Anisopliae Isolates Research Articles

Temperature-dependent modelling and spatial prediction reveal suitable geographical areas for deployment of two Metarhizium anisopliae isolates for Tuta absoluta management

Tuta absoluta is one of the most devastating pests of Solanaceae crops in Africa. We previously demonstrated the efficacy of Metarhizium anisopliae isolates ICIPE 18, ICIPE 20 and ICIPE 665 against adult T. absoluta. However, adequate strain selection and accurate spatial prediction are fundamental to optimize their efficacy and formulations before field deployment. This study therefore assessed the thermotolerance, conidial yield and virulence (between 15 and 35 °C) of these potent isolates. Over 90% of conidia germinated at 20, 25 and 30 °C while no germination occurred at 15 °C. Growth of the three isolates occurred at all temperatures, but was slower at 15, 33 and 35 °C as compared to 20, 25 and 30 °C. Optimum temperatures for mycelial growth and spore production were 30 and 25 °C, respectively. Furthermore, ICIPE 18 produced higher amount of spores than ICIPE 20 and ICIPE 665. The highest mortality occurred at 30 °C for all the three isolates, while the LT50 values of ICIPE 18 and ICIPE 20 were significantly lower at 25 and 30 °C compared to those of ICIPE 665. Subsequently, several nonlinear equations were fitted to the mortality data to model the virulence of ICIPE 18 and ICIPE 20 against adult T. absoluta using the Entomopathogenic Fungi Application (EPFA) software. Spatial prediction revealed suitable locations for ICIPE 18 and ICIPE 20 deployment against T. absoluta in Kenya, Tanzania and Uganda. Our findings suggest that ICIPE 18 and ICIPE 20 could be considered as effective candidate biopesticides for an improved T. absoluta management based on temperature and location-specific approach.

Susceptibility of Demotispa neivai (Coleoptera: Chrysomelidae) to Beauveria bassiana and Metarhizium anisopliae entomopathogenic fungal isolates.

The potential of Beauveria bassiana and Metarhizium anisopliae isolates obtained from naturally infected oil palm pests was evaluated to control Demotispa neivai as an alternative for organophosphate insecticide use in oil palm crops in Latin America. Two B. bassiana (Bb-0018 and Bb-0025) and two M. anisopliae (Ma-0002 and Ma-0003) isolates were tested against D. neivai adults for hydrophobicity, virulence, survival, adhesion to host cuticle, and mortality in semi-field conditions. Concentration-mortality bioassays demonstrate that isolates had lethal effect on D. neivai adults with Bb-0025 [median lethal concentration (LC50 )=3.45 × 107 conidia mL-1 ] and Bb-0018 (LC50 =3.75 × 107 conidia mL-1 ) being the most effective followed by Ma-0003 (LC50 =3.38 × 108 conidia mL-1 ) and Ma-0002 (5.33 × 108 conidia mL-1 ). Adult survival was 99% without exposure to fungal isolates, decreasing to 21.65% in insects exposed to Ma-0002, 19.41% with Ma-0003, 20.13% with Bb-0018, and 0.17% with Bb-0025. Mortality of D. neivai adults caused by the entomopathogenic fungal isolates was similar in both laboratory and semi-field conditions. Also, vegetative growth of the entomopathogenic fungal isolates was found in infected D. neivai adults in the field. Our data suggest that the tested entomopathogenic fungal isolates are effective against D. neivai with potential to be used as biological control agents contributing to the decrease of the use of chemical insecticides to control this oil palm pest. © 2021 Society of Chemical Industry.

Transcriptome Analysis of the Japanese Pine Sawyer Beetle, Monochamus alternatus, Infected with the Entomopathogenic Fungus Metarhizium anisopliae JEF-197.

