Pathogenic Fungus Metarhizium Research Articles

Cloning and sequence analysis of ornithine decarboxylase gene fragments from theAscomycota

Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the initial step in the biosynthesis of polyamines, the conversion of ornithine to putrescine. Based on the most conserved regions of fungal ODCs, we designed and synthesized oligonucleotides to amplify homologous fragments of three important plant pathogenic Pyrenomycete fungi (Ascomycota), Magnaporthe grisea, Colletotrichum lindemuthianum and Fusarium solani, and one insect pathogenic fungus Metarhizium anisopliae. Cloning and sequencing of the amplified fragments revealed homologies of between 37 to 88% with other fungal ODCs. The predicted peptide sequences were compared by Clustal analysis and conserved sequences corresponding to the substrate and cofactor binding sites were identified. Comparative analyses of the ODC fragments isolated in this study, revealed high homology between them (68.3-81.1%) and also with other Pyrenomycetes such as Neurospora crassa (order Sordariales; 68.6-72.9%) and Fusarium graminearum (order Hypocreales; 70.8-88.1%). Data obtained in this work revealed that these fungi constitute a compact group separated from other eukaryotic ODCs.

Evaluation of an application procedure for Metarhizium anisopliae in sugarcane ratoons for control of the white grub Dermolepida albohirtum

The biological control product BioCane™, containing spores of the fungal pathogen Metarhizium anisopliae isolate FI-1045, is available for soil application against the white grub Dermolepida albohirtum (Waterhouse) in sugarcane in Australia. Currently, BioCane™ granules can only be applied in plant crops, where they are distributed in a horizontal band in the planting furrows and covered with soil. Options for management of D. albohirtum would be broadened if M. anisopliae could also be effectively applied in ratoon crops. We evaluated the efficacy of spores of M. anisopliae sprayed as an aqueous suspension into two vertical slots cut into the soil in the rows of sugarcane ratoons. We were able to achieve vertical bands of spores, as determined by collecting soil samples at incremental depths and counting colonies after plating on artificial media, although spores were concentrated towards the bottom of the slot. Numbers of white grubs were reduced by treatment in some trials in comparison to untreated plots, but results were inferior to those obtained using the standard chemical insecticide, imidacloprid (Confidor ®). When a low application rate of M. anisopliae was combined with bifenthrin (Talstar ®), only bifenthrin was shown to contribute to efficacy with no interaction between effects of the two products, despite promising results in a previous bioassay. Application of M. anisopliae is not likely to be an economically viable treatment for sugarcane ratoons using this technique.

Colony sectorization of Metarhizium anisopliae is a sign of ageing

Spontaneous phenotypic degeneration resulting in sterile sectors is frequently observed when culturing filamentous fungi on artificial medium. Sterile sectors from two different strains of the insect pathogenic fungus Metarhizium anisopliae were investigated and found to contain reduced levels of cAMP and destruxins (insecticidal peptides). Microarray analysis using slides printed with 1730 clones showed that compared to wild-type, sterile sectors down-regulated 759 genes and upregulated 27 genes during growth in Sabouraud glucose broth or on insect cuticle. The differentially expressed genes are largely involved in cell metabolism (18.8 %), cell structure and function (13.6 %) and protein metabolism (8.8 %). Strong oxidative stress was demonstrated in sectorial cultures using the nitro blue tetrazolium assay and these cultures show other syndromes associated with ageing, including mitochondrial DNA alterations. However, genes involved in deoxidation and self-protection (e.g. heat-shock proteins, HSPs) were also upregulated. Further evidence of physiological adaptation by the degenerative sectorial cultures included cell-structure reorganization and the employment of additional signalling pathways. In spite of their very similar appearance, microarray analysis identified 181 genes differentially expressed between the two sectors, and the addition of exogenous cAMP only restored conidiation in one of them. Most of the differentially expressed genes were involved in catabolic or anabolic pathways, but the latter included genes for sporulation. Compared to the mammalian ageing process, sectorization in M. anisopliae showed many similarities, including similar patterns of cAMP production, oxidative stress responses and the involvement of HSPs. Thus, a common molecular machinery for ageing may exist throughout the eukaryotes.

