Metarhizium Anisopliae Sensu Lato Research Articles

Species Discrimination within the Metarhizium PARB Clade: Ribosomal Intergenic Spacer (rIGS)-Based Diagnostic PCR and Single Marker Taxonomy.

(1) Background: The entomopathogenic fungus Metarhizium anisopliae sensu lato forms a species complex, comprising a tight cluster made up of four species, namely M. anisopliae sensu stricto, M. pinghaense, M. robertsii and M. brunneum. Unambiguous species delineation within this "PARB clade" that enables both the taxonomic assignment of new isolates and the identification of potentially new species is highly solicited. (2) Methods: Species-discriminating primer pairs targeting the ribosomal intergenic spacer (rIGS) sequence were designed and a diagnostic PCR protocol established. A partial rIGS sequence, referred to as rIGS-ID800, was introduced as a molecular taxonomic marker for PARB species delineation. (3) Results: PARB species from a validation strain set not implied in primer design were clearly discriminated using the diagnostic PCR protocol developed. Using rIGS-ID800 as a single sequence taxonomic marker gave rise to a higher resolution and statistically better supported delineation of PARB clade species. (4) Conclusions: Reliable species discrimination within the Metarhizium PARB clade is possible through both sequencing-independent diagnostic PCR and sequencing-dependent single marker comparison, both based on the rIGS marker.

Conidial mass production of entomopathogenic fungi and tolerance of their mass-produced conidia to UV-B radiation and heat

We investigated conidial mass production of eight isolates of six entomopathogenic fungi (EPF), Aphanocladium album (ARSEF 1329), Beauveria bassiana (ARSEF 252 and 3462), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium anisopliae sensu lato (ARSEF 2341), Metarhizium pingshaense (ARSEF 1545), and Simplicillium lanosoniveum (ARSEF 6430 and 6651) on white or brown rice at four moisture conditions (75–100%). The tolerance of mass-produced conidia of the eight fungal isolates to UV-B radiation and heat (45 °C) were also evaluated. For each moisture content compared, a 20-g sample of rice in a polypropylene bag was inoculated with each fungal isolate in three replicates and incubated at 28 ± 1 °C for 14 days. Conidia were then harvested by washing the substrate, and conidial concentrations determined by haemocytometer counts. Conidial suspensions were inoculated on PDAY with 0.002% benomyl in Petri plates and exposed to 978 mW m−2 of Quaite-weighted UV-B for 2 h. Additionally, conidial suspensions were exposed to 45 °C for 3 h, and aliquots inoculated on PDAY with benomyl. The plates were incubated at 28 ± 1 °C, and germination was assessed at 400 × magnification after 48 h. Conidial production was generally higher on white rice than on brown rice for all fungal species, except for L. aphanocladii ARSEF 6433, regardless of moisture combinations. The 100% moisture condition provided higher conidial production for B. bassiana (ARSEF 252 and ARSEF 3462) and M. anisopliae (ARSEF 2341) isolates, while the addition of 10% peanut oil enhanced conidial yield for S. lanosoniveum isolate ARSEF 6430. B. bassiana ARSEF 3462 on white rice with 100% water yielded the highest conidial production (approximately 1.3 × 1010 conidia g−1 of substrate). Conidia produced on white rice with the different moisture conditions did not differ in tolerance to UV-B radiation or heat. However, high tolerance to UV-B radiation and heat was observed for B. bassiana, M. anisopliae, and A. album isolates. Heat-treated conidia of S. lanosoniveum and L. aphanocladii did not germinate.