The Japanese pine sawyer (JPS) beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), damages pine trees and transmits the pine wilt nematode, Bursaphelenchus xylophilus Nickle. Chemical agents have been used to control JPS beetle, but due to various issues, efforts are being made to replace these chemical agents with entomopathogenic fungi. We investigated the expression of immune-related genes in JPS beetle in response to infection with JEF-197, a Metarhizium anisopliae isolate, using RNA-seq. RNA samples were obtained from JEF-197, JPS adults treated with JEF-197, and non-treated JPS adults on the 8th day after fungal treatment, and RNA-seq was performed using Illumina sequencing. JPS beetle transcriptome was assembled de novo and differentially expressed gene (DEG) analysis was performed. There were 719 and 1953 up- and downregulated unigenes upon JEF-197 infection, respectively. Upregulated contigs included genes involved in RNA transport, ribosome biogenesis in eukaryotes, spliceosome-related genes, and genes involved in immune-related signaling pathways such as the Toll and Imd pathways. Forty-two fungal DEGs related to energy and protein metabolism were upregulated, and genes involved in the stress response were also upregulated in the infected JPS beetles. Together, our results indicate that infection of JPS beetles by JEF-197 induces the expression of immune-related genes.

Pathogenicity of entomopathogenic fungus, Metarhizium anisopliae MET-GRA4 isolate on dengue vectors, Aedes albopictus and Aedes aegypti mosquito larvae (Diptera: Culicidae)

Abstract Considering the rapid transmission of the dengue virus, substantial efforts need to be conducted to ward-off the epidemics of dengue viruses. The control effort is depending on chemical insecticides and had aroused undesirable conflicts of insecticide resistance. Here, we study the entomopathogenic fungus, Metarhizium anisopliae as a promising new biological control agent for vector control. The pathogenicity effects of Metarhizium anisopliae against field and laboratory strains of Aedes albopictus and Aedes aegypti larvae were tested using the larvicidal bioassay technique. The results demonstrate that the treatments using M. anisopliae isolate MET-GRA4 were highly effective and able to kill 100% of both Ae. albopictus and Ae. aegypti mosquito larvae at a conidia concentration of 1 × 10⁶/ml within 7 days of the treatment period. The fungus displayed high larvicidal activity against laboratory and field strain of Ae. aegypti larvae with LC50 values (9.6 x 103/ml, 1.3 x 103/ml) and LC95 values (1.2 x 10⁶/ml, 5.5 x 105/ml) respectively. For Ae. albopictus, LC50 values for laboratory and field strains were (1.7 x 104/ml, 2.7 x 104/ml) and the LC95 values were (2.1 x 10⁶/ml, 7.0 x 105/ml) respectively. Interestingly, the susceptibility of field strain towards M. anisopliae was higher as compared to the laboratory strain Aedes larvae. In which, the causative agents of all the dead larvae were verified by the virulence of M. anisopliae and caused morphological deformities on larval body. The findings from this study identify this isolate could be an effective potential biocontrol agent for vector mosquitoes in Malaysia.

BEHAVIORAL RESPONSE OF BEMISIA TABACI (HEMIPTERA: ALEYRODIDAE) TOWARDS METARHIZIUM ANISOPLIAE VOLATILES

The aim of this study was to investigate the behavioral response of Bemisia tabaci towards Metarhizium anisopliae (isolates PR1 and GT3) volatiles. Behavioral response of B. tabaci was investigated based on adult feeding and oviposition preference in eggplant Solanum melongena L. The highest mean number of adult (87) and egg (418) of B. tabaci were observed in the control plant, while the lowest mean number of adult (26) and egg (107) of B. tabaci were deposited in the PR1-treated plant. There were 8 and 5 compounds identified from the isolates PR1 and GT3, respectively. The highest amount of compounds of 1-Hydroxy-2-aminopropane (61.96%) and 1, 4-Dioxane-2-ol (54.18%) were released by PR1 and GT3 respectively. The results obtained so far revealed that whitefly avoided the eggplants provided with cultures of the M. anisopliae isolates emanating the volatile organic compounds and suitability largely depended upon the volatile profile.

UV-B Radiation Tolerance and Temperature-Dependent Activity Within the Entomopathogenic Fungal Genus Metarhizium in Brazil.

