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Transformants of Metarhizium anisopliae sf. anisopliae Overexpressing Chitinase from Metarhizium anisopliae sf. acridum Show Early Induction of Native Chitinase but Are Not Altered in Pathogenicity to Manduca sexta
Abstract
Extracellular chitinase activity has been implicated in the pathogenesis of several fungal infections. Following induction with chitin, the insect pathogens Metarhizium anisopliae sf. acridum ARSEF strain 324 and Metarhizium anisopliae sf. anisopliae ARSEF strain 2575 secrete 44-kDa basic and acidic isoforms of endochitinase, respectively. The gene from strain 324 (Chit1) was cloned and inserted into the genome of strain 2575 under the control of Aspergillus regulatory elements such that transgenic 2575 (2575-Chit(+)) expressed CHIT1 in a noninducing medium (i.e., not containing chitin). Isoelectric focusing followed by a zymogram technique revealed that neither wild-type 2575 nor 2575-Chit(+) produced significant amounts of the native 2575 acidic chitinase in a noninducing medium. However, in a chitin-containing medium, 2575-Chit(+) produced the native chitinase earlier than strain 2575, soon after secretion of CHIT1. We hypothesize that this is due to the production of soluble inducers following chitin hydrolysis by CHIT1 and that M. anisopliae uses enzymes expressed at low levels to sense the nature of the polymeric nutrient present in the immediate environment. However, the chitinase overproducers did not show altered virulence to caterpillars (Manduca sexta) compared to the wild-type fungus, suggesting that wild-type levels of chitinase are not limiting for cuticle penetration.
... However, the contribution of specific chitinases, particularly within the context of insect virulence, remains unclear. Overexpression of the chit1 chitinase in B. bassiana resulted in increased insect virulence (Fang et al. 2005); however, similar experiments overexpressing Machit1 (from M. acridum) in M. roberstii (formerly anisopliae) did not affect virulence, suggesting different limiting factors and differential enzymatic contributions between various insect pathogenic fungi (Screen et al. 2001). ...
... Indeed, combining chitinase-protease activities or even the addition of a chitin-binding domain to cuticle degrading proteases has been shown to increase their effectiveness in entomopathogenic fungi Fang et al. 2009). However, the effects of specific chitinases on these organisms have sometimes been obscure, and few studies have characterized the effects of loss of a chitinase on virulence, although several have examined the effects of overexpression (Fang et al. 2005;Screen et al. 2001). The Chit30 cuticle-induced chitinase in M. anisopliae has been linked to heat shock stress and virulence (da Silva et al. 2005;Staats et al. 2013), and alternative splicing of the M. anisopliae chi2 gene suggests the possibility to produce different chitinase isoforms, although the physiological consequences of this remain unknown (Baratto et al. 2006;Boldo et al. 2010). ...
The filamentous fungus, Isaria fumosorosea, is a promising insect biological control agent. Chitinases have been implicated in targeting insect cuticle structures, with biotechnological potential in insect and fungal control. The I. fumosorosea chitinase gene, Ifchit1, was isolated and determined to encode a polypeptide of 423 amino acids (46 kDa, pI = 6.53), present as a single copy in the I. fumosorosea genome. A split marker transformation system was developed and used to construct an Ifchit1 gene knockout. The ΔIfchit1 strain displayed minor alterations in mycelial growth on diverse media at 26 °C compared to the wild type and complemented (ΔIfchit1::Ifchit1) strains; however, colony morphology was affected, and the mutant strain had a temperature sensitive phenotype (32 °C). Although sporulation was delayed for the mutant, overall conidial production was almost twice than that of wild type. Biochemical assays indicated decreased chitinase activity during growth in Czapek-Dox liquid media for the ΔIfchit1 strain. Insect bioassays using diamondback moth, Plutella xylostella, larvae revealed decreased infectivity, i.e., increased LC
50
(threefold to fourfold) and a significantly delayed time to death, LT
50
from 3 to 6 days, for the ΔIfchit1 strain compared to the wild type and complemented strains. These data indicate an important role for the Ifchit1 chitinase as a virulence factor in I. fumosorosea.
... Chitinases are cuticle-degrading enzymes [1,2,3] and have been reported as pathogenicity determinants in fungi [4]. Chitinase gene has been widely studied because of its characteristic feature of degrading chitin which is a chain homopolymer of N-acetylglucosamine connected by α 1, 4 glucosidic linkages [5]. ...
... These fungi are reported to produces cuticle degrading proteins such as chitinases, proteases, and lipases [4,9,6]. Investigations on secretions of Beauveria bassiana reported production of multiple chitinases that possess different functions [1,2,3]. Difference in the functions of these chitinases suggests difference in their structure, substrate specificity or catalytic mechanism. ...
Chitinases occur in a wide range of organisms including bacteria, fungi, plants, insects, and animals. Chitinase has been widely used as one of the mycobiocontrol agent as it degrades chitin which is a chain homopolymer of N- acetylglucosamine (GlcNAc) connected by β-1,4 glucosidic linkages. Beauveria bassiana is an insect pathogenic fungus successfully used as an insect pest control agent worldwide. In this paper, 21 different chitinase, endochitinase and chit proteins / gene retrieved from Swiss-Prot database are analysed and characterized. Various Bioinformatics and molecular modeling approach were adopted to explore properties and structure of chitinase gene in Entomopathogenic fungi. Primary structure analysis predicted the physico-chemical properties such as pI, EC, AI, GRAVY and instability index and provides data about these proteins and their properties. Subcellular localization were predicted by MultiLoc software. Prediction of motifs, patterns, disulfide bridges and secondary structure were performed for functional characterization. Three dimensional structures for chitinase like proteins are not available as yet at PDB. Therefore, homology models for were developed. The modelling of the three dimensional structure of these proteins shows that models generated by Modeller were more acceptable in comparison to that by Geno3D and Swiss Model.
... Metarhizium proteases, including Pr1, are induced by protein-degradation products (29), which could result from the unique specificities of MAA_00986 and MAA_01413. Similarly, indirect induction of chitinases by the HGT proteases could follow proteolysis of the protein matrix covering cuticular chitin; the exposed chitin induces the expression of chitinases (30). Therefore, the three HGT proteases in M. robertsii are integrated into the complex cuticle-degrading enzyme system and influence the expression of other enzymes in the system. ...
Fungi are central to every terrestrial and many aquatic ecosystems, but the mechanisms underlying fungal tolerance to mercury, a global pollutant, remain unknown. Here, we show that the plant symbiotic fungus Metarhizium robertsii degrades methylmercury and reduces divalent mercury, decreasing mercury accumulation in plants and greatly increasing their growth in contaminated soils. M. robertsii does this by demethylating methylmercury via a methylmercury demethylase (MMD) and using a mercury ion reductase (MIR) to reduce divalent mercury to volatile elemental mercury. M. robertsii can also remove methylmercury and divalent mercury from fresh and sea water even in the absence of added nutrients. Overexpression of MMD and MIR significantly improved the ability of M. robertsii to bioremediate soil and water contaminated with methylmercury and divalent mercury. MIR homologs, and thereby divalent mercury tolerance, are widespread in fungi. In contrast, MMD homologs were patchily distributed among the few plant associates and soil fungi that were also able to demethylate methylmercury. Phylogenetic analysis suggests that fungi could have acquired methylmercury demethylase genes from bacteria via two independent horizontal gene transfer events. Heterologous expression of MMD in fungi that lack MMD homologs enabled them to demethylate methylmercury. Our work reveals the mechanisms underlying mercury tolerance in fungi, and may provide a cheap and environmentally friendly means of cleaning up mercury pollution.
... Esto posiblemente fue debido a la concentración de herbicida utilizada para la selección de los transformantes. Otros autores describen el uso de diferentes concentraciones de glufosinato de amonio para selección de transformantes (Cantone y Vandenberg 1999a, b;Góngora 2004;Screen et al. 2001). Esto conlleva a establecer que la concentración mínima inhibitoria de herbicida varía de acuerdo con la especie y probablemente la concentración mínima inhibitoria de glufosinato de amonio para selección de transformantes de Bb9205 monoespórica es una concentración intermedia entre 25 y 50 µg/ml. ...
Beauveria bassiana cepa Bb9205 fue transformada con genes de proteasas tipo subtilinas (pr1A, pr1J) y esterasa (ste1) aislados de Metarhizium anisopliae que están involucrados en la patogenicidad contra insectos. El objetivo del experimento fue aumentar la patogenicidad de la cepa contra la broca del café, Hypothenemus hampei. Para esto, se clonaron los genes en el plásmido pBarGPE1 que confiere resistencia al herbicida glufosinato de amonio. Se obtuvo un cultivo monoespórico de la cepa Bb9205, cuya patogenicidad fue del 78% de mortalidad contra H. hampei, usando 1 x 106 esporas/ml. Con este monoespórico se produjeron protoplastos. Con respecto a la producción de protoplastos se optimizó la metodología obteniendo una concentración de 3 x 107 protoplastos/ml después del pre-tratamiento enzimático, durante 4 horas en agitación lenta a 37°C. Se realizó la transformación con los plásmidos pBarGPE1-pr1A, pBarGPE1-pr1J y pBarGPE1-ste1 mediante los métodos de PEG y electroporación. Dos transformantes con el gen pr1A y uno con el gen ste1 resistentes a 25 µg/ml del herbicida glufosinato de amonio exhibieron incremento de actividad proteolítica y esterolítica, respectivamente. Se evaluó, en pruebas de laboratorio, la patogenicidad de las cepas de B. bassiana transformadas comparándolas con la cepa Bb9205 no transformada monoespórica. La expresión constitutiva de la proteasa en la cepa transgénica Bb9205-pr1A mejoró su actividad insecticida al demostrar un incremento de mortalidad del 21,7% y una disminución del 14,3% en el tiempo de mortalidad sobre la broca del café. El transformante Bb9205-ste1 disminuyó en un 9,5% el tiempo de mortalidad sobre broca al compararse con Bb9205 sin transformar. Los resultados indican que con el mejoramiento de cepas de Beauveria, por medio de transformación genética, se puede lograr un control biológico mejor.
... Chitinase Baratto et al. (2003Baratto et al. ( , 2006, Screen et al. (2001) ...
