Tariq M. Butt

Fungi as biocontrol agents : progress problems and potential

Fungal nutrition and pathogenesis fungal toxins genetic improvement of fungal biocontrol agents mycoparasites for the control of fungal diseases and of insect pests control of plant and animal parasitic nematodes biocontrol of weeds production, storage, formulation and application technologies biosafety.

Use of hyphomycetous fungi for managing insect pests.

1Department of Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA; 2Lethbridge Research Centre, Agriculture and Agri-Food Canada, PO Box 3000, Lethbridge, Alberta T1J 4B1, Canada; 3School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK; 4Institute of Microbiology, LeopoldFranzens University Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria

Dual Detection of Fungal Infections in Drosophila via Recognition of Glucans and Sensing of Virulence Factors

The Drosophila immune system discriminates between various types of infections and activates appropriate signal transduction pathways to combat the invading microorganisms. The Toll pathway is required for the host response against fungal and most Gram-positive bacterial infections. The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and GNBP1 that cooperate to detect the presence of infections in the host. Here, we report that GNBP3 is a pattern recognition receptor that is required for the detection of fungal cell wall components. Strikingly, we find that there is a second, parallel pathway acting jointly with GNBP3. The Drosophila Persephone protease activates the Toll pathway when proteolytically matured by the secreted fungal virulence factor PR1. Thus, the detection of fungal infections in Drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host.

Are there any risks in using entomopathogenic fungi for pest control, with particular reference to the bioactive metabolites of Metarhizium, Tolypocladium and Beauveria species?

Entomopathogenic fungi are promising alternatives to chemical insecticides. However, a major hurdle concerning the registration of these fungi as plant protection agents is the possible toxicity of secreted metabolites, especially secondary metabolites. This review summarizes data on specific secondary metabolites (destruxins, efrapeptins, oosporein, beauvericin and beauveriolides) produced by the important genera Beauveria, Metarhizium and Tolypocladium . The quantities of secondary metabolites produced by these fungi in vivo are usually much less than those secreted in nutrient rich liquid media. Methods and strategies are suggested which could standardize the risk assessment of fungal biological control agents.

Productionin vitro of appressoria by the entomopathogenic fungusMetarhizium anisopliae

Abstract Germination on complex media induced conidia of the entomopathogenMetarhizium anisopliae to produce infection structures (appressoria and penetration hyphae) when the germ tube contacted a hard surface. The morphology of the infection structures and their rate of formation are very similar to those observed for blowfly cuticle. Differentiation frequencies were greater (more than 70% as compared with less than 40%) on hydrophobic surfaces [Teflon, polyvinyl chloride, polystyrene, polypropylene, polyester (GelBond), aluminum foil] than on hydrophilic surfaces (agarose-coated polyester and cellophane). Differentiation frequencies were similar on both positively and negatively charged surfaces. Differentiationin vitro was stimulated by low levels of complex nitrogenous nutrients. Analysis of one- or multicomponent media suggested that amino acids and the lipid component of epicuticle act in combination with the hydrophobic cuticle surface to stimulate differentiation during pathogenesis. Thigmotropic and chemical stimuli for production of appressoria appear to be translated primarily during the second round of nuclear division because inhibitors of DNA and RNA synthesis do not prevent germination but block differentiation if applied before the second nuclear division. Inhibition of protein synthesis blocked both germination and differentiation.

Pathogenicity of the Hyphomycete Fungi Verticillium lecanii and Metarhizium anisopliae to the Western Flower Thrips, Frankliniella occidentalis

The pathogenicity of several isolates of the hyphomycete fungi Verticillium lecanii and Metarhizium anisopliae to Frankliniella occidentalis was investigated. Treatment of adult thrips with M. anisopliae resulted in at least 94% mortality at 7 days post-inoculation. In contrast, V. lecanii isolates only gave mortalities of between 20 and 70%. Detailed studies were made on the most virulent isolate of M. anisopliae (275) to determine its efficacy at different doses and temperatures. At 23 C the LC was ca. 3 105 conidia ml-1 after 5 days and the LTs were 50 50 3 and 4.5 days at 10 7 and 106 conidia ml-1 respectively. Temperature influenced fungal virulence to adult thrips; the LT at 18 and 20 C was ca. 4 days and at 23 or 26 C it was ca. 3 days. 50 Larvae were less susceptible to infection than adult thrips (27% versus 100% mortality), presumably due to the inoculum being shed with the exuvium during ecdysis. Conidia of M. anisopliae isolate 275 germinated rapidly on the surfaces of larvae, pupae and adults...

The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ.