Metarhizium comprises a phylogenetically diverse genus of entomopathogenic fungi. In Brazil, Metarhizium anisopliae s.str. subclade Mani 2 is predominantly isolated from insects, while M. robertsii and M. brunneum mostly occur in the soil environment. Solar radiation and high temperatures are important abiotic factors that can be detrimental to fungal propagules. We hypothesized that among 12 Brazilian isolates of Metarhizium spp., M. anisopliae Mani 2 (n = 6), being adapted to abiotic conditions of the phylloplane, is more tolerant to UV light and high temperatures than M. robertsii (n = 3) and M. brunneum (n = 3). Inoculum of each isolate was exposed to UV-B for up to 8 h and viability evaluated 48 h later. After 8 h under UV-B, most of the isolates had germination rates below 5%. Discs of mycelia were incubated at different temperatures, and diameter of colonies were recorded for 12 days. Mycelia of M. robertsii isolates grew faster at 33 °C, while M. anisopliae and M. brunneum grew most at 25 °C. Dry conidia were incubated at 20, 25 or 40 °C for 12 days, and then viabilities were examined. At 40 °C, conidia of five M. anisopliae isolates were the most tolerant. In the three experiments, considerable intra- and inter-specific variability was detected. The results indicate that conclusions about tolerance to these abiotic factors should be made only at the isolate level.

Virulence of Entomopathogenic Fungi Isolates against Green Ladybug Nezara viridula L. (Hemiptera: Pentatomidae) Eggs

Green Ladybug Nezara viridula L. spread in tropical and subtropical regions such as in America, Africa, Asia, Australia, and Europe. Imago N. viridula or nymphs attack the legume plants in the generative phase by piercing and sucking pods. So far, farmers are still using control technology in the form of chemical insecticide applications. Chemical insecticides are only able to kill the nymph and imago stages. Meanwhile, the egg stage is still able to survive and continue to grow because there are still a few chemical insecticide compounds that can thwart the hatching of eggs. Entomopathogenic fungi are one of the potential biological control agents for controlling N. viridula eggs. Because entomopathogenic fungi can infect all insect stages of eggs, nymphs or larvae as well as imago. This research was conducted using a Completely Randomized Design (CRD), with three replications and nine treatments (control, B. bassiana, M. anisopliae, and fungi isolates of Metarhizium sp., such as MetTmM, MetTrM, MetKM, MetKpP, MetKP, MetTrP). The results showed that the lowest percentage of eggs hatched due to MetTrP isolate reached 0%, but not significantly different from B. bassiana (10%) and MetKP isolate (11.67%). The highest percentage of hatched eggs was caused by MetTmM isolate which was 63.33% with the same conidia density which was 108 conidia/ml. All fungi isolate tested could infect N. viridula eggs.

Performance of Beauveria bassiana and Metarhizium anisopliae against Pyrilla (Pyrilla perpusilla) in sugarcane

Sugarcane plant hopper (Pyrilla perpusilla Walker) is a most destructive sucking pest of sugarcane in Chhattisgarh and it’s appearing sporadically on sugarcane though out the sugarcane growing areas of this state. It is responsible for low cane yield and low sugar recovery and causes quantitative and qualitative losses in sugarcane and sugar production. In the recent years, it’s appeared in severe form and become a major problem of sugarcane in this region due to heavy infestation on sugarcane. Therefore, present investigation was carried on management of sugarcane Pyrilla (Pyrilla perpusilla) with Beauveria bassiana and Metarhizium anisopliae. Results indicated that the most effective treatment was Metarhizium anisopliae Kawardha isolate @ 25%which showed highest mortality in adults and nymphs population at 15 days after 1st spray, 15 days after 2nd spray followed by M. anisopliae Bilaspur isolate @ 25% during 2015-16 and 2016-17. In case of nymphs mortality, M. anisopliae Kawardha isolate @ 25% which showed higher mortality in nymph at 15 days after 1st spray, 15, 30, 45 days after 2nd spray, respectively during both the year followed by M. anisopliae Kawardha isolate @ 15%, M. anisopliae Bilaspur isolate @ 25% andM. anisopliae Bilaspur isolate @ 15%. It’s indicated that higher concentration of Metarhizium anisopliae was found significantly superior over lower concentration against adult and nymph of Pyrilla population in sugarcane. However, both the isolates of Beauveria bassiana were not found much effective against the adults and nymphs population of Pyrilla during both the years.

Efficacy of New Isolates of Entomopathogenic Fungus, Metarhizium anisopliae (Metsch.), from Sinai Peninsula against Yellow Mealworm Tenebrio molitor L. (Coleoptera: Tenebrionidae) under Laboratory Conditions.

