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Subclusters of genes specifically upregulated on only one of the cuticles (BC, beetle; CC, cockroach; MC, Man. sexta; GC, Lymantria dispar). Samples were reordered from Fig. 1 according to the time-scale shown across the top, and genes were hierarchically clustered. Gene names and accession numbers are shown to the right of the figure.
Source publication
Metarhizium anisopliae infects a broad range of insects by direct penetration of the host cuticle. To explore the molecular basis of this process, its gene expression responses to diverse insect cuticles were surveyed, using cDNA microarrays constructed from an expressed sequence tag (EST) clone collection of 837 genes. During growth in culture con...
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Context 1
... regulated on CC, 96 (71 %) were similarly regulated on BC and 95 (70 %) were similarly regulated on GC. Among these commonly regulated genes, 64 were upregulated on all four cuticles at 24 h. The balance of genes demonstrated specific responses to different cuticles, including up-or down-regulation of genes not observed at any time-point on MC (Fig. 4). This implies that the pathogen can pre- cisely respond to different conditions. In some cases, genes coordinately upregulated on a particular cuticle were func- tionally related. Thus, several sequences upregulated at 24 h on GC, but not MC, CC or BC at this time-point, have homologues in yeast that are involved in integrating ...
Context 2
... aside, the major component of insect cuticle is chitin, and predictably therefore chitinases were upregu- lated on cuticle. Chitinase AJ274366 was expressed within 1 h on MC, but was not expressed in MM (Fig. 2). Chitosanase was only produced on GC (Fig. 4). As this coincides with the GC-specific expression of genes involved in morphogenesis, it is possible that the chitosanase may be involved in modifying cell wall components. However, five additional enzymes involved in metabolizing carbo- hydrates not known to occur in cuticle were also upregu- lated in one or more of the cuticle ...
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Citations
... The conidia virulence and the regulation of pathogenicity-related genes in entomopathogenic fungi are significantly impacted by the culture medium's composition (Freimoser et al., 2005;. Particularly, fatty acids used as supplements in production medium could affect pathogenicity according to fungal species and fatty acid type, since specific lipid assimilation systems are required, similar to those used in cuticle penetration (Keyhani 2018). ...
... The method of infection of Metarhizium anisopliae on susceptible hosts involves the direct penetration of their cuticle [33]. To simplify the explanation of this intricate process, it can be dissected into six distinct stages, as reported by Gao et al. [34]: ...
Increasing concerns over the environmental impact of chemical pesticides have prompted the exploration of alternative, and eco-friendly solutions for controlling insect pests. The use of entomopathogenic fungus (EPF) as a biological control agent is of paramount importance. We aimed to investigate the effect of different concentrations of Metarhizium anisopliae (Metschnikoff) Sorokin on larval mortality and pupal formation, of sawfly larvae, a major pest of rose plants. Our study revealed a concentration-dependent effect of M. anisopliae on sawfly larvae. At 7 days after EPF exposure, the highest concentration (1 × 109 conidia/ml) led to a mortality rate of 65.0% and at the 11th day, the larval mortality was reached up to 82.5%. The highest EPF concentration resulted in a minimal pupal formation (7.5%). This study demonstrates the potential of M. anisopliae as a valuable biological control agent against sawfly larvae infestations in rose plants.
... Thus, both, the advantages observed in the production process and the pathogenicity of conidia can be attributed to high levels of protein and fatty acids of the PKM. In fact, some of these compounds are strong inducers of the expression of proteins involved in nutrient metabolism, sporulation and virulence of the EPF (Wang et al. 2016;Freimoser et al. Content courtesy of Springer Nature, terms of use apply. Rights reserved. ...
This study aimed to evaluate the use of palm kernel meal (PKM) in the traditional solid-state fermentation system to improve the production and quality of Cordyceps javanica conidia. The impact of PKM was determined by measuring conidia yield, viability, hydrophobicity, shelf life, and conidia pathogenicity against Diaphorina citri adults. By supplementing rice grains with 5% palm kernel meal increased the conidial yield by up to 40%, without compromising conidia viability and hydrophobicity. In addition, conidia caused higher levels of mortality by mycosis against D. citri adults (90%), relative to conidia harvested from rice (52%). The conidia recovered from rice/palm kernel meal mixtures also retained viability greater than 90% after storage for 10 months at 4 °C, while the conidia produced on rice reached 80%. Thus, conidia produced in the presence of palm kernel meal can be consumed immediately or in the medium term. Some process advantages of the palm kernel meal as co-substrate in the traditional production system of C. javanica are also mentioned. These results are attractive for improving the mycoinsecticide production process, with excellent cost–benefit and minimal changes in infrastructure and process.
