Efficacy of an oil-based formulation combining Metarhizium rileyi and nucleopolyhedroviruses against lepidopteran pests of soybean

A biopesticide that combines Metarhizium rileyi and nucleopolyhedrovirus (NPV) in an oil dispersion (OD) was developed. Its efficacy against Anticarsia gemmatalis and Chrysodeixis includens was evaluated under laboratory and field conditions. First, each of the two selected fungal strains, combined or not in OD preparations with AgMNPV or ChinNPV, was tested in the laboratory against A. gemmatalis and C. includens, respectively. AgMNPV–fungus combinations had similar effect on overall mortality of A. gemmatalis compared to each pathogen used alone. C. includens was less susceptible to infection by M. rileyi strains, and ChinNPV became the major cause of larval death. The OD formulations with M. rileyi and/or NPV were applied in infested soybean fields. ChinNPV + M. rileyi caused 50.8% of overall mortality six days after spraying in a soybean crop infested by both pests, whereas for treatments with the fungus or the virus alone the percentages were 41.2% and 8.8%, respectively. In another field infested solely by A. gemmatalis, insect mortality was similar for the AgMNPV–fungus combination and the virus used alone. Larval parasitism by wasps and dipterans was not affected following biopesticide spraying. Our results suggest that the performance of a dual-action biopesticide is related to the NPV and fungal strains that are combined and the proportion of host populations simultaneously infesting the crop.     

Establishment and persistence of the entomopathogenic fungus Metarhizium anisopliae in maize fields

The entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) was applied in maize fields to control the Western Corn Rootworm Diabrotica virgifera virgifera Le Conte (Coleoptera: Chrysomelidae). Establishment and persistence of two strains of M. anisopliae were investigated after application as ‘fungal colonized barley kernels’ (FCBK) into the soil and as a spore suspension on maize leaves and on the soil surface in 2006 and 2007 at two locations in Hungary. The applied fungal strains were able to establish at both locations and a long‐term persistence of at least 15 months could be recorded in the soil. A positive correlation between density of colony forming units (CFU) in the soil and the soil inhabiting stages of the host insect D. v. virgifera could be found. M. anisopliae spores applied on maize leaves were able to survive for no longer than 3 days after application, whereas on the soil surface a noticeably increase of fungus densities were found after treatments. Molecular markers were used to identify the applied M. anisopliae strains before and after application of FCBK into the soil of the maize field.     

Mitochondrial evolution in the entomopathogenic fungal genus Beauveria

Species in the fungal genus Beauveria are pathogens of invertebrates and have been commonly used as the active agent in biopesticides. After many decades with few species described, recent molecular approaches to classification have led to over 25 species now delimited. Little attention has been given to the mitochondrial genomes of Beauveria but better understanding may led to insights into the nature of species and evolution in this important genus. In this study, we sequenced the mitochondrial genomes of four new strains belonging to Beauveria bassiana, Beauveria caledonica and Beauveria malawiensis, and compared them to existing mitochondrial sequences of related fungi. The mitochondrial genomes of Beauveria ranged widely from 28,806 to 44,135 base pairs, with intron insertions accounting for most size variation and up to 39% (B. malawiensis) of the mitochondrial length due to introns in genes. Gene order of the common mitochondrial genes did not vary among the Beauveria sequences, but variation was observed in the number of transfer ribonucleic acid genes. Although phylogenetic analysis using whole mitochondrial genomes showed, unsurprisingly, that B. bassiana isolates were the most closely related to each other, mitochondrial codon usage suggested that some B. bassiana isolates were more similar to B. malawiensis and B. caledonica than the other B. bassiana isolates analyzed.     

