Divergent immune strategies of Colorado potato beetle larvae against the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana: A comparative transcriptomic analysis.

BackgroundThe misuse of chemical insecticides has developed the phenomenon of habituation in the whitefly Bemisia tabaci (Gennadius) causing enormous economic losses under geothermal greenhouses in southern Tunisia.ResultsIn order to develop means of biological control appropriate to the conditions of southern Tunisia, the efficacy of the native strain of the predator Nesidiocoris tenuis Reuter (Hemiptera: Miridae) and two entomopathogenic fungi (EPF) Beauveria bassiana and Lecanicillium muscarium was tested against Bemisia tabaci (Gennadius). Indeed, the introduction of N. tenuis in doses of 1, 2, 3, or 4 nymphs per tobacco plant infested by the whitefly led to highly significant reduction in the population of B. tabaci, than the control devoid of predator. The efficacy of N. tenuis was very high against nymphs and adults of B. tabaci at all doses per plant with a rate of 98%. Likewise, B. bassiana and L. muscarium, compared to an untreated control, showed a very significant efficacy against larvae and adults of B. tabaci. In addition, the number of live nymphs of N. tenuis treated directly or introduced on nymphs of B. tabaci treated with the EPF remained relatively high, exceeding 24.8 nymphs per cage compared to the control (28.6).ConclusionsIt can be concluded that the native strain of N. tenuis and the EPF tested separately were effective against B. tabaci. Their combined use appears to be possible.

The effect of inoculation of strawberry roots by two entomopathogenic fungal isolates, Metarhizium robertsii (ESALQ 1622) and Beauveria bassiana (ESALQ 3375), on naturally occurring arthropod pests and plant diseases was investigated in four commercial strawberry fields during two growing seasons in Brazil. Three locations represented open-field production while strawberries were grown in low tunnels at the fourth location. Population responses of predatory mites to the fungal treatments were also assessed. Plants inoculated by the fungal isolates resulted in significantly fewer Tetranychus urticae adults compared to control plants at all four locations. The mean cumulative numbers ± SE of T. urticae per leaflet were: M. robertsii (225.6 ± 59.32), B. bassiana (206.5 ± 51.48) and control (534.1 ± 115.55) at the three open-field locations, while at the location with tunnels numbers were: M. robertsii (79.7 ± 10.02), B. bassiana (107.7 ± 26.85) and control (207.4 ± 49.90). Plants treated with B. bassiana had 50% fewer leaves damaged by Coleoptera, while there were no effects on numbers of whiteflies and thrips. Further, lower proportions of leaflets with symptoms of the foliar plant pathogenic fungi Mycosphaerella fragariae and Pestalotia longisetula were observed in the M. robertsii (4.6% and 1.3%)- and B. bassiana (6.1% and 1.3%)-treated plots compared to control plots (9.8% and 3.7%). No effect was seen on numbers of naturally occurring predatory mites. Our results suggest that both isolates tested may be used as root inoculants of strawberries to protect against foliar pests, particularly spider mites, and also against foliar plant pathogenic fungi without harming naturally occurring and beneficial predatory mites.

The Old-World bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae), is a significant threat to crops worldwide and has become resistant to traditional synthetic insecticides. The present study investigated the pathogenicity of the entomopathogenic fungi (EPF) Metarhizium robertsii (Hypocreales: Clavicipitaceae) strain WG-04 or Beauveria bassiana (Hypocreales: Cordycipitaceae) strain WG-10, and an entomopathogenic nematode (EPN) species Heterorhabditis bacteriophora against the second and fourth instar larvae of H. armigera. Both fungal species and H. bacteriophora were evaluated, singly or in combination. After 24 and 48 h post-application of the fungal spores (106 spores/mL), H. bacteriophora was introduced at a rate of 50 infective juveniles/mL, and mortality was observed at 3-, 5-, and 7-days post-treatment. Adult emergence, egg hatching, and percentage pupation were recorded. The integration of both types of biocontrol agents exhibited additive and synergistic interactions in larval stages, and enhanced mortality was recorded when EPF was used in combination with the nematodes. In the individual application of all three biocontrol agents alone, the order of efficacy was H. bacteriophora > B. bassiana > M. robertsii; however, in joint treatments, the increase in mortality and decrease in percentage egg hatching, pupation, and adult emergence was determined to be directly linked to the exposure period of the H. bacteriophora. The results of this study suggest that combining H. bacteriophora with EPF could provide a solid foundation for an economically viable method for managing H. armigera larvae in chickpea fields.

