suspensions of 1.8 × 10⁸ and 1.6 × 10⁸ conidia/ml for NI8 and GHA, respectively. These conidial levels were significantly higher than those in Triton X-100, Span 80, and Tween 80 suspensions after the same mixing time. Germination assays showed that TDA HLB 8 promoted significantly higher germination rates of strain NI8 than those observed in other commonly used laboratory surfactants. However, the germination rates of the GHA strain were unaffected by any of the surfactants tested. The efficacy of the conidial suspensions was confirmed with assays against Lygus lineolaris. Bioassay results indicated that there were no significant differences in mortalities because of surfactants. These results suggest optimization based upon HLB number will not negatively impact parameters associated with efficacy, while providing desirable physical properties.     

Sensitive and Rapid Detection of the Insect Pathogenic Fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">anisopliae</Emphasis> by Loop-mediated Isothermal Amplification

A set of six specific primers was designed by targeting intergenic spacer region (IGS) sequences. With Bst DNA polymerase, the products could be clearly amplified for 60 min at 62°C in a simple water bath. The sensitivity of the loop-mediated isothermal amplification (LAMP) for detecting Metarhizium anisopliae var. anisopliae was about 0.01 pg fungal DNA per reaction (equivalent to 27 conidia). LAMP products could be judged with agar gel or naked eye after addition of SYBR Green I. There were no cross reactions with other fungal isolates indicating high specificity of the LAMP. The LAMP could detect the presence of M. anisopliae var. anisopliae from soil. The detection limits for M. anisopliae var. anisopliae of LAMP reaction was 50 conidia per reaction in soil.     

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. Methods and Results: The conidial yields and the shelf‐life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia‐bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf‐life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. Significance and Impact of the Study: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.     

Relative susceptibility of <Emphasis Type="Italic">Spodoptera litura</Emphasis> pupae to selected entomopathogenic fungi

The pupae of Spodoptera litura (Fab.), (Lepidoptera: Noctuidae), a polyphagous pest affecting common crops in Indian subcontinent, were treated with different concentrations of conidia of four isolates of entomopathogenic fungi belonging to three species, Metarhizium anisopliae var. anisopliae (Metschnikov) Sorokin (ARSEF 7487), Lecanicillium muscarium (Petch) Zare & W Gams (two isolates ARSEF 7037 and ARSEF 6118) and Cordyceps cardinalis Sung & Spatafora (ARSEF 7193) under laboratory conditions. Suspensions (10⁸/ml) of conidia harvested from Sabouraud dextrose agar yeast extract (SDAY) plates resulted in the highest mortality (85.8%) with M. anisopliae and the lowest mortality (57.3%) with C. cardinalis. The values of LC₅₀ and LC₉₀ suggested that M. anisopliae was the most virulent fungal strain followed by L. muscarium (ARSEF 7037). However, C. cardinalis was the least virulent species among the fungi used in the bioassay. In soil bioassays, drenching the soil with conidial suspensions of ARSEF 7487 and ARSEF 7037 (10⁸ conidia/g of soil) reduced the adult emergence from pupa by 81.3% and 72.5%, respectively, while premixing the sterile soil with conidia killed lesser number of pupae (62.9% by ARSEF 7487 and 54.6% by ARSEF 7037). Our findings suggest that M. anisopliae (ARSEF 7487) and L. muscarium (ARSEF 7037) are potent entomopathogens and could be developed into biocontrol agents against rice cutworm in IPM programs. Handling editor: Helen Roy     

Insecticidal activity of entomopathogenic fungi (Hypocreales) for potato psyllid,Bactericera cockerelli (Hemiptera: Triozidae): Development of bioassay techniques,effect of fungal species and stage of the psyllid

