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One of the main negative effects of climate change on biological pest control is alteration of relationships between insect pests and their natural enemies (both entomophagous and entomopathogenic). Indeed, environmental conditions can have multiple effects on pest control success when using entomopathogenic fungi (EPF), where conidial depletion, inactivation, and loss in virulence and infectivity can all occur. Appropriate mass production and formulation strategies for EPF can partially solve these problems. However, the only strategy to guarantee high virulence and infectivity is selection of environmentally competent fungal strains that are able to persist in the host environment for the required infection period. This review examines the criteria for selection of environmentally competent EPF. While UV radiation, followed by humidity and temperature, is probably the most important propagule depletion and inactivation factors in epigeal habitats, temperature is most critical for reducing the infectivity and virulence of EPF in epigeal and hypogeal habitats. In addition, geographical origin and other biotic and abiotic factors have an important impact which may guarantee the environmental competence of selected entomopathogenic fungal strains and, therefore, farmer willingness to replace chemicals with mycoinsecticides. To achieve this, it is urgent to promote the development of microbial control solutions adapted to relatively uniform climatic zones through more simplified, targeted, and less costly EPF approval and authorization. [ABSTRACT FROM AUTHOR]

Endophytic insect pathogenic fungi have a multifunctional lifestyle; in addition to its well-known function as biocontrol agents, it may also help plants respond to other biotic and abiotic stresses, such as iron (Fe) deficiency. This study explores M. brunneum EAMa 01/58-Su strain attributes for Fe acquisition. Firstly, direct attributes include siderophore exudation (in vitro assay) and Fe content in shoots and in the substrate (in vivo assay) were evaluated for three strains of Beauveria bassiana and Metarhizium bruneum. The M. brunneum EAMa 01/58-Su strain showed a great ability to exudate iron siderophores (58.4% surface siderophores exudation) and provided higher Fe content in both dry matter and substrate compared to the control and was therefore selected for further research to unravel the possible induction of Fe deficiency responses, Ferric Reductase Activity (FRA), and relative expression of Fe acquisition genes by qRT-PCR in melon and cucumber plants.. In addition, root priming by M. brunneum EAMa 01/58-Su strain elicited Fe deficiency responses at transcriptional level. Our results show an early up-regulation (24, 48 or 72 h post inoculation) of the Fe acquisition genes FRO1, FRO2, IRT1, HA1, and FIT as well as the FRA. These results highlight the mechanisms involved in the Fe acquisition as mediated by IPF M. brunneum EAMa 01/58-Su strain. [ABSTRACT FROM AUTHOR]

The false carmine cochineal Dactylopius opuntiae has been originally used as biological control agent of the prickly pear cactus Opuntia ficus-indica Miller, considered as an exotic invasive species in many countries including Spain. However, the extremely high level of infestation has resulted in great inconvenience to the population of local residents and a serious threat to wipe out a plant that is already part of the Andalusian landscape. In the present research, different control means were evaluated towards this cochineal at field condition in a site in the province of Cadiz (Spain). The populations of the insect in the study site were confirmed to belong to D. opuntiae. We detected this insect throughout the study area with a very high population density in terms of number of colonies per cladode and the extent of the infestation. We also evaluated the efficacy of four different methods (Chlorpyrifos-methyl, Potassium soap, Cryptolaemus montrouzieri larvae, the entomopathogenic fungus Beauveria bassiana) for the control of D. opuntiae. Our results demonstrated that the Potassium soap and Chlorpyrifos-methyl were the best treatments in reducing D. opuntiae populations, with relative population density values being negative only in these two treatments, and efficacies of 91.5 and 76.7% respectively. The results of this investigation showed that the current situation of false carmine cochineal is worrisome, but nevertheless, the use of Potassium soap for its control is recommended because of its high efficacy and low environmental impact. [ABSTRACT FROM AUTHOR]

[Display omitted] • Entomopathogenic fungi (EPF) are a key component of the agroecosystem microbiota. • EPF are multipurpose microorganisms for sustainable agriculture. • EPF contribute directly and indirectly to insect pest control. • EPF establish plant relationships and produce different benefits for crops. • EPF are a source of bioactive compounds and harbor keys to a circular economy. Entomopathogenic ascomycetes (EA) are an important part of the microbiota in most terrestrial ecosystems, where they can be found regulating natural populations of arthropod pests in both epigeous and hypogeous habitats while also establishing unique relationships with plants. These fungi offer direct benefits to agriculture and human welfare. In the present work, we conducted a systematic review to comprehensively assess the range of ecosystem services provided by EA, including direct and indirect pest biocontrol, plant growth promotion, plant defense against other biotic and abiotic stresses, nutrient cycling, and the production of new bioactive compounds with agricultural, pharmaceutical and medical importance. Moreover, EA are compatible with the ecosystem services provided by other microbial and macrobial biocontrol agents. This systematic review identified the need for future research to focus on evaluating the economic value of the ecological services provided by EA with a special emphasis on hypocrealean fungi. This evaluation is essential not only for the conservation but also for better regulation and exploitation of the benefits of EA in promoting agricultural sustainability, reducing the use of chemicals that enter the environment, and minimizing the negative impacts of crop protection on the carbon footprint and human health. [ABSTRACT FROM AUTHOR]

Abstract: The susceptibility of 20 widely distributed mill and table olive varieties to Bactrocera oleae (Rossi) as affected by irrigation, and fruit diameter and oil content was evaluated in a 3‐year trial in Southern Spain. Bactrocera oleae was bivoltine life cycle in the experimental site, with significant differences among population size throughout the study. Even though the olive fruit fly damaged all varieties, significant differences in susceptibility were detected. Among the mill olive varieties “Nevadillo Blanco de Jaén” was the most susceptible, with average infestation levels ranging between 6.7% and 52.2% and between 10.3% and 69.2% under rainfed and irrigated conditions, respectively, and “Arbequina” was the least susceptible, with average infestation levels ranging between 0.6% and 12.7% and between 2.3% and 18.5% under rainfed and irrigated conditions, respectively. Among the table olive varieties, “Gordal Sevillana,” “Ascolana Tenera” and “Ocal” were the most susceptible (with average infestation levels reaching 39.7%, 36.5% and 33.3%, respectively), while “Callosina” was the least susceptible (with infestation levels of only 8.4%). Irrigation tended to promote both B. oleae infestation and its earlier occurrence compared to the rainfed condition. Even though the diameter and oil content were positively correlated with B. oleae fruit infestation (correlation coefficients ranged between 0.5 and 0.95), the present work reveals that other yet‐unknown factors may influence B. oleae oviposition preferences. The results of this study can be useful for breeding programmes to develop olive varieties resistant to B. oleae and provide key information for wide‐area olive fly pest management decisions. [ABSTRACT FROM AUTHOR]