First report of Metarhizium anisopliae s.l. action on gastrointestinal ruminant nematodes in the free-living stage and its persistence in soil

ABSTRACT The fungus Metarhizium anisopliae is one of the most studied biological control agents in parasite control programs, especially when aiming the control of arthropods. Yet, studies using this biological agent to control gastrointestinal nematodes in ruminants are scarce. The present study evaluated the efficacy of the entomopathogenic fungus Metarhizium anisopliae sensu lato (s.l.) formulated in water and mineral oil on the free-living stages of gastrointestinal nematodes (GINs) in goats, by means of in vitro tests and in semi-natural conditions. The study also evaluated the persistence of the fungus in the soil. The experiments were constituted of four groups: control water; control oil; aqueous formulation of M. anisopliae s.l.; oil formulation of M. anisopliae s.l., each group consisting of 10 pots with Urochloa brizantha cv. MG5 grass. In the in vitro test, the groups treated with the aqueous and mineral oil formulation showed efficacy in controlling gastrointestinal nematodes of goats, providing a reduction of 45.5% and 43%, respectively, and a reduction varying from 36% to 77.9% and 42.6% to 90.2%, respectively, in the experiment under semi-natural conditions. Isolate M. anisopliae s.l. proved to be effective against goat’s gastrointestinal nematodes in the free-living stage, in both in vitro and semi-natural conditions tests, by significantly reducing the number of larvae with the formulations tested and remaining active for a period up to 52 days in the pots’soil. This study suggests the potential use of M. anisopliae s.l. for the biological control of gastrointestinal nematodes in ruminants.

Potential of Cameroon-indigenous isolates of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae as microbial control agents of the flea beetle Nisotra uniformis

ABSTRACT The flea beetle Nisotra uniformis Jacoby is a widespread pest of malvaceous crops for which synthetic chemical insecticides remain the principal control tool, particularly in sub-Saharan Africa. With the ultimate aim of developing a microbial alternative for N. uniformis control, we conducted laboratory experiments (25 ± 1°C; 70 - 80% RH) to evaluate the biological attributes of three Cameroonian isolates of the entomopathogenic fungi Beauveria bassiana (Balsamo-Crivelli) Vuillemin and Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin. Spore germination rates of the isolates varied from 0.5% after 4 hrs to 100% after 24 hrs on PDA medium, with MIITAC11.3.4 spores germinating fastest. Pathogenicity tests consisted of dipping adult beetles in conidial suspensions. All isolates were pathogenic to N. uniformis, with corrected mortalities varying between 23.3 - 86.7%. BIITAC-O-2 had the lowest LC50 at 5.17 × 106 conidia/ml, while MIITAC11.3.4 and BIITAC6.2.2 had LC50 at 4.11 × 107 conidia/ml, and 2.00 × 108 conidia/ml, respectively. BIITAC6.2.2 produced the shortest time to the highest mortality rate (1.17 days) and the shortest LT50 of 1.64 days but only at the highest concentration, followed by the two highest concentrations of MIITAC11.3.4 (LT50 at 3.43 and 5.70 days). The c parameter in the Weibull model showed that BIITAC-O-2 caused the highest rate of increase in mortality (2.42) at the highest concentration, followed by MIITAC11.3.4 (1.09) and BIITAC6.2.2 (0.50). The results of our study provide the basis for including N. uniformis in the ongoing development of BIITAC6.2.2 and MIITAC11.3.4 into biopesticide while continuing with further laboratory studies on BIITAC-O-2.

High susceptibility of Tetranychus merganser (Acari: Tetranychidae), an emergent pest of the tropical crop Carica papaya, towards Metarhizium anisopliae s.l. and Beauveria bassiana strains.

