Akanthomyces diversity in Brazil and their pathogenicity to plant-sucking insects.

Currently, species within the genus Akanthomyces are poorly studied and explored compared to other hypocrealean entomopathogenic fungi employed as commercial biocontrol agents. This study aimed to molecularly identify 23 Brazilian Akanthomyces strains, most originally isolated from aphids and scales (n = 22), and one from the coffee leaf rust, and further investigate their pathogenicity to six plant-sucking insects as a means to better understand their host spectra. We also explored the capacity of A. muscarius CG935 for blastospore production via liquid fermentation. Akanthomyces dipterigenus, A. muscarius, A. lecanii, and two unidentified species were recognized as naturally occurring in Brazil. Akanthomyces dipterigenus CG829 and A. muscarius CG935 were highly virulent to nymphs of Bemisia tabaci (67.5-85.4% confirmed mortality) and the aphid Aphis fabae (74.6-75.3%), but only the first strain was virulent to the mealybug Planococcus sp. (80.9%). Akanthomyces lecanii CG824 was weakly virulent to all tested insects. None of the strains were pathogenic to the thrips Caliothrips phaseoli, and all strains showed low virulence to the wooly whitefly Aleurothrixus floccosus and the scale Duplachionaspis divergens. Submerged liquid fermentation yields varied from 1.72 × 109 (day 2) to 3.90 × 109 (day 5) blastospores mL-1. Blastospores or aerial conidia from A. muscarius CG935, at a single concentration of 1 × 107 viable propagules mL-1, resulted in 67.5-83.1% mortality of B. tabaci nymphs within 8 days post-treatment. Overall, these results encourage additional studies that could lead to the development of new mycopesticides based on Akanthomyces strains.

Diversity of anamorphic Cordyceps (formerly Isaria) isolated from Brazilian agricultural sites.

A total of 53 anamorphic strains of Brazilian Cordyceps species currently maintained in a government-owned culture collection, were reassessed for diversity and species identity using multi-loci-based phylogenetic methods. The strains used in this study were originally obtained from soil samples or were isolated from insects of the orders Hemiptera, Lepidoptera, Coleoptera and Diptera, mostly from agricultural sites. A Bayesian phylogenetic tree was constructed based on a concatenation of five loci (ITS, LSU, RPB1, RPB2 and TEF). In a few cases of ambiguity, morphological traits were also considered for species delimitations. Considerable variability within the set of strains was detected and six Cordyceps species were identified: C. amoenerosea, C. fumosorosea, C. javanica, C. tenuipes and, for the first time, C. brevistroma and C. spegazzinii are reported in Brazil. Four other taxonomically equivocal groups, closely related to other known taxa (C. amoenerosea, C. cateniannulata, C. polyarthra and C. spegazzinii), were also recognized, although further studies will be required to confirm their identifications or their descriptions as new species. Cordyceps javanica was the most common species in our dataset, originally isolated from soil and several different insect orders, and includes 17 strains from the whitefly, Bemisia tabaci. Interestingly, strains previously identified as C. fumosorosea based on morphology and growth characteristics, were shown to be C. javanica, including the active ingredients of some commercial mycoinsecticides. Cordyceps farinosa, usually mentioned in the literature as occurring in Brazil, was not found in our study. Since most strains were from insect crop pests, further studies with hosts from non-agricultural settings or from environmental samples would be advisable for a deeper understanding of the occurrence of anamorphic Cordyceps in Brazil.

Potential Impact of Chemical Fungicides on the Efficacy of Metarhizium rileyi and the Occurrence of Pandora gammae on Caterpillars in Soybean Crops.

