Susceptibility of different life stages of the European cherry fruit fly,Rhagoletis cerasi,to entomopathogenic fungi

The effects of six fungus isolates on the mortality of different life stages of the European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), were assessed in a series of laboratory experiments to find an isolate suitable for biological control. In a first step, the effects of fungus treatments on mortality, mycosis and fecundity of adult flies at a concentration of 10⁷ conidia/ml were evaluated. All fungus isolates caused mycosis but virulence varied considerably among the isolates. Beauveria bassiana and Isaria fumosorosea caused 90–100% mortality and had the strongest influence on fecundity. Metarhizium anisopliae also induced high rates of mortality, while the pathogenicity of Isaria farinosa was low. The effects of lower conidia concentrations and the influence of the age of flies were assessed in a second step. Higher conidia concentrations generally resulted in a higher mortality. B. bassiana was most efficient at low concentrations. Young flies showed lower mortality rates than older flies but, sub‐lethal effects on eclosion rate of eggs were greater in younger flies. Finally, the effects on L₃ larvae were tested: none of the fungus isolates induced mortality in more than 25% of larvae. As L₃ larvae and pupae are not susceptible to fungus infection, field control of R. cerasi should be focused on adult flies.     

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.     

Pathogenicity of entomopathogenic fungi to eggs and larvae of Spodoptera litura,the common cutworm

The eggs and second instar larvae of Spodoptera litura were treated with different concentrations of conidial suspensions of six isolates of fungi belonging to five species, Metarhizium anisopliae var. anisopliae (Metschnikov) Sorokin (ARSEF 7487), Lecanicillium muscarium (Petch) Zare & W Gams (ARSEF 7037 and ARSEF 6118), Cordyceps cardinalis (ARSEF 7193), Fusarium lateritium Nees (ARSEF 8291/MTCC 9050) and Aspergillus sp. (ARSEF 8519). In bioassay with unscaled eggs, M. anisopliae, C. cardinalis, F. lateritium and Aspergillus sp. resulted in 100% mortality above 10⁶ conidia/mL. However, with scaled egg masses, the highest hatching rate (56%) was observed with L. muscarium (ARSEF 6118) whereas the lowest hatchings were observed in the case of M. anisopliae followed by L. muscarium (ARSEF 7037), Aspergillus sp., F. lateritium, and C. cardinalis. The larvae were also found susceptible to all isolates in a dose dependent manner. Three promising isolates against larvae, viz. M. anisopliae, F. lateritium and L. muscarium (ARSEF 7037) resulted in average percent mortalities of 88, 89 and 77%, respectively at ≈10⁸ conidia/mL. When both larvae and the leaves (provided as food) were treated with ≈10⁸ conidia/mL, mortality further increased for these isolates. Based on the mortality data and the LC₅₀ values, we suggest that M. anisopliae, F. lateritium and L. muscarium (ARSEF 7037) could be developed as potential biocontrol agents against rice cutworm in IPM programs.     

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.     

Efficacy of entomopathogenic fungi against soil-dwelling life stages of western flower thrips, Frankliniella occidentalis, in plant-growing media

The potential of several entomopathogenic fungi to control soil‐dwelling stages of western flower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), was evaluated in various growing media. Two Metarhizium anisopliae (Metsch) Sorokin strains, V275 and ERL700, were the most effective, causing 85–96% mortality of thrips larvae and pupae (as measured by relative adult emergence rates), 11 days after inoculation. Mortality in other M. anisopliae‐treated media ranged from 51–84%; Beauveria bassiana (Balsamo) Vuillemin strains caused 54–84% mortality, and Paecilomyces fumosoroseus (Wize) 63–75% mortality. In contrast, mortality from the insecticide treatment (fipronil 1 g l^?1 medium) was only 15–54%. Metarhizium anisopliae V275 was selected for more in‐depth evaluation under greenhouse conditions. There was no significant difference in M. anisopliae efficacy in growing media whether it was applied as drench (84–93%) or premixed into the medium as dry conidia (85–92%). The use of M. anisopliae with reduced rates of either fipronil or imidacloprid did not significantly improve control. Overall, our study shows that M. anisopliae V275 is robust and offers much promise for the control of soil‐dwelling stages of thrips as part of an integrated pest‐management programme.     