BackgroundThe mite Tetranychus merganser is considered to be an emerging pest of various crops in tropical countries. It is one of the most detrimental pests in the papaya orchards of some regions of México. The current field control of Tetranychus spp. involves the extensive use of chemicals that have some degree of toxicity to humans or the environment and may cause selective resistance. The use of biological alternatives such as parasitoids and mite predators have limited effectiveness. In order to find effective but non-toxic alternatives for mite pest management, bio-products that are able to be mass produced and applied to large production areas have been sought, including the entomopathogen fungi. B. bassiana and M. anisopliae s.l. are the fungi most extensively used for the biological control of insect pests. Although they do not cause natural epizootic diseases in mites, there are reports that show that they infect T. urticae, and should be evaluated for use in the biological control of papaya’s mite pests.MethodsA T. merganser colony was established and the susceptibility of adult females to 30 entomopathogenic fungi strains was evaluated under laboratory conditions with an in vitro mass screening bioassay. Ten strains of Metarhizium anisopliae sensu lato (s.l.), eleven of Beauveria bassiana, nine of Lecanicillium sp. and one of Hirsutella thompsonii var. sinematosa were tested. The infectivity of adult females was evaluated calculating the percentage of mortality. To calculate the LC50 and LT50 of the most virulent strains, a bioassay was performed using serial concentrations (1×104–1×108 conidia/mL) for each strain. Strains showing ability to infect eggs laid were evaluated with a novel egg-infectivity bioassay. The internal transcribed spacer (ITS) region of the more lethal strains were sequenced.ResultsT. merganser and T. urticae were found in orchards of Carica papaya (Maradol variety and Tainung hybrid) in Campeche, México. All tested strains of M. anisopliae s.l. and B. bassiana were infectious to the adult female of T. merganser at a concentration of 1×108 conidia/mL. Six strains of M. anisopliae (Ma002, Ma003, Ma004, Ma005, Ma014 and Ma034) caused 100% mortality, and one of B. bassiana (Bb016) caused 95% mortality. The most virulent was Ma034, with an LC50 of 1.73×106 conidia/mL followed by Ma005 and Ma003. Ma005 and Ma034 were the fastest strains to reach LT50,achieving this in less than 3.7 days. Additionally, Ma034 and Ma014 strains were infectious to more than 70% of the eggs.ConclusionsT. merganser and T. urticae are present in the papaya orchards of Campeche, México. The high susceptibility of T. merganser adult females and eggs toward several M. anisopliae s.l. or B. bassiana strains suggests that these fungi are a viable alternative to control this emergent pest. The most virulent strain, Ma034, was also infective to eggs, and is the most promising to be tested in the field.

Effect of Metarhizium anisopliae s.l. on Rhipicephalus microplus free-living stage and persistence in the soil in the Brazilian Northeast

ABSTRACT The present study evaluated the pathogenicity of Metarhizium anisopliae sensu lato (s.l.) in aqueous and oil formulations against Rhipicephalus microplus in semi-field conditions and the persistence of this microorganism in the soil. Engorged females were used in a biological assay, set in vessels cultivated with Urochloa brizantha and sprayed with 80 mL of the proposed treatments. Four treatments were utilised: aqueous control; oil control; aqueous formulation and oil formulation with M. anisopliae s.l. (2.0 × 108 conidia/mL) (with 10 vessels per group). Pathogenicity against R. microplus was evaluated daily in order to identify the mortality of females and the recovery of larvae 42 days after the treatments began. Additionally, the fungus was re-isolated from deceased females and from the soil in the vessels. To evaluate the fungal viability in the soil, samples were collected before spraying and at the 3rd, 7th, 21st and 52nd days after treatments began. Oil formulation of M. anisopliae s.l. reduced the number of infesting larvae and promoted a percentage of control between 58.4% and 61.3%. Aqueous formulation promoted a percentage of control between 39.6% and 43.8%. In both groups, the persistence of the fungus in the soil was of up to 52 days. On the other hand, the control groups did not show any fungal growth. The fungal isolate, when formulated, has potential to be used in integrated control programmes against R. microplus. In addition, understanding the period of persistence of the fungus in the soil is important for the definition of protocols for fungal application in the field.

Entomopathogenic Fungi for Tick Control in Cattle Livestock From Mexico.

Ticks are one of the main economic threats to the cattle industry worldwide affecting productivity, health and welfare. The need for alternative methods to control tick populations is prompted by the high prevalence of multiresistant tick strains to the main chemical acaricides and their ecological consequences. Biological control using entomopathogenic fungi (EPF) is one of the most promising alternative options. The objective of this paper is to review the use of EPF as an alternative control method against cattle ticks in Mexico. Metarhizium anisopliae sensu lato (s.l.) and Beauveria bassiana s.l. are the most studied EPF for the biological control of ticks in the laboratory and in the field, mainly against Rhipicephalus microplus; however, evaluations against other important cattle ticks such as Amblyomma mixtum and R. annulatus, are needed. A transdisciplinary approach is required to incorporate different types of tools, such as genomics, transcriptomics and proteomics in order to better understand the pathogenicity/virulence mechanism in EPF against ticks. Laboratory tests have demonstrated the EPF efficacy to control susceptible and resistant/multiresistant tick populations; whereas, field tests have shown satisfactory control efficiency of M. anisopliae s.l. against different stages of R. microplus when applied both on pasture and on cattle. Epidemiological aspects of ticks and environmental factors are considered as components that influence the acaricidal behavior of the EPF. Finally, considering all these aspects, some recommendations are proposed for the use of EPF in integrated control schemes for livestock ticks.