Entomopathogenic fungi are microorganisms capable of infecting and killing arthropods and therefore have a great potential in pest management. As the extensive use of synthetic pesticides has led to increased resistance in insects, decreased natural enemies, and had negative impacts on environmental and human health, the search for eco-friendly control agents is urgent. Entomopathogenic fungi are promising alternatives in this regard and are attracting global attention, with increasing efforts and financial investments being made for the development, commercialization and use of fungus-based control products. Despite scientific and technological advances, there is still a need for studies to expand the number of species applicable in pest management and improve their performance in the field. There is also a need to increase user awareness regarding their correct use with the aim to establish their widespread adoption and market potential. This chapter covers the main taxonomic groups that comprise entomopathogenic fungi, their modes of action to establish insect infection and spread, and the insect’s defense mechanisms against these fungi. Furthermore, techniques of fungal isolation, selection, and production are discussed. The usage status, challenges, and prospects of mycoinsecticides are also addressed, highlighting their application potential for sustainable agricultural production.
... Chitinase Baratto et al. (2003Baratto et al. ( , 2006, Screen et al. (2001) ...
Biopesticides, using living microbial bodies and their bio-active composites against insects, are potential replacements for synthetic insecticides for safer and modern food production systems. Entomopathogenic bacteria (EPB) are important biological control agents of insect pests since the last century. Though bacterial species have been documented to be used against insects for developing symbiotic relationships, only a few of them are identified as entomopathogens. Most of these are members of the family Bacillaceae, Enterobacteriaceae, Pseudomonadaceae, Clostridiaceae, and Neisseriaceae. More than 100 bacterial species have been reported to infect various arthropods. Bacillus thuringiensis (Bt), B. sphaericus, B. cereus, and B. popilliae are the most appreciated microbial pest control agents. However, new bacterial species also need to be explored for their entomopathogenic role and materialized as new biopesticide products. The commercial biopesticides based on novel EPBs with improved genetic materials must be a part of future research for effective integrated pest management programs. This present chapter highlights the classification, infection, replication, transmission mechanisms, and important EPB in integrated pest management.
... Chitinase Baratto et al. (2003Baratto et al. ( , 2006, Screen et al. (2001) ...
The successful control of many insect-pests makes entomopathogenic nematodes (EPNs) among one of the best biocontrol agents for insect pests. Moreover, the ability of EPNs to seek out their hosts and kill them in those habitats where chemicals fail makes them even more attractive. The EPNs-bacterial mutualistic association helps them kill their hosts in a relatively shorter period than other necromenic or parasitic nematode associations. In addition to this end-user safety, hotspot application which allows minimizing treated area, natural enemies’ safety, withholding period absence, and environmental protection are a few of many advantages over chemical pesticides. Two important genera of EPNs, i.e., Heterorhabditid and Steinernematids, are associated with symbiotic bacteria Photorhabdus and Xenorhabdus, respectively, while bacterial symbiont of neosteinernamatids is yet to be described. About 21 species of Heterorhabditis and 100 species of Steinernema have been isolated and identified worldwide. With the increasing environmental concerns and low efficacy of synthetic pesticides, agriculturists and researchers have a growing interest in finding alternatives to synthetic pesticides. Several EPNs can be widely used in place of synthetic pesticides in agro-ecosystem. There is still a need to improve several aspects of EPNs, such as efficacy and efficiency, reduced costs, mass production, and formulation technology. Furthermore, their potential for recycling in the host population beckons them to be further exploited for sustainable pest control. This chapter will emphasize the use and potential of EPNs as an integral part of integrated pest management. To aid with understanding the potential of EPNs, this chapter will also provide an overview of ecology and biology, mass production, application strategies, and integration with other management tools.
... Chitinase Baratto et al. (2003Baratto et al. ( , 2006, Screen et al. (2001) ...
Modern agricultural production is dominated by the use of synthetic chemical pesticides, which account for 95% of the global market share of total pesticide use. However, this over-reliance on synthetic pesticides adversely affects and interferes with the functioning of the ecosystem. Neem (Azadirachta indica), a botanical biopesticide widely known for its bactericidal, fungicidal, insecticidal, herbicidal, and nematicidal properties, offers an eco-friendly alternative to synthetic pesticides. To date, more than 200 bioactive compounds have been extracted from neem, and several commercial formulations have been developed and registered as broad-spectrum biopesticides. More advanced strategies in the use of neem as a botanical biopesticide have been developed with a focus on developing more innovative and effective approaches. This chapter also covers current advancement on neem bioactive ingredients, their efficacy and extraction methods. In addition, stability of the bioactive compounds and environmental, health and safety issues are discussed.
... Chitinase Baratto et al. (2003Baratto et al. ( , 2006, Screen et al. (2001) ...
The growing concern over potential hazards from chemical pesticide safety among consumers and potential harm to the environment has culminated in consideration of natural management strategies of pests. Because they are complementary to most crop production systems, biopesticides based on plants can be integrated into pest management systems. Plant essential oils (EOs) can replace the more persistent non-natural pesticides in protecting the environment from the accumulation of chemicals reduce resistance and increase crop productivity. In addition, they possess low mammalian toxicity, broad-spectrum activity, and degrade rapidly in foodstuffs. In addition to exhibiting distinctive properties compared with synthetic pesticides, including high levels of pest toxicity and reduced toxicity toward non-target organisms, EOs possess contact, feeding deterrence, fumigant toxicity, oviposition, and repellent properties. In this chapter, we review the sources of EOs, their insecticidal activities, constituents, and mode of action and discuss their synergism and formulation with encapsulation for producing nanoinsecticidal products.
... Samson (Hypocreales: Clavicipitaceae), M. anisopliae, and V. lecanii have been identified with protein degrading enzymes (Sheng et al. 2006). B. bassiana has subtilisin-like serine endoprotease (Pr1 and Pr1B) and M. anisopliae has chymotrypsin (CHY1) (Screen et al. 2001). St Leger et al. (1996) constructed an engineered mycoinsecticides based on M. anisopliae by over-expressing the Pr1 toxic protease from M. anisopliae genome. ...
Background: Apprehensions about the safety and the environment regarding the insecticidal application against insect infestations have directed our attention toward advancement of biological mediators so that they are assimilated into the concept of integrated pest management stratagems to develop a more practical approach for the management of insect pests. Management of insect pests by making use of biological approaches (such as fungal entomopathogens (EPF) or others which are antagonistic to insect population) provides a substitute approach which reduces the continuous use of chemical amalgams against insect pests.
Main body: The present review provides a framework of the present status of information on EPF as it identifies with their current use as biological control of pest infestations. To utilize a variety of biological control methodologies against insect hosts, it is essential to improve our comprehension of the ecology of EPF and also their role in nature. This article may assist us with understanding the virulence and the virulence factors related with EPF and present the latest developments and accomplishments in the significant field. We focus on recent instances of studies that show the overall patterns in interactions among insect pests and EPF prompting the advancement of epizootics. Also, we sum up the topical discoveries on current status of mycoinsecticides and propose future research needs.
Conclusions: As the current mechanism of fungal pathogenesis on insects is moderately slow and needs improvement, there is likewise the requirement for additional comprehension of the interactions among entomopathogens and insect pests so as to grow soundly planned procedures by identifying potential targets and via the improvement of fungal strains for improving the adequacy of these organisms in field applications.
... Several assays of protease and chitinase activity of B. bassiana and Paecilomyces farinosus, N. rileyi indicated a correlation between the amount of secreted enzymes and the rate of virulence of individual isolates (Samsinakova et al., 1977;El-Sayed et al., 1989;Bidochka and Khachatourians, 1990;Havukkala et al., 1993;El-Sayed et al., 1993a;Barros et al., 2000). No correlation, however, was apparent between enzyme activity (lipolytic, proteolytic or chitinolytic) and pathogenicity of isolates of B. bassiana and M. anisopliae (Screen et al., 2001;Vergas et al., 12 2003). ...
The present study focuses on fungal extracellular enzymes as factors affecting fungal parasitism on the cotton leafworm, Spodoptera littoralis, especially with some new strains of entomopathogenic fungi such as Beauveria bassiana 1513, B. bassiana 3286, Paecilomyces fumosoroseus 2734 and Nomuraea rileyi 1972. Our results showed a significant increase in mortality 24 h after injection of fungi and tended to increase until pupation. The 6 th instar larvae were more susceptible to N. rileyi 1972, followed by B. bassiana 3286, and then P. fumosoroseus 2734. The larvae were least susceptible to B. bassiana 1513. At a dose of 9000 bl/insect, the median lethal times of the tested fungal isolates were between 36.5-51 h. Fungi were able to produce detectable amounts of proteases, chitinases and lipases. The activity of proteases was strong along with weak activities of chitinases and lipases. Interestingly, there appeared to be no clear correlation between pathogenicity and activity of extracellular enzymes. The extracellular enzymes always act as a unit, and everyone alone can not permit to the fungus penetration into the host. So, virulence may be the result of a set of enzymes acting together.
... Several assays of protease and chitinase activity of B. bassiana and Paecilomyces farinosus, N. rileyi indicated a correlation between the amount of secreted enzymes and the rate of virulence of individual isolates (Samsinakova et al., 1977;El-Sayed et al., 1989;Bidochka and Khachatourians, 1990;Havukkala et al., 1993;El-Sayed et al., 1993a;Barros et al., 2000). No correlation, however, was apparent between enzyme activity (lipolytic, proteolytic or chitinolytic) and pathogenicity of isolates of B. bassiana and M. anisopliae (Screen et al., 2001;Vergas et al., 12 2003). ...
The present study focuses on fungal extracellular enzymes as factors affecting fungal parasitism on the cotton leafworm, Spodoptera littoralis, especially with some new strains of entomopathogenic fungi such as Beauveria bassiana 1513, B. bassiana 3286, Paecilomyces fumosoroseus 2734 and Nomuraea rileyi 1972. Our results showed a significant increase in mortality 24 h after injection of fungi and tended to increase until pupation. The 6th instar larvae were more susceptible to N. rileyi 1972, followed by B. bassiana 3286, and then P. fumosoroseus 2734. The larvae were least susceptible to B. bassiana 1513. At a dose of 9000 bl/insect, the median lethal times of the tested fungal isolates were between 36.5-51 h. Fungi were able to produce detectable amounts of proteases, chitinases and lipases. The activity of proteases was strong along with weak activities of chitinases and lipases. Interestingly, there appeared to be no clear correlation between pathogenicity and activity of extracellular enzymes. The extracellular enzymes always act as a unit, and everyone alone can not permit to the fungus penetration into the host. So, virulence may be the result of a set of enzymes acting together.