Background and aims: Entomopathogenic mitosporic ascomycetes Beauveria, Metarhizium and Isaria sp. are commonly used for pest control but can also serve other, lesser known functions such as increasing nutrient bioavailability or promote plant growth. The objective of this work was to identify the doses of entomopathogenic fungi (EF) to be applied to soil in order to modify iron (Fe) uptake by plants and promote their growth. Methods: We used an in vitro assay to assess the ability of Beauveria bassiana, Metarhizium brunneum and Isaria farinosa to mobilize Fe from nine Fe oxides differing in composition, particle size and crystallinity, including ferrihydrite, hematite, goethite and magnetite. We also conducted an in vivo assay by applying five different doses (viz., 0, 5 × 10, 5 × 10, 5 × 10 and 5 × 10 conidia ml) of a conidial suspension of M. brunneum to the surface of a calcareous soil, which induced Fe chlorosis and a non-calcareous soil which did not induce chlorosis to explore the ability of the fungus on improving Fe nutrition and plant growth of sorghum and sunflower plants. Results: In the in vitro assay, all three EF increased Fe availability differently depending on particle size and crystallinity, and I. farinosa and B. bassiana increased the pH of the culture medium, whereas M. brunneum did not produce a great effect. In the in vivo assay, the highest dose (5 × 10 conidia ml) of M. brunneum alleviated Fe chlorosis symptoms of sorghum plants grown in the calcareous soil, and the two highest doses (5 × 10 and 5 × 10 conidia ml) increased plant height and inflorescence production of sunflower grown in both soils. Conclusions: The observed benefits of EF on plant growth and nutrition provide support for more sustainable and cost-effective use of these biocontrol agents. [ABSTRACT FROM AUTHOR]

Beauveria bassiana strain 04/01-Tip, obtained from a larva of the opium poppy stem gall wasp Iraella luteipes (Hymenoptera; Cynipidae), endophytically colonizes opium poppy (Papaver somniferum L.) plants and protects them against this pest. The goal of this study was to monitor the dynamics of endophytic colonization of opium poppy by B. bassiana after the fungus was applied to the seed and to ascertain whether the fungus is transmitted vertically via seeds. Using a species-specific nested PCR protocol and DNA extracted from surface-sterilised leaf pieces or seeds of B. bassiana-inoculated opium poppy plants, the fungus was detected within the plant beginning at the growth stage of rosette building and them throughout the entire plant growth cycle (about 120–140 days after sowing). The fungus was also detected in seeds from 50% of the capsules sampled. Seeds that showed positive amplification for B. bassiana were planted in sterile soil and the endophyte was again detected in more than 42% of the plants sampled during all plant growth stages. Beauveria bassiana was transmitted to seeds in 25% of the plants from the second generation that formed a mature capsule. These results demonstrate for the first time the vertical transmission of an entomopathogenic fungus from endophytically colonised maternal plants. This information is crucial to better understand the ecological role of entomopathogenic fungi as plant endophytes and may allow development of a sustainable and cost effective strategy for I. luteipes management in P. somniferum. [ABSTRACT FROM AUTHOR]

Abstract: The potential of a strain of Beauveria bassiana (Ascomycota: Clavicipitaceae) obtained from a naturally infected Rhynchophorus ferrugineus (Coleoptera: Curculionidae) pupa as a biological control agent against this weevil was evaluated both in the laboratory and in semi-field assays. Laboratory results indicate that this strain of B. bassiana can infect eggs, larvae and adults of R. ferrugineus (LC50 from 6.3×107 to 3.0×109 conidia per ml). However, mortality was not the only indicator of treatment efficacy because adults of either sex inoculated with the fungus efficiently transmitted the disease to untreated adults of the opposite sex, with male-to-female and female-to-male rates of transmission of 55% and 60%, respectively. In addition, treatment with B. bassiana significantly reduced fecundity (up to 62.6%) and egg hatching (32.8%) in pairing combinations with fungus-challenged males, females or both sexes. Likewise, 30–35% increase in larval mortality was observed in larvae obtained from eggs from fungus-challenged females or from untreated females coupled with inoculated males, resulting in an overall 78% progeny reduction. Semi-field preventive assays on potted 5-year old Phoenix canariensis palms, with efficacies up to 85.7%, confirmed the potential of this strain as a biological control agent against R. ferrugineus. [Copyright &y& Elsevier]

Abstract: Factors affecting the occurrence and distribution of entomopathogenic fungi in 244 soil samples collected from natural and cultivated areas in Spain were studied using an integrated approach based on univariate and multivariate analyses. Entomopathogenic fungi were isolated from 175 of the 244 (71.7%) soil samples, with only two species found, Beauveria bassiana and Metarhizium anisopliae. Of the 244 soil samples, 104 yielded B. bassiana (42.6%), 18 yielded M. anisopliae (7.3%), and 53 soil samples (21.7%) harboured both fungi. Log-linear models indicated no significant effect of habitat on the occurrence of B. bassiana, but a strong association between M. anisopliae and soils from cultivated habitats, particularly field crops. Also, irrespective of habitat type, B. bassiana predominated over M. anisopliae in soils with a higher clay content, higher pH, and lower organic matter content. Logistic regression analyses showed that pH and clay content were predictive variables for the occurrence of B. bassiana, whereas organic matter content was the predictive variable for M. anisopliae. Also, latitude and longitude predicted the occurrence of these same species, but in opposite directions. Altitude was found to be predictive for the occurrence of B. bassiana. Using principal component analysis, four factors (1 to 4) accounted for 86% of the total variance; 32.8, 22.9, 19.6 and 10.4% of the cumulative variance explained, respectively. Factor 1 was associated with high positive weights for soil clay and silt content and high negative weights for soil sand content. Factor 2 was associated with high positive weights for soil organic matter content and high negative weights for soil pH. Factor 3 was associated with high positive weights for latitude and longitude of the sampled localities and factor 4, had high positive weights only for the altitude. Bi-plot displays representing soil samples were developed for different factor combinations and indicated that, irrespective of geographical location, absence of both fungal species was determined by alkaline sandy soils with low organic matter content, whereas heaviness of soil texture, acidity and increasing organic matter content led to progressively higher percentages of samples harbouring entomopathogenic fungi. These results could aid decision-making as to whether or not a particular cultivated or natural soil is suitable for using entomopathogenic fungi as a pest control measure and for selecting the fungal species best suited to a particular soil. [Copyright &y& Elsevier]

Fruit fly ground-dwelling stages (late third instar larvae, pupae, and teneral adults) are susceptible to predation from generalist ground-dwelling predators and to infection by entomopathogenic fungi (EPF). The effect of predators can be enhanced with cover crops and that of EPF by augmentative releases. However, whether these two biological control methods could be combined has not been studied under field conditions yet. Here, we studied in the field whether the enhanced activity of predators against the medfly, Ceratitis capitata, already observed in a Lolium arundinaceum ground cover could be impaired by a soil application of Metarhizium brunneum. Our results show that C. capitata adult emergence was reduced by this EPF for up to three months after fungal application, with the combination of the cover and M. brunneum being the most effective at reducing C. capitata emergence relative to bare soil (92.5% reduction). Although M. brunneum reduced the activity density of ground-dwelling predatory beetles up to 93 days after application, it showed no clear negative effects on earwigs, no effects on spiders, and a positive effect on ants up to 65 days after application. Therefore, the combined use of a ground cover of L. arundinaceum and M. brunneum against the soil-dwelling stages of C. capitata seems to work synergistically and appears as a strong and sustainable control tactic against the medfly and other fruit orchard pests. [ABSTRACT FROM AUTHOR]