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38μM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 108 conidia mL−1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.

Evaluating the Efficacy of Entomopathogenic Fungi against the Bark Beetle, Ips stebbingi Strohmeyer (Coleoptera, Curculionidae: Scolytinae) in India

The bark beetle, Ips stebbingi Strohmeyer (Coleoptera: Curculionidae: Scolytinae) is one of the most serious pests of Pinus wallichiana A. B. Jacks (Pinaceae) in Kashmir Himalaya. In order to find an effective biocontrol agent against this pest, we determined the effectiveness of entomopathogenic fungi, viz. Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin, and Lecanicillium lecanii (Zimmerman) Zare et Gams against I. stebbingi in the laboratory conditions. Each fungal suspension contained 1.0 × 109 spores of fungi in 1 ml. The insecticide (cyclone 505 EC) was also used as positive control in the experiment. The mortality caused with these fungi was recorded in treated branches and Petri dish assay. In treated branches, B. bassiana and M. anisopliae caused higher mortality, i.e., 68% and 71.25%, respectively, 10 days after treatment, and 93.10% and 88%, respectively, 20 days after treatment. The results of Petri dish assay revealed that I. stebbingi adults were highly susceptible to both treated fungal species and insecticide. However, B. bassiana and M. anisopliae caused higher percentage mortality six days after treatment, i.e., 94.16% and 100% respectively. The percentage of mortality caused by treating with insecticide was 60%. Lecanicillium lecanii was found significantly less virulent (mortality 18.33%) in all fungal treatments. Results obtained in the present study are promising and may be used as alternative means of chemical control for management of this beetle pest; however, no recommendations concerning the potential use of these fungal pathogens for forest protection can be given, and further studies are needed in this respect, especially under field conditions.

Laboratory evaluation of entomopathogenic fungi as biological control agents against the bark beetle Pityogenes scitus Blandford (Coleoptera: Curculionidae) in Kashmir

Abstract The bark beetles (Coleoptera: Curculionidae) are widely recognised as one of the most damaging group of forest pests. Entomopathogenic fungi have shown great potential for the management of some bark beetle species. The efficacy of three entomopathogenic fungi, namely, Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin and Lecanicillium lecanii (Zimmerman) Zare and Gams was tested against the bark beetle Pityogenes scitus Blandford under the laboratory conditions. An insecticide – cyclone 505 EC, was also used as positive control in the experiment. Each fungal suspension contained 1.0×109 spores of fungi in 1 ml. In treated branches, B. bassiana and M. anisopliae caused higher percentage of mortalities, that is, 58.33% and 48%, respectively, after 10 days of treatment and 85% and 71%, respectively, after 20 days of treatment. In petri plate assay, B. bassiana, M. anisopliae and L. lecanii caused 100%, 100% and 73.33% of mortality respectively. The percentage of mortality caused by treated insecticide was 79.16%. The results obtained in the present study are promising; however, no recommendations concerning the potential use of these fungal pathogens in forest protection can be given, and further research studies are needed in this respect, especially under field conditions.

An assessment of biological control of Polygraphus major Stebbing, 1903 (Coleoptera: Curculionidae) by entomopathogenic fungi