... Metarhizium proteases, including Pr1, are induced by protein-degradation products (29), which could result from the unique specificities of MAA_00986 and MAA_01413. Similarly, indirect induction of chitinases by the HGT proteases could follow proteolysis of the protein matrix covering cuticular chitin; the exposed chitin induces the expression of chitinases (30). Therefore, the three HGT proteases in M. robertsii are integrated into the complex cuticle-degrading enzyme system and influence the expression of other enzymes in the system. ...
Significance
Recently emerged fungal diseases are contributing toward global declines in wildlife, but the mechanisms for emergence of new pathogenic fungi remain mysterious. The entomopathogen Metarhizium robertsii evolved from plant symbionts, and here we report that this host shift was facilitated by 18 horizontal gene transfers (HGTs). The necessity of breaching cuticular barriers selected for retention of these genes, as 12 are up-regulated during penetration, and their collective actions are indispensable for infection by processes including degradation of procuticular proteins and utilization of epicuticular lipids. Five of the most recently acquired HGT genes contributed to host-range expansion in some late-evolving species. Our work reveals that HGT was a key mechanism for emergence and host-range expansion of fungal entomopathogens.
... Cumulative evidence suggests that genes involved in the specificity of some entomopathogens to a narrow range of insects could control adhesion to the cuticle surface; exploitation of cuticle surface conditions (nutrients, humidity, specific recognition factors); ability to overcome structural and chemical barriers to penetration; and toxin production (48,51,69). Much research has been directed at identifying determinants of specificity and virulence, because a major goal in the development of fungal insect pathogens as biocontrol agents is to be able to control these parameters. ...
The Insect Pathogens, Page 1 of 2
Abstract
Fungi are the most common disease-causing agents of insects; aside from playing a crucial role in natural ecosystems, insect-killing fungi are being used as alternatives to chemical insecticides and as resources for biotechnology and pharmaceuticals. Some common experimentally tractable genera, such as Metarhizium spp., exemplify genetic diversity and dispersal because they contain numerous intraspecific variants with distinct environmental and insect host ranges. The availability of tools for molecular genetics and multiple sequenced genomes has made these fungi ideal experimental models for answering basic questions on the genetic and genomic processes behind adaptive phenotypes. For example, comparative genomics of entomopathogenic fungi has shown they exhibit diverse reproductive modes that often determine rates and patterns of genome evolution and are linked as cause or effect with pathogenic strategies. Fungal-insect pathogens represent lifestyle adaptations that evolved numerous times, and there are significant differences in host range and pathogenic strategies between the major groups. However, typically, spores landing on the cuticle produce appressoria and infection pegs that breach the cuticle using mechanical pressure and cuticle-degrading enzymes. Once inside the insect body cavity, fungal pathogens face a potent and comprehensively studied immune defense by which the host attempts to eliminate or reduce an infection. The Fungal Kingdom stands alone in the range, extent, and complexity of their manipulation of arthropod behavior. In part, this is because most only sporulate on cadavers, so they must ensure the dying host positions itself to allow efficient transmission.
... anisopliae, M. anisopliae var. acridum, Verticillium lecanii y Paecilomycesfumosoroseus (Jackson et al. 1985;St Leger et al. 1996;Bidochka et al. 1999;Screen et al. 2001;García-Gutiérrez 2005;Ayala-Zermeño 2006;Castellanos-Moguel 2006a) . El estudio de estas enzimas ha permitido no sólo entender la estructura de la cutícula y la forma en que ocurre su degradación, sino también seleccionar aislados por sus propiedades virulentas (St. ...
Esta obra presenta una serie de trabajos enfocados en la relación existente entre la estructura y funciona-miento de los ecosistemas y temas como la ecología microbiana, las bacterias microaeróbicas, el fitoplancton, los hongos entomopatógenos, la capacidad osmorreguladora en crustáceos y moluscos y el cambio global del clima
... It was also proved that with protease Pr1, improved chitinase expression can be attempted through genetic engineering. For example, the chit1 gene of M. anisopliae was over expressed but with no increase in the virulence against Manduca sexta [108]. An alteration in the spores, hyphae and appressorium has been observed when a constitutive promoter is used for overexpression of the chit2 gene of M. anisopliae [109]. ...
Entomopathogenic fungi are well-known biological control agents of insects that have broadly replaced the chemicals used in biopesticides for agricultural purposes. The pathogenicity of entomopathogenic fungi depends on the ability of its enzymatic equipment, consisting of lipases, proteases and chitinases, which degrade the insect's integument. Additionally, the researchers studied the content of β-galactosidase, l-glutaminase, and catalase within entomopathogenic fungi. With highly focused investigations on the use of enzymes for green technology, the group of entomopathogens are slowly gaining applications in these areas, even as phytopathogenic fungi (disease originator). This brief review will serve as a reference of the enzymes derived from entomopathogenic fungi and of their current and potential applications.
... The recombinant strain displayed significantly lower values of LC 50 and LT 50 (lethal time necessary for 50% mortality), when compared to the non-transformed (wild-type) strain and, therefore, Bbchit1 provided increased virulence. By contrast, manipulating the expression of a similar chitinase gene in M. anisopliae did not result in increased virulence in the host Manduca sexta (Screen et al., 2001). The M. anisopliae Chit1 chitinase gene was cloned from two different isolates and found to encode 44 kDa proteins differing in isoelectric points. ...
The insect exoskeleton is present as a rigid structure above a monolayer of epidermal cells and together both form the integument. The cuticle is composed of an outer, multilayered epicuticle and an inner procuticle containing the chitinous exo- and endo- cuticle. The non-living cuticle is replaced during each instar to accommodate growth and development by sequentially degrading the old cuticle and replacing it with a new one. The entire molting cycle and the shedding of the old cuticle are precisely regulated by a series of endocrine and neuroendocrine cues. This chapter addresses the various cuticular components, cuticular metabolism and cuticular biogenesis regulatory systems that have been targeted for pest management. Major classes of synthetic compounds, including benzoylphenyl ureas, benzoyl hydrazines and etoxazole that target chitin synthesis and the molting cycle are presented. In addition, we focus on recent developments toward the targeting of additional components of the cuticle or processes of cuticle biogenesis that may find future applications in pest management.
... Thus, this hybrid gene improved the virulence of the fungus to a greater extent than overexpression of either Pr1A or Bbchit1, presumably as a result of the composite structure of the insect cuticle, which contains chitin embedded in protein. WT strains of entomopathogenic fungi need to digest cuticular proteins first in order to expose the chitin and induce the expression of fungal chitinases (Screen, Hu, & St. Leger, 2001;St. Leger, Joshi, Bidochka, & Roberts, 1996b). ...
Entomopathogenic fungi have been developed as environmentally friendly alternatives to chemical insecticides in biocontrol programs for agricultural pests and vectors of disease. However, mycoinsecticides currently have a small market share due to low virulence and inconsistencies in their performance. Genetic engineering has made it possible to significantly improve the virulence of fungi and their tolerance to adverse conditions. Virulence enhancement has been achieved by engineering fungi to express insect proteins and insecticidal proteins/peptides from insect predators and other insect pathogens, or by overexpressing the pathogen's own genes. Importantly, protein engineering can be used to mix and match functional domains from diverse genes sourced from entomopathogenic fungi and other organisms, producing insecticidal proteins with novel characteristics. Fungal tolerance to abiotic stresses, especially UV radiation, has been greatly improved by introducing into entomopathogens a photoreactivation system from an archaean and pigment synthesis pathways from nonentomopathogenic fungi. Conversely, gene knockout strategies have produced strains with reduced ecological fitness as recipients for genetic engineering to improve virulence; the resulting strains are hypervirulent, but will not persist in the environment. Coupled with their natural insect specificity, safety concerns can also be mitigated by using safe effector proteins with selection marker genes removed after transformation. With the increasing public concern over the continued use of synthetic chemical insecticides and growing public acceptance of genetically modified organisms, new types of biological insecticides produced by genetic engineering offer a range of environmentally friendly options for cost-effective control of insect pests.
... Overexpression of extracellular chitinase has also been demonstrated for M. anisopliae var. acridum, but did not alter virulence to the caterpillar, M. sexta, compared to the wild-type fungus (Screen et al., 2001). 11.6.3.3. ...
... As M. robertsii breaches the epicuticle, it produces proteolytic and chitinolytic enzymes to degrade the protein-chitin procuticle (St Leger et al., 1998;Screen et al., 2001), which are downregulated when the fungus reaches the haemolymph. Among the 849 genes that were downregulated in WT, 19 GO (Gene Ontology) terms were enriched, including four in biological process, 14 in molecular function and one in cellular component ([FDR (False Discovery Rate) < 0.05]) ( Fig. 4C and D). ...
Metarhizium robertsii has been used as a model to study fungal pathogenesis in insects, and its pathogenicity has many parallels with plant and mammal pathogenic fungi. MAPK cascades play pivotal roles in cellular regulation in fungi, but their functions have not been characterized in M. robertsii. In this study, we identified the full complement of MAPK cascade components in M. robertsii and dissected their regulatory roles in pathogenesis, conidiation and stress tolerance. The 9 components of the Fus3, Hog1 and Slt2-MAPK cascades are all involved in conidiation. The Fus3- and Hog1-MAPK cascades are necessary for tolerance to hyperosmotic stress, and the Slt2- and Fus3-MAPK cascades both mediate cell wall integrity. The Hog1 and Slt2-MAPK cascades contribute to pathogenicity; the Fus3-MAPK cascade is indispensable for fungal pathogenesis. During its life cycle, M. robertsii experiences multiple microenvironments as it transverses the cuticle into the hemocoel. RNA-seq analysis revealed that MAPK cascades collectively play a major role in regulating the adaptation of M. robertsii to the microenvironmental change from the cuticle to the hemolymph. The 3 MAPKs each regulate their own distinctive subset of genes during penetration of the cuticle and hemocoel colonization, but they function redundantly to regulate adaptation to microenvironmental change.
... acridum ARSEF strain 324 and M. anisopliae sf. anisopliae ARSEF strain 2575 secrete 44-kDa basic and acidic iso-forrns of endochitinase, respectively (Screen et al., 2001). These authors found that genetically manipulating these isolates to over-produce chitinase did not result in altered virulence to caterpillars (Manduca sexta) compared to the wild-type fungus, suggesting that wild-type leve1s of chitinase are not limiting for cuticle penetration. ...
