RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-beta-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-beta-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-beta-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.

Identification of Aspergillus flavus isolates as potential biocontrol agents of aflatoxin contamination in crops

Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops

Parasitism by Ixodiphagus Wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus and Amblyomma Ticks (Acari: Ixodidae) in Three Regions of Brazil

Hymenopteran parasitoids have been shown to be of potential value in tick biocontrol. Tick parasitoids has been reported only once in Brazil, over 95 yr ago when Ixodiphagus hookeri (Howard) was reported parasitizing Rhipicephalus sanguineus (Latreille) nymphs in Rio de Janeiro. Herein, we report the occurrence of Ixodiphagus spp. in ticks from three different regions of Brazil. In the state of Maranhao, in northeastern Brazil, parasitoids were detected in R. sanguineus nymphs on three occasions, during August 2009 and September 2010 (dry season), and January 2011 (rainy season). All parasitoids found in northeastern Brazil were identified as I. hookeri. In the state of Mato Grosso do Sul (westDcentral Brazil), one Amblyomma sp. engorged nymph was shown to be parasitized by I. hookeri. In the state of Rondonia (northern Brazil), one Amblyomma sp. engorged nymph was parasitized by Ixodiphagus texanus (Howard). Because Ixodiphagus spp. are present in ecologically distinct and geographically distant areas of Brazil, they are of potential use for biocontrol in the country.

Biological and behavioral parameters of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae) are altered by the pathogen Nosema sp (Microsporidia: Nosematidae)

A major issue for mass rearing of insects concerns sanitary conditions and disease. Microsporidian infection (Nosema sp.) in laboratory colonies of Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae), used in producing the parasitoid. Cotesia flavipes Cameron (Hymenoptera: Braconidae), is representative of the problems faced by growers and industry. Although C. flavipes has been produced for several years in Brazil for biological control of D. saccharalis, we have only recently observed that the parasitoid becomes infected when developing inside hosts infected with Nosema sp. We assessed the effects of Nosema sp. on C. flavipes, including the ability to locate and select hosts, and evaluated pathogen transmission. Third instar larvae of D. saccharalis were inoculated with Nosema sp. spores at different concentrations and were parasitized when larvae reached fifth instar. Heavily infected D. saccharalis larvae did not support parasitism. Parasitoids that developed in infected D. saccharalis larvae exhibited increased duration of larval and pupal stages, decreased adult longevity and number of offspring, and reduced tibia size compared to parasitoids developing in uninfected D. saccharalis larvae. Infection by Nosema sp. reduced the ability of the C. flavipes parasitoid to distinguish between volatiles released by the sugarcane infested by healthy larvae and pure air. Uninfected parasitoids preferred plants infested with uninfected hosts. But infected C. flavipes did not differentiate between uninfected hosts and those infected with Nosema sp. The pathogen is transmitted from host to parasitoids and parasitoids to hosts. Pathogenic effects of the microsporidium in C. flavipes are sufficiently severe to justify disease management efforts, particularly considering the importance of C. flavipes as a biological control agent in sugarcane. (C) 2012 Elsevier Inc. All rights reserved.

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

Isolation, selection and characterization of root-associated growth promoting bacteria in Brazil Pine (Araucaria angustifolia)

Araucaria angustifolia, a unique species of this genus that occurs naturally in Brazil, has a high socio-economic and environmental value and is critically endangered of extinction, since it has been submitted to intense predatory exploitation during the last century. Root-associated bacteria from A. angustifolia were isolated, selected and characterized for their biotechnological potential of growth promotion and biocontrol of plant pathogenic fungi. Ninety-seven strains were isolated and subjected to chemical tests. All isolates presented at least one positive feature, characterizing them as potential PGPR. Eighteen isolates produced indole-3-acetic acid (IAA), 27 were able to solubilize inorganic phosphate, 21 isolates were presumable diazotrophs, with pellicle formation in nitrogen-free culture medium, 83 were phosphatases producers, 37 were positive for siderophores and 45 endospore-forming isolates were antagonistic to Fusarium oxysporum, a pathogen of conifers. We also observed the presence of bacterial strains with multiple beneficial mechanisms of action. Analyzing the fatty acid methyl ester (FAME) and partial sequencing of the 16S rRNA gene of these isolates, it was possible to characterize the most effective isolates as belonging to Bacillaceae (9 isolates), Enterobacteriaceae (11) and Pseudomonadaceae (1). As far as we know, this is the first study to include the species Ewingella americana as a PGPR. (C) 2011 Elsevier GmbH. All rights reserved.