Entomopathogenic fungi may play a crucial role in the regulation of caterpillar populations in soybean crops, either through natural occurrences or applied as mycopesticides. In the present work, we reported the naturally occurring entomopathogenic fungus Pandora gammae attacking the caterpillar Chrysodeixis includens, with infection rates in field trials ran in two consecutive years in the 10-35% range. As many chemicals are potentially harmful to entomopathogenic fungi, this work aimed to investigate the potential impact of two chemical fungicides (azoxystrobin + benzovindiflupyr and trifloxistrobina + prothioconazole) used to control soybean rust (Phakopsora pachyrhizi) on the natural occurrence of P. gammae and Metarhizium rileyi, as well as the efficacy of the latter fungus applied as different formulations against the soybean caterpillars Anticarsia gemmatalis and C. includens. Under laboratory conditions, fungicides used at field-recommended rates had a considerable negative impact on the germinability of M. riley on the medium surface, and all tested formulations did not protect conidia from damage by these chemicals. This harmful effect also impacted host infectivity, as the larval mortality owing to this fungus was reduced by 30-40% compared to that of the fungicide-free treatments. In field trials conducted in two subsequent years, unformulated and formulated M. rileyi conidia applied to soybean plants produced primary infection sites in caterpillar populations after a single spray. Spraying unformulated or formulated M. rileyi conidia following fungicide application on plants did not affect host infection rates over time. Moreover, the use of M. rileyi-based formulations or chemical fungicide did not interfere with the natural infection rates by P. gammae on its host, C. includens. Although a higher degree of exposure to non-selective fungicides can negatively affect fungal entomopathogens, a single foliar application of fungicides may be harmless to both M. rileyi and P. gammae in soybean fields. Additionally, this work showed that naturally occurring wasps and tachnids also play an important role in the regulation of A. gemmatalis and, notably, C. includens, with parasitism rates above 40-50% in some cases.

Evaluation of key parameters for developing a Metarhizium rileyi-based biopesticide against Spodoptera frugiperda (Lepidoptera: Noctuidae) in maize: laboratory, greenhouse, and field trials.

BACKGROUND: The entomopathogenic fungus Metarhizium rileyi is known to cause natural infections in some caterpillars. In this multiyear study, we carried out laboratory, greenhouse and three field trials with the aim of developing a sustainable option for control of the fall armyworm (FAW) in maize. Association of M. rileyi with Spodoptera frugiperda baculovirus (SfMNPV) and delivery strategies were also investigated. RESULTS: The selected isolate (CG381) was effective in the laboratory at a low concentration (~ 4 × 103 conidia cm-2 ), killing >95% of FAW larvae within 8 days. In the greenhouse assay, applications of conidia suspended in water or as dry powder in maize whorls (~ 6.3 × 106 conidia per plant) produced similar larval mortalities (88%-96%). In the field trials, conventional spraying of unformulated conidia (0.6 and 1.2 × 1012 conidia ha-1 ) caused low larval mortalities (27-31%). Simultaneous application of either unformulated or oil-based formulations of M. rileyi conidia and S. frugiperda baculovirus (SfMNPV) to plant rows caused larval mortalities comparable with each of the pathogens applied alone. However, when a formulation containing both pathogens was sprayed directly into the whorls, the overall mortality of S. frugiperda larvae due to pathogens (mostly to M. rileyi infections) reached ~ 59%, twofold higher than the other treatments with conventional spraying, whereas in the control, the overall mortality was only 1%. CONCLUSION: Optimizing exposure of S. frugiperda larvae to an inoculum of virulent entomopathogens through directed applications to maize whorls is critical to produce satisfactory mortality levels and is promising for integrated pest management.

First report on the natural occurrence of entomopathogenic fungi in populations of the leafhopper Dalbulus maidis (Hemiptera: Cicadellidae): Pathogen identifications and their incidence in maize crops.

The corn leafhopper Dalbulus maidis is one of the most important pests of maize in Latin America. Here we report, for the first time, the natural occurrence of two fungal species infecting the adult stage of this pest. In 2020, insects killed by a pale bluish green fungus in irrigated maize fields located in Northeast Brazil were found attached to the abaxial surface of leaves. Using morphological characters and multigenic phylogeny, it was identified as Metarhizium brasiliense. In the beginning of 2021, the same pathogen was seen on adults in a maize field in the Central-Western region, alongside an entomophthoralean fungus during an epizootic. The latter pathogen was molecularly identified as a species in the genus Batkoa. The number of Batkoa-infected leafhoppers, displaying the typical swollen abdomen and extended wings, reached an average of 1.88 per maize leaf (86.42% of the sampled adults). The incidence of M. brasiliense was higher in plots in the Northeastern region (0.22 and 0.53 adult per leaf) when compared to the Central-Western region (0.04 adult per leaf). The report of D. maidis adults infected by M. brasiliense in agricultural settings located in different geographic regions and over 550 km apart indicates probable widespread occurrence of this pathogen in Brazil. Moreover, this opens the possibility of more applied biological control studies and, perhaps, the development of new tools to manage D. maidis populations.