Microsclerotia (MS) produced by some species of Metarhizium can be used as active ingredients in mycoinsecticides for the control of soil-dwelling stages of geophilic pests. In this study, the MS production potential of two Metarhizium brunneum strains and one M. robertsii strain was evaluated. The three strains were able to produce MS in liquid fermentation, yielding between 4.0 × 106 (M. robertsii EAMa 01/158-Su strain) and 1.0 × 107 (M. brunneum EAMa 01/58-Su strain) infective propagules (CFU) per gram of MS. The EAMa 01/58-Su strain was selected for further investigation into the effects of key abiotic factors on their survival and conidial yield. The MS were demonstrated to be stable at different storage temperatures (−80, −18, and 4 °C), with a shelf-life up to one year. The best temperature for MS storage was −80 °C, ensuring good viability of MS for up to one year (4.9 × 1010 CFU/g MS). Moreover, soil texture significantly affected CFU production by MS; sandy soils were the best driver of infective propagule production. Finally, the best combination of soil temperature and humidity for MS germination was 22.7 °C and 7.3% (wt./wt.), with no significant effect of UV-B exposure time on MS viability. These results provide key insights into the handling and storage of MS, and for decision making on MS dosage and timing of application. [ABSTRACT FROM AUTHOR]

The present study aimed to compare some commercial traps baited with ammonium carbonate for their efficacy in monitoring Bactrocera oleae (Rossi) and their selectivity toward beneficial insects, under the conditions of olive groves located in five Mediterranean countries (Greece, Italy, Lebanon, Spain, Tunisia). The selectivity of the devices was evaluated toward several groups of non-target insects, namely lacewings (Chrysopidae), hoverflies (Syrphidae), ladybirds (Coccinellidae), bees (Apoidea) and hymenopteran parasitoids. The following devices were compared: yellow sticky panel, green sticky panel, Jackson trap with different combinations of yellow and white colours of the device and/or the panel, and McPhail trap. In most cases, the McPhail trap and the yellow panel showed the highest efficacy in monitoring male and female B. oleae flies; however, the yellow panel was most attractive for some groups of non-target insects, particularly the hymenopteran parasitoids, whereas the comparative non-target effects were sitespecific for the other arthropod groups. In the case of Chrysopidae, McPhail caught more individuals than the other traps in Italy and Spain, whereas in Lebanon and Greece, the highest number of individuals was captured in the two sticky panels. Coccinellidae were found in very low numbers only in Lebanon on yellow panels. Syrphidae were captured mainly on sticky panels in Greece and Tunisia. Apoidea were found only on yellow panels in Lebanon. The choice between the McPhail trap and the yellow panel should be made on the basis of various evaluations carried out at the site, including operational ones. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi (EF) have so far aroused little interest as plant growth promoters or nutritional enhancers while protecting the plant against insect pests and mites. In this work, we examined the performance of Triticum durum and Triticum aestivum plants pot‐grown on two unsterilized Zn‐poor calcareous Vertisols and inoculated with Beauveria bassiana or Metarhizium brunneum by application to soil or seed dressing. The effects of fungal inoculation were found to depend on crop species, soil type, sampling time and fungus application method. While the height of the control plants tended to be significantly greater than the height of inoculated plants at early and intermediate growth stages, the trend changed at more advanced plant stage of development, 56 days after sowing (DAS). This was supported by the significant increase in aerial dry matter (ADM) in T. aestivum plants treated with B. bassiana by seed dressing (17.4%) or with M. brunneum with soil application and seed dressing (20.6 and 26.9%, respectively) 69 DAS. In T. durum, at harvest, seed dressing with B. bassiana or M. brunneum had an overall positive but not significant effect on ADM or grain yield, whereas direct application to the soil slightly decreased grain yield (6.6 and 5.5%, respectively) and ADM (5.3 and 6.1%, respectively). In T. aestivum, none of the fungus–application method combinations affected yield at harvest significantly. Fungal inoculation had little influence on nutrient uptake, whereas application to soil increased Mn uptake and grain Mn concentration in T. durum. Zinc uptake was significantly increased only in T. aestivum grown on the soil with the lowest Zn content treated with M. brunneum. Grain Zn concentration tended to be inversely related to grain yield. These results support seed dressing with EF as a promising green technology for sustainable crop protection and production with no adverse effects on plant performance. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi have a high potential as microbial control agents of insect pests, both through direct application and through endophytic colonisation and the subsequent effects on the phytophagous insects. Recently, such symbiotic relationships with their host plants and compatibility with other natural enemies of insect pests have been characterized, including the modification of plant volatile emissions and the consequent effect on the insects. The present research aimed at investigating whether systemic Beauveria bassiana (Balsamo) Vuillemin colonisation of melon plants reached the floral tissues and affected flowering, pollen production and pollinator behaviour. Melon plants sprayed with B. bassiana conidial suspensions were endophytically colonised throughout the entire plant growth cycle and reached the floral tissues. In addition, the endophytically-colonized plants produced a significantly higher number of pollen grains, and flowered earlier than control plants. A foraging preference of honeybees and bumblebees for the control plants in choice assays was detected, likely due to the advanced maturation stage of colonised plants flowers. Noteworthy, changes in the emitted blend of volatiles were observed in the endophytically-colonised plants, which emitted more of the pollinator attractants D-limonene and benzeneacetaldehyde than control plants. Future research should consider the mechanisms for endophytemediated early flowering and variations in volatile profiles throughout the blooming process, together with their impact on pollination under greenhouse conditions. [ABSTRACT FROM AUTHOR]

The genus Metarhizium has an increasingly important role in the development of Integrated Pest Control against Tephritid fruit flies in aerial sprays targeting adults and soil treatments targeting preimaginals. Indeed, the soil is considered the main habitat and reservoir of Metarhizium spp., which may be a plant-beneficial microorganism due to its lifestyle as an endophyte and/or rhizosphere-competent fungus. This key role of Metarhizium spp. for eco-sustainable agriculture highlights the priority of developing proper monitoring tools not only to follow the presence of the fungus in the soil and to correlate it with its performance against Tephritid preimaginals but also for risk assessment studies for patenting and registering biocontrol strains. The present study aimed at understanding the population dynamics of M. brunneum strain EAMb 09/01-Su, which is a candidate strain for olive fruit fly Bactrocera oleae (Rossi, 1790) preimaginal control in the soil, when applied to the soil at the field using different formulations and propagules. For this, strain-specific DNA markers were developed and used to track the levels of EAMb 09/01-Su in the soil of 4 field trials. The fungus persists over 250 days in the soil, and the levels of the fungus remained higher when applied as an oil-dispersion formulation than when applied as a wettable powder or encapsulated microsclerotia. Peak concentrations of EAMb 09/01-Su depend on the exogenous input and weakly on environmental conditions. These results will help us to optimize the application patterns and perform accurate risk assessments during further development of this and other entomopathogenic fungus-based bioinsecticides. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi (EF) provide a potent biocontrol tool; also, their endophytic behavior has broadened their contribution to integrated pest management (IPM) and crop production. In this work, Beauveria bassiana and Metarhizium brunneum were applied to bread wheat (Triticum aestivum) seedlings to elucidate how fungal colonization influences plant growth and the relative expression of 24 genes involved in hormonal syntheses and plant immune mechanisms. A preliminary assay was used to determine the time needed for fungal colonization and assess its effect on wheat growth. Then, plant material collected at various times after inoculation (viz., 2, 8, 20, and 36 h and 9 and 15 days) was used to investigate gene expression by quantitative reverse transcription PCR (RT-qPCR). During the colonization time, B. bassiana and M. brunneum caused strong downregulation of most genes associated with plant immunity and the synthesis of hormones like auxin, cytokinin, and gibberellin. This effect was concomitant with a slowdown of endophytic-colonization-related plant growth until 19 days postinoculation (dpi). However, the wheat started to recover at 15 dpi, simultaneously with upregulation of auxin- and gibberellin-related genes. The results suggest that the EF trigger induced systemic resistance rather than acquired systemic resistance during early plant-microbe cross talk in wheat. Also, they confirm that the hormone and immune responses of wheat triggered by EF inoculation influenced plant growth, which can be useful with a view to optimizing management of these microorganisms for sustainable agriculture. [ABSTRACT FROM AUTHOR]