Entomopathogenic fungi secrete a range of enzymes intrinsically involved in the pathogenicity process, mainly during cuticle penetration but these enzymes also have a role during colonization of the host insect. The two most important classes of enzymes involved in cuticle penetration are the peptidases and chitinases, specifically produced by the fungus to breakdown the main components of the cuticle (protein and chitin). Other enzyme activities detected during adhesion, germination and penetration, such as that of lipases would appear to be involved in breakdown of insect defensive barriers. Trehalases may have a fundamental role in sequestering nutrients for the fungus during the haemolymph colonization phase. This review describes the role of enzymes in cuticle penetration and subsequently during the colonization process. Furthermore, the possible use of molecular modifications of the fungi in relation to expression of the key enzymes is discussed as method of genetically improving biological control agents. Finally, the industrial applications of enzymes of fungal origin are discussed.
... The recombinant strain displayed significantly lower values of LC 50 and LT 50 (lethal time necessary for 50% mortality), when compared to the non-transformed (wild-type) strain and, therefore, Bbchit1 provided increased virulence. By contrast, manipulating the expression of a similar chitinase gene in M. anisopliae did not result in increased virulence in the host Manduca sexta (Screen et al., 2001). The M. anisopliae Chit1 chitinase gene was cloned from two different isolates and found to encode 44 kDa proteins differing in isoelectric points. ...
Chitin is a polymer of N-acetyl glucosamine that forms the protective exoskeleton of all arthropods and is replaced periodically during growth and development. Chitin biosynthesis starts with the disaccharide trehalose, culminating in the polymerization of the N-acetyl glucosamine subunits by chitin synthase to produce chitin microfibrils. Chitin in the old exoskeleton is degraded by chitinases, deacetylases and hexosaminidases and recycled. Chitin synthesis has been used as a target for developing biorational insecticides such as benzoylphenyl ureas, diflubenzuron being the original such compound. Several benzoylphenyl ureas with diverse activity spectra have since been synthesized and widely used for pest control. Newer pesticides targeting not only chitinase and chitin synthase but also other novel sites are being developed. Understanding the various nuances of chitin metabolism and regulation with all the genomic resources on hand will undoubtedly pave the way for developing more target-oriented softer control agents that have minimal impact on the environment.
... The recombinant strain displayed significantly lower values of LC 50 and LT 50 (lethal time necessary for 50% mortality), when compared to the non-transformed (wild-type) strain and, therefore, Bbchit1 provided increased virulence. By contrast, manipulating the expression of a similar chitinase gene in M. anisopliae did not result in increased virulence in the host Manduca sexta (Screen et al., 2001). The M. anisopliae Chit1 chitinase gene was cloned from two different isolates and found to encode 44 kDa proteins differing in isoelectric points. ...
... Metarhizium anisopliae and Beauveria bassiana in vivo (Fang et al., 2005;Bhanu Prakash et al., 2012). The Chit1 chitinase from M. anisopliae was shown to have little or no effect on virulence, while overproduction of chitinase Bbchit1 in B. bassiana did enhance the biocontrol activity of the fungus against aphids (Screen et al., 2001;Fang et al., 2005). Chitinase chi2 of M. anisopliae has been re-ported to be responsible for virulence in a bioassay of transgenic isolates with the chitinase gene overexpressed and silenced (Boldo et al., 2009). ...
Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.
... The cuticle is mainly composed of chitin, which is degraded by endo and exochitinases through the breaking of N- et al., 2004), produced by certain fungi releasing chitinolytic enzymes (St Leger et al., 1996; Valadares-Inglis and Peberdy, 1997), which are encoded by a chitinase gene (Chit1) (Screen et al., 2001), chitinase gene ( ISSN:2395ISSN: -1079 Available online at http://www.gjms.co.in/index.php/sajms South Asia Journal of Multidisciplinary Studies SAJMS September2015, Vol. 1, No.-8 8 1993a;Chouvenc et al., 2009a;Chouvenc et al., 2009b). ...
Termites are very devastating insect pests of agricultural, ornamental crops and dry wood. They are social insect having strong inter-communication, due to which they are very active pests, with both positive and negative effects on the environment. They are found in every type of soil in the world, and have a broad range of species. Management of termites has been approached with a number of different strategies, especially chemical pesticides, which have other environmental site impacts. Microbial biological control is defined as the use, and proper adjustment, of natural enemies via microbial organisms, such as; fungi, virus, bacteria, and with the aim of suppression and management of insect populations. A broad range of species, from different groups of microbial organisms, have strong association with termites, and some have been recorded as parasites. Some species are currently used as commercial biological control agents of termites.
... Among the 10 most frequent transcripts in a strain of M. anisopliae are 3 encoding chitinases and a chitosanase (Freimoser et al., 2003a). However, when M. anisopliae was transformed to overexpress its native chitinase, the pathogenicity towards the tobacco hornworm was unaltered, suggesting that wild type levels of chitinase are not limiting for cuticle penetration (Screen et al., 2001). Another fungal species, Conidiobolus coronatus, also produces both endo-and exo-acting chitinolytic enzymes during growth on insect cuticle (Freimoser et al., 2003b). ...
This chapter highlights some of the recent and important findings obtained from studies conducted on the synthesis, structure, physical state, modification, organization, and degradation of chitin in insect tissues, as well as the interplay of chitin with chitin-binding proteins, the regulation of genes responsible for chitin metabolism, and, finally, the targeting of chitin metabolism for insect-control purposes. Chitin is the major polysaccharide present in insects and many other invertebrates as well as in several microbes, including fungi. It serves as the skeletal polysaccharide of several animal phyla, such as the Arthropoda, Annelida, Molluska, and Coelenterata. In several groups of fungi, chitin replaces cellulose as the structural polysaccharide. In insects, it is found in the body wall or cuticle, gut lining or peritrophic matrix (PM), salivary gland, trachea, eggshells, and muscle attachment points. In the course of evolution, insects have made excellent use of the rigidity and chemical stability of the polymeric chitin to assemble both hard and soft extracellular structures such as the cuticle (exoskeleton) and PM respectively, both of which enable insects to be protected from the environment while allowing for growth, mobility, respiration, and communication. All of these structures are primarily composites of chitin fibers and proteins with varying degrees of hydration and trace materials distributed along the structures.
... Kang et al (1998Kang et al ( , 1999 reported a chitinase with molecular mass of 60 kD from M. anisopliae grown in a medium containing chitin as the sole carbon source with an optimum pH of 5.0, which is different from the chitinases values previously reported by St Leger et al (1996) for endo-chitinases of 33.0, 43.5, and 45 kDa and exo-chitinases of 110 kDa. Screen et al (2001) cloned the chitinase gene (Chit1) from M. anisopliae sf. acridum ARSEF strain 324 and M. anisopliae sf. ...
An attractive alternative method to chemical pesticides is the microbial biocontrol (MBCAs) agents. They are the natural enemies devastating the pest population with no hazard effects on human health and the environment. Entomopathogenic fungi has an important position among all the biocontrol agents because of its route of pathogenicity, broad host rang and its ability to control both sap sucking pests such as mosquitoes and aphids as well as pests with chewing mouthparts, yet they only cover a small percentage of the total insecticide market. Improvements are needed to fulfill the requirements for high market share. Entomopathogenic fungi differ from other microorganisms in their infection process: they directly breach the cuticle to enter the insect hemocoel, while other microorganisms enter by ingestion through mouth and then cause disease. Insect cuticle is mainly composed of chitin and protein surrounded by wax, lipid layer or fatty acids. Fungal pathogenesis mainly starts with the secretion of cuticle degrading enzymes. Some important and well known cuticle degrading enzymes are chitinase, protease and lipase which can degrade chitin, protein and lipid of the cuticle, respectively. In this review we collected literatures from different sources and we arranged them in a such a way to better to understand the process of insect pathogenicity of entomopathogenic fungi and to find ways to improve the virulence of wild strain fungi to shorten the killing time of the pest and to commercialize the entomopathogenic fungi. In this way, the market share of the fungal entomopathogenic fungi will increase and a decrease in the usage of synthetic chemical pesticides will automatically follow.
... For instance, M. anisopliae has been genetically modified for: (1) resistance to the fungicide benomyl (Bernier et al., 1989;Goettel et al., 1990b) and the herbicides bialaphos and glufosinate ammonium (Inglis et al., 2000); (2) increased speed of kill via protease over expression (St. Leger et al., 1996); (3) chitinase overproduction (Screen et al., 2001); and (4) expression of a scorpion toxin gene (Wang and St. Leger, 2007c;Pava-Ripoll et al., 2008). Beauveria bassiana has been modified for chitinase overproduction (Fang et al., 2005;Fan et al., 2007) and for expression of: a cuticle-degrading protease from M. anisopliae (Góngora, 2004;Lu et al., 2008), a scorpion neurotoxin (Lu et al., 2008), Bacillus thuringiensis vegetative insecticidal proteins (Qin et al., 2010), a hybrid protease , and a chitinase gene from O. unilateralis (Chantasingh et al., 2011). ...
Fungal entomopathogens are important biological control agents worldwide and have been the subject of intense research for more than 100 years. They exhibit both sexual and asexual reproduction and produce a variety of infective propagules. Their mode of action against insects involves attachment of the spore to the insect cuticle followed by germination, cuticle penetration, and internal dissemination throughout the insect. During this process, which may involve the production of secondary metabolites, the internal organs of the insect are eventually degraded. Environmental factors such as ultraviolet light, temperature, and humiditycan influence the effectiveness of fungal entomopathogens in the field. Phylogenetic studies have resulted in a better understanding of associations with other fungi as well as a new classification scheme. Ecological studies have revealed fascinating aspects related to their host range, distribution, abundance, and trophic interactions. The development of fungal entomopathogens as effective biological control agents requires knowledge of bioassay methods, as well as production, formulation, and application methodologies. Some important case studies involve the gypsy moth in the USA, locusts and grasshoppers in Africa, and spittlebugs in Brazil. This chapter focuses on the biology, classification and phylogeny, ecology, and use of fungal entomopathogens as biological control agents.
... While chitinases have been mentioned as important enzymes in the process of penetration by entomopathogenic fungi (Smith et al. 1981, Coudron et al. 1984, St. Leger et al. 1986, Fan et al. 2007), Screen et al. (2001) found no differences in pathogenicity to M. sexta larvae by isolates of M. anisopliae with overexpressed chitinase production or wild isolates with little production of the enzyme, suggesting that chitinase levels were not limiting during penetration by the wild strain. All isolates in the current study showed chitinolytic activity although in small amounts, and this might be why a definite relationship with pathogenicity was not detected. ...