Trichogramma pretiosum parasitism of Pseudoplusia includens and Anticarsia gemmatalis eggs at different temperatures

Egg parasitism of Trichogramma pretiosum strain RV when presented with eggs of Anticarsia gemmatalis and Pseudoplusia includens was investigated at 18, 20, 22, 25, 28, 30 and 32 degrees C. The number of eggs parasitized per day decreased for both hosts as a function of the age of parasitoids, reaching 80% of lifetime parasitism more rapidly as temperature increased; on the 4th day at 32 degrees C and on the 12th day at 18 degrees C. The lifetime number of parasitized P. includens eggs achieved by the parasitoid maintained at 20 degrees C (44.95 +/- 3.94) differed from the results recorded at 32 degrees C (28.5 +/- 1.33). Differently, the lifetime number of A. gemmatalis parasitized eggs did not differ among the temperatures. When T. pretiosum reached 100% of lifetime parasitism, each adult female had parasitized from 28.5 +/- 1.33 to 44.95 +/- 3.94 and from 29.58 +/- 2.80 to 45.36 +/- 4.50 P. includens and A. gemmatalis eggs, respectively. Also, the longevity of these adult T. pretiosum females, for which P. includens or A. gemmatalis eggs were offered, was inversely correlated with temperature. Not only were the survival curves of those adult T. pretiosum females of type I when they were presented with eggs of A. gemmatalis but also with eggs of P. includens, i.e., there was an increase in the mortality rate with time as the temperature increased. In conclusion, T. pretiosum strain RV parasitism was impacted by temperature when on both host eggs; however, the parasitoid still exhibited high survival and, more importantly, high number of parasitized A. gemmatalis and P. includens eggs even at the extremes tested temperatures of 18 and 32 degrees C. Those results indicate that T. pretiosum strain RV might be well adapted to this studied temperature range and, thus, be potentially suitable for use in biological control programs of P. includens and A. gemmatalis in different geographical areas that fits in this range. It is important to emphasize the results here presented are from laboratory studies and, therefore, field trials still need to be carried out in the future with this strain in order to support the full development of the technology intend to use this egg parasitoid in soybean fields worldwide. (C) 2011 Elsevier Inc. All rights reserved.

Epicoccum nigrum P16, a Sugarcane Endophyte, Produces Antifungal Compounds and Induces Root Growth

Background: Sugarcane is one of the most important crops in Brazil, mainly because of its use in biofuel production. Recent studies have sought to determine the role of sugarcane endophytic microbial diversity in microorganism-plant interactions, and their biotechnological potential. Epicoccum nigrum is an important sugarcane endophytic fungus that has been associated with the biological control of phytopathogens, and the production of secondary metabolites. In spite of several studies carried out to define the better conditions to use E. nigrum in different crops, little is known about the establishment of an endophytic interaction, and its potential effects on plant physiology. Methodology/Principal Findings: We report an approach based on inoculation followed by re-isolation, molecular monitoring, microscopic analysis, plant growth responses to fungal colonization, and antimicrobial activity tests to study the basic aspects of the E. nigrum endophytic interaction with sugarcane, and the effects of colonization on plant physiology. The results indicate that E. nigrum was capable of increasing the root system biomass and producing compounds that inhibit the in vitro growth of sugarcane pathogens Fusarium verticillioides, Colletotrichum falcatum, Ceratocystis paradoxa, and Xanthomomas albilineans. In addition, E. nigrum preferentially colonizes the sugarcane surface and, occasionally, the endophytic environment. Conclusions/Significance: Our work demonstrates that E. nigrum has great potential for sugarcane crop application because it is capable of increasing the root system biomass and controlling pathogens. The study of the basic aspects of the interaction of E. nigrum with sugarcane demonstrated the facultative endophytism of E. nigrum and its preference for the phylloplane environment, which should be considered in future studies of biocontrol using this species. In addition, this work contributes to the knowledge of the interaction of this ubiquitous endophyte with the host plant, and also to a better use of microbial endophytes in agriculture.