Entomopathogenic fungi were isolated from soil samples collected from North and South Sinai governorates, by the use of Galleria bait method (GBT). The isolates were identified using the conventional methods. The virulence of nine entomopathogenic fungi Metarhizium anisopliae isolates (M1, M2, M3, M4, M5, M6, M7, M8 and M9)were tested against Tenebrio molitre L. Results proved that (M1) was more effective against larvae compared with all other isolates. the highest concentration of 1×108 spores/g revealed 71,56,47,31,37,52,41,46 and 64% mortality percentage for M1, M2, M3, M4, M5, M6 M7, M8, and M9, respectively at tenth day after treatment.

Isolation, Molecular Identification and Host Range Evalution of Metarhizium anisopliae Egyptian Isolate

Metarhizium anisopliae is the most popular entomopathogenic fungi used successfully in microbial control. Isolation, identification, and estimation of M. anisopliae Egyptian isolate host range has been done in this study. Our isolate was identified molecularly by amplification of Internal transcribed spacer (ITS) region using polymerase chain reaction technique (PCR). The sequence of ITS was compared with other M. anisopliae isolates published in gene bank and our isolate showed 98% homology with most published M. anisopliae isolates so submitted in gene bank database as a new Egyptian isolate with accession no MT102079.The pathogenicity was evaluated by applying five spore suspension concentrations against four insect pests, Spodoptera littoralis (2nd and 4th instar larvae), Galleria mellonella (3rd and 5th instar larvae), Tetranychus urticae (nymph) and Rhynchophorus ferrugineus (3rd and 5th instar larvae), and our results revealed that M. anisopliae was highly effective against all tested insects. The highest concentration used (109) caused a high mortality rate after 10 days post-treatment reached 96% for T. urticae nymph which appears to be highly sensitive insect tested in this study followed by S, littoralis 2nd and 4th instar larvae which showed 96 and 90% mortality rate respectively. High pathogenicity of the tested isolate also noticed in G. mellonella in which the highest concentration used (109) induced 93 and 86% for 3rd and 5th instar larvae respectively, while the same instars of R. ferrugineus were moderately affected and 73 and 66% mortality rate was achieved by the same concentration after 10 days post-treatment.

Entomopathogenic Fungi as Endophytes for Biological Control of Subterranean Termite Pests Attacking Cocoa Seedlings.

This study was conducted in the scope of developing a sustainable effective approach against subterranean termite pests using entomopathogenic and endophytic fungus-based biopesticides. Termites, Odontotermes spp. workers, were tested for their susceptibility to 15 entomopathogenic fungal isolates through the direct spraying of conidia suspensions at 1 × 108 conidia/mL. In general, all the isolates screened were pathogenic, with 100% mortality 4–7 days post-inoculation. However, the most virulent isolates were Metarhizium brunneum Cb15-III; the M. anisopliae isolates ICIPE 30 and ICIPE 60; Hypocrea lixii F3ST1; and the Beauveria bassiana isolates ICIPE 279, ICIPE 706 and ICIPE 662. These isolates were further tested for their endophytic colonization of cocoa seedlings using seed soaking, soil drench and foliar spray at 1 × 108 conidia/mL. The colonization of the plant tissues by the fungi was determined using a culture-based technique. Only the B. bassiana isolates ICIPE 706 and ICIPE 279, and H. lixii F3ST1 colonized the cocoa seedlings, with varied colonization rates among isolates and inoculation methods. Three naturally occurring endophytes—Trichoderma asperellum, Fusarium solani and F. redolens—were also isolated from the cocoa seedling tissues. These findings suggest that cocoa seedlings are conducive to endophytic fungal growth either occurring naturally or from artificial inoculation Our findings could possibly lead to an innovative approach to the management of herbivory and subterranean termite pests in cocoa agroforests.

Entomopathogenic Fungi against Cabbage Aphids, Brevicoryne brassica L.