The influence of treatment on the number of larvae that produced a cocoon was analyzed with an ANCOVA, where the factors were the age of the larvae treated and the treatment, and the covariate was the number of larvae that survived the treatment. The effect of the I B. bassiana i treatment was detectable from the third day on the L2-L3 larvae (Figure 2A), from the fourth day on the L4-L5 larvae (Figure 2B) and from the fifth day on the L6 larvae (Figure 2C). The strain of I B. bassiana i obtained from the larvae collected in the forest was sequenced and aligned with the I B. bassiana i species and has shown a different virulence against I C. perspectalis i larvae depending on the age of the larvae. Several larvae from the bioassays with L2-L3 and L4-L5 larvae prepared a cocoon (Table 5), remaining inside it as diapausing larvae, ceasing feeding and development. [Extracted from the article]

Entomopathogenic fungi are known due to their ability to kill insects and besides they can play other roles, such as promoting plant growth or improving nutrient uptake. The main aim of this work was to assess the potential of Beauveria bassiana strain EABb 04/01-Tip for improving iron (Fe) nutrition in plants grown on calcareous substrates. Plants of a dicot (tomato) and a monocot (wheat) were pot-grown on an artificial substrate consisting of 0–240 g kg −1 Fe oxide-coated sand (FOCS), 250 g kg −1 calcium carbonate sand and quartz sand. The plants were subjected to two different treatments, namely seed dressing, which involved inoculating seeds with a conidial suspension of B. bassiana before sowing, and a control without inoculation. Leaf chlorophyll concentrations (SPAD) and Fe uptake by plants were correlated with the FOCS content of the substrate, therefore the experimental design was appropriate to see different levels of Fe chlorosis symptoms. B. bassiana was able to colonise both tomato and wheat plants without a negative effect on plant height, plant dry weight, root development in tomato or grain production in wheat. In addition, B. bassiana alleviated Fe chlorosis symptoms (described for first time) in both crops during early growing stages (<50 days after sowing) but the intensity of the effect depended on the plant species and available Fe on substrate. Nutritional alterations in K due to fungal application were detected in tomato and wheat plants. [ABSTRACT FROM AUTHOR]

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived fromMetarhizium anisopliaestrain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC50values differed by up to ~10-fold (5.3×106−4.5×105conidia/ml) and LT50values 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 vitroproduction and intrahemoceol injection usingG. mellonellaas 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 bothin vitroand 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 thatin vitroscreening for insect toxins can mimicin vivo(i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology. [ABSTRACT FROM AUTHOR]

Highlights: [•] The tillage effect on entomopathogenic fungi (EF) associated to Prays oleae was evaluated. [•] No significant differences were found between the different soil management practices. [•] Seasonal variation on abundance and diversity of EF was detected. [•] Beauveria bassiana was the most abundant entomopathogenic fungi in both soil types. [•] Results provide new insights to the dynamics of EF in the olive grove ecosystem [Copyright &y& Elsevier]

Viruses have been discovered in numerous fungal species, but unlike most known animal or plant viruses, they are rarely associated with deleterious effects on their hosts. The knowledge about viruses among entomopathogenic fungi is very limited, although their existence is suspected because of the presence of virus-like double-stranded RNA (dsRNA) in isolates of several spedes. Beauveria bassiana is one of the most-studied species of entomopathogenic fungi; it has a cosmopolitan distribution and is used as a biological control agent against invertebrates in agriculture. We analyzed a collection of 73 isolates obtained at different locations and from different habitats in Spain and Portugal, searching for dsRNA elements indicative of viral infections. The results revealed that the prevalence of viral infections is high; 54.8% of the isolates contained dsRNA elements with viral characteristics. The dsRNA electropherotypes of infected isolates indicated that virus diversity was high in the collection analyzed and that mixed virus infections occurred in fungal isolates. However, a hybridization experiment indicated that dsRNA bands that are similar in size do not always have similar sequences. Particular virus species or dsRNA profiles were not associated with locations or types of habitats, probably because of the ubiquity and efficient dispersion of this fungus as an airborne species. The sequence of one of the most common dsRNA elements corresponded to the 5.2-kbp genome of a previously undescribed member of the Totiviridae family, termed B. bassiana RNA virus 1 (BbRV1). [ABSTRACT FROM AUTHOR]

This study delves into the compatible use of a parasitoid with multifunctional endophytic Entomopathogenic Ascomycetes (EA) in IPM under greenhouse conditions. The parasitoid Hyposoter didymator was evaluated against Spodoptera littoralis in a multitrophic system with melon plants that were endophytically colonized by one of three EA strains (Metarhizium brunneum [one] or Beauveria bassiana [two]). In the first scenario, plants were inoculated by three different methods, and after infestation with noctuid larvae, the parasitoid was released at a 1:20 ratio. Microbiological and molecular techniques allowed the identification of progressive colonization throughout the whole plant life cycle, and for B. bassiana, approximately 20% of seeds from new fruits were colonized. The parasitoid was shown to be compatible with all strains and application methods, with total mortality rates ranging from 11.1 to 77.8%. Significant lethal and sublethal effects, a decrease in pupal weight and mortality of pupae showing abnormalities and an extension of the immature developmental times were observed for different strain–application method combinations. Additionally, the fungal treatments improved crop growth, as revealed by the significant gains in plant weight. In a second scenario (by inoculating plants with the fungi only by leaf spraying), and after infestation with noctuid larvae, the parasitoid was released at a 1:10 ratio, which revealed the remote fungal effect from the inoculation point and confirmed the compatibility of the parasitoid-EA-based strategy. These findings underscore the compatible use of a parasitoid with endophytic EA for S. littoralis control that can additionally exploit their multifunctionality for sustainable crop production. [ABSTRACT FROM AUTHOR]