Recently the use of fungal entomopathogens against bark beetles has gained increasing attention throughout the world and researchers continue to seek highly pathogenic fungal isolates for controlling beetle pests. In the present study, the efficacy of three entomopathogenic fungi, namely Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin and Lecanicillium lecanii (Zimmerman) Zare & Gams, was tested against Polygraphus major Stebbing, 1903 under laboratory conditions. Each fungal suspension contained 1.0 × 10<sup>9</sup> spores of fungi in 1 ml. An insecticide – Cyclone was also used as positive control in the experiment. The mortality caused by these fungi was recorded in treated branches and petri plate assay. In treated branches, B. bassiana and M. anisopliae s. l. caused higher mortality, i.e. 57.77 and 46%, respectively, after 10 days of treatment and 98 and 92.77%, respectively, after 20 days of treatment. The results of the petri plate assay revealed that P. major adults were highly susceptible to both applied fungal species and insecticide. However, B. bassiana and M. anisopliae s. l. caused higher percentage mortalities after six days of treatment, i.e. 100 and 91.66%, respectively. The percentage mortality caused by application of the insecticide was 69%. L. lecanii was observed to be significantly less virulent (mortality 46.66%) in all fugal treatments. After observing the promising nature of the three entomopathogenic fungi by testing them, we arrive at the conclusion that the tested fungi have a potential for the control of P. major, and further field experiments are warranted to investigate their efficacy under more practical conditions.

Metarhizium anisopliae s.l. modulation of lipid metabolism during tick infection is independent of AMPK and ERK pathways.

Despite the importance of fat body in metabolism of arthropods, studies in ticks are scarce. This study evaluated the lipid composition and activation of extracellular signal-regulated protein kinase (ERK) and AMP-activated protein kinase (AMPK) enzymes in Rhipicephalus microplus fat body after infection with different isolates of the fungus Metarhizium anisopliae sensu lato (Metschnikoff, 1879) Sorokin, 1883. The isolates CG 32, GC 112, GC 148, GC 347, and GC 629 were inoculated as viable or non-viable conidia in the ticks. The engorged females were dissected, and their fat bodies were collected 24 and 48h after infection. The lipid composition was assessed by thin layer chromatography, and enzyme activation was detected by Western blotting with antibodies against p-AMPK and p-ERK. The study showed increased levels of triacylglycerol 24 and 48h and fatty acid after 48h after inoculation with different isolates of viable fungi in the tick's hemocoel. Detection of the active form of ERK was demonstrated only after inoculation with non-viable conidia of all isolates tested. The active form of AMPK, only isolate CG 112 was able to activate with viable or non-viable conidia, whereas isolates CG 32 and CG 629 were able to activate with non-viable conidia. This study provides the first report about changes in important metabolic pathways in ticks infected with entomopathogenic fungi and suggests that the lipid content is modulated by non-usual pathways. However, further studies may be necessary for a better elucidation of this interaction.

Susceptibility of lesser mealworm, Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae) to entomopathogenic fungi isolated from poultry houses litter and nearby soil

The lesser mealworm Alphitobius diaperinus is present in great numbers in poultry houses. These insects are especially dangerous as a potential carriers of pathogens such as bacteria, viruses and parasites. We explored the possibility of using local strains of entomopathogenic fungi isolated from litter and from soil to control lesser mealworm populations. Isolated fungi showed low pathogenicity to lesser mealworm beetles. Infection with a suspension at a concentration of 1 × 108 spores/ml resulted in only 4 Metarhizium anisopliae sensu lato isolates showing the highest insect mortality in the range of 30–36%. Still lower pathogenicity was found in isolates of Beauveria bassiana, with only 4 isolates of B. bassiana causing a mortality of 17–26%. Isolates of Isaria fumosorosea and I. farinosa did not cause mortality in beetles that differed significantly from that in the control variant. The larvae were more susceptible to infection. Except for I. fumosorosea, all species caused 100% mortality in larvae. For further studies, the B. bassiana 3K isolate (from the litter) could be selected because of its high mortality (100%) and high larval infectivity (50% overgrown with mycelium).

Fitness costs to Helicoverpa armigera after exposure to sub-lethal concentrations of Metarhizium anisopliae sensu lato: Study on F1 generation

The entomopathogenic fungus, Metarhizium anisopliae (Metsch.) Sorokin is a valuable biocontrol agent attacking larval stages of many lepidopteran pests including Helicoverpa armigera (Hübner). Sub-lethal effects of M. anisopliae sensu lato (s.l.) (isolate M14) were investigated on life table parameters of offspring from treated larvae of H. armigera. Duration of different life stages was significantly affected by fungal treatments. Fecundity was decreased in females derived from H. armigera larvae treated with M. anisopliae s.l. Sub-lethal concentrations of the entomopathogen reduced the net reproduction rate (R0) of F1 insects for all treatments compared with the control. Similar reductions were observed for the intrinsic and the finite rates of increase (rm and λ, respectively). The mean generation time (T) and the doubling time (DT) were statistically higher in offspring of individuals exposed to some fungal concentrations than control insects. Our results indicated that there was a significant decrease in the F1 population of H. armigera derived from larvae that were exposed to sub-lethal concentrations of M. anisopliae s.l.