The nematophagous fungus Verticillium chlamydosporium secreted several proteases in submerged culture in which soya peptone was the sole carbon and nitrogen source. One protease, VCP1 (M(r) 33,000, pI 10.2), was purified 14-fold from culture filtrates to apparent homogeneity using preparative isoelectric focusing in free solution, and shown to rapidly hydrolyse the chymotrypsin substrate Suc-(Ala)2-Pro-Phe-pNA and elastin. VCP1 had a Km for Suc-(Ala)2-Pro-Phe-pNA of 4.3 x 10(-5) M and a kcat of 5.8 s-1. It was highly sensitive to PMSF and TPCK, but only moderately sensitive to chicken egg-white and soya bean trypsin inhibitors. VCP1 degraded a wide range of polymeric substrates, including Azocoll, hide protein, elastin, casein and albumin, and accounted for most of the non-specific protease activity detected in culture filtrates. The purified enzyme hydrolysed proteins in situ from the outer layer of the egg shell of the host nematode Meloidogyne incognita and exposed its chitin layer. VCP1 was secreted by several isolates of V. chlamydosporium and V. lecanii, pathogens of nematodes and insects respectively, but not plant-pathogenic species of Verticillium. These observations suggest that VCP1 or similar enzyme(s) may play a role in the infection of invertebrates.

Insect natural products and processes: New treatments for human disease

In this overview, some of the more significant recent developments in bioengineering natural products from insects with use or potential use in modern medicine are described, as well as in utilisation of insects as models for studying essential mammalian processes such as immune responses to pathogens. To date, insects have been relatively neglected as sources of modern drugs although they have provided valuable natural products, including honey and silk, for at least 4-7000 years, and have featured in folklore medicine for thousands of years. Particular examples of Insect Folk Medicines will briefly be described which have subsequently led through the application of molecular and bioengineering techniques to the development of bioactive compounds with great potential as pharmaceuticals in modern medicine. Insect products reviewed have been derived from honey, venom, silk, cantharidin, whole insect extracts, maggots, and blood-sucking arthropods. Drug activities detected include powerful antimicrobials against antibiotic-resistant bacteria and HIV, as well as anti-cancer, anti-angiogenesis and anti-coagulant factors and wound healing agents. Finally, the many problems in developing these insect products as human therapeutic drugs are considered and the possible solutions emerging to these problems are described.

Inter- and intra-specific variation in destruxin production by insect pathogenic Metarhizium spp., and its significance to pathogenesis.

Inter- and intra-specific variation in destruxin production was detected in Metarhizium and may be important in determining virulence and/or specificity against insects. Strains of M. anisopliae var. anisopliae produced different amounts of destruxins A, B and E, but strain V220 did not produce any destruxins. M. anisopliae var. majus, M. flavoviride and M. album which are reported to be specific towards Coleoptera, Orthoptera and Hemiptera, respectively, had different destruxin profiles with destruxin A predominant. In time course studies on M. anisopliae var. anisopliae V245, destruxin E levels declined with time while destruxin A levels increased. The most virulent strains, Ma23 and V245, produced large quantities of destruxins but some low toxin producers were also virulent, suggesting that destruxins are not the only pathogenicity determinants. Some weakly to moderately pathogenic strains were highly pathogenic when injected into Galleria mellonella larvae, demonstrating the importance of the cuticle as a barrier to fungal infection. Only trace amounts of destruxin A or a combination of A and B could be detected in Galleria larvae infected with M. anisopoliae var. anisopliae or M. anisopliae var. majus. No destruxins were detected in larvae infected with M. flavoviride. Destruxin production may be influenced by the nutrients in insects or culture media.

Pathogenicity of the entomogenous fungi Metarhizium anisopliae and Beauveria bassiana against crucifer pests and the honey bee

The susceptibility of the cabbage stem flea beetle (Psylliodes chrysocephala; Coleoptera: Chrysomelidae) to different isolates of the entomogenous, hyphomycete fungi Beauveria bassiana and Metarhizium anisopliae, all from heterologous hosts, was investigated. The most pathogenic isolates were of M. anisopliae (V208, V233, V234, V242, V245, and V248). Studies were conducted to determine the virulence of V208 and V245, and their specificity for three other crucifer pests, Phaedon cochleariae, Myzus persicae and Lipaphis erysimi and a beneficial insect, Apis mellifera. Flea beetle mortality rates increased with dose; the estimated LC50 S of V208 and V245 at 14 days post‐inoculation were c. 106 conidia ml‐1. The respective estimated LT50 values of V208 and V245 for P. chrysocephala at 1 × 107 conidia ml‐1 were 10.0 and 9.3 days, and at 1 × 1010 conidia ml LT50s were 3.8 and 4.2 days. Both V208 and V245 were pathogenic for P. cochleariaeM. persicae and L. erysimi. Myzus persicae and L. erysimi died within 4 da...