To understand the relationship between pathogenicity and enzymatic activity of fungal isolates associated with Diaphorina citri Kuwayama, three isolates of Beauveria bassiana (Bals.) Vuill. and six of Hirsutella citriformis Speare from southeastern and Central Mexico were characterized enzymatically. Pathogenicity against D. citri was evaluated in four of them. They killed >90%, and no differences among isolates were found; however, by the change rate (rL) of the logistic model, a trend of more mortality (1.12) was confirmed in the Bb-Ch isolate of B. bassiana obtained from adult D. citri from Quintana Roo State, while the Y-2 isolate of H. citriformis from Yucatan State resulted in least mortality (0.74). Enzymatic production, both intra and inter-specific, varied, but greatest variability was found in proteases, and there were strains without proteolytic activity. A positive relationship (r = 0.84 to 0.98) was found only between intracellular proteases versus pathogenicity; isolates with more and least production were Bb-Ch (3.15) and Y-2 (0.12), respectively, suggesting this methodology for detecting enzymes might be useful in selecting the most pathogenic isolates. Based on pathogenicity and intracellular enzymatic activity, strains with the greatest potential for managing D. citri were Bb-Ch and Bb-Cat of B. bassiana. Based on enzymatic activity only, Y-5 and Camp-1 could be the H. citriformis strains more pathogenic to D. citri.
... Chitinase and protease act concurrently, synchronicity in the action of these enzymes is supported in our study by the fact that highest levels of chitinolytic and proteolytic activity occurred on 4 th to 6 th day. This is in accordance with Screen et al (2001) reported that when cuticular proteins are degraded by proteases then cuticular chitin exposed and able to induce the expression of fungal chitinases. This is supported by Fang et al (2009) ...
Nine native fungal isolates were isolated from soil and insect cadavers collected from different districts of Punjab. Four procured isolates of Beauveria bassiana viz. MTCC 4495, MTCC 2028, MTCC 6291, NBAII-11 along with nine native fungal isolates were characterized on the basis of morphology, enzyme activity and virulence against Pieris brassicae larvae. On the basis of morphological characters viz. colony morphology, spore shape, size and conidial arrangement F1 was presumptively identified as Zoophthora and F4, F6 were presumptively identified as Beauveria spp. F3 and F7 were presumptively identified as Metarhizium spp. and F2, F5, F8 and F9 may be identified as Fusarium spp. Four procured B. bassiana and three native (F4, F6 and F9) fungal isolates were evaluated for their cuticle degrading enzyme activities and variability was observed both in the pattern of enzyme secretion and level of enzyme activity among various fungal isolates. Among seven fungal isolates maximum mean protease activity (1.27 U/ml) was recorded in F4, maximum mean chitinase activity (0.64 U/ml) was recorded in F6 and maximum mean lipase activity (1.47 U/ml) was recorded in B. bassiana MTCC 4495. Among the days the maximum mean protease activity (1.59 U/ml), maximum mean chitinase activity (0.73 U/ml) and maximum mean lipase activity (1.72 U/ml) was recorded at sixth day of incubation. In bioassay studies against second and third instar larvae of P. brassicae fungal isolate B. bassiana MTCC 4495 recorded 84.43 and 72.22 per cent mean cumulative mortality against second and third instar larvae of P. brassicae after ten days respectively. Among native fungal isolates F4 recorded 85.55 and 63.33 per cent mean cumulative mortality against second and third instar larvae after ten days of treatment respectively. Three fungal isolates (B. bassiana MTCC 4495, F4 and F6 @108spores ml-1) along with commercial formulation of B. bassiana (Biopower 1.5% @ 1litre/ 200 lite of sprayable volume/ac) and chemical control (Spinosad 2.5 SC @ 240 ml/ac) were evaluated against P. brassicae. Chemical treatment (Spinosad 2.5 SC/ac @ 240 ml/ac) was significantly betterthan all other treatments recorded maximum yield (87.20 q/ac). This was followed by the commercial formulation of B. bassiana (Biopower) which recorded (79.29 q/ac) yield which was at par with B. bassiana MTCC 4495 recorded yield of 76.74 q/ac.
... Kang et al (1998Kang et al ( , 1999 reported a chitinase with molecular mass of 60 kD from M. anisopliae grown in a medium containing chitin as the sole carbon source with an optimum pH of 5.0, which is different from the chitinases values previously reported by St Leger et al (1996) for endo-chitinases of 33.0, 43.5, and 45 kDa and exo-chitinases of 110 kDa. Screen et al (2001) cloned the chitinase gene (Chit1) from M. anisopliae sf. acridum ARSEF strain 324 and M. anisopliae sf. ...
An attractive alternative method to chemical pesticides is the microbial biocontrol (MBCAs) agents. They are the natural enemies devastating the pest population with no hazard effects on human health and the environment. Entomopathogenic fungi has an important position among all the biocontrol agents because of its route of pathogenicity, broad host rang and its ability to control both sap sucking pests such as mosquitoes and aphids as well as pests with chewing mouthparts, yet they only cover a small percentage of the total insecticide market. Improvements are needed to fulfill the requirements for high market share. Entomopathogenic fungi differ from other microorganisms in their infection process: they directly breach the cuticle to enter the insect hemocoel, while other microorganisms enter by ingestion through mouth and then cause disease. Insect cuticle is mainly composed of chitin and protein surrounded by wax, lipid layer or fatty acids. Fungal pathogenesis mainly starts with the secretion of cuticle degrading enzymes. Some important and well known cuticle degrading enzymes are chitinase, protease and lipase which can degrade chitin, protein and lipid of the cuticle, respectively. In this review we collected literatures from different sources and we arranged them in a such a way to better to understand the process of insect pathogenicity of entomopathogenic fungi and to find ways to improve the virulence of wild strain fungi to shorten the killing time of the pest and to commercialize the entomopathogenic fungi. In this way, the market share of the fungal entomopathogenic fungi will increase and a decrease in the usage of synthetic chemical pesticides will automatically follow.
... acridum overexpression. Nonetheless, there was no increase in the virulence against Manduca sexta [59]. The fact remains that, when a constitutive promoter was used for the chi2 gene of M. anisopliae overexpression, the time to death was reduced, even when alterations in the spores, hyphae and appressorium were detected [7]. ...
... Kang et al. (1998Kang et al. ( , 1999 reported a chitinase with molecular mass of 60 kD from M. anisopliae grown in a medium containing chitin as the sole carbon source with an optimum pH of 5.0, which is different from the chitinases values previously reported by St Leger et al. (1996) for endo-chitinases of 33.0, 43.5, and 45 kDa and exo-chitinases of 110 kDa. Screen et al. (2001) cloned the chitinase gene (Chit1) from M. anisopliae sf. Acridum ARSEF strain 324 and M. anisopliaes ARSEF strain 2575 (Chit1) using the pro-moter of Aspergillus (gpd) for constitutive expression. ...
The development of mosquito vector control technology with fungi has a new generation. However, fungi have numerous benefits over the microbes for preparation in insecticides as mosquito have a vigorous lethal of existence in fungal products. The fungal spores, metabolites, protein, toxins, enzymes, and nanoparticles have been shown significant efficacies against adults and its developmental stages of mosquitoes. The developments and formulations have used of new technology that produce new larvicides and adulticides. The marketable perfection of entomopathogenic fungi for mosquito control has been delayed by unacceptable action comparative to chemical compound. However, the new technologies are urgent need for their separation and preservation postponements the significance. Recently several chemical based strategies have shown favourable results in the field. This article propose new prospective on fungal infection used for mosquitoes control present to future based alternative.
... cuticle degrading protease (St. Leger et al. 1992), chitinase genes (Bogo et al. 1998;Screen et al. 2001;Bagga et al. 2004;Fang et al. 2005), adhesin that assists spore attachment (Wang and St. Leger 2007), and a cell protective coat protein helping evasion the host immunity recognition (Wang and St Leger 2006). Modification of these genes may improve the virulence of fungal strains and shorten the killing time, which may be achieved by transformation systems (Bernier et al. 1989;Goettel et al. 1990;St. ...
The use of microbial organisms as biological control agents has progressed significantly since Metschnikoff launched the first attempt at microbial insect control with Metarhizium anisopliae in 1879. Following the lead of Metschnikoff, entomopathogenic nematodes, fungi, bacteria and viruses have been extensively studied for commercialization and practical use as biopesticides in inundative releases against insect pests in various cropping systems. However, compared with chemical insecticides, these microbial products represent less than 2% of the total insecticide market share. Factors such as control efficacy, cost, formulation, shelf life, application techniques, and persistence have limited the commercial use of these microbial control agents in insect pest management. This review discusses research advances for entomopathogens, especially commercialization, formulation and application techniques, for microbial biocontrol of insect pests in the horticultural ecosystem.
... Overexpression of extracellular chitinase, an enzyme important in the cuticular penetration of insects by entomopathogenic fungi, has also been demonstrated for M. anisopliae var. anisopliae (Screen et al. 2001). They expressed a chitinase gene from M. anisopliae var. ...
... Several assays of protease and chitinase activity of B. bassiana and Paecilomyces farinosus, N. rileyi indicated a correlation between the amount of secreted enzymes and the rate of virulence of individual isolates (Samsinakova et al., 1977;El-Sayed et al., 1989;Bidochka and Khachatourians, 1990;Havukkala et al., 1993;El-Sayed et al., 1993a;Barros et al., 2000). No correlation, however, was apparent between enzyme activity (lipolytic, proteolytic or chitinolytic) and pathogenicity of isolates of B. bassiana and M. anisopliae (Screen et al., 2001;Vergas et al., 12 2003). ...
The present study focuses on fungal extracellular enzymes as factors affecting fungal parasitism on the cotton leafworm, Spodoptera littoralis, especially with some new strains of entomopathogenic fungi such as Beauveria bassiana 1513, B. bassiana 3286, Paecilomyces fumosoroseus 2734 and Nomuraea rileyi 1972. Our results showed a significant increase in mortality 24 h after injection of fungi and tended to increase until pupation. The 6th instar larvae were more susceptible to N. rileyi 1972, followed by B. bassiana 3286, and then P. fumosoroseus 2734. The larvae were least susceptible to B. bassiana 1513. At a dose of 9000 bl/insect, the median lethal times of the tested fungal isolates were between 36.5-51 h. Fungi were able to produce detectable amounts of proteases, chitinases and lipases. The activity of proteases was strong along with weak activities of chitinases and lipases. Interestingly, there appeared to be no clear correlation between pathogenicity and activity of extracellular enzymes. The extracellular enzymes always act as a unit, and everyone alone can not permit to the fungus penetration into the host. So, virulence may be the result of a set of enzymes acting together.
