Imperfect match between radiation exposure times required for conidial viability loss and infective capacity reduction attenuate UV-B impact on Beauveria bassiana.

BACKGROUND: UV-B radiation represents a significant challenge for the widespread use of entomopathogenic fungi in pest management. This study focused on research of the asynchronous response between virulence and conidial viability against Ceratitis capitata adults using specific statistical models. Moreover, it was also investigated whether the observed differences in susceptibility to UV-B radiation in in vitro assays among three selected isolates of Beauveria bassiana were reflected in the above-mentioned asynchrony. RESULTS: While the irradiation of the three isolates of B. bassiana was associated with a significant loss of conidial viability, their virulence was not significantly affected compared to nonirradiated treatments when exposed to 1200 mW m-2 for 6 h before or after the inoculation of C. capitata. In fact, the irradiation time needed to reduce the mortality to 50% compared to the controls was 34.69 h for EABb 10/225-Fil, 16.36 h for EABb 09/20-Fil, and 24.59 h for EABb 09/28-Fil. Meanwhile, the irradiation time necessary to reduce conidial viability to 50% was 9.89 h for EABb 10/225-Fil, 8.74 h for EABb 09/20-Fil, and 4.71 h for EABb 09/28-Fil. CONCLUSION: These results highlight the importance of modeling the response of entomopathogenic fungi virulence and conidial susceptibility when exposed to UV-B radiation for the selection of environmentally competent isolates, regardless of the results obtained in previous in vitro assays on conidial germination. This strategic approach is critical in overcoming the challenges posed by UV-B radiation and holds the key to realizing the full potential of entomopathogenic fungi in pest management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Ecosystem services of entomopathogenic ascomycetes.

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.

Persistence of Metarhizium brunneum (Ascomycota: Hypocreales) in the Soil Is Affected by Formulation Type as Shown by Strain-Specific DNA Markers.

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.

Entomopathogenic Fungi-Mediated Solubilization and Induction of Fe Related Genes in Melon and Cucumber Plants.

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.

Survey of Natural Enemies of the Invasive Boxwood Moth Cydalima perspectalis in Southwestern Mediterranean Europe and Biocontrol Potential of a Native Beauveria bassiana (Bals.-Criv.) Vuill. Strain.

Cydalima perspectalis (Lepidoptera: Crambidae), a species native to East Asia, has been especially devastating in the Mediterranean region and Catalonia, northeast Spain, where Buxus sempervirens is an essential component of the natural forest. As an invasive species, the lack of biotic mortality factors in the arrival region has been one of the main factors allowing its expansion. Therefore, this study aimed to collect and identify possible indigenous natural enemies adapting to the new species in the boxwood of the southwest Mediterranean region. Later, the efficacy of some of the collected species for controlling C. perspectalis larvae was tested in laboratory conditions. The larval collection was carried out in successive years in the boxwood of the region. Several collected larvae were infected with an entomopathogen, Beauveria bassiana, or parasitized by Compsilura concinnata, both common in native Lepidoptera caterpillars. The B. bassiana strain was found to be highly virulent against the developed larvae of C. perspectalis, which suggests that B. bassiana may be an effective treatment in parks and gardens when the first overwintering larvae are detected. The biology of the parasitoid identified is not very well known in Europe, which suggests the necessity of studying its biology and alternative hosts in the region in order to improve its population.

Elucidating the Effect of Endophytic Entomopathogenic Fungi on Bread Wheat Growth through Signaling of Immune Response-Related Hormones.

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. IMPORTANCE Microbial control of insect and mite pests is a key tool to develop integrated pest management (IPM) and sustainable agriculture. Entomopathogenic fungi (EF) may have associations with the plants, playing additional ecological roles in the rhizosphere, in the phylloplane, and as plant endophytes. Beauveria bassiana 04/01TIP and Metarhizium brunneum 01/58Su are two strains that showed very good results either in pest control or plant growth promotion and would be good candidates to develop mycoinsecticides as an alternative to pesticides. However, deep knowledge about their interaction with the plant would let farmers optimize their use and understand the plant response, enhancing and promoting their broader contribution to IPM and crop production.

Production of Microsclerotia by Metarhizium sp., and Factors Affecting Their Survival, Germination, and Conidial Yield.

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.

Endophytic Colonization by the Entomopathogenic Fungus Beauveria Bassiana Affects Plant Volatile Emissions in the Presence or Absence of Chewing and Sap-Sucking Insects.