Graphical abstract
... Genetic transformation of single gene or double genes makes it be more inefficient to obtain new and more genes directly correlated with appressorium formation. Numerous virulence genes in entomogenous fungi can be obtained by comparing the differential expression genes when they grew in vitro and host insects (Freimoser et al. 2005). Further, we can narrow the DEGs library and obtain contractive virulence genes mainly related to rudimentary destruction of insect cuticle (appressorium and extracellular enzyme) by comparing the differential expression model between the hemocoel Fig. 6 qRT-PCR verification of selected M. rileyi DEGs probably involved in the destruction of insect cuticle injection and topical infection. ...
Insect pathogenic fungi, also known as entomopathogenic fungi, are one of the largest insect pathogenic microorganism communities, represented by Beauveria spp. and Metarhizium spp. Entomopathogenic fungi have been proved to be a great substitute for chemical pesticide in agriculture. In fact, a lot of functional genes were also already characterized in entomopathogenic fungi, but more depth of exploration is still needed to reveal their complicated pathogenic mechanism to insects. Metarhizium rileyi (Nomuraea rileyi) is a great potential biocontrol fungus that can parasitize more than 40 distinct species (mainly Lepidoptera: Noctuidae) to cause large-scale infectious diseases within insect population. In this study, a comparative analysis of transcriptome profile was performed with topical inoculation and hemolymph injection to character the infectious pattern of M. rileyi. Appressorium and multiple hydrolases are indispensable constituents to break the insect host primary cuticle defense in entomopathogenic fungi. Within our transcriptome data, numerous transcripts related to destruction of insect cuticle rather growth regulations were obtained. Most importantly, some unreported ribosomal protein genes and novel unannotated protein (hypothetical protein) genes were proved to participate in the course of pathogenic regulation. Our current data provide a higher efficiency gene library for virulence factors screen in M. rileyi, and this library may be also useful for furnishing valuable information on entomopathogenic fungal pathogenic mechanisms to host.
... Metarhizium anisopliae (10 7 conidia/mL) was cultured in 70 mL basal medium (0.6% NaNO 3 , 0.2% glucose, 0.2% peptone, 0.05% yeast extract w/v) containing 0.7% R. microplus cuticles and 0.05% cholesteryl stearate (RM), as induced infection condition (Beys-da- . This strategy is widely used in studies with M. anisopliae (Beys-da- Silva et al., 2010Freimoser et al.,2005;Manalil et al., 2010). A culture medium containing 1% glucose instead of cuticle and cholesteryl stearate was used as a control (C). ...
Although Metarhizium anisopliae is one of the most studied fungal biocontrol agents, its infection mechanism is far from being completely understood. Using multidimensional protein identification technology (MudPIT), we evaluated the differential secretome of M. anisopliae E6 induced by the host Rhipicephalus microplus cuticle. The proteomic result showed changes in the expression of 194 proteins after exposure to host cuticle, such as proteins involved in adhesion, penetration, stress and fungal defense. Further, we performed a comparative genomic distribution of differentially expressed proteins of the M. anisopliae secretome against another arthropod pathogen, using the Beauveria bassiana ARSEF2860 protein repertory. Among 47 analyzed protein families, thirty were overexpressed in the M. anisopliae E6 predicted genome compared to B. bassiana. An in vivo toxicity assay using a Galleria mellonella model confirmed that the M. anisopliae E6 secretome was more toxic in cattle tick infections compared to other secretomes, including B. bassiana with cattle ticks and M. anisopliae E6 with the insect Dysdereus peruvianus, which our proteomic results had also suggested. These results help explain molecular aspects associated with host infection specificity due to genetic differences and gene expression control at the protein level in arthropod-pathogenic fungi.
... Metarhizium species produce a variety of metabolites belonging to several chemical classes, such as toxins, particularly destruxins (widely studied molecules produced by these fungi), proteases and other enzymes. These substances aid tissue penetration, and are toxic to a wide range of animals and insects as well as to microorganisms such as fungi (Freimoser et al., 2005;Pal et al., 2007;Schrank & Vainstein, 2010). Several authors have described the antagonistic effect of Metarhizium on phytopathogens, both in vitro and in whole plants, such as Collelotrichum graminicola (Lopez, 1991), Fusarium solani f. sp. ...
