Ciência Rural, SantaBiological Maria, insectv.44, n.4, control p.645-651, using Metarhizium abr, 2014 anisopliae: morphological, molecular, and ecological aspects. 645 ISSN 0103-8478
Biological insect control using Metarhizium anisopliae: morphological, molecular, and ecological aspects
Controle biológico de insetos utilizando Metarhizium anisopliae: aspectos morfológicos, moleculares e ecológicos
Patricia Vieira TiagoI* Neiva Tinti de OliveiraI Elza Áurea de Luna Alves LimaI
ABSTRACT INTRODUCTION
Microbial control of insects is based on the rational use of pathogens to maintain environmentally balanced pest Biological control consists of the population levels, and Metarhizium anisopliae has been the introduction of benefi cial predatory or parasitic most studied and most utilized fungal species for that purpose. species into cultivation systems where they were The natural genetic variability of entomopathogenic fungi is previously absent or present only at low population considered one of the principal advantages of microbial insect control. The inter- and intraspecifi c variability and the genetic levels. This technique is designed to negatively diversity and population structures of Metarhizium and other affect specifi c target species that could otherwise entomopathogenic fungi have been examined using ITS-RFLP, become pests or infectious agents (GLIESSMAN, ISSR, and ISSP molecular markers. The persistence of M. anisopliae in the soil and its possible effects on the structures of 2001). Susceptibility to pests is a general refl ection resident microbial communities must be considered when selecting of plant health, which can be negatively infl uenced isolates for biological insect control. by poor soil fertility management (NICHOLLS & Key words: biological control, Metarhizium anisopliae, molecular ALTIERI, 2007). One of the objectives of biological markers. control is to assure that the benefi cial organism to be introduced can complete its lifecycle at the site, RESUMO and then reproduce with suffi cient effi ciency to become a permanent resident of the agrosystem. O controle microbiano consiste na utilização racional de patógenos, visando à manutenção da população de Frequently, however, the niche conditions available insetos em equilíbrio no ambiente. Metarhizium anisopliae é a to the benefi cial introduced organism do not espécie mais estudada e utilizada no controle biológico de insetos. fully satisfy its long-term needs, requiring its A variabilidade genética dos fungos entomopatogênicos pode ser considerada uma das principais vantagens no controle microbiano reintroduction (GLIESSMAN, 2001). Changes in de insetos e pode ser detectada por meio de marcadores production practices and the use of agricultural moleculares, como ITS-RFLP, ISSR e ISSP. Esses marcadores additives are often necessary for biological control são usados para a caracterização inter e intraespecífi ca de Metarhizium e outros fungos entomopatogênicos e poderão to be successful. Integrated Pest Management (IPM) auxiliar na compreensão da diversidade genética e da estrutura is an alternative to unilateral intervention strategies das populações destes fungos. A persistência de M. anisopliae no using agrochemicals, with a wider focus on the solo e seu possível efeito na estrutura da comunidade microbiana ecology of the insect pests as well as the crop plants, deste solo são características importantes e pouco estudadas, que devem ser consideradas no processo de seleção de isolados para o based on the use of complementary tactics and the controle biológico de insetos. adoption of cultivation techniques that favor plant diversity. Pest control in this type of approach is Palavras-chave: controle biológico, Metarhizium anisopliae, marcadores moleculares. initially based on natural agents such as pathogens,