Efficacy of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against Thaumetopoea pityocampa Shiff. (Lepidoptera: Thaumatopoeidae)

Abstract

In this study, Metarhizium anisopliae TR 106 and Beauveria bassiana TR 217 was tested against fourth instar larvae of Thaumetopoea pityocampa. The LT₅₀ and LT₉₀ of 1?×?10⁶ concentration of M. anisopliae against T. pityocampa were 3.60 and 4.11 for direct application, while these were 2.87 and 3.60?days, respectively in leaves application. The LT₅₀ and LT₉₀ of the 1?×?10⁸ concentration of the same isolate were 2.50 and 2.95?days for direct application, and 2.98 and 3.74?days for leaves application. The LT₅₀ of insect and leaves application for 1?×?10⁶ of B. bassiana were 3.75 and 3.49?days, respectively. The LT₉₀ of same concentration for insect application was 4.48?days, while LT₉₀ for leaves application was 4.63?days. Similarly, LT₅₀ of insect and leaves application for 1?×?10⁸ of B. bassiana were 3.03 and 3.31?days, while LT₉₀ were 3.68 and 4.29?days, respectively. Approximate 100% mycosis was observed in all treatments.     

Interaction of fungicides with the entomopathogenic fungus Isaria fumosorosea

The effects of eight commonly used fungicides: propamocarb, fenarimol, triadimefon, procimidone, azoxystrobin, carbendazim, cooper oxychloride and Trichoderma harzianum on germination, mycelial growth and virulence of Isaria fumosorosea (Wize) Brown & Smith (Ascomycota: Hypocreales) was studied. The greatest effect on germination was observed by azoxystrobin, followed by carbendazim, triadimefon and procimidone. Propamocarb, fenarimol, cooper oxychloride and T. harzianum did not affect conidial germination with germinations of 95, 93, 79 and 84%, respectively. Virulence was evaluated against early fourth instar nymphs of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). The mix containing fungal conidial suspensions plus cooper oxychloride or fenarimol presented a significant reduction in the mortality in comparison with spore suspension itself. This study suggests that the most appropriate fungicides for use in Integrated Pest Management Programs against T. vaporariorum in combination with I. fumosorosea are propamocarb and T. harzianum.     

Effects of entomopathogenic nematodes on the spruce web‐spinning sawfly Cephalcia arvensis panzer and its parasitoids in the field

Field tests were conducted in the Asiago Forest, Venetian Prealps (Italy) to evaluate the efficacy of nematode strains (Heterorhabditis sp. HL 81 Steinernema carpocapsae IS 230 S. feltiae C = bibionis,) IS 389 S. kraussei SK) against the spruce web‐spinning sawfly Cephalcia arvensis. Soil applications of 100 juveniles cm^‐2 of S. feltiae and S. kraussei resulted in 56% and 36.4% reduction of emergences of sawfly, respectively, when performed before the mature larvae drop and enter the soil. The effectiveness of S. feltiae becomes 32.3% if the nematodes are applied when the larvae have already prepared their chambers. S. feltiae parasitized more females and long‐term diapausing individuals than S. kraussei. The two most effective strains (IS 389 and SK) seem to be well adapted to low temperature, which is likely to be the most important limiting factor for nematode activity in the mountain spruce forests. An ichneumonid parasitoid (Xenoschesis fulvipes,) was strongly affected by S. feltiae, resulting in 66.6% reduction of emergences. Another ichneumonid Ctenopelma lucifer, seems to be less affected than X. fulvipes by the nematode application.     

An investigation on Lecanicillium muscarium as a biocontrol agent of stem borer pests of Rosa damascena in Kerman Province of Iran

The Rosa damascena has organic production and this plant is the most important economic crop in Kerman province. Roses have been used since the earliest times in rituals, cosmetics, perfumes, medicines and aromatherapy. The rose stem sawfly (Hartigia trimaculata) and rose stem girdler (Agrilus aurichalceus) are new and major pests of R. damascena in the Lalehzar region of Kerman province. These pests cause severe damage to plants by feeding stems and new management strategies for their control are continually being investigated. To investigate appropriate biological control agent in the region during 2005–2007, 184 isolates of fungi were collected from these pests. Isolation of fungi was achieved using standard methods. In this study, H. trimaculata and A. aurichalceus from R. damascena for the first time were recorded in Iran. Lecnicillium muscarium from H. trimaculata, L. muscarium from A. aurichalceus, Acremonium kiliense from H. trimaculata, and A. egyptiacum from A. aurichalceus have for the first time been recorded, and L. muscarium has been introduced as a suitable biological agent for control of these pests.     