• Direct exposure of Philaenus to Metarhizium brunneum reduces its survivorship. • M. brunneum successfully colonises the host plant and reduces survival of Philaenus. • Philaenus reaches the xylem faster on M. brunneum -colonised plants. • Xylem sap ingestion by Philaenus is similar on non-colonised and colonised plants. • Contact with M. brunneum does not alter the egestion pattern of Philaenus. The most effective way to contain spread of the plant pathogen Xylella fastidiosa in Europe is to manage populations of its main insect vector, Philaenus spumarius L. (Hemiptera: Aphrophoridae). The action of some entomopathogenic endophytic fungi (EEF) on insect pests, directly through contact or indirectly by internal colonization of host plant tissues, may offer an excellent control option within integrated pest management (IPM) programmes. This study evaluated the impact of two EEF strains as control agents of P. spumarius (Metarhizium brunneum strain EAMa 01/58-Su and Beauveria bassiana strain EABb 01/33-Su [Hypocreales: Clavicipitaceae and Cordycipitaceae, respectively]); both strains were known to be excellent control agents of many other serious chewing and sap-sucking agricultural pests. First, the pathogenicity of strains applied directly to P. spumarius adults was evaluated, resulting in 50.0% and 32.0% mortality and a mean survival time of 4.7 and 6.2 days for M. brunneum and B. bassiana , respectively. Furthermore, colonization of Sonchus oleraceus by EEFs was observed after both soil and foliar applications. Greatest colonization (91.0%) was observed in plants treated with foliar applications of M. brunneum. Based on these results, we evaluated the feeding behaviour of P. spumarius on S. oleraceus plants previously colonized by M. brunneum using the 'Electrical Penetration Graph' technique. P. spumarius fed in a similar way on EEF-colonized and non-colonized S. oleraceus plants, with the exception that insects feeding on colonized plants reached xylem vessels faster than insects feeding on non-colonized plants. In addition, significantly higher mortality of P. spumarius was observed when feeding on plants treated by M. brunneum compared with the control. The duration of xylem ingestion events indicates that P. spumarius preferred to feed on basal leaves of the plant compared with apical leaves. Our results indicate strong potential for strain EAMa 01/58-Su of M. brunneum to be used as part of an X. fastidiosa IPM programme. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • M. acridum IMI 330189 was more virulent than B. bassiana EABb 90/2-Dm at low dosages. • Most treatments reduced per capita feeding (32–51%) during the interval of no thermoregulation. • Only IMI 330189 reduced per capita feeding (50%) when insects were allowed to thermoregulate. • Insect mortality suggested additive interaction in the lethal effects between the two strains. • For sublethal effects (feeding and fecundity) a potential antagonistic interaction was detected. Abstract The effects of single and mixed infections with Beauveria bassiana (EABb 90/2-Dm) and Metarhizium acridum (IMI 330189) strains on survival, feeding and reproduction of thermoregulating Dociostaurus maroccanus were evaluated. Adult locusts (2–3 days post fledging) were treated with low dosages of both fungal pathogens alone and in mixture (total dosage for single treatments and combinations = 1 × 102 and 1 × 103 spores per insect). M. acridum IMI 330189 was more virulent than B. bassiana EABb 90/2-Dm at both dosages. In the mixed infections, in which half of the infective units of the more virulent pathogen were replaced by the less virulent pathogen, the analysis of the cumulative insect mortality after 30 days suggested additive interaction in the lethal effects between the two strains. All fungal treatments, except EABb 90/2-Dm at 1 × 102 spores per insect showed reduction in per capita feeding, as indicated by fecal production per insect per day when insects were maintained at 27 ± 2 °C (32–51% of reduction compared with the control); but only IMI 330189 caused significant reduction in per capita feeding (50%) when those insects were allowed to thermoregulate. Both strains and their mixtures caused a significant reduction of locust fecundity, with a 21–53% reduction in the number of egg-pods per female, and 30–65% reduction in the number of fertile eggs per female. In both sublethal effects (feeding and fecundity) a potential antagonistic interaction between the fungal strains was detected. Locust fecundity (egg-pods per female) and per capita feeding were positively correlated (r = 0.783). Implications of these findings on the potential use of both strains to control D. maroccanus populations are discussed. [ABSTRACT FROM AUTHOR]

Studies conducted over the last decades have shown the potential of entomopathogenic fungi for the biocontrol of some insect pests. Entomopathogenic fungi infect their host through the cuticle, so they do not need to be ingested to be effective. These fungi also secrete secondary metabolites and proteins that are toxic to insect pests. In this context, we analyzed the pathogenicity of Metarhizium anisopliae (Metschn.) strains IBCB 384 and IBCB 425 and Beauveria bassiana (Bals.-Criv.) Vuill. strains E 1764 and E 3158 against Galleria mellonella (Linn.) larvae, during pre-invasion and post-invasion phases. The results showed M. anisopliae, especially strain IBCB 384, was most virulent in the pre-invasion phase against G. mellonella, whereas B. bassiana, especially strain E 1764, was most virulent in the post-invasion phase. During in vivo development and in the production of toxic serum, B. bassiana E 3158 was the most virulent. Different fungal growth (or toxin) strategies were observed for studied strains. Metarhizium anisopliae IBCB 425 prioritizes the growth strategy, whereas strain IBCB 384 and B. bassiana strains E 1764 and E 3158 have a toxic strategy. All strains have pathogenicity against G. mellonella, indicating their possible use for biocontrol. [ABSTRACT FROM AUTHOR]

Although entomopathogenic fungi (EPF) are best known for their ability to protect crops against insect pests, they may have other beneficial effects on their host plants. These effects, which include promoting plant growth and conferring resistance against abiotic stresses, have been examined in recent years to acquire a better understanding of them. The primary purposes of the present study were (i) to ascertain in vitro whether three different strains of EPF (viz., Metarhizium, Beauveria and Isaria) would increase the Fe bioavailability in calcareous or non-calcareous media containing various Fe sources (ferrihydrite, hematite and goethite) and (ii) to assess the influence of the EPF inoculation method (seed dressing, soil treatment or leaf spraying) on the extent of the endophytic colonization of sorghum and the improvement in the Fe nutrition of pot-grown sorghum plants on an artificial calcareous substrate. All the EPFs studied were found to increase the Fe availability during the in vitro assay. The most efficient EPF was M. brunneum EAMa 01/58–Su, which lowered the pH of the calcareous medium, suggesting that it used a different strategy (organic acid release) than the other two fungi that raised the pH of the non-calcareous medium. The three methods used to inoculate sorghum plants with B. bassiana and M. brunneum in the pot experiment led to differences in re-isolation from plant tissues and in the plant height. These three inoculation methods increased the leaf chlorophyll content of young leaves when the Fe deficiency symptoms were most apparent in the control plants (without fungal inoculation) as well as the Fe content of the above-ground biomass in the plants at the end of the experiment. The total root lengths and fine roots were also increased in response to fungal applications with the three inoculation methods. However, the soil treatment was the most efficient method; thus, its effect on the leaf chlorophyll content was the most persistent, and the effects on the total root length and fine roots were the most apparent. In conclusion, EPF improved the Fe nutrition of the sorghum plants, but their effects depended on the inoculation method. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated the presence of Metarhizium species on the epigeal areas of weeds and woody plants in various Mediterranean ecosystems, and the question arises whether isolates from the phylloplane, which experiences greater exposure to environmental UV-B radiation than soil isolates do, could have better UV-B radiation tolerance. The in vitro response of 18 Metarhizium strains isolated from phylloplane and soil of several Mediterranean ecosystems to UV-B radiation and the in vitro and in vivo effects of UV-B radiation on the viability and virulence of a selected M. brunneum strain against C. capitata were determined. The conidial germination, culturability and colony growth of these strains exposed to 1200 mW m −2 for 2, 4 or 6 h were evaluated. Germination rates below 30% and poor conidia recovery rates were observed for all strains. However, no relationship between the Metarhizium species or isolation habitat and the effect of UV-B radiation was found. Strain EAMa 01/58-Su, which showed a high tolerance to UV-B inactivation in terms of relative germination, was subsequently selected to investigate the UV-B related effects on virulence toward C. capitata adults. In a series of bioassays, the virulence and viability was determined using pure dry conidia, which were irradiated with 1200 mW m −2 for 6 h prior or after adult flies were inoculated, which resulted in a significant 84.7–86.4% decrease in conidial viability but only a slightly significant reduction of virulence, with 100.0% and 91.4% adult mortality rates and 4.6 and 5.9 days average survival time for the no UV-B and UV-B treatments, respectively. A second series of experiments was performed to determine whether the UV-B effects on strain EAMa 01/58-Su were dose- or exposure time-dependent. Adult flies were inoculated with five doses (1.0 × 10 4 –1.0 × 10 8 conidia ml −1 ) and then irradiated at 1200 mW m −2 for 6 h, and similar LC 50 values, 3.8 × 10 7 and 4.3 × 10 7 conidia ml −1 , were determined for the UV-B and no UV-B treatments, respectively. However, the LT 50 values for flies inoculated with 1.0 × 10 8 conidia ml −1 and with1.0 × 10 7 conidia ml −1 were 15.1% and 30.8% longer for UV-B treatments than no UV-B treatments, respectively. Next, adult flies were treated with 1.0 × 10 8 conidia ml −1 and then exposed to 1200 mW m −2 for 0, 6, 12, 24, 36 and 48 h, and the relationships among exposure time and conidia viability and fly mortality losses were determined. The exposure time for adult flies at 1200 mW m −2 to achieve a 50% reduction in fly mortality was 47.2 h, which was longer than that of 5.6 h required for a 50% reduction in conidia viability. Our results show that the UV-B radiation significantly affected the virulence of EAMa 01/58-Su strain against C. capitata adults, with this effect being dependent on the exposure time but not related to fungal dosage. [ABSTRACT FROM AUTHOR]