... As indicated by the analysis of some fungal genomes, proteases and chitinases are required for the fungal pathogens to tackle the protein-and chitin-rich insect cuticles (Gao et al., 2011). Since the fungal chitinases usually lack chitin-binding domain (CBD), genetic engineering of B. bassiana to over-express hybrid chitinases carrying an insectderived CBD resulted in a significant increase in fungal virulence (Fan et al., 2007) while the integration of native chitinases into M. anisopliae and M. acridum failed to alter fungal pathogenicity to Manduca sexta (Screen et al., 2001). Not surprisingly, an endogenous subtilisin-like protease CDEP1 or M. anisopliae Pr1A over-expressed in B. bassiana also enhanced substantially the fungal virulence Fang et al., 2009). ...
Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae, are environmentally friendly biocontrol agents (BCAs) against various arthropod pests. We provide an overview to the past-decade advances in fungal BCA research and application in China. Since 1960s, fungal BCAs have been mass-produced for application and at present, thousands of tons of their formulations are annually applied to control forest, agricultural, greenhouse and grassland insect pests throughout the country. Apart from technical advances in mass production, formulation and application of fungal BCAs, basic studies on the genomics, molecular biology, genetic engineering and population genetics of fungal entomopathogens have rapidly progressed in the past few years in China. The completed genomic studies of M. anisopliae, Metarhizium acridum, B. bassiana and Cordyceps militaris provide profound insights into crucial gene functions, fungal pathogenesis, host–pathogen interactions and mechanisms involved in fungal sexuality. New knowledge gained from the basic studies has been applied to improve fungal virulence and stress tolerance for developing more efficacious and field-persistent mycoinsecticides by means of microbial biotechnology, such as genetic engineering. To alleviate environmental safety concerns, more efforts are needed to generate new data not only on the effects of engineered BCAs on target and non-target arthropods but also on their potential effects on gene flow and genetic recombination before field release.
... The recombinant strain displayed significantly lower values of LC 50 and LT 50 (lethal time necessary for 50% mortality), when compared to the non-transformed (wild-type) strain and, therefore, Bbchit1 provided increased virulence. By contrast, manipulating the expression of a similar chitinase gene in M. anisopliae did not result in increased virulence in the host Manduca sexta (Screen et al., 2001). The M. anisopliae Chit1 chitinase gene was cloned from two different isolates and found to encode 44 kDa proteins differing in isoelectric points. ...
Entomopathogens, employed as biological control, were formulated as eco-friendly substitutes for chemical insecticide. Nevertheless, microbial-based insecticides were implicated to be commercially inadequate based on their indiscriminate efficacy and low virulence. The resistance, pathogenicity, and virulence of entomopathogens to adverse conditions were greatly enhanced by genetic engineering. Improvement of virulence was achieved by modifying entomopathogens to express insecticidal proteins/peptides. The continued use of synthetic chemical insecticides remains an apprehension by the public as well as their approval of genetically modified organisms including inventive biological insecticides which provide diverse beneficial and environmentally sustainable options to control insect pests.
BACKGROUND
Dialeurodes citri is an important pest in citrus‐producing areas of the world. Lecanicillium attenuatum parasitizes D. citri and kills it, suggesting a potential approach for the biological control of pests. However, the low virulence of the fungus and its slow rate of killing have limited its commercial competitiveness. The objective reason for these disadvantages is immunological rejection by the host. Our strategy was to use fungi to express the dsRNA of the host immune genes. The fungal hyphae release siRNA at the time of infection, thus interfering with the expression of immune genes in the host and facilitating fungal invasion.
RESULTS
We selected prophenoloxidase (DcPPO), prophenoloxidase‐activating factor (DcPPO‐AF), and lysozyme (DcLZM) as target genes to construct intron‐splicing hairpin RNA expression vectors and to successfully obtain transgenic fungi. Two days after infection, the immune genes of D. citri showed varying degrees of silencing compared with those in the positive control group. The LC50 (spores/mL) values of La::GFP, La::DcPPO, La::DcPPO‐AF, and La::DcLZM were 9.63 × 10⁴, 2.66 × 10⁴, 1.21 × 10⁵, and 3.31 × 10⁴, respectively. The LT50 values of these fungi were 5.15, 3.60, 5.34, and 4.04 days, respectively. The virulence of La::DcPPO and La::DcLZM increased 3.62‐ and 2.91‐fold, respectively, and their LT50 decreased by 30.10% and 21.55%, respectively.
CONCLUSIONS
The results indicate that this method, which uses tens of thousands of hyphae to inject dsRNA to improve the virulence of transgenic fungi, can play a greater role in the prevention and control of pests in the future.
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Beauveria bassiana strain 9205 (Bb9205) was transformed with protease genes (prlA and priJ) and one sterase gene (ste 1) isolated from Metarhizium anisopliae that are involved in insect pathogenicity. The goal of this research was to increase the pathogenicity of the strain against the coffee berry borer, Hypothenemus hampci. Tb do this, genes were cloned into the plasmid pBarGPEl that confers resistance to the herbicide ammonium-gluphosinate. A monosporic line of strain Bb9205 was obtained with pathogenicity of 78% mortality against M. hampci, using 1 x 10ft spore/ml. Protoplasts were produced from this monosporic line. The methodology for the protoplast production was optimized obtaining 3x 107 protoplasts/ml after enzymatic pretreatment for 4 hours under slow agitation at 37°C. Transformation was carried out with the plasmids pBarGPEl- priA, pBarGPE 1-priJ and pBarGPE l-s(el by PEG and electroporation. TWo strains transformed with the prlA gene and one strain transformed with the stel gen were obtained, that showed resistance to 25 μq/m\ of ammonium glufosinate. These strains showed increased proteolytic and sterolytic activity, respectively. Under laboratory conditions, pathogenicity tests of B. bassiana transformants was compared to untransformed monosporic Bb9205. Constitutive over-expression of protease in the transgenic strain Bb9205-prJA improved its insecticidal activity. The transformed strain showed a 21,7% increase in the insect mortality rate with 14,3% reduction in the time of mortality. The Bb9205-sfel transformant strain showed a 9,5% reduction of the time of mortality compared with the untransformed strain. The results indicate that breeding of Beauueria strains by genetic transformation can be achieved for purposes of better biological control. © 2005, Sociedad Colombiana de Entomologia. All rights reserved.
Isaria fumosorosea is one of important entomopathogenic fungi showed a good potential in controlling Bemisia tabaci. The effects of I. fumosorosea ⊿Ifchit1 mutant (Ifchit1 gene deletion mutant) on the mortality, oviposition, and host immunological response of B. tabaci, on Brassica campestris L. plant, were evaluated under laboratory conditions. The wild-type fungal strain infection significantly increased insect mortality and reduced the oviposition effeciency of B. tabaci, whereas the ⊿Ifchit1 mutant was much less effective, resulting in higher survival and ovipositing of B. tabaci. The activities of four insect enzymes were examined during a time course of fungal infection. Insect phenoloxidase, perioxidase, and catalase activities were decreased in whiteflies treated with the wild type and mutant I. fumosorosea strain at 12–36 h post treatment. However, these enzyme activities increased in fungal-treated whiteflies as compared to controls between 36 and 60 h post-infection, reaching peak values. Superoxide dismutase activity in fungal-treated whiteflies was higher than that in controls during the entire experimental time course examined. The overall enzyme activity profiles in ⊿Ifchit1 mutant-treated whiteflies were significantly different from wild-type strain treatments. Our results showed that loss of the Ifchit1 gene in I. fumosorosea affects whitefly mortality, ovipositioning and various antioxidant enzyme activities, providing new insights into the role of chitinases in I. fumosorosea-insect host–pathogen interactions.
In this study we reported the screening of ten different fungal isolates for higher chitinase yield and its effect against the stored pest Corcyra cephalonica as natural bio-control agent without toxic effects. Different fungal strains were cultured and screened the chitinase activity for the production of chitinase enzyme. The majority of the farmers profoundly rely on synthetic pesticides for the eradication of stored food pests. These synthetic pesticides are leaving a very large amount of toxic residue in the environment which is harmful to human beings. Due to these toxic effects of synthetic pesticide we attempted to find the biological control agent against stored pests. From the fungal populations, ten different fungal species were used to screen for the higher production of chitinase. The chitinase was partly purified by ammonium sulfate precipitation method and was evaluated for its anti-feedant and growth inhibitory activities against the stored food pest Corcyra cephalonica Stainton at 62.5, 125, 250, 500 and 1000 ppm enzyme concentrations. Trichoderma viride shows a maximum chitinase activity of 2.75±0.029 U.mL-1 colloidal chitin on the sixth day. Next to Trichoderma viride, Metarhizium anisoplea and Candida albicans shows a chitinase activity of 2.51±0.031 and 2.23±0.070 U.mL-1 Colloidal chitin respectively. Treatment of Corcyra cephalonica with the partially purified chitinase from Trichoderma viride reduced the normal larval development according to a dose-dependent manner. This study shows Trichoderma viride chitinase can affect the growth of Corcyra cephalonica larvae. The outcome necessitates further insect bioassays and detection of underlying mechanisms.
Chitinolytic fungal strains were isolated from soil in Thailand. They were screened as chitinase producers by testing their shrimp shell digestion ability on potato dextrose agar plates. The chitinase activity was tested with colloidal chitin in culture medium C and basal medium. There was greater activity in culture medium C than in the basal medium. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis from the culture filtrate of medium C showed three protein bands at about 40 kDa, 46 kDa and 56 kDa. The chitinase gene was sequenced from genomic DNA. The obtained sequence consisted of 713 bp upstream, a 1,499 bp open reading frame that was interrupted by three introns and 1,698 bp downstream sequences. The intron lengths were 63 bp, 57 bp and 110 bp, respectively. The sequence was found to be the most similar to the chitinase gene of Paecilomyces lilacinus (EF183511). Pairwise alignment of the 1,499 bp and P. lilacinus resulted in 72.5% DNA sequence identity, while alignment of the 1,269 bp coding sequence and P. lilacinus resulted in 78.5% cDNA sequence identity and 83.5% amino acid sequence identity. The protein structure contained two conserved domains of the putative substrate binding site (S-I-G-G) and catalytic domain (D-G-I-D-L-D-W-E), suggesting that this fungal chitinase belonged to the glycosyl hydrolases family 18 chitinase (GH18). Phylogenetic analysis of the chitinase gene from the nematopathogenic fungi suggested that this chitinase sequence was class V chitinase.