Virulence of Metarhizium anisopliae and Beauveria bassiana isolates and the effects of fungal infection on the reproduction potential of Rhiphicephalus microplus engorged females

The aim of the study was to assess the virulence of five Metarhizium anisopliae (Ma) and three of Beauveria bassiana (Bb) isolates, and the effect of the fungal infection to the reproduction of engorged females from two colonies of Rhiphicephalus microplus; one colony was collected from naturally infested cattle (Native) and the other one from a laboratory colony (Media Joya). Virulence was evaluated using the immersion technique at a concentration of 1?×?10⁸ conidia/ml; control groups received a water suspension with Tween 80 (0.1%). The Reproductive Efficiency Index ‘REI’ (eggs laid/engorged female weight) and the Reproductive Aptitude Index ‘RAI’ (eggs hatched as larvae/engorged female weight) were calculated for both groups. This experiment shows that two entomopathogenic fungal isolates, Bb115 and Ma136, caused high mortality from 5 days post-treatment (PT), reaching mortality rates of 99–100% at 15 days PT in both R. microplus colonies. The Bb115 isolate caused 98 and 79% reduction in egg oviposition in the field and laboratory colonies, respectively, while the reduction in egg hatchability was 98 and 89% in the field and laboratories colonies, respectively. In the case of Ma136, the egg oviposition was reduced in 73% in the field colony and 64% in laboratory colony, while in the field and laboratory colonies, with a reduction in egg hatchability of 73% and 86%, respectively. These results indicate the potential of Bb115 and Ma136 isolates as possible biological control agents of R. microplus.     

Ecological fitness of the biocontrol agent Fusarium oxysporum Fo47 in soil and its impact on the soil microbial communities

Some nonpathogenic strains of Fusarium oxysporum can control Fusarium diseases responsible for severe damages in many crops. Success of biological control provided by protective strains requires their establishment in the soil. The strain Fo47 has proved its efficacy under experimental conditions, but its ecological fitness has not been carefully studied. In a series of microcosm studies, the ability of a benomyl-resistant mutant Fo47b10 to establish in two different soils was demonstrated. One year after its introduction at two concentrations in the disinfected soils, the biocontrol agent (BCA) established at similar high population densities, whereas in the nondisinfected soils it survived at lower densities, related to the initial concentrations at which it was introduced. The BCA behaved similarly in the two soils at temperatures ranging from 5 to 25 °C and soil water potentials between −0.01 and −1.5 MPa. In addition, terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA showed that the structures of the bacterial and fungal communities evolved with time but were not significantly affected by the introduction of the BCA. Overall, the results showed that Fo47 is potentially a good BCA, able to establish in different soil environments without perturbing the investigated microbial structures.     

A review of the hymenopterous natural enemies of cockroaches with emphasis on biological control

The biology and control potential of hymenopterous natural enemies of cockroaches in the familiesAmpulicidae, Evaniidae, Pteromalidae, Eulophidae, Eupelmidae, andEncyrtidae were reviewed. Emphasis was placed on parasitoids of major domiciliary cockroach species, especially those that have had experimental usage in integrated pest management programs. The most promising parasitoids wereAprostocetus hagenowii (Ratzeburg) againstPeriplaneta species, andComperia merceti (Compere) againstSupella longipalpa (F.). Future research should focus on practical augmentation techniques that will be compatible with other control methodologies in the urban environment.      

Lethal effects of Gliocladium virens,Beauveria bassiana and Metarhizium anisopliae on the malaria vector Anopheles albimanus (Diptera: Culicidae)

Control of Anopheles albimanus, the main vector of malaria on the coast of the State of Chiapas, is based mainly on application of chemical insecticides, which has resulted in resistance to most registered insecticides. Strategies for biological control may provide sustainable alternatives. We report on the lethal effects of a native isolate of Gliocladium virens on An. albimanus larvae and adults, compared to that of strains of Beauveria bassiana and Metarhizium anisopliae. Conidial suspensions of G. virens, B. bassiana and M. anisopliae cultured on Sabouraud agar were tested in bioassays with An. albimanus larvae and adults. Mosquito larvae were more susceptible to all fungi, compared to adults. On early and late instar larvae, M. anisopliae showed the most pathogenic effect (LC₅₀ of 1.4×10⁵ conidia/mL in early instars; 1.1×10⁵ conidia/mL in late instars), followed by G. virens (LC₅₀ of 3.3×10⁵ conidia/mL in early instars and 3.5×10⁶ conidia/mL in late instars). Metarhizium anisopliae sensu lato and the native G. virens could be considered good choices for An. albimanus control in southern Mexico.     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.     

