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Subtilisin gene expression during growth on cuticle from Man. sexta compared with expression during nutrient deprivation. The curves show average expression ratios for different subtilisins in minimal medium (MM) and on Man. sexta cuticle (MC): &, Pr1A; %, Pr1B; #, Pr1I; N , Pr1E; +, Pr1J; n, Pr1D; m, Pr1C; x, Pr1K.
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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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... enzymes. It had been shown previously that total subtilisin activity is produced in response to nutrient deprivation, but that production is enhanced by the addition of cuticle to media (Paterson et al., 1994). Consistent with this, subtilisins Pr1A and Pr1B were upre- gulated on MM, and to a greater extent on insect cuticles (Fig. 5). Increased induction by cuticle compared with nutrient deprivation alone suggests that subtilisin produc- tion is controlled by multiple regulatory systems evoked under different environmental conditions. In contrast, Pr1C, Pr1D, Pr1E, Pr1F, Pr1I and Pr1J were down- regulated at most time-points in MM. Of these, Pr1C and Pr1D were ...
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... 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.
... Two trypsin-related proteases, previously reported to be less efficient in degrading insect cuticles compared to Pr1 (Rosas-García et al., 2014), were noticed and might contribute in the early stages of colonization thus helping invasion (Manalil et al., 2010;St Leger et al., 1987). Taken as a pattern of evolution (Gao et al., 2020) and a potential virulence marker for R. microplus (Perinotto et al., 2014a), the rise in Pr1 expression (Freimoser et al., 2005;Staats et al., 2014), as well as its enzymatic activity over time (Perinotto et al., 2014b), have been demonstrated in in vitro studies. Although Pr1 isoforms are well-known (Andreis et al., 2019), the specific up-regulation of Pr1D and Pr1K observed here endorses their importance during growth and corroborates with the superior Pr1 activities found in our protease assays. ...
... Although Pr1 isoforms are well-known (Andreis et al., 2019), the specific up-regulation of Pr1D and Pr1K observed here endorses their importance during growth and corroborates with the superior Pr1 activities found in our protease assays. These isoforms were also highly expressed in the presence of Manduca sexta cuticle (Freimoser et al., 2005) and, we speculate that MND somehow modulated their increased differential expression. Accordingly, MND might potentially evoke a positive regulation of some specific isoforms, Table 3 Differentially expressed proteins identified in Metarhizium anisopliae s.l. ...
Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38µM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 10⁸ conidia mL⁻¹. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.
... Likewise, the limitation of nutrients in the fungus was decisive to induce conidiation, and affect its virulence. In fact, the nutrient limitation has been described as a strong inducer of the expression of genes involved in sporulation [23,24], in the expression of enzymes involved in virulence, and in nutrient metabolism [7,10,25]. ...
... The results obtained in the present study show that culture media formulations based on insect cuticle (Sph®) and a reduction of nutrients (¼ SDAY) were more convenient than commercial culture media to enhance the pathogenicity of B. bassiana, C. javanica, and M. robertsii; they also turn out to be cheaper, easy to prepare, and with multiple advantages in various fungal applications. Our findings suggest that the culture media provide the nutrients (proteins, chitin, lipids, sugars, and amino acids) in the concentrations required (Sph® 1%) for the development of fungi and the induction of the enzymes (i.e., a twofold increase in Pr1 activity; Supporting Information) involved in their pathogenicity [25]. On the contrary, to make a convincing and reliable choice of the nutrient source, we suggest the use of PCA as an exploratory tool to find a correlation of variables, with which strong selection criteria can be established. ...
Media formulated with insect cuticle (0.5% and 1%; Sphequit Sph®), with a reduction in nutrients (¼ Sabouraud dextrose agar + yeast [SDAY]) and commercial media (potato dextrose agar, Sabouraud dextrose agar) were evaluated for the cultivation of Beauveria bassiana, Cordyceps javanica (Isaria javanica [Bally] Samson & Hywel‐Jones), and Metarhizium robertsii. By using principal component analysis, it was determined that the ¼ SDAY and Sph formulations have greater advantages than commercial media for the development of fungi. The ¼ SDAY and Sph (0.5% and 1%) improved hydrophobicity, radial growth rate, germination, conidia yield, and virulence in B. bassiana; in M. robertsii, they favored conidia yield, germination, and virulence, and in C. javanica, the ¼ SDAY and Sph 0.5% media enhanced conidia yield, germination, radial growth rate, and virulence. We suggest that these formulations are an alternative to commercial culture media as they are cheaper and appropriate to improve the growth characteristics and virulence of the three strains evaluated. Some applications of culture media are suggested, and the importance of multivariate analysis as an exploratory tool to carry out the choice of culture media in a suitable way for the development of mycoinsecticides is also discussed.
... The large secretomes of insect pathogens probably reflect the many microhabitats they must adapt to in insecta, including the cuticle and the haemolymph, as well as additional environmental habitats in the soil and with plants. These complex lifestyles are reflected in transcriptional reprogramming involving hundreds of differentially expressed genes as Metarhizium strains rapidly adapt to host cuticles, haemolymph or root exudate [61,62]. The ability to recognize appropriate hosts, and penetrate their cuticle, are among the necessary steps for the transition from either saprophyte or root colonizer to pathogen. ...