Entomopathogenic fungi are gaining acceptance in Integrated Pest Management (IPM) systems as effective and environmental safety biological control agents to protect a great variety of crops against pest insects. Many of these insect-pathogenic fungi can establish themselves as endophytes and thereby may induce the plant immune system. The activation of plant defenses by the fungal endophytic colonization can have a direct impact on herbivores and plant pathogens. An integral component of many plant defense responses is also the release of volatile organic compounds, which may serve as an indirect defense by attracting the natural enemies of herbivores. Here we investigated the effect of endophytic colonization by the entomopathogenic fungus Beauveria bassiana on the volatile emission by melon and cotton plants, either unharmed or after being damaged by sap-sucking aphids or leaf chewing caterpillars. We found that when the plants are colonized by B. bassiana they emit a different blend of volatile compounds compared to uncolonized control plants. Some of the emitted compounds have been reported previously to be released in response to herbivory and have been implicated in natural enemy attraction. Several of the compounds are also known to have antimicrobial properties. Therefore, endophytic colonization by B. bassiana might help to not only direct control insect pests but also increase the resistance of plants against agronomically important pests and phytopathogens.

Delving into the Causes and Effects of Entomopathogenic Endophytic Metarhizium brunneum Foliar Application-Related Mortality in Spodoptera littoralis Larvae.

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.

Compatibility between the endoparasitoid Hyposoter didymator and the entomopathogenic fungus Metarhizium brunneum: a laboratory simulation for the simultaneous use to control Spodoptera littoralis.

BACKGROUND: The cotton leafworm, Spodoptera littoralis, is one of the most destructive pests in the Mediterranean basin, being predominantly controlled using synthetic chemical pesticides. Strain EAMa 01/58-Su of the fungus Metarhizium brunneum and the parasitoid Hyposoter didymator are promising biological control agents for this pest. In this study, we assessed the compatibility between these two agents to control S. littoralis under joint attack scenarios. RESULTS: Firstly, the direct and indirect effects of the fungus towards parasitoid adults were studied. The fungus significantly decreased life expectancy of the parasitoid (mortality = 62.5%; mean lethal concentration = 1.85 × 106 conidia ml-1 ; average survival time = 92.2 h) when applied at high concentrations (108 conidia ml-1 ), whereas it did not affect the reproductive potential of the parasitoid females during the 3 days after treatment. Secondly, the combinations between the two agents to control S. littoralis under different simultaneous use scenarios (inoculation of S. littoralis larvae with the fungus before being exposed to parasitoid females and vice versa) were investigated, with additive effect in all cases. A significant effect on fitness (preimaginal development time and reproductive potential) of the F1 parasitoid generation were detected. Moreover, parasitization significantly reduced the total hemocytes in S. littoralis hemolymph compared with the control, promoting fungal infection. Finally, parasitoids showed a significant preference for non-inoculated S. littoralis larvae. CONCLUSIONS: We demonstrated compatibility (additive effect) between fungus and parasitoid under different joint attack scenarios to control S. littoralis in laboratory conditions. However, this will be supported by our ongoing greenhouse and field studies. © 2019 Society of Chemical Industry.

Effects of Endophytic Entomopathogenic Ascomycetes on the Life-History Traits of Aphis gossypii Glover and Its Interactions with Melon Plants.

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.

Sublethal effects of mixed fungal infections on the Moroccan locust, Dociostaurus maroccanus.

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 ±â€¯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.

Mycoviral Population Dynamics in Spanish Isolates of the Entomopathogenic Fungus Beauveria bassiana.

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.

Development of an attract-and-infect system to control Rhynchophorus ferrugineus with the entomopathogenic fungus Beauveria bassiana.

BACKGROUND: A new Beauveria bassiana-based attract and infect device (AID) to control Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) was developed. The virulence and persistence of the fungal formulation used in the AID were evaluated in the laboratory. Semi-field and field trials were carried out to validate the results and establish the potential of this device as a control tool. RESULTS: In laboratory conditions, a 50% lethal time (LT50 ) of 4.33 days was obtained when adults (7-10 days old) were exposed to the inoculation tunnel (IT) containing 1 × 1010 conidia g-1 in an oil-based fungal formulation. This formulation maintained conidium viability at 50% for up to 2 months. Moreover, when adults were exposed to 2.5-month field-aged ITs, mortality still reached 50% 40 days after exposure. In addition, no differences were observed between ITs aged in early spring and those aged in summer, suggesting that the fungal formulation is not strongly affected by environmental factors in Mediterranean basin conditions. Semi-field assays showed that the device allowed an easy transit of weevils through the IT, which were effectively attracted and infected. Using the AIDs in 4-ha plot field trials, a reduction of >50% in the percentage of infested sentinel palms was obtained. CONCLUSION: Based on the results obtained in terms of the efficacy and persistence of this new AID in the field and its potential in reducing R. ferrugineus populations and palm infestation, this device could become a key tool for the management of R. ferrugineus. © 2018 Society of Chemical Industry.

