Morphology and RAPD analysis of certain potentially entomopathogenic isolates of Metarhizium anisopliae Metsch. (Deuteromycotina: Hypocreales)

Diversity within the entomopathogenic fungi have been traditionally analyzed using morphological features but morphology alone can lead to ambiguity pertaining to identification at species level. Therefore utilization of molecular methods to detect the level of polymorphism among species helps minimize the problem. Nine isolates of Metarhizium were morphologically characterized by assessing their colour, media pigmentation, size and shape of conidia. Molecular characterization was carried out by random amplified polymorphic DNA analysis. The RAPD - PCR assay for nine isolates were performed by amplifying random sequences using three RAPD primers. The amplification products for the different isolates were compared with each other and were screened for the presence or absence of specific bands. The scored band data was subjected to cluster analysis. A genetic similarity matrix was constructed using Jaccard’s coefficient method. Colony colour varied from pale green to blackish green. Pigment production was observed for four isolates. Average width of spores ranged from 2.10 - 4.10μm and length 3.20 - 7.69 μm. The spores were grouped as either oval, round or elongated. The three primers generated a total of 166 reproducible distinct bands among the 9 isolates and the similarity was estimated on the basis of number of shared bands. The Jaccard’s similarity coefficient between isolate pairs ranged from 0.00 to 0.70 indicating a high genetic diversity. The maximum similarity was noticed between isolates MIS13 and MIS18. A dendrogram was generated from RAPD patterns of the Metarhizium isolates. Grouping of isolates into clusters correlated with similarities in their RAPD DNA patterns

Dissemination of Metarhizium anisopliae infection among the population of Odontotermes obesus (Isoptera: Termitidae) by augmenting the fungal conidia with attractants

Long-term biocontrol strategies for termite management have limited success as the behavioral response exhibited by termites in the presence of entomopathogenic fungi was different. To minimize these responses, a study was conducted to attract the termites towards the treated area by augmenting fungal conidia with a mixture of attractants such as sugarcane bagasse, sawdust and cardboard powder. In laboratory experiments, mortality due to the horizontal transmission of Metarhizium anisopliae infection among Odontotermes obesus workers and soldiers was 50 to 98% and 16 to 78% for the five isolates tested. The foraging activity of workers and soldiers increased from 23 to 58% for IWST-Ma13 when conidia were mixed with attractants. In a field study, the weight loss of stakes treated with conidial attractant decreased to 10.9 g compared to dry conidial treatments (19.3 g) for IWST-Ma13 after five months. Similarly, the width of mud galleries covering the tree bark was reduced when treated with conidial baits and gunny bags containing conidial attractant. In treated mounds, as the Acoustic emission signal voltage decreased from the threshold voltage level, the relative magnitude signal (RMS) remains low after five months of treatments with that of the control. From this study it was observed that the mixing of conidia with attractants could augment the dissemination potential of fungal infection among the termites. By adapting attractant based Metarhizium baits and dusting of conidial attractant mixture inside mounds may augment the dissemination potential in epizootic transmission of fungal infection

Virulence and mycotoxic effects of Metarhizium anisopliae on Mahogany shoot borer, Hypsipyla robusta (Lepidoptera: Pyralidae)

Developing appropriate control measures for the Mahogany shoot borer, Hypsipyla robusta Moore has become increasingly important due to the severe damaging effect of the pest on the establishment of the saplings of Swietenia mahagoni Jacq (Sapindales: Meliaceae). Existing management methods are largely limited to silvicultural practices and spraying of chemical insecticides. To identify a potential fungal biocontrol agent, we compared the virulence of six native and two standard ARSEF isolates of Metarhizium anisopliae Metsch. against this pest. The average survival time and conidial yield of IWST-Ma7 was higher (6.2 to 7.3 days and 4.9 to 4.7×105 conidia/ml) than the standards. Significant difference in sporulation on the cadavers between isolates, doses and incubation periods were substantiated for the selection of potential strain. The mycotoxic effects of crude soluble protein extract when incorporated in the artificial diet, the ARSEF 2596 and ARSEF 3603 showed LD50 value of 3.7% and 5.6%. However, IWST-Ma7 was highly lethal with significant lowest LD50 value of 2.6%. The enzyme activity of IWST-Ma7 was highest for chitinase, CDA, protease and lipase viz., 1.90 U/mg, 1.80 U/mg, 0.98 U/mg and 0.80 U/mg respectively. However the enzyme activity of chitinase and Chitin deacetylase assay for all the isolates was significantly higher than protease and lipase activity. The ITS regions (5.8S rDNA and 28S rDNA) of seven isolates of M. anisopliae were amplified using the ITS1 and ITS4 primers which was a unique fragment of approximately 550 bp. Based on ITS regions, phylogenetic tree have been constructed and the isolates have been grouped in to 5 clades. The virulence and mycotoxic effects of different isolates could rationally be used to employ them for the management of the mahogany borer