An analytical strategy based on a hybrid quadrupole-Orbitrap mass spectrometry was proposed for the simultaneous screening of known destruxins and characterization of potential members of this class of secondary metabolites, in order to evaluate the metabolite production of entomopathogenic fungi used as biocontrol agents. Initially, the fragmentation pathway of the known and commercially available destruxin A was established combining high resolution mass spectrometry (HRMS) and multiple stage MS data in order to obtain the strategy for the characterization of other destruxins for which reference standards were not available. Nineteen known destruxins including A, B, C, D, Ed, F, A, B, Ed, A, B, D, A, DesmA, DesmB, DesmC, DesmB, and two chloro-derivatives (Cl and E chlorohydrin) were unequivocally identified in Metarhizium brunneum using the proposed strategy. In addition, four unknown destruxins, namely C, Ed, G, and G, were structurally elucidated and characterized for the first time in this fungal strain. [ABSTRACT FROM AUTHOR]

The response of entomopathogenic mitosporic ascomycete (EMAs) to abiotic stresses might be adapted to the microhabitats in which they inhabit. In phylloplane, these organisms are more exposed to such stresses than they are in soil, which may have led to adaptation to this environment. In the present work, we investigate whether Beauveria bassiana genotype or isolation habitat, i.e., soil or phylloplane, within the same geographic area influences their responses to key environmental stresses, such as temperature, moisture and ultraviolet radiation (UV-B), which can affect their successful use in microbial control. Twenty isolates of B. bassiana obtained from the soil and phylloplane in two ecosystems from southern Spain (holm oak dehesa and a reforested area) were selected to study the population distribution of these isolates and evaluate their thermal, humidity and UV-B requirements. Molecular characterization was conducted by using elongation factor-1α (EF-1α), the intergenic nuclear region Bloc and 15 microsatellite primers. The cluster analysis based on concatenated EF-1α and Bloc sequences grouped the 20 isolates into five clades within B. basiana , with Clades a, b, d and e containing both soil and phylloplane isolates and Clade c including three phylloplane isolates. The dendrogram and the minimal spanning network generated from the genetic distances among multilocus genotypes showed four divergent groups corresponding to the five clades obtained based on the sequence data (Clades b and d were represented in the same group), with a high degree of shared alleles within groups and few alleles shared among groups. Although no relationship was found between MLG and the habitat (soil or phylloplane) of isolation, isolates grouped into Clade c, all of which were collected from phylloplane, formed a separate group of MLGs. To investigate our hypothesis, the responses to temperature (germination and colony growth evaluated in the range 15–35 °C), water activity (conidia germination evaluated against values of a w between 1 and 0.862) and UV-B exposure (conidia exposed to 920 or 1200 mW m −2 for 2, 4 or 6 h) of the soil and phylloplane isolates from the five clades were investigated. No associations of isolate-specific genetic or physiological characteristics with isolate habitat, i.e., soil or phylloplane, were found. These results provide no support for the hypothesis that EMAs strains from the phylloplane have evolved to resist unfavourable environmental conditions. [ABSTRACT FROM AUTHOR]

Background and aims: Entomopathogenic fungi are known for their insecticidal properties but possess other, lesser known attributes as plant growth promoters and nutrient bioavailability improvers. In this work, their effect on iron availability and ability to alleviate iron chlorosis symptoms were examined. Methods: Three strains of common entomopathogenic fungi ( Beauveria bassiana, Isaria farinosa and Metarhizium brunneum) were studied in an in an in vitro experiment involving three iron oxides of different nature. Then, lupin, sorghum, wheat and sunflower pot-grown on three calcareous soils with a low iron availability and one non-calcareous sandy soil with a high iron availability, were studied. Metarhizium brunneum was applied to the soil surface to compare with untreated soils. Results: Metarhizium brunneum EAMb 01/58-Su was the most efficient strain in dissolving iron from iron oxides in the in vitro experiment; also, it increased leaf chlorophyll content in sorghum and wheat, plant height in wheat, and flower production in sunflower grown on two of the three calcareous soils. However, the fungus decreased dry weight in wheat and sunflower production in plants grown on the sandy soil. No effect on lupin plants -the most sensitive to iron chlorosis- was observed. Conclusions: M. brunneum alleviated iron chlorosis symptoms in sorghum, wheat and sunflower grown on calcareous soils but had a negative effect on two of the four plant species grown on the sandy soil. These encouraging results with calcareous soils suggest giving priority to this soil dwelling pest control strategy in such conditions to take advantage of this new added value. [ABSTRACT FROM AUTHOR]