The potato psyllid, Bactericera cockerelli (?ulc), is a pest of potato, tomato, and some other solanaceous vegetables and has also been incriminated in the transmission of a bacterial pathogen, Candidatus Liberibacter solanacearum, resulting in a serious disease known as ‘zebra chip’. Although there are several reports of fungal pathogens in psyllids, there are none from B. cockerelli, nor have any fungi been evaluated against it. Five isolates of fungi, one Beauveria bassiana, two Metarhizium anisopliae and two Isaria fumosorosea, were bioassayed against B. cockerelli on potato leaves under ideal conditions for the fungi. All applications were made with a Potter spray tower. With the exception of concentration-effect studies, all other applications were made using 10⁷ conidia/mL in a 2-mL aqueous suspension. All isolates except B. bassiana, produced 95–99% mortality, corrected for control mortality, in adults 2–3 days after application of conidia and 91–99% in nymphs 4 days after application. The corrected mortalities for adults and nymphs treated with B. bassiana were 53 and 78%, respectively, 4 days after application. I. fumosorosea Pfr 97 produced 95% corrected mortality in both first and late third instar nymphs. M. anisopliae (F 52) produced 96% corrected mortality in first and third instar nymphs. Pfr 97 and F 52 were evaluated for insecticidal activity against third instar B. cockerelli using 10⁵, 10⁶, and 10⁷ conidia per mL. Mortality produced by I. fumosorosea Pfr 97 ranged from 83 to 97% and that of M. anisopliae F 52 was 88 to 95% at these concentrations.     

Evaluation of Metarhizium anisopliae for the control of Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae), the principal vector of bluetongue virus in Australia

Four isolates of the entomopathogenic fungus Metarhizium anisopliae were tested for their potential to control the biting midge Culicoides brevitarsis, the principal vector of bluetongue virus in Australia. Adult C. brevitarsis died three to eight days after walking on paper substrate treated with 0.7 g/m² conidia of any of the isolates, indicating that M. anisopliae has potential as a surface treatment or topical application control strategy. Incorporation of the fungus into freshly excreted cattle dung at rates of between 0.25 and 1 g conidia/kg reduced the emergence of adult midges by up to 98.5% compared to untreated dung indicating that M. anisopliae has the potential to control C. brevitarsis larvae in cattle dung. Three of the isolates produced similar mortality rates on adult and immature C. brevitarsis while the fourth isolate produced lower, but still significant, mortality rates on adult and immature stages.     

Resistance of the termite, <Emphasis Type="Italic">Coptotermes formosanus</Emphasis> Shiraki to <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> due to grooming

Termites, Coptotermes formosanus, reared individually, were highly susceptible to the entomopathogenic fungus, Metarhizium anisopliae, while termites reared in␣groups were highly resistant. When reared in groups, the termites treated with M.␣anisopliae conidia on the body surface were groomed by their nestmates and more than 80% of the conidia were removed from the cuticle within 3 h. However, there was not a significant reduction in the numbers of conidia on the body surfaces of termites reared individually. For the termites maintained in groups, conidia were found in foreguts, midguts and hindguts, but very few conidia were detected in the guts of termites reared individually. Conidia in the alimentary tracts did not germinate, but some of were alive. As a result, it seems that the removal of foreign bodies, such as fungal conidia, from the␣cuticle is one function of termite mutual grooming behavior and that conidia removed from the cuticle are eliminated through alimentary tracts. This study indicates that mutual grooming behavior is very effective in protecting these termites from M.␣anisopliae infection.     