The entomopathogenic fungus Beauveria (Cordyceps) bassiana holds much promise as a pest biological control agent. B. bassiana produces at least three in vitro single cell infectious propagules, including aerial conidia, vegetative cells termed blastospores and submerged conidia, that display different morphological, biochemical and virulence properties. Populations of aerial conidia, blastospores and submerged conidia were produced on agar plates, rich liquid broth cultures and under conditions of nutrient limitation in submerged cultures, respectively. cDNA libraries were generated from mRNA isolated from each B. bassiana cell type and approximately 2,500 5' end sequences were determined from each library. Sequences derived from aerial conidia clustered into 284 contigs and 963 singlets, with those derived from blastospores and submerged conidia forming 327 contigs with 788 singlets, and 303 contigs and 1,079 contigs, respectively. Almost half (40-45 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. The expressed sequence tag dataset also included sequences representing a significant portion of proteins in cellular metabolism, information storage and processing, transport and cell processes, including cell division and posttranslational modifications. Transcripts encoding a diverse array of pathogenicity-related genes, including proteases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites, were also identified. Comparative analysis between the libraries identified 2,416 unique sequences, of which 20-30 % were unique to each library, and only approximately 6 % of the sequences were shared between all three libraries. The unique and divergent representation of the B. bassiana transcriptome in the cDNA libraries from each cell type suggests robust differential gene expression profiles in response to environmental conditions.

The effect of a number of plant extracts on the susceptibility of experimental insects to enthomopathogenic microorganisms has been studied. It is shown that the weight of the wax moth Galleria mellonella larvae tends to decrease by 30–50% under the treatments of polar and nonpolar extracts from the ledum sprouts and the reindeer lichen, while the crude hemlock extract had the opposite effect, contributing to the larva weight gain by approximately 30%. The treatment with the reindeer lichen extract causes synergistic effects on mortality from both the nuclear polyhedrosis virus and the fungal infection in the gypsy moth Lymantria dispar and the wax moth, respectively. It has been determined that the main components of this extract are perlatolic acid, usnic acid, and a third component whose exact chemical identity is still unknown. The usnic acid is the most prospective additive component to entomopathogenic microorganisms. The treatment with the usnic acid caused the increase in mortality from the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana in the Colorado potato beetle Leptinotarsa decemlineata and the wax moth. However, the maximum effect occurs only after the treatment with the crude extract, which can be explained by either the combined effects of all the extract components or the change in a range of the properties of the components in the presence of the other crude extract components.

Fungal volatile organic compounds (VOCs) with biological activity produced by entomopathogenic fungi (Beauveria bassiana and Metarhizium robertsii) isolated from banana fields (Canary Islands) and the nematophagous fungus Pochonia chlamydosporia, used in biocontrol of root-knot nematodes, repel the banana weevil (BW), Cosmopolites sordidus (Germar, 1824) under laboratory conditions. BW is the main pest of banana (Musa spp. (Linnaeus, 1753)). Its cryptic behavior makes it difficult to manage. Repellent VOCs alter BW behavior and can thus be used in sustainable pest management strategies. We evaluated fungal VOCs styrene (C1), benzothiazole (C2), 1,3-dimethoxybenzene (C5) and 2-cyclohepten-1-one (C7) in Canary Islands banana fields naturally infested with BW. 1,3-dimethoxybenzene (C5) significantly reduced the attraction of BW adults to sordidin (BW aggregation pheromone) in banana fields. C5 was detected in the field using GC-MS. C1 and C2 had a mild repellent effect influenced by seasonal changes. C7 VOC did not repel BW in the field. Site and season affected VOCs repellence to BW. Climate may influence VOCs evaporation and therefore their repellent efficacy. VOCs modify BW spatial ecology under field conditions. The inverse distance weighted (IDW) interpolation technique showed changes in BW infestation patterns after application of VOCs in the field. In conclusion, VOCs from biocontrol fungi reduced BW attraction to its aggregation pheromone in banana fields. These responses to experimental BW repellents were influenced by weather and BW population size. BW repellents have potential to be used in "push-pull" strategies to manage BW sustainably in banana crops.

