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This study aimed to isolate and characterize a native strain of Beauveria bassiana, coded as Bv065, showcasing its potential as a biological control agent targeting the palm weevil Dynamis borassi. Originating from a naturally infected D. borassi specimen collected in southwestern Colombia, the fungus underwent molecular identification and was identified as B. bassiana, exhibiting high sequence similarity with known reference strains. The physiological characterization revealed that Bv065 thrived within a temperature range of 25 to 30 °C and a pH range of 6 to 9. Moreover, the key carbon sources that allow optimal growth of the strain were identified through metabolic profiling, including sucrose, D-mannose, and γ-amino-butyric acid. These findings offer strategic insights for scalability and formulation methodologies. Additionally, enzymatic analyses unveiled robust protease activity within Bv065, crucial for catalysing insect cuticle degradation and facilitating host penetration, thus accentuating its entomopathogenic potential. Subsequent evaluations exposed Bv065's pathogenicity against D. borassi, causing significant mortality within nine days of exposure, albeit exhibiting limited effectiveness against Rhynchophorus palmarum. This study underscores the importance of understanding optimal growth conditions and metabolic preferences of B. bassiana strains for developing effective biopesticides. The findings suggest Bv065 as a promising candidate for integrated pest management strategies in neotropical regions, particularly for controlling palm weevil infestations in coconut and peach palm cultivation. Future research avenues include refining mass production methodologies, formulating novel delivery systems, and conducting comprehensive field efficacy trials to unlock the full potential of Bv065 in fostering sustainable pest management practices. Overall, this study contributes to the growing body of knowledge on entomopathogenic fungi and their pivotal role in biological control, offering nuanced perspectives on eco-friendly alternatives to conventional insecticidal interventions.

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Increased attention is being focused on the biological control of agricultural pests using microorganisms, owing to their potential as a viable substitute for chemical control methods. Insect cadavers constitute a potential source of entomopathogenic microorganisms. We tested whether bacteria and fungi isolated from Spodoptera frugiperda (JE Smith) cadavers could affect its survival, development, egg-laying pattern, and hatchability, as well as induce mortality in Anthonomus grandis Boheman adults. We isolated the bacteria Enterobacter hormaechei and Serratia marcescens and the fungi Scopulariopsis sp. and Aspergillus nomiae from fall armyworm cadavers and the pest insects were subjected to an artificial diet enriched with bacteria cells or fungal spores to be tested, in the case of S. frugiperda, and only fungal spores in the case of A. grandis. Enterobacter hormaechei and A. nomiae were pathogenic to S. frugiperda, affecting the survival of adults and pupae. The fungus Scopulariopsis sp. does not affect the survival of S. frugiperda caterpillars and pupae; however, due to late action, moths and eggs may be affected. Aspergillus nomiae also increased mortality of A. grandis adults, as well as the development of S. frugiperda in the early stages of exposure to the diet, as indicated by the vertical spore transfer to offspring and low hatchability. Enterobacter hormaechei and A. nomiae are potential biocontrol agents for these pests, and warrant further investigation from a toxicological point of view and subsequently in field tests involving formulations that could improve agricultural sustainability practices.

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Coffee fruit rot (CFR) is a well-known disease worldwide, mainly caused by Colletotrichum spp., the most important species being C. kahawae subsp. kahawae. In Puerto Rico, Colletotrichum spp. were identified as pathogens of coffee fruits. The coffee berry borer (CBB) was shown to be a dispersal agent of these fungi, and interaction of Fusarium with Colletotrichum affecting coffee fruits was suggested. In this study, we demonstrated that Fusarium spp. also cause CFR in Puerto Rico. Fusarium spp. are part of the CBB mycobiota, and this insect is responsible for spreading the pathogens in coffee fields. We identified nine Fusarium spp. (F. nirenbergiae, F. bostrycoides, F. crassum, F. hengyangense, F. solani-melongenae, F. pseudocircinatum, F. meridionale, F. concolor, and F. lateritium) belonging to six Fusarium species complexes isolated from CBBs and from rotten coffee fruits. Pathogenicity tests showed that F. bostrycoides, F. lateritium, F. nirenbergiae, F. solani-melongenae, and F. pseudocircinatum were pathogens causing CFR on green coffee fruits. F. bostrycoides was the predominant species isolated from the CBB mycobiota and coffee fruits with symptoms of CFR, suggesting a close relationship between F. bostrycoides and the CBB. To our knowledge, this is the first report of F. bostrycoides, F. solani-melongenae, F. pseudocircinatum, and F. nirenbergiae causing CFR worldwide and the first report of F. lateritium causing CFR in Puerto Rico. Understanding the CFR disease complex and how the CBB contributes to dispersing different Fusarium spp. on coffee farms is important to implement disease management practices in Puerto Rico and in other coffee-producing countries.