Assessment of the diversity of Brazilian entomopathogenic fungi in the genus Beauveria.

We combined matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) along with sequencing of the B locus intergenic region (Bloc) to assess the diversity of Brazilian species within the anamorphic genus Beauveria. A total of 121 strains maintained in a government-owned culture collection and isolated from a range of hosts/substrates over a long time span (1981-2015) were assessed. Strains were collected in five of six Brazilian biomes, mostly in the Atlantic Forest (42.2%) and Cerrado (29.8%), primarily from insect pests of crops. All strains were subjected to MS, and those not accurately identified by this technique were genomically analyzed. Among the outcomes of this study, four taxa from the genus Beauveria were recognized, with the great majority of strains belonging to B. bassiana s.str. (93.4%), followed by B. caledonica (2.5%), B. pseudobassiana (2.5%) and B. amorpha (1.6%). B. bassiana s.str. was found in all biomes and isolated from a wide range of hosts/substrates. Due to low numbers, associations of the remaining Beauveria species with specific hosts or habitats/biomes were not clear, except that all three B. caledonica strains were found only in the Cerrado biome and were associated with adults of the banana weevil, Cosmopolites sordidus (Col.:Curculionidae). B. pseudobassiana is reported for the first time on the South American continent, in a subtropical region and from two insect orders not yet associated with this taxon. We also showed that some strains previously ascribed to B. brongniartii were misidentifications. The biodiversity of Beauveria analyzed in our study was comparatively low. The geographic origins of strains used in our study were biased towards biomes with intense human interventions. Future surveys on more conserved, less environmentally disturbed biomes, such as Caatinga, Pampa, Pantanal, and Amazon are needed for a more comprehensive picture of the diversity of Beauveria and related genera in Brazil.

Within-host interactions of Metarhizium rileyi strains and nucleopolyhedroviruses in Spodoptera frugiperda and Anticarsia gemmatalis (Lepidoptera: Noctuidae).

Members of the family Baculoviridae have been quite successfully used as biocontrol agents against some lepidopterans. Likewise, a number of fungi are important natural enemies of these pests. An interesting approach to increase control efficacy could be the combination of a given nucleopolyhedrovirus (NPV) and a fungus, since they possess distinct modes of action. As a first step towards this goal, we assessed the interaction between NPV (either AgMNPV-79 or SfMNPV-6nd) and the entomopathogenic fungus Metarhizium rileyi (either CG1153 or CG381), using Anticarsia gemmatalis and Spodoptera frugiperda as hosts. In sequential applications of these pathogens, per os inoculation of an NPV (leaf discs with 2.5 × 104 occlusion bodies) either two days before or two days post-spraying of its counterpart fungal strain (5 × 103 conidia.cm-2 sprays) usually resulted in an antagonistic effect. When both pathogens were simultaneously applied at different combined dosages, usually an additive effect was seen. Interestingly, a number of dead larvae showing signs of co-infections (partially with soft integument and partially mummified) were recorded. However, mixes with lower dosages of both pathogens did not cause significantly higher insect mortalities compared to low dosages of the fungus applied alone. The advantages and disadvantages of the simultaneous applications of NPV and M. rileyi aiming at the management of either A. gemmatalis or S. frugiperda were discussed.

Conidial vigor vs. viability as predictors of virulence of entomopathogenic fungi.

We tested the hypothesis that debilitated conidia exhibiting slow-germination (requiring>16h to germinate) are less virulent than vigorous conidia exhibiting fast germination (requiring⩽16h to germinate). Preparations of Beauveria bassiana s.l. strain CG 1027 with variable ratios of vigorous to debilitated conidia were assayed against third-instar larvae of Spodoptera frugiperda. As the proportion of debilitated conidia in test preparations increased, LC50 expressed in terms of total viable conidia increased, while LC50 expressed solely in terms of vigorous conidia remained constant, indicating that vigorous conidia were responsible for nearly all mortality observed in the assays. Larvae treated with conidia from low-quality batches (with high proportions of debilitated conidia) survived consistently longer than those treated with comparable doses of conidia from high-quality batches. These results confirm our previous hypotheses that inclusion of debilitated conidia in viability assessments can lead to overestimation of the quality (potency) of mycoinsecticide preparations and support our recommendation for use of short incubation periods for assessing viability whenever viability is relied upon as an indicator of product quality.

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.