IDepartamento de Micologia, Centro de Ciências Biológicas (CCB), Universidade Federal de Pernambuco (UFPE), Av. Prof. Nelson Chaves s/n, 50670-420, Recife, PE, Brasil. E-mail: [email protected] *Autor para correspondência. Received 01.29.13 Approved 10.10.13 Returned by the author 01.27.14 CR-2013-0122.R2 Ciência Rural, v.44, n.4, abr, 2014. 646 Tiago et al. parasites and predators, with the use of agrotoxins structures or blastospores and appressoria are produced being contemplated only as a last resort. However, as by M. anisopliae through mycelial differentiation. biological pest control methods do not demonstrate Blastospores can function in certain cases as immediate results in agro-industrial systems with reproductive units and are produced in submerged large-scale production and commercialization goals cultures (JACKSON & JARONSKI, 2009) and in (as agrotoxins), commercial groups tend to avoid the hemolymph of insect hosts (ALVES, 1998). The the costs and labor related to their development appressoria, formed at the extremity of the hyphae, and perfection. Nonetheless, growing energy costs, may be involved in fungus pathogenicity and have environmental degradation, and infl ation all reinforce the function of initiating epicuticular and procuticle the argument that immediate fi nancial gains should penetration of the insect tegument (ALVES, 1998). not be the principal motivating force in agricultural The production of microsclerotia by isolates of M. production (ALTIERI, 2002). In spite of the strong anisopliae has been observed after cultivation in liquid economic pressure on agricultural production, many media with different concentrations of carbon and farmers are making the transition to practices that are carbon-nitrogen (JACKSON & JARONSKI, 2009). more environmentally friendly and have the potential The fungal-host relationship occurs to contribute to long-term agricultural sustainability through the adhesion and germination of conidia with biological control being one of th e principal tools on the surface of the insect, followed by hyphae in this conversion process (GLIESSMAN, 2001). penetration through the cuticle. The process of host Microbial control is an aspect of biological colonization initiates after penetration, with the insect control and consists of the rational use of penetrating hyphae becoming thicker and ramify pathogens to maintain pest balances in agricultural within the tegument and the hemocoel of the insect, environments, with increases in the numbers of forming blastospores. The hyphae continue to grow other natural enemies often being observed in and invade various internal organs after the death of fi elds where microbial control has been used. the host and will subsequently emerge from the insect Successful programs of microbial control using body and produce conidia that disseminate and infect entomopathogenic fungi to combat arthropod pests in other individuals (ALVES, 1998). soils and aquatic environments have been developed, Molecular studies of the processes of principally utilizing the genera Metarhizium, host infection have shown them to be complex and Beauveria, Sporothrix, Lecanicillium, Nomuraea, multifactorial. The adhesion and penetration steps Hirsutella, Aschersonia, Isaria, Paecilomyces, and have been most closely examined and appear to be Enthomophthora (ALVES & LOPES, 2008). Species decisive to infection. The participation of an adhesin within the genus Metarhizium are pathogenic fungi coded by the gene Mad1 in the adhesion of conidia having broad ranges of insect hosts. M. anisopliae to the cuticle of Manduca sexta Linnaeus larva was was found to be a species complex composed of nine demonstrated using mutants in which this gene was species based on multilocus phylogeny (BISCHOFF deleted, with these mutants demonstrating signifi cant et al., 2009). The objective of this study was to analyze some morphological, molecular and ecological decreases in conidial germination, suppression of aspects of M. anisopliae. the formation of blastospores, and reduced virulence (WANG & St. LEGER, 2007a). COSENTINO- Metarhizium anisopliae GOMES et al. (2013) described that the inhibition of Metarhizium anisopliae, a anamorphic phosphatase activity in the conidia of M. anisopliae fungus which belong to the phylum Ascomycota, reduced adhesion to the integument of Dysdercus is the most intensively studied species of the genus peruvianus (Hemiptera: Pyrrhocoridae) and Metarhizium, considering that the teleomorph (indirectly) its infection. Cordyceps brittlebankisoides [= Metacordyceps The participation of perilipin (proteins brittlebankisoides (Liu, Liang, Whalley, Yao & Liu) that surround lipidic droplets in the cell interior) Sung, Sung, Hywel-Jones & Spatafora] was isolated in appressoria differentiation in M. anisopliae has from insect larva (Coleoptera: Scarabaeidae) and also been reported. The deactivation of the Mpl1 identifi ed as M. anisopliae var. majus [= M. majus gene in some strains generates defi ciencies in the (Johnston) Bischoff, Rehner & Humber] (LIU et al., infection process due to the formation of appressoria 2001). The reproductive structures of M. anisopliae with lower concentrations of lipidic droplets and (the anamorph, the most commonly encountered form) resultantly lower levels of osmotic pressure - resulting comprise conidiophores and conidia. Leveduriform in diffi culties in terms of hyphal penetration (WANG