Production of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae

ABSTRACTThe spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media increased the spore yield in solid-state from three to sevenfold: A. clavatus produced 48.4 ± 5.2 and 15.7 ± 1.6 × 108 spores/g, A. flavus produced 22.3 ± 4.1 and 3.1 ± 2.5 × 108 spores/g, and M. anisopliae produced 39.6 ± 6.5 and 13.1 ± 2.6 × 108 spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC50) of 1.1, 1.8, and 1.3 × 108 spores/ml against Culex quinquefasciatus Say larvae in 72 h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognised entomopathogenic fungus.

Effects of pupal treatment with Proteus® and Metarhizium anisopliae sensu lato on functional response of Habrobracon hebetor parasitising Helicoverpa armigera in an enclosed experiment system

ABSTRACTThe ectoparasitoid wasp, Habrobracon hebetor Say (Hymenoptera: Braconidae) is a valuable biocontrol agent attacking larval stages of many lepidopteran pests including Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). The possibility of combined application of this parasitoid wasp with other biological control agents and chemical insecticides is necessary for the success of an Integrated Pest Management (IPM) programme. One of the most important behaviours of a parasitoid refers to the number of hosts attacked by per parasitoid as a function of host density. In this research, the single and joint effects of the insecticide, Proteus® and the entomopathogenic fungus, Metarhizium anisopliae sensu lato (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) (isolate M14) were studied on the functional response of H. hebetor. Newly mated females (<24 h) from treated pupae of H. hebetor were used to study the functional response of this parasitoid wasp to different densities of H. armigera larvae. A type II functional response was observed in all treatments. The results revealed that control and Proteus® treatments developed the lowest (0.5599 ± 0.0373 h) and the highest (0.5709 ± 0.0443 h) handling time, respectively. The most and the least values of attack rate were observed in control (0.0996 ± 0.0164 h−1) and Proteus® (0.0776 ± 0.0119 h−1) treatments, respectively. The maximum theoretical parasitism rate (T/Th) was estimated to be 42.86 for female control wasps. After conducting field experiments on functional response along with life table parameters, M. anisopliae sensu lato (isolate M14) may be used as a compatible biocontrol agent with H. hebetor in IPM programmes of H. armigera.

Phylogenetic status and pathogenicity of Metarhizium majus isolated from a fruit beetle larva in Japan

Some isolates of Metarhizium anisopliae sensu lato with large conidia (MALC), which was formerly known as M. anisopliae var. majus, from a scarabaeid host, such as isolates from the coconut rhinoceros beetle (Oryctes spp.), have a narrow host range and are particularly adapted to their original host. Despite recent taxonomic revisions of the genus Metarhizium on the basis of DNA sequences, variations in host preferences of MALC have not been linked to DNA sequence variations. This study focused on the phylogenetic status and pathogenicity of MALC isolated from the fruit beetle larva Protaetia orientalis submarumorea (Pos) in Japan to investigate the relationship between virulence and nucleotide sequence variation among MALC. On the basis of the results of the phylogenetic analyses of DNA sequences from five loci, we identified an isolate from Pos (Hn1) as M. majus, which comprised many isolates with large conidia from scarabaeid insects. In the virulence assay of genetically diverse Metarhizium isolates, only Hn1 and its most closely related isolates from soil showed pathogenicity to Pos; however, these Pos-pathogenic isolates showed weak virulence against silkworms. In the analysis of the intergenic spacer region of rDNA, the Pos-pathogenic isolates displayed unique sequence variation and were clearly distinguished from closely related lineages, including isolates from Oryctes sp., which formed a separate monophyletic group. These results indicate that the Pos-pathogenic isolates from Japan are particularly adapted to Pos and can be genetically distinguished from M. majus isolates from different scarabaeid insects.