Toxicity testing of destruxins and crude extracts from the insect-pathogenic fungus Metarhizium anisopliae

Increasing sensitivity towards secondary metabolites from fungal biological control agents (BCAs) has prompted the toxicological risk assessment of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae. Viability studies on one human and one insect cell line were used to compare the two approaches of testing individual metabolites (destruxins A, B and E) or the complete crude extract from liquid cultures. Furthermore, crude extract was separated into fractions, which did not contain the main destruxins A, B and E. Evaluation of the cytotoxic activity of these different compounds suggested that a wide range of metabolites with synergistic or adverse effects are present in the crude extract. The results indicate that identification and toxicological assessment of each individual metabolite produced by a BCA is not only time and cost-intensive, but also does not convey the whole picture. Testing of the crude extract offers an alternative approach and is recommended when assessing the risks of metabolites for registration purposes.

Influence of nutrition on the production and physiology of sectors produced by the insect pathogenic fungus Metarhizium anisopliae

Metarhizium anisopliae strains V245 and V275 differed in their stability when grown on different nutrient media. V275 produced fewer sectors than V245 irrespective of the cultural conditions. Both strains produced more sectors on nutrient rich media. At least four distinct types of sectors were produced in vitro. Most sectors were sterile or sporulated poorly and produced significantly lower quantities of virulence determining enzymes like Pr1. Real-time PCR confirmed differential expression of the pathogenicity-related genes pr1 A, ste 1, try 1, and chy 1 encoding for the subtilisin Pr1A, esterase, trypsin and chymotrypsin, respectively. API-ZYM revealed that the enzyme profiles of sectors differed from those of the parent cultures and also from other sectors. Sectors of M. anisopliae also produced less destruxins than the parent cultures independent of the strain.

Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on insects and artificial substrates

Solar ultraviolet radiation (UV-A and UV-B) is a major factor in failure of programs using the insect pathogenic fungus Metarhizium anisopliae as a biological control agent. Studies were conducted to determine if growth conditions, viz. artificial (agar media or rice grain) or natural (infected insects) substrates for conidial production affect two traits that directly influence performance of conidia after field application: tolerance to UV-B radiation and conidial germination speed. Conidia of two isolates (ARSEF 23 and ARSEF 2575) of M. anisopliae var. anisopliae produced on potato dextrose agar plus yeast extract (PDAY) or on fungus-killed larvae of two insect species, Galleria mellonella and Zophobas morio, were inactivated by exposure to UV-B radiation. Conidia of both isolates when produced on insect cadavers were significantly more sensitive to UV-B radiation than conidia produced on PDAY. Also, conidia from insect cadavers germinated slower than those from PDAY cultures. A comparison of conidia from artificial substrates showed that conidia produced on Czapek’s and Emerson’s YpSs agar media or rice grains had higher tolerance to UV-B radiation and germinated faster than conidia raised on PDA and PDAY. Accordingly, the growth substrate and nutritional environment in which conidia are produced influences M. anisopliae conidial UV-B tolerance and speed of germination; and manipulation of these variables could be used to obtain conidia with increased tolerance to UV-B radiation and shorter germination times.

Nucleotide sequence variation does not relate to differences in kinetic properties of neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

Genetic variability in a putative virulence factor, the neutral trehalase ( Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., Km, Vmax, and Kcat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower Kcat/Km. However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.