The tick Rhipicephalus (Boophilus) microplus is a bovine ectoparasite that causes severe economic losses in herds in tropical and subtropical areas due to parasitism and disease transmission. Tick control is currently based mainly on the use of chemicals. However, alternative strategies are required due to the ability of ticks to develop resistance, the demands of consumers for chemical-free foods, and the environmental impact of chemical acaricides. In accordance with this scenario, the use of fungi to biocontrol ticks is an efficient and important tool. The entomopathogenic and acaricidal fungus Metarhizium anisopliae is the most studied fungus for the biocontrol of R. microplus. Various studies on different aspects of the use of M. anisopliae to control ticks and other pests have been published during the decade. Such knowledge is crucial in the search for more efficient strains and for optimizing new formulations for large-scale use of this efficient, economic and environmentally safer form of tick control. Here we focus on the biocontrol of the tick R. microplus based on the use of M. anisopliae, including the different fungal isolates, their virulence, the biochemical and molecular aspects of the pathogenesis and the control strategies.
The present study aims to offer new insights into the use of new fungal isolates as biocontrol agents and to investigate the outcome of interactions between the cotton leafworm, Spodoptera littoralis and the hyphomycete entomopathogens (Beauveria bassiana 1513 and 3286, Paecilomyces fumosoroseus 2734 and Nomuraea rileyi 1972). The mechanisms affecting success or failure of fungal infection in the cotton leafworm were investigated.
Based on the results derived from median lethal spore concentration and time after infection, our data demonstrated that tested fungi caused fatal infection to the immature stages of S. littoralis.
Infection with all tested fungi reduced pupal and adult survivals; fungal infection did not affect adult longevity and egg-production, but eventually caused no hatchability.
Our data showed that B. bassiana, P. fumosoroseus and N. rileyi produced the extracellular enzymes proteases, chitinases and lipases. The activity of proteases was strong, while weak activities of chitinases and lipases were detected. Interestingly, the present study could not identify a correlation between enzyme activity and virulence of the fungal strains.
Drastic changes in haemolymph carbohydrates, lipids and proteins concentrations were observed after fungal injection. Electrophoretic analysis of the haemolymph proteins demonstrated that after injection of fungi into the haemocoel of S. littoralis, some proteins were changed in their percentages; others were induced to be synthesized. Moreover, injection with N. rileyi appeared to prevent expression of some proteins. Phenoloxidase is a key enzyme in cellular and humoral immune responses. Significant decrease in phenoloxidase activities was observed at 24 h in insects injected with B. bassiana, whereas an increase was detected at 48 h post-injection with N. rileyi. No marked changes in lysozyme activity in insects injected with B. bassiana were detected at all time intervals post-injection. In contrast, significant decrease in lysozyme activity at the end of observation was detected.
Differential haemocyte counts showed drastic changes in all haemocyte types following injection with each fungus, especially in plasmatocytes and granular haemocytes. Following injection with B. bassiana blastospores, the total haemocyte count decreased significantly, however, the number of haemocytes remained constant after injection with N. rileyi.
In B. bassiana-injected insects, peaks of phagocytosis and nodule formation were observed, and later the rate of phagocytosis tended to decrease. In contrast, phagocytosis and nodulation rates increased gradually to a maximum at 72 h after injection with N. rileyi.
The conclusion derived from the haematological and immunological studies indicated that the immune reactions against B. bassiana were quite effective than N. rileyi.
Synthesis of chitinase and chitosanase by the entomopathogenic fungus Metarhizium anisopliae is regulated by products of chitin and chitosan degradation through an inducer-repressor mechanism. Slow-feeding with sugars or alanine (about 20 pg ml-l h-I) in a carbon deficient medium to prevent catabolite repression (restricted cultures) demonstrated that the most effective inducers of chitinase and chitosanase were the principal monomeric constituents of chitin (N-acetylglucosamine) and chitosan (glucosamine) respectively. Increasing the rate of release of N-acetylglucosamine decreased chitinase synthesis by about 87 % while causing a sevenfold increase in growth. In batch cultures high chitinase activities were present only in chitin-containing medium. There was a negative correlation between accessibility and amount of chitin substrates, levels of free N-acetylglucosamine in culture fluids and chitinase production. Addition of carbohydrates, lipid or proteins to chitin-grown cultures repressed chitinase production. Basal levels of chitinase were produced in non-inducing media. Production of chitobiase ( N-acetylglucosaminidase) was enhanced from high basal levels by amino sugars, but was less inducible and less susceptible to catabolite repression than chitinase.
ABSTRACT Nine transformants of Trichoderma longibrachiatum with extra copies of the egl1 gene were studied for mitotic stability, endoglucanase production, and biocontrol activity against Pythium ultimum on cucumber seedlings. The transformants showed a significantly higher level of expression of the egl1 gene in comparison to the wild type under both inducing and noninducing growth conditions. Transformants with the egl1 gene under the control of a constitutive promoter had the highest enzymatic activity. Both the endoglucanase activity and the transforming sequences were stable under nonselective conditions. When applied to cucumber seeds sown in P. ultimum-infested soil, T. longibrachiatum transformants with increased inducible or constitutive egl1 expression generally were more suppressive than the wild-type strain.
Different classes of cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens inhibited spore germination of Botrytis cinerea in a bioassay in vitro. The addition of any chitinolytic or glucanolytic enzyme to the reaction mixture synergistically enhanced the antifungal properties of five different fungitoxic compounds against B. cinerea. The chemicals tested were gliotoxin, flusilazole, miconazole, captan and benomyl. Dose response curves were determined for each combination of toxin and enzyme, and in all cases the ED50 values of the mixtures were substantially lower than ED50 values of the two compounds used alone. For instance, the addition of endochitinase from T. harzianum at a concentration of 10 micrograms ml-1 reduced the ED50 values of toxins up to 86-fold. The level of synergism appeared to be higher when enzymes were combined with toxins having primary sites of action associated with membrane structure, compared with pesticides having multiple or cytoplasmic sites of action. Among enzymes tested, the highest levels of synergism with synthetic fungicides were detected for the endochitinase from T. harzianum strain P1, which, when used alone, was the most effective chitinolytic enzyme against phytopathogenic fungi of those tested. The use of hydrolytic enzymes to synergistically enhance the antifungal ability of fungitoxic compounds may reduce the impact of some chemical pesticides on plants and animals.
Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might he achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.
Transformation systems developed for Trichoderma spp. were utilized to improve the biocontrol efficiency of the mycoparasitic fungus Trichoderma harzianum by increasing the copy number of the basic proteinase gene prb1. The transformants were stable and carried from two to ten copies of prb1. High levels of expression of prb1 during fungus-fungus interaction were detected when T. harzianum and Rhizoctonia solani were confronted in vitro. In liquid cultures the proteinase was induced by cell walls of R. solani. Under greenhouse conditions, incorporation of T. harzianum transformants into pathogen-infested soil significantly reduced the disease caused by R. solani in cotton plants.
We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks
trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction
schemes and can easily be applied on genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal
signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW
server.
Disease resistance in transgenic plants has been improved, for the first time, by the insertion of a gene from a biocontrol fungus. The gene encoding a strongly antifungal endochitinase from the mycoparasitic fungus Trichoderma harzianum was transferred to tobacco and potato. High expression levels of the fungal gene were obtained in different plant tissues, which had no visible effect on plant growth and development. Substantial differences in endochitinase activity were detected among transformants. Selected transgenic lines were highly tolerant or completely resistant to the foliar pathogens Alternaria alternata, A. solani, Botrytis cinerea, and the soilborne pathogen Rhizoctonia solani. The high level and the broad spectrum of resistance obtained with a single chitinase gene from Trichoderma overcome the limited efficacy of transgenic expression in plants of chitinase genes from plants and bacteria. These results demonstrate a rich source of genes from biocontrol fungi that can be used to control diseases in plants.
There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome.
A hidden Markov model of signal peptides has been developed. It contains submodels for the N-terminal part, the hydrophobic region, and the region around the cleavage site. For known signal peptides, the model can be used to assign objective boundaries between these three regions. Applied to our data, the length distributions for the three regions are significantly different from expectations. For instance, the assigned hydrophobic region is between 8 and 12 residues long in almost all eukaryotic signal peptides. This analysis also makes obvious the difference between eukaryotes, Gram-positive bacteria, and Gram-negative bacteria. The model can be used to predict the location of the cleavage site, which it finds correctly in nearly 70% of signal peptides in a cross-validated test--almost the same accuracy as the best previous method. One of the problems for existing prediction methods is the poor discrimination between signal peptides and uncleaved signal anchors, but this is substantially improved by the hidden Markov model when expanding it with a very simple signal anchor model.
The role of the Trichoderma harzianum endochitinase (Ech42) in mycoparasitism was studied by genetically manipulating the gene that encodes Ech42, ech42. We constructed several transgenic T. harzianum strains carrying multiple copies of ech42 and the corresponding gene disruptants. The level of extracellular endochitinase activity when T. harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains as compared with the wild type, whereas gene disruptants exhibited practically no activity. The densities of chitin labeling of Rhizoctonia solani cell walls, after interactions with gene disruptants were not statistically significantly different than the density of chitin labeling after interactions with the wild type. Finally, no major differences in the efficacies of the strains generated as biocontrol agents against R. solani or Sclerotium rolfsii were observed in greenhouse experiments.