Inhibition of the entomopathogenic fungus Metarhizium anisopliae sensu lato in vitro by the bed bug defensive secretions (E)-2-hexenal and (E)-2-octenal

The two major aldehydes (E)-2-hexenal and (E)-2-octenal emitted as defensive secretions by bed bugs Cimex lectularius L. (Hemiptera: Cimicidae), inhibit the in vitro growth of an isolate of Metarhizium anisopliae sensu lato (s.l.) (Metsch.) Sokorin (Hypocreales: Clavicipitaceae) (ARSEF 1548). These chemicals inhibit fungal growth by direct contact and via indirect exposure (“fumigation”). Fumigation with (E)-2-octenal for as little as 0.5 h was sufficient to inhibit all fungal growth. Bed bugs placed on filter paper treated with an isolate of M. anisopliae s.l. conidia in the absence of (E)-2-octenal exhibited 99 % mortality after one week. However, bed bugs placed on fungal-treated filter paper and exposed to (E)-2-octenal at 1 h experienced 10 % mortality. The inhibition of fungal growth by bed bug aldehydes is discussed in the context of other biotic and abiotic barriers to infection.

Susceptibility of adults of the cerambycid beetle Hedypathes betulinus to the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, and Purpureocillium lilacinum.

The cerambycid beetle Hedypathes betulinus (Klug) (Coleoptera: Cerambycidae) causes severe damage to yerba mate plants ( Ilex paraguariensis (St. Hilaire) (Aquifoliales: Aquifoliaceae)), which results in large losses of production. In this study, the pathogenicity of entomopathogenic fungi of the species Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae), Metarhizium anisopliae sensu lato (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae), and Purpureocillium lilacinum (Thom) Luangsa-ard, Hywel-Jones, Houbraken and Samson (Hypocreales: Ophiocordycipitaceae) on yerba mate were evaluated. Fifteen isolates of B. bassiana , two of M. anisopliae , and seven of P. lilacinum on H. betulinus adults were analyzed under laboratory conditions. The raw mortality rate caused by B. bassiana isolates varied from 51.1 to 86.3%, and their LT 50 values varied between 8.7 and 13.6 d. The isolates of M. anisopliae caused 69.6‒81.8% mortality, and their LT 50 values varied between 7.4 and 7.9 d. In contrast, isolates of P. lilacinum were not pathogenic. M. anisopliae and B. bassiana isolates were pathogenic against H. betulinus adults, suggesting that they may be useful in biological control programs for insect pests of yerba mate.

Naturally occurring entomopathogenic fungi infecting stored grain insect species in Punjab, Pakistan.

The occurrence of entomopathogenic fungi isolated from stored grain insect pests sampled from various geographical regions of Punjab, Pakistan, was investigated. In total, 25,720 insects from six different species were evaluated, and 195 isolates from 24 different fungal species were recovered. These included the Ascomycetes Beauveria bassiana sensu lato (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) , Metarhizium anisopliae sensu lato (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae), Purpureocillium lilacinum (Thorn) Samson (Hypocreales: Ophiocordycipitaceae), and Lecanicillium attenuatum (Zare and W. Gams) (Hypocreales: Clavicipitaceae). The cadavers of red flour beetle Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae) were significantly infected with the fungi followed by rice weevil Sitophilus oryzae (L.) (Coleoptera: Curculionidae), lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), rusty grain beetle Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae), and cowpea weevil Callosobruchus maculatus (F.) (Coleoptera: Bruchidae); however, the least were recovered from khapra beetle Trogoderma granarium (Everts) (Coleoptera: Dermestidae). The geographical attributes (altitude, longitude, and latitude) greatly influenced the occurrence of entomopathogenic fungi with highest number of isolates found from >400 (m) altitude, 33°–34′ N latitude, and 73°–74′ E longitude. The findings of the current surveys clearly indicated that the entomopathogenic fungi are widely distributed in the insect cadavers, which may later be used in successful Integrated Pest Management programs.