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Ambrosia beetles from the scolytine tribe Xyleborini (Curculionidae) are important to the decomposition of woody plant material on every continent except Antarctica. These insects farm fungi on the walls of tunnels they build inside recently dead trees and rely on the fungi for nutrition during all stages of their lives. Such ambrosia fungi rely on the beetles to provide appropriate substrates and environmental conditions for growth. A small minority of xyleborine ambrosia beetle-fungal partnerships cause significant damage to healthy trees. The xyleborine beetle Coptoborus ochromactonus vectors a Fusarium (Hypocreales) fungus that is lethal to balsa (Ochroma pyramidale (Malvaceae)) trees in Ecuador. Although this pathogenic fungus and its associated beetle are not known to be established in the United States, several other non-native ambrosia beetle species are vectors of destructive plant diseases in this country. This fact and the acceleration of trade between South America and the United States demonstrate the importance of understanding fungal plant pathogens before they escape their native ranges. Here we identify the fungi accompanying Coptoborus ambrosia beetles collected in Ecuador. Classification based ribosomal internal transcribed spacer 1 (ITS) sequences revealed the most prevalent fungi associated with Coptoborus are Fusarium sp. and Graphium sp. (Microascales: Microascaceae), which have been confirmed as ambrosia fungi for xyleborine ambrosia beetles, and Clonostsachys sp. (Hypocreales), which is a diverse genus found abundantly in soils and associated with plants. Phylogenetic analyses of the Fusarium strains based on ITS, translation elongation factor (EF1-α), and two subunits of the DNA-directed RNA polymerase II (RPB1 and RPB2) identified them as Fusarium sp. AF-9 in the Ambrosia Fusarium Clade (AFC). This Fusarium species was previously associated with a few xyleborine ambrosia beetles, most notably the species complex Euwallacea fornicatus (Eichhoff 1868) (Curculionidae: Scolytinae: Xyleborini). Examination of ITS and EF1-α sequences showed a close affinity between the Graphium isolated from Coptoborus spp. and other xyleborine-associated Graphium as well as the soil fungus Graphium basitruncatum. This characterization of ambrosia fungi through DNA sequencing confirms the identity of a putative plant pathogen spread by Coptoborus beetles and expands the documented range of Fusarium and Graphium ambrosia fungi.

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AIMS: Entomopathogenic Metarhizium fungi are widely recognized for their biological control potential. In Cuba, several fungus-based bio-insecticides have been developed and are produced as part of integrated pest management (IPM) programmes for economically relevant agricultural pests. Screening of fungal isolates from the INISAV strain collection was used for the development of bio-insecticides against important pest insects as, for example the sweet potato weevil, Cylas formicarius. METHODS AND RESULTS: Six fungal isolates from Cuba were microscopically, morphologically and molecular-taxonomically characterized using marker sequences ef1a, rpb1 and rpb2, and the 5TEF region of the ef1a gene. Five isolates were assigned to the species Metarhizium anisopliae sensu stricto and one isolate to Metarhizium robertsii. The pathogenic potential was evaluated against adults of C. formicarius, and growth and conidial production on different nutritional media were determined. Metarhizium anisopliae strain LBM-267 displayed pronounced virulence against the sweet potato weevil and abundant conidia production on several culture media. CONCLUSIONS: Entomopathogenic fungal isolates from Cuba were assigned to the taxonomic species M. anisopliae sensu stricto and M. robertsii. Virulence assessment with respect to C. formicarius led to the identification of two M. anisopliae isolates holding biocontrol potential. Isolate LBM-11 has previously been developed into the bio-insecticide METASAVE-11 that is widely used to control several species of plant pathogenic weevils, Lepidoptera and thrips in Cuba. Isolate LBM-267 has not been employed previously but is as virulent against C. formicarius as LBM-11; its growth and conidial production capacities on different nutritional media will likely facilitate economically feasible bio-insecticide development. SIGNIFICANCE AND IMPACT OF THE STUDY: Metarhizium anisopliae isolate LBM-267 has been selected as a promising candidate for biocontrol of the sweet potato weevil, an economically important agricultural pest in Cuba, and for further R&D activities within the framework of the Biological Control Program of Cuba.