The arrival of invasive pests in recent years has been facilitated due to advances in globalization. Species are considered invasive when individuals arrive and remain in a new location different from their original location. Therefore, Drosophila suzukii and Zaprionus indianus are considered invasive pests that recently arrived in Brazil. Both D. suzukii and Z. indianus are polyphagous pests. Therefore, they use different fruits as a food source, thus having several substrates in common. By sharing food and space for the development of their individuals, the species are subjected to different types of interaction, such as competitive interaction. Competition is a natural process that can occur in different ways, such as intraspecific, interspecific, and apparent competition. Intraspecific competition involves individuals of the same species exploiting the same resources. Interspecific competition occurs between individuals of different species, and apparent competition is an indirect interaction between individuals sharing a common natural enemy. In this way, we investigated the effects of different competitive interactions between invasive species and their effect on biological and behavioral parameters and their effect on the control of these pests. Firstly, we confirmed the co-occurrence of invasive species in the field. In laboratory tests, we explored the effect of intra and interspecific competition on development, reproduction, behavior, and projection of this interaction over time. D. suzukii, a primary pest, was not affected by the presence of its competitor while Z. indianus showed high reproductive rates when in the presence of D. suzukii, reflecting the densities found in the field. The effect of intraspecific density on the biological control due the indirect interaction of endophytic entomopathogenic fungi in strawberry crops was also explored. The interaction was investigated for pests from different parts of the plant (foliar parts and fruit parts) in two strawberry cultivars (Camarosa and San Andreas) treated with two different entomopathogenic fungi (Beauveria bassiana and Metarhizium robertsii). The control capacity proved to be dependent on the cultivar and pest density since overcompensation was observed for treatments involving the combination of two stresses: fungal inoculation and high density. This shows that control efficiency depends on monitoring pest densities in the field, the cultivar used and the frequency of inoculation processes that must be carried out to guarantee the control capacity of this technique. Finally, the existence of apparent competition between the co-occurring pests D. suzukii and Z. indianus sharing the natural enemy Metarhizium spp was assessed. Although direct interactions between the fungus and individual pests have shown promise for control, it has not been possible to observe strong evidence of apparent competition occurring when pests are interacting with the natural enemy. The results highlight the importance of studying competitive interactions to better understand the pest development and to evaluate the best ways to control them.

Simple SummaryEndoparasites such as Ascaris suum can pose a serious threat to the health of livestock and, consequently, humans. One promising way of controlling the threat is the use of natural enemies/antagonistic fungi. In this study, we examined the effects of entomopathogenic fungi (naturally attacking insects) as a bioregulator in the invasive stages of the parasitic nematode A. suum. The conducted study indicates that none of the fungal strains tested have nematocidal activity against A. suum eggs, and they do not meet the criteria required for use in the bioregulation of the parasite’s dispersal stages. Among the strains tested, Isaria fumosorosea and Metarhizium robertsii stood out, combining the highest metabolic activity with nematocidal activity against A. suum.The aim of the study was to evaluate the potential of using five selected species of entomopathogenic fungi (Beauveria bassiana, B. brongniartii, Conidiobolus coronatus, Isaria fumosorosea, and Metarhizium robertsii) in the bioregulation of the dispersive stages of the parasitic nematode—Ascaris suum. Experimental cultures of each of the selected entomopathogenic fungi, as well as a control culture without fungi, were incubated with A. suum eggs at 26 °C for 28 days. Development of the A. suum eggs was observed using a light microscope on the 7th, 14th, 21st, and 28th days of incubation. The API-ZYM® test was used to determine, semiquantitatively, the activity of 19 hydrolytic enzymes from the entomopathogenic fungi. The cytotoxicity of the fungi was determined using tetrazole salt MTT. It was found that none of the five tested strains of entomopathogenic fungi showed an ovicidal effect, and none of them colonized the A. suum egg shells. However, ovistatic activity was observed mainly until the 14th day of incubation by I. fumosorosea, M. robertsii, and B. bassiana. In the MTT test, M. robertsii showed moderate cytotoxicity, while the other species showed low cytotoxicity. Among the strains tested, I. fumosorosea showed the highest spectrum of hydrolase production (13 out of 19 enzymes gave a positive reaction from 3 to 5; 20–40 nM or more). The absence of morphological changes in the A. suum egg shells suggests that the antagonistic effect of the studied entomopathogenic fungi may be due to their cytotoxicity, associated with the production of secondary metabolites—toxins (M. robertsii) and enzymatic activity (I. fumosorosea).