Metarhizium anisopliae, a potential agent for the control of grape phylloxera

This investigation deals with the control effects of the insect pathogenic fungus Metarhizium anisopliae var. anisopliae on Daktulosphaira vitifoliae. In pot experiments, the soil surrounding phylloxera-infected grapes was inoculated with barley colonised with M. anisopliae. After thirty-two days, ineight of ten Metarhizium-applied pots nofresh phylloxera infections could be observed.In two of ten plants, a few fresh nodositiessingly occupied with phylloxera or phylloxeraeggs could be found. In all untreated plants,fresh nodosities with either single (two of sixplants) or multiple (four of six plants)occupation with phylloxera could be observed.M. anisopliae could be re-isolated in aconcentration of <1 × 103 CFU g$minus;1 soil dry weight from those pots with phylloxera-infected plants that had been treated with the fungus. The potential role of M. anisopliae in grape phylloxera management is discussed.

Laboratory Evaluation of the Entomopathogenic Fungus, Metarhizium anisopliae Var. major against the Subterranean Termite, Odontotermes guptai Roonwal and Bose

Infectivity of the soil mixed conidia of the fungal pathogen Metarhizium anisopliae var. major was evaluated against the termite, Odontotermes guptai Roonwal and Bose under laboratory conditions. Four concentrations, viz ., 0.8x10 7 , 1.2x10 7 , 1.6x10 7 , and 2x10<SUP7 conidia/g soil caused complete mortality of the test termite in 192, 156, 132 and 120 hours, respectively. The LC 50 value at 12 th was calculated as 9.07x10 6 conidia/g soil. The LT 50 value for the most effective dose was 86.84h. However, the test termites became less active within 48 hours of incubation.

Fever and phenotype: transgenerational effect of disease on desert locust phase state

AbstractNatural enemy attack can cause transgenerational shifts in phenotype such that offspring are less vulnerable to future attack. Desert locusts (Schistocerca gregaria) show density‐dependent variation in their resistance to pathogens, such that they are less vulnerable to pathogens when in the high‐density gregarious phase state (when they would probably be more exposed to pathogens) than when in the solitarious phase state. We therefore hypothesized that infected gregarious parents would maintain this phenotype in their offspring. We infected gregarious desert locust nymphs with the fungal pathogen Metarhizium anisopliae var. acridum, and allowed them to survive to reproduction by means of behavioural fever. The phase state of the locust offspring was assessed by their colouration and behavioural assays. Contrary to our hypothesis, we found an increase in solitarization in the infected population (14.6% solitarious offspring from infected parents, vs. &lt;2% from uninfected counterparts at equivalent density). In a second experiment, we simulated behavioural fever temperatures and obtained a similar result (13.6% solitarious offspring vs. 4.4% from controls), implying that the phenomenon is probably a side‐effect of the hosts’ fever response. Identification of this novel environmental factor affecting locust phase state could have important implications for the biological control of these major pests.

Evidence for differential selection and potential adaptive evolution in the worker caste of an inquiline social parasite

Social parasites exploit the socially managed resources of social insect colonies in order to maximise their own fitness. The inquilines are among the most specialised social parasites, because they are dependent on being fully integrated into their host's colony throughout their lives. They are usually relatives of their host and so share ancestral characteristics (Emery's rule). Closely related inquiline-host combinations offer a rare opportunity to study trade-offs in natural selection. This is because ancestral adaptations to a free-living state (e.g. the production of a worker caste) become redundant and may be replaced by novel, parasitic traits as the inquiline becomes more specialised. The dynamics of such pro- cesses are, however, unknown as virtually all extant inquiline social parasites have completely lost their worker caste. An exception is Acromyrmex insinuator, an incipient permanent social parasite of the leaf-cutting ant Acromyrmex echinatior. In the present study, we document the size distribution of parasite and host workers and infer how selection has acted on A. insinuator to reduce, but not eliminate, its investment in a worker caste. We show that the antibiotic producing metapleural glands of these parasite workers are signif- icantly smaller than in their host counterparts and we deduce that the metapleural gland size in the host represents the ancestral state. We further show experi- mentally that social parasite workers are more vulnerable to the general insect pathogenic fungus Metarhizium than are host workers. Our findings suggest that costly disease resistance mechanisms are likely to have been lost early in inquiline evolution, possibly because active selection for maintaining these traits became less when parasite workers had evolved the ability to exploit the collective immune system of their host societies.