The present investigation was carried out during two successive Cabbage seasons (2016-2017 and 2017- 2018), to study the impact of entomopathogenic fungi on Brevicoryne brassica L. The aphid Populations were evaluated in the field early in the season in December 2016 which began to appear on cabbage plants. Thereafter number of aphids increased gradually to reach a peak of abundance during December 2016 & 2017 and January 2017 & 2018). Three concentrations were used (1 x105, 1 x 106 and 1 x 107 spores/ ml.). Under laboratory conditions, the results showed that V. lecanii, M. anisopliae, and B. bassiana have high toxicity and mortality rates occurred after 3rd day from treatment. The maximum percent of mortality (100 %) occurred after the 10th day from treatment with the 3rd concentration in V. lecanii. The 3rd concentration (1 x 107spores/ ml.) was highly toxic in V. lecanii, B. bassiana, and M. anisopliae to the adult of Brevicoryne brassica L. compared with the other two concentrations.Under field conditions, the third concentration (1 x 107) also, was the best concentration against Brevicoryne brassica L. after the third application in V. lecanii, B. bassiana, and M. anisopliae. The percent of reduction was ranged between 93.3 to 99.2% and 92.0 to 97.7 in the high concentrations, in the two seasons, respectively. V. lecanii and B. bassiana were highly effective than M. anisopliae against Brevicoryne brassica L. These results confirmed that V. lecanii, B. bassiana, and M. anisopliae isolates are promising agents for Brevicoryne brassica L. control in the field.

Tolerance of Metarhizium anisopliae Sorokin isolates to selected insecticides and fungicides

An experiment was conducted to assess the compatibility of the popular insecticides like spinosad, cypermethrin, imidacloprid and chlorantraniliprole as well as fungicides copper oxychloride, carbendazim and hexaconazole with native isolates of M. anisopliae (MC 2, MC 4, MC 7). Among the isolates, MC 2, MC 7 and MC 4 were found compatible with insecticides spinosad, imidacloprid and chlorantraniliprole as well as fungicide copper oxychloride. Isolates MC 2 and MC 7 exhibited highest growth with only 3.70 and 5.18 per cent inhibition in the PDA medium amended with highest dose of copper oxychloride (0.30 g/ l ) when compared to MC 4 (7.03 % inhibition). Among the three isolates tested, the isolate MC 7 was more compatible with highest growth at all higher doses of chlorantraniliprole (0.35 ml/L), spinosad (0.38ml/ l) and imidacloprid (0.15g/ l) by recording least per cent growth inhibition (11.00, 11.41 and 14.44 per cent inhibition respectively). The insecticide cypermethrin was slightly toxic to all the isolates of M. anisopliae and fungicides, carbendazim and hexaconazole were not compatible with the M. anisopliae isolates.

Control of the Banded Fruit Weevil, Phlyctinus callosus (Schönherr) (Coleoptera: Curculionidae), Using Entomopathogenic Fungi

Phlyctinus callosus (Schönherr) (Coleoptera: Curculionidae), the banded fruit weevil (BFW), is a sporadic pest, causing damage to fruits, leaves and roots of deciduous fruits and grapevines in the Western Cape Province. Using trunk barriers to prevent the flightless weevils from reaching the leaves and fruits is labour-intensive. Entomopathogenic fungi (EPF) are effective biocontrol agents for the control of soil-borne and above-ground insect pests. Different EPF isolates were screened at 1 × 106 conidia/ml for virulence against different life stages (larvae, pupae and adults) of the BFW. The results indicated that unformulated, laboratory-cultured Beauveria bassiana 1 (Broadband®) and Metarhizium anisopliae 1 (Meta 69®) resulted in significantly higher BFW larval mortality of 97 % and 93 %, respectively, than B. bassiana 2 (Eco-Bb®) with 58 % mortality. Beauveria bassiana 1 (92 %) resulted in significantly higher pupal mortality than B. bassiana 2 (68 %) and M. anisopliae 1 (66 %). Beauveria bassiana 1 caused 90 % adult BFW mortality, which differed significantly from that obtained with B. bassiana 2 (69 %) and M. anisopliae 1 (65 %). A local EPF isolate (EA2) was compared with the commercial EPF isolates, and B. bassiana 1 (92 %) again caused significantly higher mortality of BFW adults compared to B. bassiana 2 (65 %), M. anisopliae 1 (60 %) and M. anisopliae isolate EA2 (64 %). In a sand bioassay using final instar BFW larvae, B. bassiana 1 (85 %) resulted in higher mortality than both B. bassiana 2 (55 %) and M. anisopliae 1 (70 %). The results indicated that all the commercial EPF isolates were effective against the immature and adult stages of the BFW, but with clear differences between isolates. Beauveria bassiana 1 consistently caused the highest BFW mortality under laboratory conditions. Large-scale field trials with the formulated commercial products are recommended to further demonstrate the potential use of this biocontrol agent in integrated pest management programmes.