Metarhizium brunneum (Ascomycota; Hypocreales) Treatments Targeting Olive Fly in the Soil for Sustainable Crop Production.

Soil treatments with Metarhizium brunneum EAMa 01/58-Su strain conducted in both Northern and Southern Spain reduced the olive fly (Bactrocera oleae) population density emerging from the soil during spring up to 70% in treated plots compared with controls. A model to determine the influence of rainfall on the conidial wash into different soil types was developed, with most of the conidia retained at the first 5 cm, regardless of soil type, with relative percentages of conidia recovered ranging between 56 and 95%. Furthermore, the possible effect of UV-B exposure time on the pathogenicity of this strain against B. oleae adults coming from surviving preimaginals and carrying conidia from the soil at adult emergence was also evaluated. The UV-B irradiance has no significant effect on M. brunneum EAMa 01/58-Su pathogenicity with B. oleae adult mortalities of 93, 90, 79, and 77% after 0, 2, 4, and 6 of UV-B irradiance exposure, respectively. In a next step for the use of these M. brunneum EAMa 01/58-Sun soil treatments within a B. oleae IPM strategy, its possible effect of on the B. oleae cosmopolitan parasitoid Psyttalia concolor, its compatibility with the herbicide oxyfluorfen 24% commonly used in olive orchards and the possible presence of the fungus in the olive oil resulting from olives previously placed in contact with the fungus were investigated. Only the highest conidial concentration (1 × 108 conidia ml-) caused significant P. concolor adult mortality (22%) with enduing mycosis in 13% of the cadavers. There were no fungal propagules in olive oil samples resulting from olives previously contaminated by EAMa 01/58-Su conidia. Finally, the strain was demonstrated to be compatible with herbicide since the soil application of the fungus reduced the B. oleae population density up to 50% even when it was mixed with the herbicide in the same tank. The fungal inoculum reached basal levels 4 months after treatments (1.6 × 103 conidia g soil-1). These results reveal both the efficacy and environmental and food safety of this B. oleae control method, protecting olive groves and improving olive oil quality without negative effects on the natural enemy P. concolor.

Different strategies to kill the host presented by Metarhizium anisopliae and Beauveria bassiana.

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.

Entomopathogenic fungi-based mechanisms for improved Fe nutrition in sorghum plants grown on calcareous substrates.

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.

UV-B radiation-related effects on conidial inactivation and virulence against Ceratitis capitata (Wiedemann) (Diptera; Tephritidae) of phylloplane and soil Metarhizium sp. strains.

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 1200mWm-2 for 2, 4 or 6h 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 1200mWm-2 for 6h 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 5.9days 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 (1.0×104-1.0×108conidiaml-1) and then irradiated at 1200mWm-2 for 6h, and similar LC50 values, 3.8×107 and 4.3×107conidiaml-1, were determined for the UV-B and no UV-B treatments, respectively. However, the LT50 values for flies inoculated with 1.0×108conidiaml-1 and with1.0×107conidiaml-1 were 15.1% and 30.8% longer for UV-B treatments than no UV-B treatments, respectively. Next, adult flies were treated with 1.0×108conidiaml-1 and then exposed to 1200mWm-2 for 0, 6, 12, 24, 36 and 48h, and the relationships among exposure time and conidia viability and fly mortality losses were determined. The exposure time for adult flies at 1200mWm-2 to achieve a 50% reduction in fly mortality was 47.2h, which was longer than that of 5.6h 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.

Analytical strategy for determination of known and unknown destruxins using hybrid quadrupole-Orbitrap high-resolution mass spectrometry.

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, A1, B1, Ed1, A2, B2, D2, A3, DesmA, DesmB, DesmC, DesmB2, and two chloro-derivatives (Cl and E2 chlorohydrin) were unequivocally identified in Metarhizium brunneum using the proposed strategy. In addition, four unknown destruxins, namely C1, Ed2, G, and G1, were structurally elucidated and characterized for the first time in this fungal strain.