Entomopathogenicity of beauveria bassiana and metarhizium anisopliae to the cowpea aphid,aphis craccivora koch (homoptera: Aphididae)

The pathogenicity of four isolates each of the entomopathogenic fungi, Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. to apterous adult Aphis craccivora Koch was evaluated in the laboratory at 4 concentrations of conidia. All fungi isolates tested were found to be pathogenic to the insect but their virulence varied among species and isolates within species. Three isolates, B. bassiana CPD 11 and M. anisopliae CPD 4 and 5 caused significantly higher mortality than the other isolates at the various concentrations tested causing mortality of between 58–91%, 64 to 93% and 66–100%, respectively, at 7 days post treatment. At the highest concentration of 1 × 10⁸conidiaml^‐1, these isolates produced the shortest LT₅₀s of 3.5, 3.6 and 3.4 days, respectively. Their LC₅₀s were 6.8 × 10⁵, 3.1 × 10⁵ and 2.7 × 10⁵ conidia ml^‐1, respectively. The results indicate that these isolates are promising candidates for the control of the cowpea aphid but their pathogenicity to various aphid non‐target beneficial organisms within the cowpea agroecosystem warrant further investigation before initiating field control.     

Management of pine wilt disease vectoring Monochamus alternatus adults using spray and soil application of Metarhizium anisopliae JEF isolates

Chemical control is widely used to control the Japanese pine sawyer beetle, Monochamus alternatus, but strong chemical regulations require an environmentally sound management strategy. In this work, we investigated the use of entomopathogenic fungi and their application as a means of practical pest management. Thirty-two diverse species of fungal isolates were assayed against adult pine sawyer beetles using a contact method under laboratory conditions, and four isolates showed over 70% virulence consequently. These isolates, two each of Beauveria bassiana and Metarhizium anisopliae were sprayed on the adult beetles at 1 × 10⁷ conidia/ml in plastic containers, respectively. The M. anisopliae-treated adult beetles showed 67% mortality. M. anisopliae isolates JEF-197 and JEF-279 demonstrated dosage-dependent insecticidal activity. Following the laboratory experiments, semi-field trials were conducted in young pine trees under high (RH 94%) and low (RH 35%) humidity conditions. In the high humidity conditions, most of the adult beetles stayed on the top of the branches. When the two M. anisopliae isolates were sprayed on the beetles, they showed ca. 50–70% insecticidal activity 11 days after application. In contrast, in low humidity conditions, the adult beetles tried to move off the branches and onto the soil. When the beetles reached the JEF-197 and JEF-279-treated soil, we measured >90% insecticidal activity. This work suggests that M. anisopliae was the most virulent entomopathogenic fungus against adult Japanese pine sawyer beetles, and this forest insect could be ecologically controlled by the spray and soil application of the M. anisopliae isolates.     

Performance of a semiochemical‐baited autoinoculation device treated with Metarhizium anisopliae for control of Frankliniella occidentalis on French bean in field cages

The performance of an autoinoculation device was evaluated in field cage experiments for control of Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), in French bean, Phaseolus vulgaris L. var. Samantha (Fabaceae). Treatments consisted of a fungus‐treated device with and without semiochemical (Lurem‐TR), and a fungus‐free device as control; the fungus used was Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae). The overall mean number of conidia acquired by a single insect was higher in the fungus‐treated semiochemical‐baited device than in the device without semiochemical: 5.0 ± 0.6 × 10⁴ vs. 2.2 ± 0.4 × 10⁴ conidia per insect. The overall thrips mortality was also higher in the semiochemical‐baited device than in the device without the semiochemical: 59.3 ± 3.9 vs. 41.7 ± 3.5%. Conidial viability was not affected in the M. anisopliae‐treated device without semiochemicals, 7 days after treatment, whereas it was considerably decreased – from 81.0 ± 1.3 to 6.5 ± 1.1%, 2 and 7 days post‐inoculation – in the M. anisopliae‐treated semiochemical‐baited device. Thrips mortality was positively correlated with M. anisopliae conidial viability, and conidial viability was negatively correlated with conidial acquisition. This suggests that the semiochemical volatiles reduced the conidial viability, which in turn resulted in significant reduction in thrips mortality, despite the highest conidial acquisition in the semiochemical‐baited fungus‐treated device. Thrips density per plant was significantly reduced in both fungus‐treated treatments (with semiochemical: 8.7 ± 1.7 adults per plant; without semiochemical: 6.6 ± 1.4 adults per plant) compared with the fungus‐free control (19.8 ± 2.6 adults per plant). These results demonstrate the potential for an autoinoculation device strategy for the control of thrips, particularly in screenhouses.     