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Most beetle-fungus symbioses do not represent a threat to agricultural and natural ecosystems; however, a few beetles are able to inoculate healthy hosts with disease-causing fungal symbionts. Here, we report the putative nutritional symbionts associated with five native species of ambrosia beetles colonizing commercial avocado trees in four locations in Michoacán. Knowing which beetles are present in the commercial orchards and the surrounding areas, as well as their fungal associates, is imperative for developing a realistic risk assessment and an effective monitoring system that allows for timely management actions. Phylogenetic analysis revealed five potentially new, previously undescribed species of Raffaelea, and three known species (R. arxi, R. brunnea, R. fusca). The genus Raffaelea was recovered from all the beetle species and across the different locations. Raffaelea lauricola (RL), which causes a deadly vascular fungal disease known as laurel wilt (LW) in Lauraceae species, including avocado, was not recovered. This study points to the imminent danger of native ambrosia beetles spreading RL if the pathogen is introduced to Mexico's avocado orchards or natural areas given that these beetles are associated with Raffaelea species and that lateral transfer of RL among ambrosia beetles in Florida suggests that the likelihood of this phenomenon increases when partners are phylogenetically close. Therefore, this study provides important information about the potential vectors of RL in Mexico and other avocado producing regions. Confirming beetle-fungal identities in these areas is especially important given the serious threat laurel wilt disease represents to the avocado industry in Mexico.

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Xyleborus sp beetles are types of ambrosia beetles invasive to the United States and recently also to Mexico. The beetle can carry a fungus responsible for the Laurel Wilt, a vascular lethal disease that can host over 300 tree species, including redbay and avocado. This problem has a great economic and environmental impact. Indeed, synthetic chemists have recently attempted to develop new neonicotinoids. This is also due to severe drug resistance to "classic" insecticides. In this research, a series of neonicotinoids analogs were synthesized, characterized, and evaluated against Xyleborus sp. Most of the target compounds showed good to excellent insecticidal activity. Generally, the cyclic compounds also showed better activity in comparison with open-chain compounds. Compounds R-13, 23, S-29, and 43 showed a mortality percent of up to 73% after 12 h of exposure. These results highlight the enantioenriched compounds with absolute R configuration. The docking results correlated with experimental data which showed both cation-π interactions in relation to the aromatic ring and hydrogen bonds between the search cavity 3C79 and the novel molecules. The results suggest that these sorts of interactions are responsible for high insecticidal activity.

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Parthenogenesis is widely distributed in Metazoa but it is especially frequent in weevils (Coleoptera, Curculionidae) with one fifth of all known cases. Previous studies have shown that in the tribe Naupactini parthenogenetic reproduction most likely originated with an infection of the endoparasitic bacterium Wolbachia pipientis. In particular, Pantomorus postfasciatus possess a mixed reproductive mode: some populations have males while in others they are absent, and females produce clones by thelytoky. To better understand this scenario, we studied the population structure and infection status in 64 individuals of P. postfasciatus from Argentina and Brazil. We sequenced two mitochondrial (COI and COII) and one nuclear (ITS-1) fragments and obtained two very divergent haplogroups, one corresponding to the sexual populations uninfected with Wolbachia, and another conforming a monophyletic parthenogenetic (or presumptively parthenogenetic) and infected clade. Each of these haplogroups was identified as an independently evolutionary unit by all species delimitation analyses accomplished: multilocus *BEAST and BP&P, and single locus GMYC and K/θ rule. Additionally, present evidence suggests that Wolbachia infection occurred at least twice in all-female populations of P. postfasciatus with two different bacterial strains. Speciation mediated by Wolbachia is a recently described phenomenon and the case of P. postfasciatus is the first known case in a diplo-diploid insect. A model that describes how thelytoky-inducing phenotypes of Wolbachia could generate new lineages is discussed.