The diversity of entomopathogenic Hypocreales from the soil and phylloplanes in five Mediterranean cropping systems with different degrees of management [organic olive orchard conventional olive orchard, holm oak reforestation, holm oak dehesa (a multifunctional agro-sylvo-pastoral system), and sunflower plantation] was studied during four seasons. A total of 697 entomopathogenic fungal isolates were obtained from 272 soil samples, 1608 crop phylloplane samples and 1368 weed phylloplane samples. The following nine species were identified: Beauveria amorpha, B. bassiana, B. pseudobassiana, B. varroae, Metarhizium brunneum, M. guizhoense, M. robertsii, Paecilomyces marquandii and lilacinum using EF-1α gene sequences. All the fungal entomopathogenic species were found in both the soil and phylloplane samples, with the exception of M. robertsii , which was only isolated from the soil. The species richness, diversity (Shannon–Wiener index) and evenness (Pielou index) were calculated for each cropping system, yielding the following species ranking, which was correlated with the crop management intensity: holm oak reforestation > organic olive orchard > conventional olive orchard > holm oak dehesa > sunflower plantation. The number of fungal species isolated was similar in both phylloplane habitats and dissimilar between the soil and the crop phylloplane habitats. The ISSR analysis revealed high genotypic diversity among the B. bassiana isolates on the neighbourhood scale, and the isolates were clustered according to the habitat. These results suggest that the entomopathogenic Hypocreales in the phylloplane could result from the dispersal of fungal propagules from the soil, which might be their habitat of origin; a few isolates, including EABb 09/28-Fil of Beauveria bassiana , inhabit only the phylloplane. [ABSTRACT FROM AUTHOR]

Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host. [ABSTRACT FROM AUTHOR]

Abstract: Fusarium oxysporum, the causal agent of vascular wilt disease, affects a wide range of plant species and can produce disseminated infections in humans. F. oxysporum f. sp. lycopersici isolate FGSC 9935 causes disease both on tomato plants and immunodepressed mice, making it an ideal model for the comparative analysis of fungal virulence on plant and animal hosts. Here we tested the ability of FGSC 9935 to cause disease in the greater wax moth Galleria mellonella, an invertebrate model host that is widely used for the study of microbial human pathogens. Injection of living but not of heat-killed microconidia into the hemocoel of G. mellonella larvae resulted in dose-dependent killing both at 30°C and at 37°C. Fluorescence microscopy of larvae inoculated with a F. oxysporum transformant expressing GFP revealed hyphal proliferation within the hemocoel, interaction with G. mellonella hemocytes, and colonization of the killed insects by the fungus. Fungal gene knockout mutants previously tested in the tomato and immunodepressed mouse systems displayed a good correlation in virulence between the Galleria and the mouse model. Thus, Galleria represents a useful non-vertebrate infection model for studying virulence mechanisms of F. oxysporum on animal hosts. [Copyright &y& Elsevier]

Background: The entomopathogenic anamorphic fungus Beauveria bassiana is currently used as a biocontrol agent (BCA) of insects. Fifty-seven Beauveria bassiana isolates -53 from Spain- were characterized, integrating group I intron insertion patterns at the 3'-end of the nuclear large subunit ribosomal gene (LSU rDNA) and elongation factor 1-alpha (EF1-α) phylogenetic information, in order to assess the genetic structure and diversity of this Spanish collection of B. bassiana. Results: Group I intron genotype analysis was based on the four highly conserved insertion sites of the LSU (Ec2653, Ec2449, Ec2066, Ec1921). Of the 16 possible combinations/genotypes, only four were detected, two of which were predominant, containing 44 and 9 members out of 57 isolates, respectively. Interestingly, the members of the latter two genotypes showed unique differences in their growth temperatures. In follow, EF1-a phylogeny served to classify most of the strains in the B. bassiana s.s. (sensu stricto) group and separate them into 5 molecular subgroups, all of which contained a group I intron belonging to the IC1 subtype at the Ec1921 position. A number of parameters such as thermal growth or origin (host, geographic location and climatic conditions) were also examined but in general no association could be found. Conclusion: Most Spanish B. bassiana isolates (77.2%) are grouped into a major phylogenetic subgroup with wordwide distribution. However, high phylogenetic diversity was also detected among Spanish isolates from close geographic zones with low climatic variation. In general, no correlation was observed between the molecular distribution and geographic origin or climatic characteristics where the Spanish B. bassiana isolates were sampled. [ABSTRACT FROM AUTHOR]

[Display omitted] • B. bassiana endophytically colonized melon, tomato and cotton plants. • Cotton was the the least suitable host plant for endophytic colonization. • B. bassiana re-isolation rate from cotton plants increased after the first passage. • This is the first report an of improvement of endophytic capacity by passaging. • Virulence stability after successive passage through a host plant was demonstrated. Systemic crop protection using endophytic isolates of entomopathogenic fungi is at the forefront of IPM. Nonetheless, any potential trade-offs between virulence and endophytic behaviour must be elucidated if they are to be effectively used in pest management strategies. Here we investigated endophytic adaptation in an isolate of Beauveria bassiana following successive passage through melon, tomato and cotton tissues. Plants were sprayed with a suspension of B. bassiana endophytic isolate EABb 04/01-Tip to initiate endophytic colonization. Once colonization was established, the fungus was re-isolated from the plant, applied to another plant and re-isolated again; this was repeated to achieve three passages. After each passage, a conidial suspension of each isolate was used in bioassays to evaluate both virulence against 4th instar larvae of the model insect Galleria mellonella and to quantify the extent of endophytic activity in each respective host plant species. When sprayed leaves were inspected for fungal colonization, differences in percentage tissue colonization amongst the plant species were detected after the first re-isolation. Endophytic colonization rates in melon and tomato, which varied from 70 to 100%, were higher than those observed in cotton, which ranged from 40 to 50%; endophytic colonization in cotton increased to 75–100% after the third passage. This improvement in endophytic behaviour in cotton, an apparently suboptimal plant for fungal colonization, suggests an evolutionary adaptation to localized or transient endophytic colonization, while further assays are needed. Meanwhile, when endophytic colonization of non-sprayed leaves distant from the sprayed ones was investigated, endophytic activity was evident in all three crop species suggesting that movement within plants after successive passage increased the extent of endophytic colonization from transient to systemic. The present research highlights the potential for adaptation to endophytic behaviour in crops that are less suitable for endophytic colonization. Furthermore, we demonstrated stability in virulence after multiple passages through host plants. This is a key result for the development of IPM strategies based on endophytic entomopathogenic fungi. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work has been to evaluate in the laboratory the potential of entomopathogenic fungi against adults and larvae of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) through fiber band application and a potted plant bioassay with soil application, respectively. Our previous findings revealed that Metarhizium anisopliae EAMa 01/58-Su isolate was the most virulent against neonate larvae of the buprestid. In the present work, M. anisopliae EAMa 01/58-Su isolate has been also shown to be highly virulent against adult beetles by immersion in a conidial suspension; thus it was selected to accomplish our objectives. When adult beetles were stimulated to climb 100×200mm non-woven commercial fiber bands impregnated with conidia of M. anisopliae EAMa 01/58-Su isolate, total mortality rates varied from 85.7% to 100.0%; whereas no significant correlation was detected between the time needed to cross the band (mean value 648.7±22.4s) and the time of death, with mean average survival time ranging between 10.3 and 16.0 days, compared to 28 days of the controls. Potted seedlings (5–6 months old) of cherry plum (Prunus myrobalana Lois.), a commonly used apricot rootstock, were used to study the efficacy of soil treatment with M. anisopliae EAMa 01/58-Su isolate against neonate C. tenebrionis larvae. The soil inoculation with M. anisopliae EAMa 01/58-Su isolate had a significant effect on the mean number of dead larvae recovered from the roots, with mean mortality ranging from 83.3% to 91.6%; whereas no significant differences were detected between the three fungal doses. In all cases, dead larvae found within roots exhibited external signs of fungal growth. Hence, it may be possible to use M. anisopliae EAMa 01/58-Su isolate in a biocontrol strategy targeting both adults and larvae of C. tenebrionis. [Copyright &y& Elsevier]