Infectivity of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) isolates to the arboreal termite Odontotermes sp. (Isoptera: Termitidae)

Infectivity of the entomopathogenic fungus Metarhizium anisopliae against workers of the arboreal termite Odontotermes sp. was assayed under laboratory conditions. Test isolates were collected from different sources, including soil from varied locations and insect hosts from the orders Lepidoptera, Coleoptera, Hemiptera and Orthoptera. All the 23 isolates tested and the standard (ARSEF 7413) were pathogenic to the workers of Odontotermes sp. at a concentration of 107 conidia/ml, with mean mortality ranging from 57.5 to 100%. Two of the isolates (Ma2, Ma13) and the standard caused 100% mortality in the termite species. A detailed bioassay was subsequently conducted with the five most promising isolates, namely Ma1, Ma2, Ma13, Ma16 and Ma17, at concentrations ranging from 104 to 107 conidia/ml. The lethal concentrations (LC50) of these isolates ranged from 0.01 to 0.46 £ 105 conidia/ml. The average survival time (AST) for the termites treated with the most virulent isolate (Ma2) varied from 4.2 to 5.7 days across the four spore loads, while AST with the standard isolate ranged from 5.3 to 6.3 days. Two of the isolates, Ma2 and Ma13, were found to be significantly more pathogenic to Odontotermes sp. workers than all the others, including the standard

Biocontrol Potential of Metarhizium anisopliae (Metsch.) Sorokin (Deuteromycotina: Hyphomycetes) against Ailanthus Defoliator, Eligma narcissus (Cram.)

Eligma narcissus is recognised as a serious pest of Ailanthus in Southern India and defoliation of Ailanthus by this pest causes apparent loss of growth increment. The common control methods for this pest is mostly insecticides and the concern about the environmental effects of chemical insecticides, has emphasised the use of environmentally more benign microbial agents. Among entomopathogens, Fungi are the most explored and often act as important natural control agents that limit insect populations. On this point of view, Bio efficacy of 25 isolates of Metarhizium anisopliae was assessed to establish their virulence against E. narcissus in the laboratory and effective formulations of two potent isolates were subsequently evaluated in the field. MIS7 and MIS13 were more effective among the different isolates evaluated against E. narcissus. The median lethal concentration (LC50) of all the isolates ranged from 6.46×105 conidia/ml to 628.92×105 conidia/ml. Median lethal concentration of (LT50) of 4.9 and 5.4 days were recorded for MIS7 and MIS13 respectively at a concentration of 1× 107 conidia/ml. Virulence tests of the isolates MIS7 and MIS13 and 0.5% Pongamia pinnata seed oil, individually and in different combinations, indicated improved efficacy of the isolates when used in combination and also when combined with seed oil. Formulations composed of “MIS7+MIS13+0.5% Pongamia pinnata seed oil” and “MIS7+MIS13” proved to be superior against E. narcissus, causing 76.30% and 93.93% mortality, respectively. Field evaluation of the formulation MIS7+MIS13+0.5% Pongamia pinnata seed oil recorded 5.79 larvae per plant resulting in 60.53% reduction of infestation while the formulation, MIS7+MIS13 showed 53.76% reduction of infestation with 6.56 larvae per plant. The observations from this study suggest the prospects of using the entomopathogenic fungus, M. anisopliae for the control of E. narcissus

Attempts at controlling Teak Defoliator (Hyblaea puera Cramer, Lepidoptera, Hyblaeidae) with the entomopathogenic fungus, Metarhizium anisopliae (Metsch.): laboratory, nursery and field trials