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BACKGROUND: The Ambrosia Fusarium Clade phytopathogenic Fusarium fungi species have a symbiotic relationship with ambrosia beetles in the genus Euwallacea (Coleoptera: Curculionidae). Related beetle species referred to as Euwallacea sp. near fornicatus have been spread in California, USA and are recognized as the causal agents of Fusarium dieback, a disease that causes mortality of many plant species. Despite the importance of this fungi, no transcriptomic resources have been generated. The datasets described here represent the first ever transcripts available for these species. We focused our study on the isolated species of Fusarium that is associated with one of the cryptic species referred to as Kuroshio Shot Hole Borer (KSHB) Euwallacea sp. near fornicatus. RESULTS: Hydrogen concentration is a critical signal in fungi for growth and host colonization, the aim of this study was to evaluate the effect of different pH conditions on growth and gene expression of the fungus Fusarium sp. associated with KSHB. An RNA-seq approach was used to compare the gene expression of the fungus grown for 2 weeks in liquid medium at three different pH levels (5.0, 6.0, and 7.0). An unbuffered treatment was included to evaluate the capability of the fungus to change the pH of its environment and the impact in gene expression. The results showed that the fungus can grow and modulate its genetic expression at different pH conditions; however, growth was stunted in acidic pH in comparison with neutral pH. The results showed a differential expression pattern in each pH condition even when acidic conditions prevailed at the end of the experiment. After comparing transcriptomics data from the three treatments, we found a total of 4,943 unique transcripts that were differentially expressed. CONCLUSIONS: We identified transcripts related to pH signaling such as the conserved PAL/RIM pathway, some transcripts related to secondary metabolism and other transcripts that were differentially expressed. Our analysis suggests possible mechanisms involved in pathogenicity in this novel Fusarium species. This is the first report that shows transcriptomic data of this pathogen as well as the first report of genes and proteins involved in their metabolism identifying potential virulence factors.

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Dendroctonus bark beetles are a worldwide significant pest of conifers. This genus comprises 20 species found in North and Central America, and Eurasia. Several studies have documented the microbiota associated with these bark beetles, but little is known regarding how the gut bacterial communities change across host range distribution. We use pyrosequencing to characterize the gut bacterial communities associated with six populations of Dendroctonus valens and D. mexicanus each across Mexico, determine the core bacteriome of both insects and infer the metabolic pathways of these communities with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to evaluate whether these routes are conserved across geographical locations. Our results show that the ß-diversity with UniFrac unweighted varies among locations of both bark beetles mainly due to absence/presence of some rare taxa. No association is found between the pairwise phylogenetic distance of bacterial communities and geographic distance. A strict intraspecific core bacteriome is determined for each bark beetle species, but these cores are different in composition and abundance. However, both bark beetles share the interspecific core bacteriome recorded previously for the Dendroctonus genus consisting of Enterobacter, Pantoea, Providencia, Pseudomonas, Rahnella, and Serratia. The predictions of metabolic pathways are the same in the different localities, suggesting that they are conserved through the geographical locations.

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An isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) was tested for its ability to reduce survival and reproduction of spruce beetle, Dendroctonus rufipennis (Kirby) (Coleoptera: Scolytinae), under laboratory and field conditions. Conidial suspension applied directly to adults or to filter papers that adults contacted had a median survival time of 3-4 d in laboratory assays and beetles died more rapidly when exposed to conidial suspension than when treated with surfactant solution only. In the field, conidial suspension was applied to the surface of felled and pheromone-baited Engelmann spruce (Picea engelmannii) trees using a backpack sprayer. Mortality of colonizing parent beetles (F0), reproduction (abundance of F1 offspring in logs), and emergence of F1 beetles from logs was compared between treated and nontreated logs. Application of spore suspension increased mortality of F0 adults by 36% on average. Total F1 reproduction was reduced by 17% and emergence from logs was reduced by 13% in treated logs, but considerable variability in reproduction and emergence was observed. Viable spores were re-isolated from treated logs up to 90 d after application, indicating that spores are capable of long-term persistence on the tree bole microhabitat. Subsequent in vitro tests revealed that temperatures below 15°C and exposure to spruce monoterpenes likely limit performance of B. bassiana under field conditions, but exposure to low-intensity light or interactions with spruce beetle symbiotic fungi were not strongly inhibitory. It is concluded that matching environmental tolerances of biocontrol fungi to field conditions can likely improve their usefulness for control of spruce beetle in windthrown trees.