Evaluation of the sea anemone Anthothoe albocincta as an augmentative biocontrol agent for biofouling on artificial structures

Augmentative biocontrol, defined as the use of indigenous natural enemies to control pest populations, has not been explored extensively in marine systems. This study tested the potential of the anemone Anthothoe albocincta as a biocontrol agent for biofouling on submerged artificial structures. Biofouling biomass was negatively related to anemone cover. Treatments with high anemone cover (>35%) led to significant changes in biofouling assemblages compared to controls. Taxa that contributed to these changes differed among sites, but included reductions in cover of problematic fouling organisms, such as solitary ascidians and bryozoans. In laboratory trials, A. albocincta substantially prevented the settlement of larvae of the bryozoan Bugula neritina when exposed to three levels of larval dose, suggesting predation as an important biocontrol mechanism, in addition to space pre-emption. This study demonstrated that augmentative biocontrol using anemones has the potential to reduce biofouling on marine artificial structures, although considerable further work is required to refine this tool before its application.     

First record of Aspergillus oryzae (Eurotiales: Trichocomaceae) as an entomopathogenic fungus of the locust,Locusta migratoria (Orthoptera: Acrididae)

A novel entomopathogenic fungus of Locusta migratoria was identified as Aspergillus oryzae using a comparative sequence analysis of the internal transcribed spacer regions, aflatoxin B₁ detection and morphological analysis. The fungus isolated from a dead locust collected in northwestern China was found to be pathogenic to the insect. Phylogenetic experiments revealed a 99% similarity between the fungus and those of three species, A. oryzae, Aspergillus flavus and Aspergillus parvisclerotigenus which are in the same branch of the Flavi section of the genus Aspergillus. Tests to detect aflatoxin B₁ demonstrated that this fungus is a non-aflatoxin B₁ producer, unlike A. parvisclerotigenus. Furthermore, morphological comparison with A. oryzae and A. flavus revealed that Aspergillus sp. XJ-1 belongs to A. oryzae, and named as A. oryzae XJ-1. The results of bioassays against third-instar locusts showed that mortality was dose-dependent and its median lethal concentrations were 3.3 × 10⁸, 1.7 × 10⁷ and 7.2 × 10⁶ conidia/ml on the 10th-, 13th- and 15th-day post-inoculation. Therefore, the A. oryzae XJ-1 may have biocontrol potential against locusts.     

Introduction of Beauveria bassiana as a biological control agent for Tribolium castaneum in Kerman province

Barley, Hordeum vulgare, one of the important crops in the word, is used in malting, feed and food industries. The red flour beetle, Tribolium castaneum, was found wherever grains or other dried foods are stored. Disinfestations of barley using chemical methods to kill insects, in this research, for the first time we isolated the pathogenic KB512 of entomopathogenic fungi Beauveria bassiana from soil and insects, which produced aerial and submerged conidia and blastospores in laboratory conditions. We investigated the best conditions for the production and utilisation of spore suspension to spray the larvae of T. castaneum, which is one of the important pests in Kerman province (Iran). One hundred and eighty isolates that naturally infected by T. castaneum were reared during spring and summer seasons 2010–2011. The pathogenicity test was carried out with direct spray. To bioassay the isolates, three concentrations of the spore suspension were prepared as follows: 1?×?10⁶ and 1?×?10⁸ conidia/ml. The pests were sprayed by aerial conidial suspension, which was prepared by 0.01% Tween 80 in distilled water, and the controls were sprayed by 0.01% Tween 80 in distilled water. After spraying the pests, the plates were incubated at 25?±?1?°C and 80% of relative humidity. Then, the treated pests were monitored every day for the fungal growth and mortality.     