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     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

Interactions among vegetable-infesting aphids,the fungal pathogen Metarhizium anisopliae (Ascomycota: Hypocreales) and the predatory coccinellid Cheilomenes lunata (Coleoptera: Coccinellidae)

Entomopathogenic fungi are among biocontrol agents being considered for the control of aphids on a variety of crops. Predatory coccinellids, although generalist, are also among important natural enemies that must be conserved for aphid management. Laboratory studies were carried out to investigate the interaction between three vegetable-infesting aphids, Metarhizium anisopliae isolate ICIPE 62 and the coccinellid predator Cheilomenes lunata. At a concentration of 1?×?10⁸?conidial?ml^–1, the fungus was found to cause mortality of 7.5% to C. lunata, compared to 2.5% mortality in the control at 10?days post-treatment. Female adult C. lunata to which fungus-infected aphids were offered as prey never accepted them as food source in non-choice bioassays. However, live and dead non-infected aphids were fed upon. In choice bioassay, a total of 1–3 out of 24 infected non-sporulating aphids per species (average of 0.1–0.4 aphids per arena) were consumed by 48?h-starved C. lunata within a period of 60?min, but avoided sporulating cadavers. Foraging adult C. lunata enhanced the spread of conidia of M. anisopliae from infected cadavers to fourth instars Aphis gossypii feeding on okra (0.8–15.0% mortality), Brevicoryne brassicae (3.3–15.0% mortality) and Lipaphis pseudobrassicae (0.8–14.2% mortality) on kale plants. Results of this study demonstrate compatibility between M. anisopliae and C. lunata, and could provide a sustainable strategy for effective management of aphids on crucifers and okra cropping systems.     

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.     

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).     

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.     

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.     

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.     

金龟子绿僵菌在森林土壤中的分布及对松墨天牛致病性测定

松墨天牛是重大森林植物检疫性病害——松材线虫病的主要媒介昆虫。本研究于2005年8月～2006年8月从福建、江西两省共110个林分样区(其中松林88个样区)采集土壤样品330份,采用选择性培养基分离土壤中的金龟子绿僵菌。从21个样区的26份土样中分离出的金龟子绿僵菌占采集样区的19．1％和样品的7．9％,成菌落数(CFU)500～72500CFU/g,表明金龟子绿僵菌在森林土壤中有较为广泛的分布。对分离到的9个产孢量高的菌株,采用浸渍法(1×10~7孢子/mL)接种3～4龄健康松墨天牛幼虫,采用跗节接种法接种2～15日龄健康成虫,测定其致病力。结果表明,MaYTTR-03、MaYTTR-04菌株对松墨天牛幼虫和成虫均有较高致病力,表现出良好的生防潜力。     

金龟子绿僵菌在森林土壤中的分布及对松墨天牛致病性测定

松墨天牛是重大森林植物检疫性病害——松材线虫病的主要媒介昆虫。本研究于2005年8月～2006年8月从福建、江西两省共110个林分样区（其中松林88个样区）采集土壤样品330份,采用选择性培养基分离土壤中的金龟子绿僵菌。从21个样区的26份土样中分离出的金龟子绿僵菌占采集样区的19.1%和样品的7.9%,成菌落数（CFU）500～72500CFU/g,表明金龟子绿僵菌在森林土壤中有较为广泛的分布。对分离到的9个产孢量高的菌株,采用浸渍法（1×10⁷孢子/mL）接种3～4龄健康松墨天牛幼虫,采用跗节接种法接种2～15日龄健康成虫,测定其致病力。结果表明,MaYTTR-03、MaYTTR-04菌株对松墨天牛幼虫和成虫均有较高致病力,表现出良好的生防潜力。     

Virulence of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against red palm weevil,Rhynchophorus ferrugineus (Olivier)

Nineteen different isolates of the entomopathogenic fungi Beauveria bassiana sensu lato (s.l.) and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers were tested against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) at two different spore concentrations (1 × 10⁷ and 1 × 10⁸ conidia mL^?1). Three isolates of B. bassiana and two of M. anisopliae gave >88 % larval and >75 % adult mortality of R. ferrugineus on their highest dose rate respectively. More sporulating cadavers (mycosis) resulted from a high dose rate compared to low dose on both life stages of R. ferrugineus. The current study confirmed the lethal action of B. bassiana and M. anisopliae isolates with mortality levels usually directly proportional to the conidial concentration. This study further confirmed that the isolates recovered from R. ferrugineus dead cadavers gave more mortality compared to the other sources. In the virulence assay two isolates of B. bassiana caused the highest percentage of both larval and adult mortality at all exposure intervals which suggest that they may be the most promising for use in sustainable management programs aimed at microbial control in date palm orchards.     

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.