Cosmopolites sordidus (Germar) susceptibility to indigenous Cameroonian Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates

AbstractManagement of the banana root borer (BRB), Cosmopolites sordidus (Germar; Coleoptera: Curculionidae), remains a challenge in banana and plantain production worldwide. Synthetic pesticides remain the most widely used solution while mycoinsecticides are increasingly being recommended. In this study, we selected indigenous isolates of Beauveria bassiana and Metarhizium anisopliae collected from plantain fields in Cameroon, and tested them in the laboratory for their viability, pathogenicity and virulence against all C. sordidus life stages. Of 13 isolates initially screened for spore germination and pathogenicity to adult weevils in conidial suspension of 3.2 × 108 conidia/ml, eight isolates with high to moderate germination and highest weevil mortality were selected for dose–response bioassays with four concentrations per isolate: 3.2 × 102, 3.2 × 104, 3.2 × 106 and 3.2 × 108 conidia/ml. The virulent isolates from adult bioassays were tested with eggs, larva and pupae in conidial suspension of 3.2 × 108 conidia/ml. Isolates performance depended on insect life stage with significantly high pathogenicity and virulence against larval, pupa and adult stages. The Beauveria isolate BIITAC6.2.2 caused the highest mortality rates followed by MIITAC1.1.5. Lethal times and lethal concentrations were relatively low for the three M. anisopliae isolates and three B. bassiana isolates which were the best isolates in almost all insect life stages. Apart from being effective in multiple life stages, these isolates were transmitted horizontally from one stage to another when eggs and pupae were treated. The implication of these findings for integrated management of the BRB, and potential biopesticides development and commercialization are discussed.

An effective entomopathogenic fungus Metarhizium anisopliae for the simultaneous control of Aedes albopictus and Culex pipiens mosquito adults

Abstract The purpose of this study was to select the most effective Metarhizium anisopliae isolates for the simultaneous control of Aedes albopictus and Culex pipiens mosquito adults. The selection was performed using 65 isolates of M. anisopliae, an entomopathogenic fungus isolated from Korean soils. The selection process was carried out through evaluations of conidial thermotolerance, UV-B tolerance, pathogenicity and virulence against adults of two mosquito species and conidial production on grain medium. The thermotolerance of 65 M. anisopliae isolates ranged from 0% to 99%, and their UV-B tolerance ranged from 7.1% to 94.8%. Among them, 30 isolates with high thermotolerance were selected for evaluation of their pathogenicity and virulence against adults of the two mosquito species. Metarhizium anisopliae isolates were more susceptible to Cx. pipiens adults than Ae. albopictus adults because 11 isolates were pathogenic to Ae. albopictus and 21 isolates were pathogenic to Cx. pipiens. Virulence and conidial production were evaluated using 6 isolates with similar pathogenicity against the two species of mosquito. As a result, the accumulated mortality was 74–100% for Ae. albopictus and 78–100% for Cx. pipiens. LT50 values ranged from 2.3 to 5.2 days for Ae. albopictus and from 3.2 − 4.7 days for Cx. pipiens. Comparison of conidial production using three kinds of grain media showed various results according to isolate and medium. Based on the above results, M. anisopliae CN6S1W1 was finally selected as the most effective fungal isolate for the simultaneous control of Ae. albopictus and Cx. pipiens mosquito adults.

Exploration of indigenous strains of the green muscardine fungus from soils and their pathogenicity against the tobacco caterpillar, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

In the recent years, the keen interest has been increased towards biological control-based management practices using entomopathogenic fungi (EPF) for the control of many economically important insects. In this management practices, selection of right strains of EPF is an important criteria to control the target pests in effective manner. In this context, this research work was formulated to find out environmentally effective strains of EPF in the soils of costal districts of Andhra Pradesh. Out of 135 soil samples collected, 8 soil samples yielded 8 strains of Metarhizium anisopliae. Among all 8 M. anisopliae isolates, the least median lethal concentration (LC50) for 2nd instar of the tobacco caterpillar, Spodoptera litura (Lepidoptera: Noctuidae) was 1.24 × 105 spores ml-1 in M-7, followed by 1.72 × 105 spores ml-1 in M-5 and 1.86 × 105 spores ml-1 in M-4 in an increasing order. Likewise, the median lethal time (LT50) values of different isolates of EPF against 2nd instar of S.litura were calculated for a uniform highest dose of 1 × 108 spores ml−1. Among all 8 M. anisopliae isolates, the lowest LT50 was observed as 186.44 hours post infection (hpi) with M-5 and 189.34 hpi with M-7.