Eicosanoid involvement in the regulation of behavioral fever in the desert locust, Schistocerca gregaria.

The desert locust Schistocerca gregaria behaviorally thermoregulates in order to try and maintain a favoured "set point" body temperature. Locusts infected with the deuteromycete fungal pathogen Metarhizium anisopliae var acridumchoose a significantly elevated temperature. This "behavioral fever" greatly delays the progress of mycosis. We have confirmed this phenomenon and shown that desert locusts also fever when infected with the bacterial pathogen Serratia marcescens. Elevation in the prefered environmental temperature occurs also upon injection with laminarin and lipopolysaccharide (microbial cell wall components). Since such treatments also stimulate the immune system it would appear that "behavioral fever" is probably a feature of the immune response. The eicosanoid biosynthesis inhibitor dexamethasone prevented laminarin invoked fever. This effect was reversable by arachidonic acid. Therefore in common with the febrile response in mammals behavioral fever in insects may be mediated locally by circulating eicosanoids.

Detection and characterisation of pr1 virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae

In an initial attempt to characterise single-spore isolates from Metarhizium anisopliae wild-type strain V275, three genetically identical spontaneous pr1A- and pr1B-deficient mutants were detected. These mutants were identified by Nested-PCR amplification and verified by Southern hybridisation. Sequencing and comparison of the highly conserved sequences of 28S rDNA domains 9–11 confirmed that the mutant isolates were Metarhizium anisopliae, but each had both morphological and genetic differences from the wild-type parent. RAPD analyses indicated that the overall genetic similarity between wild-type strain and the mutant was less than 70%. Enzyme assays proved that the mutant isolates had significantly lower Pr1 and elastase activities. Bioassays showed reduced lethal activity of the mutants (20%) on Tenebrio molitor, but lethal activities were similar to those of the wild-type parent on the larvae of Galleria mellonella. This paper shows for the first time that a stable mutant strain lacking the most important pr1 genes is still able to infect its respective hosts. Our observations suggest that it is not uncommon for Metarhizium anisopliae to lose its virulence genes during maintenance on artificial medium.

Molecular cloning, characterisation, and expression of a neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

A neutral trehalase gene (NTH1) was isolated from a lambdaEMBL3 genomic library of the insect pathogenic fungus Metarhizium anisopliae. Sequencing of the gene revealed extensive homology with other fungal neutral trehalases. The NTH1 gene exists as a single copy in the genome. Two STREs exist in the 5'UTR of NTH1, which may mediate transcriptional activation of the NTH1 gene in response to various stresses. The NTH1 gene encodes a protein of 737 amino acids with a calculated M(r) of 83.1kDa. A cyclic adenosine 3',5'-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site were found in the amino-terminal domain of NTH1, consistent with a regulatory enzyme. Expression of the trehalase cDNA was achieved in Saccharomyces cerevisiae. Southern blot analysis of RT-PCR products indicated that the neutral trehalase gene is transcribed in vitro in cell-free haemolymph of the tobacco hornworm Manduca sexta and in vivo in the early stage of infection.

Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect host's immune system.

Laboratory shelf-life of oil-formulated conidia of the locust and grasshopper fungal pathogen Metarhizium flavoviridae Gams & Rozsypal, in mixtures with the pyrethroid insecticide lambda-cyhalothrin

The addition of 20 ppm lambda-cyhalothrin pyrethroid insecticide to Metarhizium flavoviridae (Fungi: Deuteromycetes) dry conidia in oil kept germination above 80% for 2 and 12 months at temperatures of approximately 30 and 8°C respectively. At 30°C, conidia in oil alone maintained viability for only a few weeks, but this was doubled at 8°C. Even dry conidia retained good viability for only 5 months at the higher temperature. The extended viability of conidia given by adding the pyrethroid to the oil formulation is regarded as very valuable for areas where the fungus is to be used against locusts and grasshoppers.