Ciência Rural, v.44, n.4, abr, 2014. Biological insect control using Metarhizium anisopliae: morphological, molecular, and ecological aspects. 647
& St. LEGER, 2007b). Defective appressoria were regions composed of 18S, 5.8S and 28S genes that are also observed after the deletion of the mapka1 gene transcribed and processed to generate mature rRNA, (catalytic subunit 1 of the protein kinase A) (FANG but are separated by variable intergenic spacer regions et al., 2009). Subtilisin-type proteases have been denominated ITS1 and ITS2. The genetic aggregate intensively studied in penetration processes, and 10 that codes for rRNA appears to be repeated hundreds genes are known to code for different isoforms of of times in the fungus genome and demonstrates these enzymes (Pr1A - Pr1J) and appear to refl ect both highly conserved and variable regions, allowing specifi city in relation to different hosts (BAGGA et scientists to analyze variations at different taxonomic al., 2004). MOS1 is another protein with an apparent levels. The 18S region is the most highly conserved, role in the adaptation of fungi to the high osmotic and is therefore only used in comparisons between pressure encountered in insect hemolymph (WANG distantly related organisms. The 28S region is more et al., 2008). Other genes, such as Mcl1 (collagen- variable and therefore appropriate for comparing like protein), Cag8 (which regulates the G protein different genera (or different species, in some cases). signaling pathway), chi2 (endochitinase), chi3 (endo- ITS regions evolve relatively rapidly and can be used and exochitinase), and Mpk1 (phosphoketolase) are to distinguish closely related species or even varieties known to be involved in the host infection processes within the same species (FUNGARO, 2000). of M. anisopliae, with reductions in virulence if they DNA samples digested with restriction are inactivated (FANG et al., 2007; BOLDO et al., enzymes (RFLP) can identify polymorphisms based 2009; DUAN et al., 2009). SU et al. (2013) undertook on the numbers and sizes of the fragments produced, comparative proteomic analyses of the conidia and which allows the differentiation of species and mycelia of M. anisopliae (Ma1291). The proteins isolates of Metarhizium based on the presence or identifi ed as exclusive to the conidia were involved absence of rDNA restriction sites (PIPE et al., 1995). in protective processes, appressorium formation, In the present study, isolates of M. anisopliae could and the degradation of the host cuticle and exclusive be grouped according to their geographical origins, proteins to mycelia were involved in biosynthetic and although no signifi cant correlations were observed energy-generating metabolic processes, such as UTP- in terms of their hosts. VELÁSQUEZ et al. (2007) glucose-1-phosphate uridylyltransferase and heat- observed that there were no associations between the shock protein 70. diversity of isolates of M. anisopliae from different regions in Chile and their geographic origins. ITS- Molecular characterization RFLP was used to defi ne specifi c primers that could Molecular markers can represent the be used to detect and identify M. anisopliae var. phenotype of an expressed gene or a DNA segment anisopliae (DESTÉFANO et al., 2004). corresponding to a non-expressed region of the Eukaryotic chromosomes contain genes genome. Advances in molecular biology have that are separated by non-coding regions (introns) as resulted in the development of various methods for well as regions with coding information represented detecting genetic polymorphism at the DNA level by proteins (exons). Introns can be separated into and have aided our understanding of genetic diversity four basic