Virulence and proteolytic activity of entomopathogenic fungi against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae)

The virulence and proteolytic activity of some entomopathogenic fungi isolates, viz., Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, and Trichoderma harzianum, against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), were evaluated. Common maize plants (Zea mays L.) infested with females of T. urticae were treated in vivo by spraying with suspensions of 1 × 108 conidia ml−1 concentration of selected isolates. Lethal effects of fungal isolates were assessed as percentages of daily mortalities of mites, compared to the mortality in control. Virulence of the fungi isolates was estimated based on the LC50 values calculated by probit analysis for the individuals treated by 1 × 105 conidia ml−1 concentration. Proteolytic activity of isolates was assayed on casein substrate to reflect their virulence towards T. urticae. The mite mortality rates increased with increasing conidial concentrations as well as days after treatment. The mortality rate caused by M. anisopliae isolate varied from 18.75 to 85%, with LC50 value of 4.6 × 105 conidia/ml and LC90 value of 2.4 × 108 conidia/ml during 7 days, respectively. The isolate of B. bassiana caused 15 to 70% mortality, and its LC50 and LC90 values estimated 3.3 × 106 and 7.8 × 109 conidia/ml, respectively. However, V. lecanii isolate caused 11.25 to 72.50% mortality with LC50 of 5.2 × 106 conidia/ml, while T. harzianum was potentially less virulent than other isolates causing 8.75 to 63.75% mortality rate to T. urticae with LC50 of 9.4 × 106 conidia/ml. M. anisopliae showed the highest proteolytic activity at all concentrations, followed by B. bassiana in 3rd, 5th, and 7th day post treatment. These findings recommend the selection of virulent fungal isolates for use as natural and environmentally safe agents in biological control programs to combat mite pests.

Isolation and identification of entomopathogenic fungus from Eastern Ghats of South Indian forest soil and their efficacy as biopesticide for mosquito control.

The repeated usage of chemical insecticides, responsible for insecticide resistance in mosquitoes and environmental toxicity. Currently effective and environmental-safe control strategies are needed for the control disease-vector mosquitoes. Entomopathogens can be an effective alternative to chemical insecticide. Herein we isolated and tested 46 soil-borne entomopathogenic fungi belonging to six genera, namely Beauveria sp., Metarhizium sp., Fusarium sp., Aspergillus sp., Trichoderma sp., and Verticillium sp., fungi conidia were tested on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus larvae. Bioassays results show that M. anisopliae fungal isolate causes a 100%, 98.6% and 92% mortality within six days, on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus, respectively. M. anisopliae treated three mosquito larvae have lower lifetime with LT50 values in A. stephensi, 2.931days; A. aegypti, 2.676days and C. quinquefasciatus, 3.254days. 18s rDNA sequence analysis confirmed that the isolated fungus are belonging to the genus of M. anisopliae-VKKH3, B. bassiana-VKBb03, and V. lecanii-VKPH1. Our results clearly show that M. anisopliae has good potential, as a low-cost, environmentally safe tool for the control of A. aegypti, A. stephensi, and C. quinquefasciatus mosquitoes.

Physiology Characterization of Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae on Different Carbohydrate Sources

Insect pathogens that are often used to control insect pests are fungi Beauveria bassiana and Metarhizium anisopliae. One of the agar media, which is often used in the entomopathogenic function of propagation in the laboratory, is PDA (potato dextrose agar). The price of instant PDA is quite high, starting from Rp. 750,000 per 250 g doing the research costs quite large. Cheap and easily obtained abundant natural resources can be used as an alternative medium for microorganism growth to reduce the overall costs to be incurred in research. Carbohydrate sources used in this study were potatoes, cassava, sweet potatoes, corn, and rice, which were made into the agar media. Observation parameters were viability rates, colony growth rates, and sporulation of B. bassiana and M. anisopliae fungi isolates. The experiments are used in a completely randomized design (CRD) with two treatment factors. The data obtained were processed with variance and continued with the DNMRT test at 5% significance level. The results showed that cassava agar caused the highest germination at 24 hours. The highest diameter was fungi grown on potato dextrose agar. The highest sporulation was fungi grown on corn agar.