Pathogen alarm behavior in a termite: A new form of communication in social insects

Dampwood termites, Zootermopsis angusticollis, show an alarm response after detecting the presence of spores of the pathogenic fungus Metarhizium anisopliae. Termites in direct contact with a high concentration of spores (10(7) spores/ml) show a striking vibratory display which appears to convey information about the presence of pathogens to nearby unexposed nestmates through substrate vibration. Nestmates not directly in contact with spores that perceive the vibrational signal increase significantly their distance from the spore-exposed vibrating termites, apparently to escape from the source of infection. The fleeing response is not induced by the presence of the spores alone or by pheromones, and requires the perception of the vibrations propagated through the substrate. This "pathogen alarm behavior" appears to be a previously unrecognized communication mechanism that allows termites to reduce disease risks within the nest.

The effect of plant growth and topography on the acquisition of conidia of the insect pathogen Metarhizium anisopliae by larvae of Phaedon cochleariae

The susceptibility of mustard beetle, Phaedon cochleariae , larvae to the insect pathogenic fungus Metarhizium anisopliae , was influenced by the topography of the host plant, the dosage and the larval instar. At high doses (> 10 9 conidia ml −1 ) more insects were likely to acquire conidia. Most of the inoculum adhered to the mid-ventral region of the abdomen. Inoculum applied to the leaf became diluted during leaf expansion and this decreased beetle mortality. Leaf expansion was slower at 10°C than at alternating temperatures of 23/10° or when constant at 23°. Slightly more larvae acquired conidia when fed on oilseed rape than Chinese cabbage or turnip. Most conidia adhered to the legs and the mid-ventral region of the abdomen. Mortality was usually less on oilseed rape than Chinese cabbage and turnip. These observations suggest that fungistatic compounds of oilseed rape were interfering with the infection process.

Control of Western flower thrips (Frankliniella occidentalis Pergande) pupae in compost

SummaryWestern flower thrips (Frankliniella occidentalis) develops from an active larval stage through to a non‐feeding, almost immobile, pre‐pupal and pupal stage. This generally occurs in the compost or soil below the plant on which the larvae fed. Control of thrips at this stage in their development offers a chance of utilising pathogens or pesticides not suitable for use in an integrated control programme aimed at adult and larval stages. Trials were done with F. occidentalis pupae and pre‐pupae in a soil/peat based compost using 11 pesticides, three fungal pathogens and four species of insect parasitic nematodes. The pesticides malathion and chlorpyrifos‐methyl gave the most promising result with 97.5% and 96.5% control, respectively. The fungal pathogen Metarhizium anisopliae proved better when applied as a pre‐pupation rather than as a post‐pupation treatment (74.5% : 26.6% control). The insect parasitic nematode Steinernema carpocapsae gave 76.6% control when applied at 25 times 104 nematodes litre‐1 of compost. The results are discussed in relation to control of thrips in glasshouses.

Cloning and characterisation of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae

Metarhizium anisophilae (Ma) secretes a range of proteases when grown in vitro on insect cuticle. A trypsin-like serine protease, PR2, was purified from culture filtrates by anion exchange chromatography and the N-terminal sequence determined. Using oligodeoxyribonucleotide probes based on this sequence and that of the highly conserved trypsin active site, a gene was isolated from a λEMBL3 genomic library of Ma isolate ME1. Sequencing of the gene and RT-PCR revealed that the gene contains two introns which are 94 and 40 bp long. The deduced protein consists of 254 amino acids, has a putative signal sequence to allow transport into the endoplasmic reticulum and probably undergoes a second proteolytic processing step at its N terminus to yield the mature enzyme. The putative mature enzyme has extensive homology with other serine proteases of the trypsin subclass and, in particular, with the trypsin characterised from Fusarium oxysporum.