categories according to their structural and the population structures of fungi populations characteristics and self-splicing mechanisms: group I, (FALEIRO, 2007). II, nuclear pre-mRNA, and nuclear tRNA. The introns The polymerase chain reaction (PCR) of groups I and II are classifi ed according to their technique, allied to methodologies of cloning and DNA internal organizations and have the intrinsic capacity sequencing, have allowed the rapid accumulation of self-splicing; the latter two intron groups can be of information relating to genome structure and the used as molecular markers in intra- and interspecifi c discovery of repetitive DNA sequences (which are studies of diversity (HAUGEN et al., 2005). rich sources of genetic polymorphism). A number Group I introns are encountered in of methodologies have been described for analyzing eukaryotic organisms such as fungi, protists, and polymorphisms based on PCR, including ITS-RFLP green algae in nuclear, mitochondrial, and chloroplast (Internal Transcribed Spacer - Restriction Fragment genomes. Group I introns are encountered in the Length Polymorphism), ISSP (Intron Splice Site eukaryotic nuclear genome in rDNA genes at specifi c Primer), ISSR (Inter Simple Sequence Repeats), and sites that code for the larger and smaller rRNA SSR (Simple Sequence repeats) (FALEIRO, 2007). subunits. These introns are autonomous genetic The DNAs coding for rRNA are arranged elements characterized by their capacity to transfer as genetic aggregates with three genetically conserved from one allele to another (as some are mobile
Ciência Rural, v.44, n.4, abr, 2014. 648 Tiago et al. elements – transposons) and by their ability to self- with fl uctuations in its population levels. Additional splice from RNA transcripts (HAUGEN et al., 2005). studies will therefore be necessary to determine if the Group I introns are generally irregularly distributed plant rhizosphere can truly be considered a refuge – being present in some isolates but absent in others (a locality where the fungus can survive outside its – and thus can serve as markers of genetic variability. insect host) for M. anisopliae in the soil. MEYLING Genetic diversity among M. anisopliae isolates have & EILENBERG (2007) suggested that plant been observed in studies of genes associated with associations are important to the biological cycle of the largest ribosomal subunit and with four insertion M. anisopliae in temperate regions. It is possible sites of group I introns, and the presence/absence of that this fungus has multiple functions in terms of these introns allow the delimitation of seven groups plant protection, with antagonistic effects against (MÁRQUEZ et al., 2006). phytopathogenic fungi. Microsatellite DNAs show numerous A number of studies have examined the short, repeated, tandem sequences, and their analyses molecular mechanisms involved in the capacity of involve replicating fragments containing those M. anisopliae to adhere to both insects and roots, repetitions through the use of oligonucleotides that resulting in the identifi cation of adhesins MAD1 bind to the regions which fl ank them (SSR sites). and MAD2. Adhesin MAD1 is involved in insect These markers were used to examine polymorphism pathogenicity and MAD2 with fungal adhesion to in M. anisopliae, but the primers used to examine plant roots (WANG & St. LEGER, 2007a), and a samples derived from soil cores from different study by WANG et al. (2005) examining genetic regions in Chile (VELÁSQUEZ et al., 2007) and expression demonstrated that M. anisopliae could numerous countries in Asia and Europe (FREED et act as both a pathogen (growing on the cuticle and in al., 2010) were not effi cient in detecting informative the hemolymph of insect hosts) and a saprophyte in polymorphisms. To the contrary of microsatellite the rhizosphere (growing on the bean root exudates). analysis, the ISSR technique amplifi es fragments WYREBEK & BIDOCHKA (2013) amplifi ed and located between two repetitive regions present cloned the full Mad1 and Mad2 genes in fourteen in various genomes (FALEIRO 2007), known as isolates of seven different species of Metarhizium to inter-microsatellite regions. This ISSR marker did assess their genetic variability. Phylogenetic analyses demonstrate differences among different isolates of 5’ EF-1α (which is used for species identifi cation), of M. anisopliae var. anisopliae of the same origin Mad1, and Mad2 indicated that the evolution of the and from the same host, principally when using Mad2 gene was more congruent with the phylogeny the primers (GACA)4 and (GTG)5, providing DNA of 5’ EF-1α than of Mad1. This suggests that Mad2 fi ngerprints for a number of isolates (TIAGO et al., diverged among the Metarhizium lineages and 2011). The genetic structure of Metarhizium spp. contributed to clade- and species-specifi c variations, (M. anisopliae and its sister species, M. robertsii), while Mad1 was largely conserved. pathogens found in Chinese burrower bugs Studies have shown that some insect populations (Schiodtella formosana), were assessed pathogenic endophytic fungi, such as Metarhizium, using ISSR. They differentiated into two main clades are able to transfer insect-derived nitrogen to including over 71% of all strains causing epizootics, plant roots, probably in exchange for plant sugars. with a similarity of 83% (LUAN et al., 2013). Metarhizium has a phylogenetic heritage of plant symbiosis (i.e., the genus is closely related to other The soil ecology of Metarhizium anisopliae endophytes) and has also evolved as a generalist Metarhizium anisopliae demonstrates insect pathogen (BEHIE et al 2013). considerable metabolic and ecological versatility A number of workers have investigated the and has been observed colonizing the rhizosphere persistence of M. anisopliae in the soil, with greater and adhering to the surfaces of plant roots, and it fungal survival being observed in sandy-clay soils may signifi cantly infl uence this ecological niche by and in soils with average compaction density values repelling and killing soil insects (HU & St. LEGER, (LANZA et al., 2004). High average numbers of 2002). BRUCK (2005) observed that the conidia of colonies of M. anisopliae could be recovered 30 days M. anisopliae demonstrated greater persistence in the after inoculation, and viability for up to 120 days was rhizosphere of Picea abies Linnaeus than in the soil observed in previously sterilized soils (GUERRA alone. On the other hand, a pilot study by St. LEGER et al., 2009) and for up to 216 days after fi eld (2008) in a pasture site indicated that M. anisopliae inoculation (MARTINS et al., 2004). A study based could survive for various years in the soil, although on quantitative PCR (qPCR) demonstrated that
Ciência Rural, v.44, n.4, abr, 2014. Biological insect control using Metarhizium anisopliae: morphological, molecular, and ecological aspects. 649
Metarhizium Clade 1 (M. majus, M. guizhouense, a plant symbiont that can act as a saprophyte in the M. pinghaense, M. anisopliae, M. robertsii, M. rhizosphere but has also evolved as a generalist insect brunneum) was present at high densities in soil pathogen. As such, the paradigm that M. anisopliae samples from pastures and improved fi eld margins, is principally an insect pathogen is questionable, indicating that both of these semi-natural habitat and additional studies will be necessary to better types provide potential refuges for these species understand its ecological role in the soil. (SCHNEIDER et al., 2012). The introduction of exogenous REFERENCES microorganisms into natural and agricultural ecosystems may affect the soil microbial community ALTIERI, M. Agroecologia: bases científi cas para uma agricultura sustentável. Guaíba: Agropecuária, 2002. 400p. and, consequently, diverse ecological processes in those environments. The effects of the introduction ALVES, S.B. Controle microbiano de insetos. Piracicaba: of entomopathogenic fungi into soil microbial FEALQ, 1998. 1163p. communities represent an ecological intervention that ALVES, S.B.; Lopes, R.B. Controle microbiano de pragas na has not yet been extensively examined. A study by América Latina: avanços e desafi os. Piracicaba: FEALQ, 2008. SCHWARZENBACH et al. (2009) using ribosomal 414p. internal spacer analysis (RISA) to examine the effects of B. brongniartii on fungal community structures in BAGGA, S. et al. Reconstructing the diversifi cation of subtilisins soil microcosms indicated that its presence in the soil in the pathogenic fungus Metarhizium anisopliae. Gene, v.324, p.159-169, 2004. Available from:
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