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Plant rhizobacteria have been successfully used as biocontrol agents against fungal phytopathogens. However, their potential to control two important avocado diseases, namely Fusarium dieback (FD) and Phytophthora root rot (PRR), has been poorly studied. FD is an emerging disease triggered by fungi associated with two ambrosia beetle species (Euwallacea fornicatus species complex), while PRR is caused by Phytophthora cinnamomi, a soil-borne oomycete. In the present work, the antifungal activity of bacteria isolated from avocado rhizosphere was tested in dual culture assays against Fusarium euwallaceae, Graphium euwallaceae and Graphium sp., causal agents of FD, and against P. cinnamomi. In 2015, rhizosphere soil samples of FD infested and non-infested avocado trees were collected from a commercial avocado orchard in Escondido, California. In an initial screening, 72 of the 168 assessed bacterial isolates reduced mycelial growth of F. euwallaceae by up to 46%. Eight bacterial isolates showing inhibition percentages larger than 40% were then selected for further antagonism assays against the other fungal pathogens. Five bacterial isolates, determined by 16S rDNA sequencing to belong to the Bacillus subtilis/Bacillus amyloliquefaciens species complex, successfully inhibited the mycelial growth of both Graphium species by up to 30%. The same isolates and an additional isolate identified as Bacillus mycoides, inhibited the growth of P. cinnamomi by up to 25%. This is the first report of avocado rhizobacteria with antifungal activity against pathogens responsible for FD and PRR in avocado.

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Stem bleeding disease, caused by the fungus Thielaviopsis paradoxa is one of the most important diseases of coconut palm Cocos nucifera worldwide and some insect species have been found in association with T. paradoxa infected coconut palms. The present research investigated whether coconut palm-infesting bark beetles (Coleoptera: Curculionidae: Scolytinae) could disseminate stem bleeding disease. Adult bark beetles collected from coconut palms presenting stem bleeding symptoms were inoculated in PDA culture medium. Results showed no germination of the fungus T. paradoxa from either cuticle or digestive tract of bark beetles. Thus, we found no evidence that bark beetles carry viable spores of T. paradoxa that could infect healthy coconut palms and disseminate stem bleeding disease.(AU)

A resinose, causada pelo fungo Thielaviopsis paradoxa, é uma das doenças mais importantes do coqueiro Cocos nucifera em todo o mundo, e algumas espécies de insetos têm sido encontradas em associação com coqueiros infectados por T. paradoxa. O presente trabalho investigou se os besouros escolitíneos (Coleoptera: Curculionidae: Scolytinae) poderiam disseminar a resinose a partir de coqueiros infectados. Escolitíneos adultos coletados de coqueiros com sintomas da resinose foram inoculados em meio de cultura BDA. Os resultados mostraram que não houve germinação do fungo T. paradoxa da cutícula ou do trato digestivo dos insetos. Assim, não encontramos evidências de que besouros escolitíneos carreguem esporos viáveis de T. paradoxa que pudessem infectar coqueiros e disseminar a doença resinose.(AU)

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Stem bleeding disease, caused by the fungus Thielaviopsis paradoxa is one of the most important diseases of coconut palm Cocos nucifera worldwide and some insect species have been found in association with T. paradoxa infected coconut palms. The present research investigated whether coconut palm-infesting bark beetles (Coleoptera: Curculionidae: Scolytinae) could disseminate stem bleeding disease. Adult bark beetles collected from coconut palms presenting stem bleeding symptoms were inoculated in PDA culture medium. Results showed no germination of the fungus T. paradoxa from either cuticle or digestive tract of bark beetles. Thus, we found no evidence that bark beetles carry viable spores of T. paradoxa that could infect healthy coconut palms and disseminate stem bleeding disease.