Phakopsora pachyrhizi causes Asian soybean rust, resulting in severe losses of soybean yield. This study assessed the biocontrol efficacy of cell-free culture filtrates and conidial suspensions of Metarhizium anisopliae (MABR-01) and M. humberi (MHBR-03) against P. pachyrhizi in vitro and in planta. A 50% concentration of culture filtrates of M. anisopliae and M. humberi inhibited the germination of P. pachyrhizi urediniospores by 85 and 96%, respectively, compared to the 50% potato-dextrose broth (PDB) control. The conidial suspensions caused no inhibition of P. pachyrhizi in vitro, but the conidial suspension of M. anisopliae controlled the rust disease in plants with a 51% efficiency. Soybean plants colonised by M. anisopliae MABR-01 and exposed to P. pachyrhizi showed less severe disease in the shoots compared to plants not colonised by M. anisopliae; the disease was reduced by 40%. Spraying plants with 50 and 75% culture filtrates of M. humberi and a 75% concentration of M. anisopliae significantly reduced rust disease, by an average of 86%. Plants pretreated with Metarhizium culture filtrates showed a 30% reduction in P. pachyrhizi colonisation based on qPCR quantification of the pathogen genomic DNA. These results indicate a high biotechnological and sustainable potential of these two Metarhizium species for control of Asian soybean rust.
... Comparing the virulence results of the A. fijiensis GDIZM-1 strain to the nymphs and adults of D. citri, it was found that its insecticidal ability against younger nymphs of D. citri was higher than that against older nymphs and adults, which may be related to the nutrition and structure of the integument, defense mechanism and microflora composition on the body surface at different developmental stages of D. citri [67][68][69]. The nymphs of D. citri have weak activity on twigs or buds and are easily infected by fungal spores. ...
The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the most widespread and devastating pest species in citrus orchards and is the natural vector of the phloem-limited bacterium that causes Huanglongbing (HLB) disease. Thus, reducing the population of D. citri is an important means to prevent the spread of HLB disease. Due to the long-term use of chemical control, biological control has become the most promising strategy. In this study, a novel highly pathogenic fungal strain was isolated from naturally infected cadavers of adult D. citri. The species was identified as Aspergillus fijiensis using morphological identification and phylogenetic analysis and assigned the strain name GDIZM-1. Tests to detect aflatoxin B1 demonstrated that A. fijiensis GDIZM-1 is a non-aflatoxin B1 producer. The pathogenicity of the strain against D. citri was determined under laboratory and greenhouse conditions. The results of the laboratory study indicated that nymphs from the 1st to 5th instar and adults of D. citri were infected by A. fijiensis GDIZM-1. The mortality of nymphs and adults of D. citri caused by infection with A. fijiensis increased with the concentration of the conidial suspension and exposure time, and the median lethal concentration (LC50) and median lethal time (LT50) values gradually decreased. The mortality of D. citri for all instars was higher than 70%, with high pathogenicity at the 7th day post treatment with 1×108 conidia/mL. The results of the greenhouse pathogenicity tests showed that the survival of D. citri adults was 3.33% on the 14th day post-treatment with 1 × 108 conidia/mL, which was significantly lower than that after treatment with the Metarhizium anisopliae GDIZMMa-3 strain and sterile water. The results of the present study revealed that the isolate of A. fijiensis GDIZM-1 was effective against D. citri and it provides a basis for the development of a new microbial pesticide against D. citri after validation of these results in the field.
... Recent research found that high concentrations of Butyl Benzyl Phthalate (BBP) significantly downregulated ribosomal RNA transcription on Chironomus riparius [68]; therefore, ribosomal gene expression in TF6 was inhibited, most likely due to some plant allelochemicals in S. tuberosum. Other studies have reported many insect ribosomal genes to be downregulated, including Metarhizium anisopliae [69], C. suppressalis [50], and Anopheles gambiae [70], in response to unsuitable diets or virus infection. Paracoccus marginatus fed C. papaya grew faster than those fed S. tuberosum (Table 1) [15], indicating that C. papaya is a more suitable plant for P. marginatus. ...
Paracoccus marginatus (Hemiptera: Pseudococcidae) is an invasive pest with a diverse host range, strong diffusion, and high fecundity. It has been observed that P. marginatus feeding on Carica papaya have a higher survival rate, fecundity, and longer lifespan than P. marginatus feeding on Solanum tuberosum, indicating their successful adaptation to C. papaya; however, the mechanisms underlying host plant adaptation remain unclear. Therefore, RNA-seq was performed to study the transcriptional responses of P. marginatus feeding on C. papaya and S. tuberosum plants. A total of 408 genes with significant differential expression were defined; most of them were downregulated in S. tuberosum, including those of digestive enzymes, detoxifying enzymes, ribosomes, and reproductive-related genes, which may result from the adaptation of the host to nutritional needs and changes in toxic chemical levels. Enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes showed that lysosome and longevity regulating pathways related to digestion, detoxification, and longevity were enriched. We suggest that C. papaya is a more suitable host than S. tuberosum, and downregulated target genes may have important effects on the adaptation of P. marginatus to host transfer.
