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[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 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]

• 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]

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]

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]

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]

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]

[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]