A resinose, causada pelo fungo Thielaviopsis paradoxa, é uma das doenças mais importantes do coqueiro Cocos nucifera em todo o mundo, e algumas espécies de insetos têm sido encontradas em associação com coqueiros infectados por T. paradoxa. O presente trabalho investigou se os besouros escolitíneos (Coleoptera: Curculionidae: Scolytinae) poderiam disseminar a resinose a partir de coqueiros infectados. Escolitíneos adultos coletados de coqueiros com sintomas da resinose foram inoculados em meio de cultura BDA. Os resultados mostraram que não houve germinação do fungo T. paradoxa da cutícula ou do trato digestivo dos insetos. Assim, não encontramos evidências de que besouros escolitíneos carreguem esporos viáveis de T. paradoxa que pudessem infectar coqueiros e disseminar a doença resinose.

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Dendroctonus bark beetles comprise 20 taxonomically recognized species, which are one of the most destructive pine forest pests in North and Central America, and Eurasia. The aims of this study were to characterize the gut bacterial diversity, to determine the core bacteriome and to explore the ecological association between these bacteria and bark beetles. A total of five bacterial phyla were identified in the gut of 13 Dendroctonus species; Proteobacteria was the most abundant, followed by Firmicutes, Fusobacteria, Actinobacteria and Deinococcus-Thermus. The α-diversity was low as demonstrated in previous studies and significant differences in ß-diversity were observed. The core bacteriome was composed of Enterobacter, Pantoea, Pseudomonas, Rahnella, Raoultella, and Serratia. The tanglegram between bacteria and bark beetles suggests that members of bacterial community are acquired from the environment, possibly from the host tree. These findings improve the knowledge about the bacterial community composition, and provide the bases to study the metabolic functions of these bacteria, as well as their interaction with these bark beetles.

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The knowledge of the spatiotemporal dynamics of pathogens and their vectors is an important step in determining the pathogen dispersion pattern and the role of vectors in disease dynamics. However, in the case of mango wilt little is known about its spatiotemporal dynamics and the relationship of its vector [the beetle Hypocryphalus mangiferae (Stebbing 1914)] to these dynamics. The aim of this work was to determine the spatial-seasonal dynamic of H. mangiferae attacks and mango wilt in mango orchards and to verify the importance of H. mangiferae in the spatiotemporal dynamics of the disease. Two mango orchards were monitored during a period of 3 yr. The plants in these orchards were georeferenced and inspected monthly to quantify the number of plants attacked by beetles and the fungus. In these orchards, the percentage of mango trees attacked by beetles was always higher than the percentage infected by the fungus. The colonization of mango trees by beetles and the fungus occurred by colonization of trees both distant and proximal to previously attacked trees. The new plants attacked by the fungus emerged in places where the beetles had previously begun their attack. This phenomenon led to a large overlap in sites of beetle and fungal occurrence, indicating that establishment by the beetle was followed by establishment by the fungus. This information can be used by farmers to predict disease infection, and to control bark beetle infestation in mango orchards.

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Bark beetles (Curculionidae: Scolytinae) feed on the xylem and phloem of their host, which are composed of structural carbohydrates and organic compounds that are not easily degraded by the insects. Some of these compounds might be hydrolyzed by digestive enzymes produced by microbes present in the gut of these insects. In this study, we evaluated the enzymatic capacity of bacteria (Acinetobacter lwoffii, Arthrobacter sp., Pseudomonas putida, Pseudomonas azotoformans, and Rahnella sp.) and yeasts (Candida piceae, Candida oregonensis, Cyberlindnera americana, Zygoascus sp., and Rhodotorula mucilaginosa) isolated from the Dendroctonus rhizophagus gut to hydrolyze cellulose, xylan, pectin, starch, lipids, and esters. All isolates, with the exception of C. piceae, showed lipolytic activity. Furthermore, P. putida, P. azotoformans, C. americana, C. piceae, and R. mucilaginosa presented amylolytic activity. Esterase activity was shown by A. lwoffii, P. azotoformans, and Rahnella sp. Cellulolytic and xylanolytic activities were present only in Arthrobacter sp. and P. azotoformans. The pectinolytic activity was not recorded in any isolate. This is the first study to provide evidence on the capacity of microbes associated with the D. rhizophagus gut to hydrolyze specific substrates, which might cover part of the nutritional requirements for the development, fitness, and survival of these insects.