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Background and aims Endophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants. Methods A total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique. Results The putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA. Conclusion Endophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86 % as Trichoderma asperellum, 33.33 % as Trichoderma harzianum, 14.29 % as Trichoderma tomentosum, 4.76 % as Trichoderma koningiopsis, and 4.76 % as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen. (C) 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Isolation and characterisation of aerobic endospore forming Bacilli from sugarcane rhizosphere for the selection of strains with agriculture potentialities

Eighteen aerobic endospore forming strains were isolated from sugarcane rhizosphere in N-free medium. A phenotypic description and analysis of the 5' end hypervariable region sequences of 16S rRNA revealed a high diversity of Bacillus and related genera. Isolates were identified, and four genera were obtained: seven strains belonged to Bacillus (Bacillaceae family), four belonged to Paenibacillus, six belonged to Brevibacillus and one strain was identified as Cohnella (Paenibacillaceae family). Four Brevibacillus strains showed in vitro inhibitory activity against plant pathogens fungi Curvularia and Fusarium. Seventy-four percent of the isolated bacteria grew on pectin as the only carbon source, showing polygalacturonase activity. Pectate lyase activity was detected for the first time in a Brevibacillus genus strain. All isolates showed endoglucanase activity. Calcium phosphate solubilisation was positive in 83.3% of the isolates, with higher values than those reported for Bacillus inorganic phosphate solubilising strains. High ethylene plant hormone secretion in the culture medium was detected in 22% of the bacteria. This is the first report of ethylene secretion in Paenibacillaceae isolates. Indole-3-acetic acid production was found in a Brevibacillus genus isolate. It was reported for the first time the presence of Cohnella genus strain on sugarcane rhizosphere bearing plant growth promoting traits. The sugarcane isolate Brevibacillus B65 was identified as a plant growth inoculant because it showed wider spectra of plant stimulation capabilities, including an antifungal effect, extracellular hydrolases secretion, inorganic phosphate solubilisation and plant hormone liberation. In this work, sugarcane was shown to be a suitable niche for finding aerobic endospore forming 'Bacilli' with agriculture biotechnological purposes.

Epicolactone - Natural Product Isolated from the Sugarcane Endophytic Fungus Epicoccum nigrum

The endophytic fungus Epicoccum nigrum was isolated from sugarcane and the bioguided fractionation of the ethyl acetate extract led to the isolation of epicolactone, mellein, and 4,5-dimethylresorcinol. Characterization of epicolactone by MS, NMR and X-ray crystallography revealed a new natural product with an unusual carbon skeleton. The production of this secondary metabolite decreased in mutants of Epicoccum nigrum transformed by Agrobacterium tumefaciens. Additionally, these mutants produced 4-hydroxymellein.

Identification of differentially expressed genes from Trichoderma harzianum during growth on cell wall of Fusarium solani as a tool for biotechnological application

Background: The species of T. harzianum are well known for their biocontrol activity against many plant pathogens. However, there is a lack of studies concerning its use as a biological control agent against F. solani, a pathogen involved in several crop diseases. In this study, we have used subtractive library hybridization (SSH) and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum genes expression during growth on cell wall of F. solani (FSCW) or glucose. RT-qPCR was also used to examine the regulation of 18 genes, potentially involved in biocontrol, during confrontation between T. harzianum and F. solani. Results: Data obtained from two subtractive libraries were compared after annotation using the Blast2GO suite. A total of 417 and 78 readable EST sequence were annotated in the FSCW and glucose libraries, respectively. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on FSCW or glucose. We identified various genes of biotechnological value encoding to proteins which function such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. Fifteen genes were up-regulated and sixteen were down-regulated at least at one-time point during growth of T. harzianum in FSCW. During the confrontation assay most of the genes were up-regulated, mainly after contact, when the interaction has been established. Conclusions: This study demonstrates that T. harzianum expressed different genes when grown on FSCW compared to glucose. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against F. solani. The identification and evaluation of these genes may contribute to the development of an efficient biological control agent.