Differential susceptibility of Amblyomma maculatum and Amblyomma americanum (Acari:Ixodidea) to the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae

Nymph and adult ticks from Ambylomma americanum and Ambylomma maculatum were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (90517) and Metarhizium anisopliae (20500). Fungal suspensions of conidia harvested from potato dextrose plates containing 10⁸ conidia/ml caused greater than 90% mortality in adult A. maculatum but less than 10% mortality in adult A. americanum over a 28 day time course. Similarly, infection with M. anisopliae (10⁸ conidia/ml) resulted in 60 and 15% mortality in A. maculatum and A. americanum, respectively. Nymphs of both tick species were more susceptible to fungal infection reaching mortality rates of almost 100% for A. maculatum and over 35% for A. americanum. Scanning electron microscopy of infected ticks showed rapid attachment, germination, and proliferation of fungal spores on A. maculatum cuticles, and to a much lesser extent on A. americanum cuticles. Pentane extracts of A. americanum cuticle hydrocarbons inhibited germination and hyphal growth of B. bassiana conidia, whereas no inhibition was observed using A. maculatum extracts. Significant mortality towards A. americanum was observed (>60%, 28 days) only when the ticks were treated with B. bassiana directly from the growth medium (10⁷ blastospores/ml, grown for 3–4 days in Sabouraud dextrose + 0.5% yeast extract liquid media). These results indicate tick species display differential susceptibility to the entomopathogenic fungi B. bassiana and M. anisopliae, and that the ability to overcome fungistatic compounds present in the tick epicuticle may determine the likelihood of successful infection and virulence.     

Impact of culture age on conidial germination,desiccation and UV tolerance of entomopathogenic fungi

The impact of culture age on conidial yields, germination and tolerance to UV exposure of freshly harvested and dry conidia produced by five entomopathogenic fungal (EPF) isolates was studied. Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Lecanicillium muscarium were grown on potato dextrose agar (PDA) medium for 7 or 14 days at 25°C. While the age of cultures had a significant impact on the germination rate of conidia produced by isolates L. lecanii CBS 122.175 and B. bassiana LMSA 1.01.093, other EPF isolates germinated at the same rate regardless of the culture age. When exposed to UV radiation, conidia produced by all isolates germinated at a lower rate compared to the non-irradiated conidia, although this decrease in germination (20–80% decrease) was unaffected by the culture age. Air-drying had only a slight impact on conidial germination (0–60% decrease). Under the conditions of this study, the stability of irradiated conidia produced by M. anisopliae LMSA 1.01.197 and B. bassiana CBS 110.25 was significantly increased when conidia were dried prior to UV exposure. This increase in tolerance to stress of dried conidia might be caused, at least partially, by the low metabolic activity associated with dehydration.     

The mechanism of the mycoinsecticide diluent on the efficacy of the oil formulation of insecticidal fungus

Adverse conditions, including low humidity, UV irradiation, and high temperature, appreciably affect the efficacy of mycoinsecticides. Oil formulation increased the virulence of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against locusts and grasshoppers by reducing the dependence on saturated water. A mycoinsecticide diluent (a water-in-oil emulsion) has been widely used to dilute the oil formulation of M. anisopliae in China. The aim of our study was to elucidate the mechanism by which the mycoinsecticide diluent improves the virulence of M. anisopliae. We investigated the effects of the mycoinsecticide diluent on the virulence, invasion speed, and viability of the conidia under various adverse conditions. The results demonstrated that the mycoinsecticide diluent significantly improved the virulence of conidia at low humidity (68, 75, and 84%). In particular, at an RH of 68%, the LT₅₀ for locusts treated with the emulsion was 5.4 days and was 31.6% lower than the value for locusts treated with an oil formulation. In addition, the concentration of the hyphal bodies found in the haemolymph of locusts treated with emulsion was about 27-fold higher than that in locusts treated with oil formulation four days after inoculation. This result was further confirmed by determining the concentration of M. anisopliae var. acridum DNA in locust haemolymph using quantitative PCR. The percentage germination of conidia in the emulsion was also significantly higher than that in oil at 68% RH. There was no significant difference in percentage germination between conidia treated with the emulsion and oil when exposed to irradiation with ultraviolet-B (UV-B) or high temperature. These results demonstrate that the mycoinsecticide diluent enhances the virulence of M. anisopliae formulated in oil at low humidity by providing adequate water for germination without interfering with the UV tolerance and thermotolerance of the conidia.