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Bark beetles (Curculionidae: Scolytinae) and associated microorganisms must overcome a complex tree's defence system, which includes toxic monoterpenes, to successfully complete their life cycle. A number of studies have suggested these microorganisms could have ecological roles related with the nutrition, detoxification, and semiochemical production. In particular, in filamentous fungi symbionts, cytochrome P450 (CYP) have been involved with terpenoid detoxification and biotransformation processes. Candida oregonensis has been isolated from the gut, ovaries, and frass of different bark beetle species, and it is a dominant species in the Dendroctonus rhizophagus gut. In this study, we identify, characterise, and infer the phylogenetic relationships of C. oregonensis CYP genes. The results indicate that the cytochrome P450 complement (CYPome) is composed of nine genes (CYP51F1, CYP61A1, CYP56D1, CYP52A59, CYP52A60, CYP52A61, CYP52A62, CYP5217A8, and CYP5217B1), which might participate in primary metabolic reactions such as sterol biosynthesis, biodegradation of xenobiotic, and resistance to environmental stress. The prediction of the cellular location suggests that these CYPs to be anchored to the plasma membrane, membranes of the endoplasmic reticulum, mitochondria, and peroxisomes. These findings lay the foundation for future studies about the functional role of P450s, not only for yeasts, but also for the insects with which they interact.

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The coffee berry borer (CBB; Hypothenemus hampei) is a major pest of coffee responsible for significant crop losses worldwide. The entomopathogenic fungus Beauveria bassiana represents a natural means of controlling this insect pest; however, little is known concerning the molecular determinants that contribute to the virulence of the fungus towards the CBB. In order to examine genes involved in insect virulence, two expressed sequence tag (EST) libraries, representing germinating conidia and growing hyphae/mycelia of B. bassiana cells grown on cuticular extracts of the CBB were constructed and analysed. In total, 4186 cDNA transcripts were obtained, which included 2141 from the cuticle-germinated conidia and 2045 from the cuticle-grown mycelium libraries, respectively. The average sequence length obtained was 470 bp and transcript assembly resulted in a set of 1271 and 1305 unique gene sequences for the conidial and mycelia libraries, respectively. Around 50 % of the sequences in each library could be annotated by gene ontology terms. An analysis of the two generated libraries as well as a previously reported EST library of B. bassiana grown on chitin was performed. Between the cuticle-germinated conidia and the cuticle-grown mycelia libraries, 322 unique gene sequences were shared, of which 90 % could be annotated, leaving 949 unique cuticle-germinated conidial genes and 983 unique growing hyphae/mycelia genes of which around 65 % were annotated. ESTs shared between the libraries indicated a basic response pattern for B. bassiana against H. hampei, which included genes implicated in pathogenicity. The expression profiles of four genes were evaluated with a cyclophilin, an alkaline-like serine protease and a mitogen-activated protein kinase (MAPK), showing elevated expression during initial phases of infection, i.e. conidia germinating on insect extracts. These data provide clues and gene candidates for further exploration concerning the biology and molecular mechanisms of entomopathogenicity by this fungus.

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The boll weevil (Anthonomus grandis) is the main pest of cotton in the Americas. The aim of this work was to evaluate isolates of the entomopathogenic fungi Beauveria bassiana sensu lato and Metarhizium anisopliae sensu lato virulent against A. grandis. Screening was performed to evaluate the pathogenicity of 28 isolates of M. anisopliae s.l. and 66 isolates of B. bassiana s.l. against boll weevil adults. To select the isolates, LC(50) values of the most virulent isolates were calculated, and compatibility between the fungi and insecticides was studied. In addition, the effects of these isolates on the feeding behavior of the adults were evaluated. Isolates Ma 50 and Ma 20 were the most virulent against A. grandis and their LC(50) values were 1.13×10(7) and 1.20×10(7) conidia/ml, respectively. In addition, these isolates were compatible with pyrethroid insecticides, but none with endosulfan. On the other hand, infected females reduced the damage caused by feeding on the cotton squares and their weight gain. This shows that entomopathogenic fungi cause mortality in the insects, but also these fungi could influence the feeding behavior of the females. In summary, these results indicate the possibility of the use of M. anisopliae s.l. as a microbiological control agent against boll weevils. Also, this species could be included in an Integrated Pest Management program.

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