Abstract: We used a newly developed bioassay method to demonstrate for the first time the potential of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to be used for the control of neonate larvae of Capnodis tenebrionis, a major threat to stone-fruit orchards in several countries. Four B. bassiana and four M. anisopliae isolates were all pathogenic for neonate larvae of C. tenebrionis; mortality rates 10 days after inoculation by dipping in a suspension with 108 conidia/ml varied from 23.5% to 100%. Three of the four M. anisopliae isolates caused 100% mortality. In most cases, postmortem hyphal growth and sporulation of M. anisopliae or B. bassiana was observed covering the larvae in their galleries. The eight isolates were also evaluated for pathogenicity to C. tenebrionis eggs at the same dosage. Only two B. bassiana isolates caused significant egg hatching reduction of 84.5% and 94.5%. Our results indicate that M. anisopliae and B. bassiana may be considered as promising for a new approach to prevent larval infestations by C. tenebrionis. [Copyright &y& Elsevier]

The present work describes sequence and transcription of three Spodoptera frugiperda genes encoding 6-cysteine-rich peptides. Sequence alignments indicate that the predicted peptides belong to the insect defensin family, although phylogenetic analyses suggest they form a cluster distinct from that of other neopteran insect defensins. The three genes were identified in a non-immune-challenged Sf9 cells cDNA (DNA complementary to RNA) library (Landais et al., Bioinformatics, in press) and were named spodoptericin, Sf-gallerimycin and Sf-cobatoxin. Spodoptericin is a novel defensin-like gene that appears to be weakly up-regulated following injection of bacteria and fungi. Interestingly, no sequence motif clearly homologous to cis regulatory element involved in the regulation of antimicrobial genes was found. An homologue of the spodoptericin gene was identified in the SilkBase Bombyx mori cDNA library. Sf-gallerimycin is related to the Galleria mellonella gallerimycin gene and is induced after immune challenge by injection of bacteria in the larval fat body as well as in hemocytes. In silico analysis of the sequence upstream from the cDNA reveals the presence of at least one motif homologous to a nuclear factor kappaB (NF-κB) binding site. Finally, Sf-cobatoxin is related to the G. mellonella cobatoxin-like gene. Despite high levels of constitutive expression compared to the two previous genes, transcription of Sf-cobatoxin is increased after immune, in particular, bacterial challenge. We therefore confirm that these three genes encode potential candidate molecules involved in S. frugiperda innate humoral response. [Copyright &y& Elsevier]

The aim of the current study was to delve into the causes of mortality of Spodoptera littoralis larvae feeding on Metarhizium-colonized plants in the absence of fungal outgrowth on the cadavers as previous studies reported and to elucidate the possible indirect effects of this fungus-colonized diet. The effect was evaluated in experiments conducted using leaf discs of colonized plants and in planta using fungus-colonized whole plants. The mortality rates of larvae fed on Metarhizium-colonized melon leaves were 45.0% and 87.5%, and the average survival times were 6.6 and 3.1 days in experiments performed with discs and in planta, respectively. Notably, these mortality levels were not associated with observed apoptosis mediated by caspases 1, 3-7 and 8; thus, further investigation into the possible immune system reaction of the insect after the ingestion of colonized plants is required. The leaf consumption of S. littoralis larvae fed on melon-colonized leaves was lower than that on control plants in the disc experiments but not in experiments conducted in planta. In this regard, in experiments performed in planta, plant damage increased larval mortality in both fungally challenged and control larvae. There was also a meaningful effect of exposure to Metarhizium-colonized melon leaf discs on S. littoralis fitness, with significant reductions in 39.0% and 22.0% in female fecundity and egg fertility, respectively, detected in females emerging from pupae developing from larvae surviving exposure to colonized plant discs; all larvae died in the in planta experiments. Hence, the present work presents new findings revealing the high potential of endophytic entomopathogenic fungi to improve the outcome of foliar applications against chewing insects in the short, mid- and long term, by the reduction of the reproductive potential of surviving adults and reveals new insights into the development of bioassays with whole plants for more detailed evaluation of the impact of these fungi as endophytes used for plant protection. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi are sprayed commercially for aphid control in greenhouses. Recently, their ability to grow endophytically within plants was discovered, offering the opportunity for systemic biological control. Endophytic colonization of host plants could also influence life-table parameters and behavior of herbivores. We investigated lethal and pre-mortality effects of Beauveria bassiana and Metarhizium brunneum on Aphis gossypii; aphids either received inoculum while feeding on recently sprayed leaves (surface inoculum and endophytically-colonized) or while feeding on unsprayed but endophytically-colonized leaves. We used choice assays to identify any preferences for endophytically-colonized or control plants. Volatile emissions from endophytically-colonized plants and control plants were also compared. Aphid mortality rates ranged between 48.2 and 56.9% on sprayed leaves, and between 37.7 and 50.0 on endophytically-colonized leaves. There was a significant effect of endophytic colonization on the rate of nymph production, but this did not result in an overall increase in the aphid population. Endophytic colonization did not influence host-plant selection even though there were qualitative and quantitative differences in the blend of volatiles released by endophytically-colonized and control plants. Although endophytic colonization did not change herbivore behavior, plants still benefit via indirect defense, resistance to plant pathogens or abiotic stress tolerance. [ABSTRACT FROM AUTHOR]

The use of mycoviruses to manipulate the virulence of entomopathogenic fungi employed as biocontrol agents may lead to the development of novel methods to control attacks by insect pests. Such approaches are urgently required, as existing agrochemicals are being withdrawn from the market due to environmental and health concerns. The aim of this work is to investigate the presence and diversity of mycoviruses in large panels of entomopathogenic fungi, mostly from Spain and Denmark. In total, 151 isolates belonging to the genera Beauveria, Metarhizium, Lecanicillium, Purpureocillium, Isaria, and Paecilomyces were screened for the presence of dsRNA elements and 12 Spanish B. bassiana isolates were found to harbor mycoviruses. All identified mycoviruses belong to three previously characterised species, the officially recognised Beauveria bassiana victorivirus 1 (BbVV-1) and the proposed Beauveria bassiana partitivirus 2 (BbPV-2) and Beauveria bassiana polymycovirus 1 (BbPmV-1); individual B. bassiana isolates may harbor up to three of these mycoviruses. Notably, these mycovirus species are under distinct selection pressures, while recombination of viral genomes increases population diversity. Phylogenetic analysis of the RNA-dependent RNA polymerase gene sequences revealed that the current population structure in Spain is potentially a result of both vertical and horizontal mycovirus transmission. Finally, pathogenicity experiments using the Mediterranean fruit fly Ceratitis capitata showed no direct correlation between the presence of any particular mycovirus and the virulence of the B. bassiana isolates, but illustrated potentially interesting isolates that exhibit relatively high virulence, which will be used in more detailed virulence experimentation in the future. [ABSTRACT FROM AUTHOR]