Tetranychus urticae (Acari: Tetranychidae) is a widespread and serious mite pest that infests tomato plants and causes economic losses worldwide. We investigated the acaricidal efficacy of two isolates of entomopathogenic fungi (EPF) Metarhizium robertsii (WG-7) and Beauveria bassiana (WG-12) alone and in combination with abamectin when applied topically to tomato leaf discs in the laboratory against T. urticae. We also evaluated the establishment and proliferation of T. urticae mite life stages on tomato plants in the greenhouse after application of each of the above treatments. The combination of abamectin with each EPF caused 100% mortality in T. urticae immatures after 2 days while each EPF or abamectin alone caused moderate mortality, not exceeding 74.2% 3 days post-exposure. Complete (100%) mortality of adults was observed after 5 days in leaf discs treated with M. robertsii plus abamectin whereas B. bassiana plus abamectin caused 100% mortality after 7 days. The mean number of eggs, emerged immatures, and adults were significantly reduced on both sides of the leaves (i.e., abaxial and adaxial sides) after using the combined application of M. robertsii or B. bassiana plus abamectin, compared to abamectin alone and controls. Our results reveal that the acaricidal efficacy of abamectin combined with either EPF was significantly better in managing the T. urticae life stages than either treatment alone under greenhouse conditions.

One of the ways to suppress harmful phytophages is a microbiological method of protection. Entomopathogenic fungi are promising agents of biological control in the fight against insects - pests of agriculture and forestry. Biopreparations, based on entomopathogenic fungi Beauveria bassiana, lead to mass reproduction and distribution resulting in epizootics occurrence. A wide range of biological activity of ascomycete Beauveria bassiana and a safety in relation to animals, humans and plants allows the use of mycoinsecticides based on the fungi in agriculture and forestry. The migratory locust, the Colorado beetle and Ips hauseri are highly sensitive to fungi cultures isolated from the bark beetles. A similar susceptibility to the entomopathogenic fungus Beauveria bassiana is observed among these pests. The main purpose of the work is to assess the biological activity of the studied strains on insects from different systematic groups, as well as the evaluation of mycelial growth in the temperature range of 15-30°C in isolates of the entomopathogenic fungus Beauveria bassiana. Two new strains of Beauveria genus, that have a sufficiently high virulence for test insects, have been selected as a promising producer strains for the production of mycoinsecticides. In addition, temperature preferences are determined for the studied fungal cultures and optimal titers of the working suspension are selected. Key words: entomopathogenic fungi, Beauveria bassiana, virulence, thermotolerance, mycoinsecticide.

In the accompanying paper [Cho, E.-M., Liu, L., Farmerie, W. & Keyhani, N. O. (2006). Microbiology 152, 2843-2854], the analysis of expressed sequence tag (EST) libraries derived from homogeneous single-cell populations of aerial conidia, in vitro blastospores and submerged conidia of the entomopathogenic fungus Beauveria (Cordyceps) bassiana has been reported. Here an extended EST analysis is presented of complex cell mixtures derived from fungal cells sporulating on chitin or grown under culture conditions inducing the production of the B. bassiana secondary metabolite, oosporein. Fungal material used for the construction of the libraries included germinating conidia and blastospores, mycelia, as well as cells in various developmental stages. Approximately 2,500 5' end sequences were determined from random sequencing of clones from each library, and were clustered into 277 contigs with 1,069 singlets, and 306 contigs with 1,064 singlets, for the chitin and oosporein libraries, respectively. Almost half (45-50 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. Approximately 20-25 % of the sequences in each library could be annotated by gene ontology terms. A comparative analysis between the two libraries, as well as the libraries in the accompanying paper, is presented. A set of 4,360 clustered and unique sequences was characterized. The data are indicative of a highly plastic gene expression repertoire being available to B. bassiana for growth during different environmental and developmental conditions, and provides a dataset for gene discovery and genome annotation.