... The ability to recognize appropriate hosts, and penetrate their cuticle, are among the necessary steps for the transition from either saprophyte or root colonizer to pathogen. After sensing an appropriate host, Metarhizium adapts to it by formation of infection structures and secretion of different specific effector cocktails, i.e. appressoria and a first set of effectors for the penetration stage, followed by blastospores and a different sets of organ-specific effectors for infecting insect haemolymph and tissues [62]. Many genes associated with the lifestyle royalsocietypublishing.org/journal/rsob Open Biol. ...
... Similarly, analysis of the other large family of proteases, the subtilisins, revealed that one enzyme (Pr1A) was induced by nutrient deprivation, whereas other subtilisins are induced at different times as the cuticle is colonized [62]. The subtilisins differ in their secondary substrate specificities, adsorption properties to cuticle and alkaline stability [85]. ...
The genus Metarhizium and Pochonia chlamydosporia comprise a monophyletic clade of highly abundant globally distributed fungi that can transition between long-term beneficial associations with plants to transitory pathogenic associations with frequently encountered protozoans, nematodes or insects. Some very common ‘specialist generalist’ species are adapted to particular soil and plant ecologies, but can overpower a wide spectrum of insects with numerous enzymes and toxins that result from extensive gene duplications made possible by loss of meiosis and associated genome defence mechanisms. These species use parasexuality instead of sex to combine beneficial mutations from separate clonal individuals into one genome (Vicar of Bray dynamics). More weakly endophytic species which kill a narrow range of insects retain sexuality to facilitate host–pathogen coevolution (Red Queen dynamics). Metarhizium species can fit into numerous environments because they are very flexible at the genetic, physiological and ecological levels, providing tractable models to address how new mechanisms for econutritional heterogeneity, host switching and virulence are acquired and relate to diverse sexual life histories and speciation. Many new molecules and functions have been discovered that underpin Metarhizium associations, and have furthered our understanding of the crucial ecology of these fungi in multiple habitats.
... seem to fulfill most of the requirements in an ecofriendly agricultural system. However, some enzymatic differences have been found in alcohol dehydrogenase, acetyl CoA synthetase, pyruvate decarboxylase, and aldehyde dehydrogenase between Trichoderma reesei and the entomopathogenic fungus, Metarhizium anisopliae [40]. As the metabolic engineering seems rather difficult and time-consuming, the superior isolates of Trichoderma spp. ...
The current world of agriculture suffers from many limitations and limiting factors, and Trichoderma fungi as biologically beneficial agents can play considerable roles in soil and crop residue management, biological control of plant diseases and pests even in the niches hardly accessible by chemical pesticides, agricultural environment maintenance, as well as plant growth and yield promotion. Therefore, the creation of Trichoderma fungi via genetic transformation of superior isolates by the genetic constructs for the expression and secretion of insect-specific insecticidal peptides (Trichodermans) can improve their effectiveness in the control of insect pest targets.
... seem to fulfill most of the requirements in an ecofriendly agricultural system. However, some enzymatic differences have been found in alcohol dehydrogenase, acetyl CoA synthetase, pyruvate decarboxylase, and aldehyde dehydrogenase between Trichoderma reesei and the entomopathogenic fungus, Metarhizium anisopliae [40]. As the metabolic engineering seems rather difficult and time-consuming, the superior isolates of Trichoderma spp. ...
... It was stated that the secretion of protease, chitinase, and lipases by M. anisopliae are associated with the process of infection [37]. These enzymes are considered to assist host tissue penetration by degrading the host's exterior layers (cuticles of nematode, insects, and cell wall of fungi) or utilizing the host's proteins for nutrition [38]. For example, previous studies reported that chitinase from Trichoderma harzianum and Clonostachys rosea enhanced the inhibition of B. cinerea [39][40][41][42]. ...
Gray mold disease caused by Botrytis cinerea is a devastating disease that leads to serious financial loss. In this study, the entomopathogenic fungus Metarhizium anisopliae that acts against the gray mold pathogen B. cinerea was evaluated. M. anisopliae produced a significant inhibition zone in front of the B. cinerea colony in the dual culture test. In addition, volatile organic compounds generated by M. anisopliae were shown to have an inhibitory effect on B. cinerea mycelia growth and reduced 41% of gray mold severity of postharvest tomatoes. The 10% concentration of the culture filtrate of M. anisopliae inhibited 88.62% of colony radial growth as well as 63.85% of sclerotia germination and all conidia germination of B. cinerea. Furthermore, the culture filtrate of M. anisopliae retained its inhibitory effect against the radial growth of B. cinerea even after heating for 15 min at 100 °C. Feasible mechanisms of M. anisopliae involved in the control of B. cinerea were explored, and it was demonstrated that the plasma membrane of B. cinerea conidia was damaged by the product of metabolism of M. anisopliae. In addition, after treating with culture filtrate of M. anisopliae, the B. cinerea phenotype was shown to be abnormal, and cell organelles of B. cinerea mycelia were damaged significantly. A significant control efficacy of M. anisopliae against tomato gray mold was detected on both the detached leaf assay (84.24%) as well as the whole plant (72.38%). In addition, a 78% reduction in tomato fruit mold was detected at a 10% treated concentration of M. anisopliae. These findings suggest that M. anisopliae possesses potential as a biocontrol agent against tomato gray mold in the greenhouse and during the postharvest stage.