To date zinc carboxypeptidases have only been found in animals and actinomycete bacteria. A cDNA clone (MeCPA) for a novel fungal (Metarhizium anisopliae) carboxypeptidase (MeCPA) was obtained by using reverse transcription differential display polymerase chain reaction to identify
pathogenicity genes. MeCPA resembles pancreatic carboxypeptidases in being synthesized as a precursor species (418 amino acids)
containing a large amino-terminal fragment (99 amino acids). The mature (secreted) form of MeCPA shows closest amino acid
identity to human carboxypeptidases A1 (35%) and A2 (37%). MeCPA was expressed in an insect cell line yielding an enzyme with
dual A1 + A2 specificity for branched aliphatic and aromatic COOH-terminal amino acids. However, in contrast to the very broad
spectrum A + B-type bacterial enzymes, MeCPA lacks B-type activity against charged amino acids. This is predictable as key
catalytic residues determining the specificity of MeCPA are conserved with those of mammalian A-type carboxypeptidases. Thus,
in evolutionary terms the fungal enzyme is an intermediate between the divergence of A and B forms and the differentiation
of the A form into A1 and A2 isoforms. Ultrastructural immunocytochemistry of infected host (Manduca sexta) cuticle demonstrated that MeCPA participates with the concurrently produced endoproteases in procuring nutrients; an equivalent
function to digestive pancreatic enzymes.
Extracellular chitinases have been suggested to be virulence factors in fungal entomopathogenicity. We employed isoelectric focusing and a set of three fluorescent substrates to investigate the numbers and types of chitinolytic enzymes produced by the entomopathogenic fungi Metarhizium anisopliae, Metarhizium flavoviride, and Beauveria bassiana. Each species produced a variety of N-acetyl-(beta)-d-glucosaminidases and endochitinases during growth in media containing insect cuticle. M. flavoviride also produced 1,4-(beta)-chitobiosidases. The endochitinases could be divided according to whether they had basic or acidic isoelectric points. In contrast to those of the other two species, the predominant endochitinases of M. anisopliae were acidic, with isoelectric points of about 4.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the acidic chitinases of M. anisopliae into two major bands (43.5 and 45 kDa) with identical N-terminal sequences (AGGYVNAVYFY TNGLYLSNYQPA) similar to an endochitinase from the mycoparasite Trichoderma harzianum. Use of polyclonal antibodies to the 45-kDa isoform and ultrastructural immunocytochemistry enabled us to visualize chitinase production during penetration of the host (Manduca sexta) cuticle. Chitinase was produced at very low levels by infection structures on the cuticle surface and during the initial penetration of the cuticle, but much greater levels of chitinase accumulated in zones of proteolytic degradation, which suggests that the release of the chitinase is dependent on the accessibility of its substrate.
Solid media are described on which the production of the extracellular enzymes amylase, lipase, DNA- and RNAase, pectinase, protease, urease, and chitinase were detected. The media were tested with seven plant pathogenic and six saprophytic fungi, as well as a sample of leaf compost. Antibiotics were examined for their ability to suppress bacterial growth in the fungal media. The effects of antibiotics and pH on fungal growth and extracellular enzyme production were examined. The solid media described could be useful for evaluating individual fungi and for rapid screening of genetic variants for the presence or absence of enzyme production, as well as for ecological studies and possible chemotaxonomic differentiation.
Fourth instar larvae of the tobacco hornworm, Manduca sexta, were inoculated with conidia of Metarhizium anisopliae then fed a diet containing the insect chitin synthesis inhibitor Dimilin. Cuticle of Dimilin-treated insects provided reduced resistance to penetration by hyphae of M. anisopliae. Widespread histolysis of postecdysial (Dimilin-affected) cuticle occurred. In addition, although lamellate preecdysial cuticle was not affected by Dimilin, the majority of the cuticle in the vertical cuticular columns was laid down at the same time as the postecdysial cuticle. Therefore, the vertical cuticular columns were areas of weakness in the preecdysial cuticle which as a consequence failed to provide a mechanical barrier to the penetrating fungus.
Trichoderma harzianum strain P1 produces a variety of chitinolytic enzymes including N-acetyl-β-D-glucosaminidases, chitin 1,4-β-chitobiosidases, and an endochitinase. Chitabiosidases and an endochitinase were purified from dialyzed, concentrated culture filtrates using gel filtration, chromatofocusing, and isoelectric focusing. Three protein bands were evident in the purified chitobiosidase preparation, representing different levels of N-glycosylation of the same protein. The pI of all purified proteins was ∼3.9 []
Two chitinolytic enzymes from Trichoderma harzianum strain P1 were tested for their antifungal activity in bioassays against nine different fungal species. Spore germination (or cell replication) and germ tube elongation were inhibited for all chitin-containing fungi except T. harzianum strain P1. The degree of inhibition was proportional to the level of chitin in the cell wall of the target fungi. For most of the fungi tested, the ED 50 values for the endochitinase and the chitobiosidase were 35-135 μg ml -1 and 62-180 μg ml -1 , respectively. Complete inhibition occurred at 200-300 μg ml -1 []
Extracellular fluids from Metarhizium anisopliae grown on chitin as the sole carbon source contained distinct chitinase (pH optimum 5.3, MW 33 kDa) and chitobiase (pH optimum 5, MW 110 kDa, pI 6.4) activities. Chitinase activity was stabilized against extremes of pH and temperature by the presence of its chitin substrate. Chitinase fractions eluted from a sephadex column had activity against chitosan as well as locust cuticle chitin, crystalline chitin, and colloidal chitin. Products of enzymolysis were analyzed by descending paper chromatography. Chitinase had no activity against chitobiose, only trace activity against chitotriose, but preferentially cleaved chitotetraose in the middle bond releasing chitobiose. N-Acetyglucosamine was the major (vs colloidal chitin) or only (vs crystalline chitin) product detected following chitin hydrolysis. The chitobiase (also called β-N-acetylglucosaminidase) possessed simple glycosidase activity against p-nitrophenyl-acetylglucosaminide and hydrolyzed di-, tri-, and tetrasaccharides to N-acetylglucosamine. Chitobiase activity was significantly inhibited by its reaction product, N-acetylglucosamine. The mechanism of action of chitinolytic enzymes is discussed.
A carboxymethyl-substituted soluble chitin and a reprecipitated colloidal chitin derivative were covalently linked with Remazol Brilliant Violet 5R. Both conjugates are new dye-labelled substrates suitable for the assay and detection of chitinase and lysozyme activity. A reliable colorimetric microassay adapted to microtitre plates was established, based on the precipitability of carboxymethyl-chitin-Remazol Brilliant Violet 5R from buffered solutions with hydrochloric acid. Furthermore, sensitive plate-clearing assays were developed for the isolation and detection of chitinolytic microbial populations, and for the screening of pure cultures.
Extracellular fluids from Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii grown on cuticle as the sole carbon source released amino acids and N-acetylglucosamine from protein and chitin, respectively, in comminuted locust cuticle. An endoprotease, chitinase, and N-acetyl-β-glucosaminidase were each purified from culture filtrates of M. anisopliae until free of other cuticle-degrading enzymes and tested singly, in combination, or in sequence against “whole cuticle” (containing tanned and untanned proteins) and exuviae (tanned cuticle). The protease hydrolyzed ca. 25–30% of cuticle proteins (), releasing peptides (mean chain length, 4.7) containing all 15 amino acids found in locust cuticle. Small amounts of amino sugars were also liberated following protein solubilization. Chitinase tested separately released monomeric N-acetylglucosamine (equivalent to 3–4% of cuticle chitin); however, when combined simultaneously with protease, N-acetylglucosamine release was increased × 1.5. Pretreatment with protease considerably enhanced chitinase activity (ca. × 3.5) compared to controls (preincubated with autoclayed protease). This implies that cuticular chitin is shielded by protein. N-acetyl-β-glucosaminidase showed no detectable activity against cuticle either alone or in combination with protease or chitinase. Exuvia was comparatively resistant to both proteolytic and chitinolytic attack; pretreatment with protease had no effect on subsequent chitinase activity. The results are discussed in relation to cuticle structure and the role of host and fungal enzymes in degrading cuticle during molting or infection.
Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as composite carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular enzymes corresponding to the major components of insect cuticle, e.g., endoproteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase, and N-acetylglucosaminidase. Considerable variations occurred in levels of production between species and even within a species, but endoproteases were exceptional as production of them was high with all the isolates. Cuticle-degrading enzymes were produced rapidly and sequentially in culture. The first activities to appear (<24 hr) were those of the proteolytic complex; chitinases were always produced substantially later. Chitin was probably masked by protein as a fluorescent chitin-specific lectin and calcofluor (binds to β-glucans) stained mainly cuticles from which protein had been removed (by protease or KOH).
ABSTRACT Transformants of the biocontrol agent Trichoderma harzianum strain CECT 2413 that overexpressed a 33-kDa chitinase (Chit33) were obtained and characterized. Strain CECT 2413 was cotransformed with the amdS gene and its own chit33 gene under the control of the pki constitutive promoter from T. reesei. Southern blotting indicated that the chit33 gene was integrated ectopically, mostly in tandem. Some transformants showed the same restriction pattern, indicating preferable sites of integration. There was no correlation between the number of integrated copies and the level of expression of the chit33 gene in the transformants. When grown in glucose, the extracellular chitinase activity of the transformants was up to 200-fold greater than that of the wild type, whereas in chitin, the activity of both the transformants and the wild type was similar. Under both conditions, the transformants were more effective in inhibiting the growth of Rhizoctonia solani as compared with the wild type. Similar results were obtained when culture supernatants from the transformants and the wild type were tested against R. solani.
This review article deals with recent developments in molecular and physiological aspects of chitinases from plants, fungi, bacteria, insects and fishes.
The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.
Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/l in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopicus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi.
Chitinases are produced by a wide variety of pathogenic and parasitic microbes and invertebrates during their attack on chitin-containing organisms. Examples discussed include enzymes of insect and algal viruses, of yeast killer toxin plasmids, of bacterial and fungal pathogens of fungi and insects, and of parasitic protozoa. These chitinases play roles in penetration of fungal cell walls, and of exoskeletons and peritrophic membranes of arthropods. Salivas of some invertebrate predators have chitinolytic activity which may be involved in their attack on their prey. Chitinases play a major defensive role in all plants against attack by fungi, and perhaps also against attack by insect pests. The plant chitinases form a very large and diverse assemblage of enzymes from two families of glycosyl hydrolases. At least some vertebrates, including fish and humans, also may utilise chitinases in their defence against pathogenic fungi and some parasites.
The use of ignite (Basta; glufosinate; phosphinotri-cin) to select transformants of bar-containing plasmids in Neuro-spora crassa Cuticle-degrading enzymes of entomopathogenic fungi: Cuticle degrada-tion in vitro by enzymes from entomopathogens
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Charac-terization of chitinase and chitobiase produced by the ento-mopathogenic fungus Metarhizium anisopliae
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