Background Among the phytosanitary problems, affecting tomato crops in greenhouses heated by geothermal water in southern Tunisia, the tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is gaining prominence from year to year. Results This study determined the susceptibility of eggs, second-instar larvae of T. absoluta to commercial formulations of the entomopathogenic fungi (EPF) Beauveria bassiana and Lecanicillium muscarium at different doses (1010, 109, 108, 107 and 106 spores/ml). Pathogenicity tests were carried out under controlled laboratory conditions at 27 ± 1 °C, and RH of 75 ± 5%, showed that the three commercial formulations of B. bassiana R444 (Bb-Protec®), B. bassiana ATCC 74,040 (Naturalis®) and L. muscarium Ve6 (Mycotal®) were effective against eggs and second instar larvae of T. absoluta. Mortality rates of eggs and larvae were high with increasing the concentrations of the fungi. B. bassiana ATCC 74,040 was the most effective one, with an egg mortality rate of (71.42%) and larval mortality was greater than (80%), at the highest concentration of 1010 spores/ml. The lethal concentration required to kill 50% of eggs was 1.56 × 108, 1.73 × 109 and 1.09 × 1010 conidia/ml for B. bassiana ATCC, B. bassiana R444, and L. muscarium, respectively. After 7 days, the median lethal concentration (LC50) values against larvae were the lowest for B. bassiana ATCC with 2.63 × 107 and 4.76 × 106 conidia/ml by way 1 and way 2 treatments, respectively. Thus, these three formulations affected the emergence of pest’s adults. The fungi significantly reduced pupation and adult emergence. Only 50.25, 60.15 and 69.16% of T. absoluta adults emerged in the treatments with B. bassiana ATCC, B. bassiana R444, and L. muscarium, respectively. Conclusions Biological performances of the EPF showed the role of a biological control agent that can be played against T. absoluta within the framework of an Integrated Pest Management program.

Crocidolomia pavonana is one of the most prominent pest that cause damage to vegetables especially Brassicaceae such us cabbage, broccoli, mustard greens and turnips, these vegetable have been widely consumed and cultivated in Indonesia. The invation of this pest might created high risk of cultivated failure. Enviromentally pest control efforts by utilizing biological control agents such us biopesticides of plants and entomopathogenic fungi have been carried out, but the work was relatively long and strongly influenced by environmental factors. The purpose of this study was to combine biopesticide of Mirabilis jalapa and entomopathogenic fungi Beauveria bassiana to look at mortality of C. pavonana larvae observing by histological incision and scanning electron microscope. Concentration treatments of extracts M. jalapa was (control; 0,1; 0,2; 0,4 and 0,8 gr/ml) and the result showed that the effective concentration was 0,8 g/ml which affect significantly (P<0,05) in reduce pupa weight, improve pupasi time, lowering percentage of emergence imago and improve the long phase of pupa which differ significantly with control. The combination of biopesticides proved to accelerate the mortality of larvae. Histological incision observed at hour 24, 48, 72 and 96, where the biggest damage occurred at hour 96. Observation by scanning electron microscope showed fungus spores that attach to the body surface of larvae subsequently penetrate into the body. Thus the combination use of biopesticides M. jalapa and fungi B. bassiana, can be used as an alternative pest control C. pavonana.

In vivo expression of genes in the entomopathogenic fungus Beauveria bassiana during infection of lepidopteran larvae

In the field, substantial quantities of insect pathogenic fungal conidia (5 × 1012/ha) are usually applied for the control of pests. In this regard, attempts are being made to obtain higher yields of conidia to make the process viable. One of the approaches is to induce microcycle conidia (MC) production. In a solid-state fermentation on rice, the SYB-grown inoculum with more pseudomycelia of B. bassiana enhanced MC production almost 5 times compared to the aerial conidia (AC) within 10 days. A chitosan (CNP) and alginate-chitosan (ACNP) nanocoating of MC increased the overall temperature and UV stability. The % cumulative mortalities of Spodoptera litura larvae over 10 d were 83 ± 8.0, 90 ± 5.0, 83 ± 5.0, and 90 ± 6 for AC-, MC-, CNP- coated MC and ACNP-coated MC, respectively. Using probit analysis, the LT50 values were 5.8, 6.0, 7.5, and 6.3 d for AC, MC, CNPs-MC, and ACNPs-MC, respectively. It was observed that chitin deacetylase (CDA) plays a significant role in increasing MC formation. The higher relative proportion of total CDA over chitosanase activity (higher CDA: chitosanase activity ratio) was found to be correlated with the microcycle conidiation.