... Lipolytic enzymes including lipases, act primarily on the epicuticle, followed by proteases and chitinases, according to the presence of polymeric substrates in the different portions of the cuticle (Beys da Silva et al., 2010a,b). Among the proteases that can act at this level are subtilisins, trypsins, chymotrypsins, metallopeptidases, aspartyl peptidases, and exopeptidases (Semenova et al., 2020); where their expression from fungi such as M. anisopliae will depend specifically on the composition of the cuticle and hemolymph (Freimoser et al., 2005). EPF such as M. anisopliae and B. bassiana can express up to 11 different subtilisins, one of the most important being the Pr1 subtilisin-like peptidases, which intervene in the arthropod pathogenesis, causing hydrolysis of the cuticle and providing nutrients to the fungus (Gao et al., 2020;Semenova et al., 2020). ...
Ticks are one of the main economic threats to the cattle industry worldwide affecting productivity, health and welfare. The need for alternative methods to control tick populations is prompted by the high prevalence of multiresistant tick strains to the main chemical acaricides and their ecological consequences. Biological control using entomopathogenic fungi (EPF) is one of the most promising alternative options. The objective of this paper is to review the use of EPF as an alternative control method against cattle ticks in Mexico. Metarhizium anisopliae sensu lato (s.l.) and Beauveria bassiana s.l. are the most studied EPF for the biological control of ticks in the laboratory and in the field, mainly against Rhipicephalus microplus ; however, evaluations against other important cattle ticks such as Amblyomma mixtum and R . annulatus , are needed. A transdisciplinary approach is required to incorporate different types of tools, such as genomics, transcriptomics and proteomics in order to better understand the pathogenicity/virulence mechanism in EPF against ticks. Laboratory tests have demonstrated the EPF efficacy to control susceptible and resistant/multiresistant tick populations; whereas, field tests have shown satisfactory control efficiency of M . anisopliae s.l. against different stages of R . microplus when applied both on pasture and on cattle. Epidemiological aspects of ticks and environmental factors are considered as components that influence the acaricidal behavior of the EPF. Finally, considering all these aspects, some recommendations are proposed for the use of EPF in integrated control schemes for livestock ticks.
... Leger et al. 1987, 1988, 1992. The other subtilisins showed differences in regulation that could probably allow these virulence determinants to target different hosts and stages (Freimoser et al. 2005). Using liquid chromatography/tandem mass spectrometry (LC-MS/MS), Santi and co-workers detected carboxypeptidase and Pr1A protease produced by M. anisopliae and induced by the Rhipicephalus microplus cuticle. ...
During its history, humankind has been affected by three factors: food deficiency, health problems, and environmental issues. With world’s population increasing at a high rate, our requirement for food is increasing. Consequently, agricultural practices that maximize crop productivity are necessary. These include the development of new agronomic technologies and new plant varieties, the use of fertilizers, pesticides and herbicides, in order to minimize losses due to plant predators and weeds, respectively. Thus, a continued need for pest management in agriculture became evident, with pressure to efficiently produce more food using less land. To solve this issue, conventional chemical pesticides have been widely used in agriculture despite presenting risks to human health, hazards to the environment as well as affecting non-target species. Therefore, the use of biopesticides is desired due to their target specificity and low environmental damage. They encompass different types of molecules, usually produced by microbial biosynthesis, and are widely used for pest control. Biocontrol, which depends on microorganisms or their products such as hydrolytic enzymes, became a promising alternative to conventional pest control. Microbial hydrolytic enzymes such as proteases, chitinases, lipases, and glucanases are attractive for this purpose, since they present toxic properties, acting synergistically to control pest attacks. Proteases act on the insect cuticles, since proteins constitute the majority of this structure. These enzymes also can act in the insect midgut and hemocoel. Proteases can also be used in the biological control of other noxious agents, such as bacteria, fungi, and nematodes. Chitinases can degrade the peritrophic matrix and cuticle of insects, as well as the fungal cell wall. Lipases hydrolyze lipoproteins, waxes and fats present in the insect integument, causing its disruption. Glucanases affect fungal cell wall development, differentiation, and mycoparasitism, because glucan is a major cell wall component. In this chapter we cover details about enzymes structure, biochemistry, mechanisms of action, applications, and perspectives in this field.