Defoliator pests often cause severe damage to commercially valuable timber trees, such as teak, grown in plantations, affecting the quality and quantity of the wood produced. Twenty-five isolates of an entomopathogenic fungus, Metarhizium anisopliae (Metsch.), derived from different forest habitats were tested for their efficacy against the Teak Defoliator, Hyblaea puera Cram. The median lethal concentration (LC50) for the isolates ranged from 0.65 to 947.41 × 105 conidia mL−1. Four isolates, viz. MIS2, MIS19, MIS7 and MIS1, were promising, having low LC50 values. A median lethal time (LT50) of 3.8 d was recorded for the isolates MIS2 and MIS7 when the moth larvae were exposed to a spore load of 107 conidia mL−1. Formulations composed of “MIS2 + MIS7 + 0.5% Pongamia pinnata seed oil” and “MIS2 + MIS7” proved to be superior against the Teak Defoliator, causing 94.73% and 93.93% mortality, respectively. Application of the formulation “MIS2 + MIS7 + 0.5% P. pinnata oil” resulted in 63.6% and 56.2% reduction in infestation in nurseries and plantations, respectively, while the formulation “MIS2 + MIS7” recorded 56.18% and 51.60% reduction. Our results indicate that M. anisopliae formulations could be an effective means of managing Teak Defoliator in plantations, particularly when combined with P. pinnata oil

Virulence of MetarhiziumM isolates against the polyphagous defoliator pest, Spilarctia obliqua (Llepidoptera: Aarctiidae)

Species of the entomopathogenic fungus Metarhizium are some of the most promising biocontrol agents against Lepidopteran pests. Laboratory bioassays were conducted to study the virulence of five isolates of Metarhizium sp. against larvae of the polyphagous pest, Spilarctia obliqua (Lepidoptera: Arctiidae). The Metarhizium isolates used in the study were recovered from soil and various insect hosts belonging to Lepidoptera, Coleoptera and Isoptera. Initially, a preliminary assay was carried out involving 18 Metarhizium isolates at a spore concentration 107 conidia ml-1 and five isolates, MIS 1, MIS 2, MIS 3, MIS 8 and MIS 9, which caused more than 50% mortality were chosen for the detailed bioassay. The assay was carried out with four different spore concentrations of each isolate, ranging from 104-107 conidia ml-1. The lethal concentration LC50 of these five isolates ranged from 2.1 × 105 to 38.9 × 105 conidia ml-1. The lethal time LT50 of the isolates ranged from 7.0-8.1, 6.0-7.7, 5.1-6.7, 4.6-5.4 days at concentrations 1 × 104, 1 × 105, 1 × 106, 1 × 107 conidia ml-1 respectively. The most pathogenic among the five isolates was the MIS 2 isolate with the lowest LC50 of 2.11 × 105 conidia ml-1 and LT50 of 4.6, 5.1, 6.0 and 7.0 days respectively at concentrations 1 × 107, 1 × 106, 1 × 105, 1 × 104 conidia ml-1. Isolate MIS 3 was second in effectiveness. These two isolates showed promise for use as biocontrol agents against S. obliqua

Cuticle degrading enzyme production by some isolates of the entomopathogenic fungus, Metarhizium anisopliae (Metsch.)

Entomopathogenic fungi have been recognized as viable alternate options to chemicals in insect pest control. Unlike other potential biocontrol agents, fungi do not have to be ingested to infect their hosts but invade directly through the cuticle. Entry into the host involves both enzymic degradation of the cuticle barrier and mechanical pressure. Production of a range of cuticle degrading enzymes is an important event in the interaction of entomopathogenic fungi and host. Enzyme secretion is believed to be a key contributor for the virulence of a fungal isolate

Pathogenicity of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) Isolates to the Ailanthus Webworm, Atteva fabriciella (Lepidoptera: Yponomeutidae) Under Laboratory and Field Conditions

The virulence of 25 Metarhizium anisopliae isolates was tested under laboratory conditions and the two most effective isolates were evaluated in the field for control of the Ailanthus defoliator, Atteva fabriciella. A bioassay was carried out to determine the dose and time mortality responses. The LC50 of the isolates ranged from 3.16 to 647.81 × 105 conidia mL−1. Toxicity tests of the isolates MIS7 and MIS13 and 0.5% Pongamia pinnata seed oil, individually and in different combinations, indicated improved efficacy of the isolates when used in combination and also when combined with seed oil. Evaluation of these formulations in the field showed 66.36% reduction of infestation with MIS7 + MIS13 + 0.5% P. pinnata seed oil and 61.15% reduction with MIS7 + MIS13. The study indicated a possibility of employing combined formulations of M. anisopliae and also combination with P. pinnata seed oil for augmenting the effectiveness of the fungus