Molecular phylogeny of the entomopathogenic fungi of the genus Cordyceps (Ascomycota: Clavicipitaceae) and its evolutionary implications

Abstract Cordyceps is an endoparasite ascomycetous genus containing approximately 450 species with a diversity of insect hosts, traditionally included in the family Clavicipitaceae of Ascomycota. Establishing the relationships among species with a varied range of morphologies and hosts is of importance to our understanding of the phylogeny and co‐evolution of parasites and hosts in entomopathogenic ascomycetes. To this end, we used a combination of molecular index and morphological characters from 40 representative species to carry out comprehensive molecular phylogenetic analyses. Based on the phylogenetic tree, we used the program DISCRETE for inferring the rates of evolution and finding ancestral states of morphological character. The phylogenetic analyses revealed two important points. (i) Types of perithecia attached to stroma reflected an evolutionary trend in Cordyceps. The vertically immersed perithecia form was the ancestral state, superficial and obliquely immersed perithecia were derived characters, obliquely immersed was irreversible. Species with obliquely immersed perithecia were in a closely related group and were the derived group. (ii) A strong correlation between fungal relatedness and the microhabitat supported the hypothesis that the host jumps through commingling in soil microhabitats. Based on the results of these analyses, host switching explains the diversity of entomopathogenic fungi of the genus Cordyceps.     

Molecular phylogeny of the entomopathogenic fungi of the genus Cordyceps (Ascomycota:Clavicipitaceae) and its evolutionary implications

Cordyceps is an endoparasite ascomycetous genus containing approximately 450 species with a diversity of insect hosts,traditionally included in the family Clavicipitaceae of Ascomycota.Establishing the relationships among species with a varied range of morphologies and hosts is of importance to our understanding of the phylogeny and co-evolution of parasites and hosts in entomopathogenic ascomycetes.To this end,we used a combination of molecular index and morphological characters from 40 representative species to carry out comprehensive molecular phylogenetic analyses.Based on the phylogenetic tree,we used the program DISCRETE for inferring the rates of evolution and finding ancestral states of morphological character.The phylogenetic analyses revealed two important points.(i) Types of perithecia attached to stroma reflected an evolutionary trend in Cordyceps.The vertically immersed perithecia form was the ancestral state,superficial and obliquely immersed perithecia were derived characters,obliquely immersed was irreversible.Species with obliquely immersed perithecia were in a closely related group and were the derived group.(ii) A strong correlation between fungal relatedness and the microhabitat supported the hypothesis that the host jumps through commingling in soil microhabitats.Based on the results of these analyses,host switching explains the diversity of entomopathogenic fungi of the genus Cordyceps.     

Potential of an antagonistic bacterium isolate obtained fromLentinus lepideus basidiospores as a biocontrol agent

TheBurkholderia sp. isolate 87-11 obtained from basidiospores ofLentinus lepideus was antagonistic against severalPythium andRhizoctonia isolates. The bacterium was tested against soilborne diseases of five plants caused byP. aphanidermatum andR. solani by soil and seed application, and its potential as a biocontrol agent is discussed.     

An entomopathogenic fungus and nematode prove ineffective for biocontrol of an invasive leaf miner Profenusa thomsoni in Alaska

A non-native invasive sawfly, the amber-marked birch leaf miner Profenusa thomsoni (Konow), was first detected in south-central Alaska in 1996 and is now widely distributed throughout urban and wild birch trees in Alaska. Impacts have been considered primarily aesthetic because leaf miners cause leaves of birch trees (Betula spp.) to senesce prematurely, but the leaf miners likely also reduce birch vigour and thereby increase susceptibility to diseases and other insects. We tested the ability of commercially available biological control agents to control P. thomsoni. The entomopathogenic fungus Beauveria bassiana (Bals.-Criv.) Vuillemin GHA strain and the entomopathogenic nematode Steinernema carpocapsae (Weiser) were applied in aqueous suspension to the soil/litter surface beneath infested birch trees in Alaska at one site in 2007 and 2008 and two sites in 2010. There was no evidence the fungus or nematode controlled P. thomsoni. Instead, there was evidence the fungus increased the density of this pest insect at two sites, likely by reducing its predators. As tested, B. bassiana and S. carpocapsae do not appear effective as biological controls of P. thomsoni.