Efficient Biocontrol Agent Research Articles

Molecular characterization and functional properties of deep-soil-inhabiting actinobacteria for combating Fusarium dieback disease in tea crop

Dieback, caused by Fusarium solani, severely damages tea foliage and decreases both the quality and quantity of harvestable shoots. The management of dieback disease through integrated approach has been suggested and the use of microbial biocontrol agents is one of the alternative methods. The search for efficient microbial biocontrol agents is also essential for overcoming the drawbacks of synthetic fungicides. Among the microbial biocontrol agents, actinobacteria have promising role in disease management. In the present study, 18 native isolates of actinobacteria were isolated from tea rhizosphere soil collected from 12 tea gardens. Evaluation of 18 isolates of actinobacteria against F. solani by dual culture technique revealed that isolates MBK05, MBK21, MBK31, and MBK34 were potential antagonists. Among them, MBK05 and MBK21 were fast growing isolates, hence selected for detailed study like morpho-molecular characterization, for antifungal traits as well as their efficacy in disease control under field conditions. Based on the 16S rRNA, the isolates MBK05 and MBK21 were putatively identified as Kitasatospora putterlickiae and Streptomyces bungoensis, respectively. Both isolates possessed multifarious physiological activities and were found to produce cellulase, protease, chitinase (except MBK05), and β-glucanase. The liquid formulations of both isolates in 10 and 20 % concentrations (1 × 108 CFU/mL) were evaluated in two seasons against Fusarium dieback under field conditions. Results revealed that 20 % concentration of both isolates gave maximum percent disease reduction over control, i.e., 72.25 % with MBK05 and 70.3 % with MBK21. Among the synthetic fungicides evaluated, copper oxychloride 50 %WP (1 %) showed maximum disease reduction (75.46 %). These results support that K. putterlickiae and S. bungoensis are promising microbial biocontrol agents against Fusarium dieback in tea crop.

Entomopathogenic bacteria Photorhabdus luminescens as natural enemy against the African migratory locust, Locusta migratoria migratorioides (Reiche & Fairmaire, 1849) (Orthoptera: Acrididae)

BackgroundThe African Migratory Locust, Locusta migratoria migratorioides (Reiche & Fairmaire, 1849) (Orthoptera: Acrididae), is a major threat to agricultural crops and food security on a worldwide scale; hence, maintaining control over it is crucial. Photorhabdus luminescens bacteria can accomplish the efficient biocontrol agent criteria. As a result, the aim of this study was to assess the efficacy of the P. luminescens (EGAP3) strain and its cell-free filtrate against L. migratoria migratorioides, as well as to investigate changes in the activity of carbohydrates hydrolyzing enzymes, amylase, invertase, and trehalase in whole-body homogenates of the 5th nymphal instar under laboratory conditions and to investigate the histopathological changes in the midgut of the locust.ResultsThe virulence of entomopathogenic bacteria was determined at different densities of 4 × 107, 4 × 106, 4 × 105, and 4 × 104 colony-forming units (CFU)/ml at different exposure times as well as different concentrations of its cell-free filtrate, undiluted cell-free filtrate (100, 50, 25, and 12.5%). The results indicated that higher-density cell suspension up to 4 × 107 cells.ml−1 and undiluted cell-free filtrate (100%) were the most effective insecticidal fluids, reaching up to 76.7 and 80%, respectively, after 7 days. The estimated LC50 value was 2.7 × 106 cells.ml−1 for bacteria, and the estimated LC50 value for cell-free filtrate was the 2nd dilution (50%).ConclusionsThe data clarified the toxicological and histopathological effects and carbohydrate hydrolyzing enzyme activities of the host insect L. migratoria migratorioides, following bacteria and cell-free filtrate infection; that provides an overview of the efficiency of bacteria and their cell-free filtrate on the host. In conclusion, P. luminescens (EGAP3) and its toxins can be an optimal option for bio-controlling of L. migratoria migratorioides.

AMPK Is Involved in Regulating the Utilization of Carbon Sources, Conidiation, Pathogenicity, and Stress Response of the Nematode-Trapping Fungus Arthrobotrys oligospora.

ABSTRACTAMP-activated protein kinase (AMPK), a heterotrimeric complex, can sense energy and nutritional status in eukaryotic cells, thereby participating in the regulation of multiple cellular processes. In this study, we characterized the function of the catalytic α-subunit (SNF1) and the two regulatory β- and γ-subunits (GAL83 and SNF4) of AMPK in a representative nematode-trapping fungus, Arthrobotrys oligospora, by gene knockout, phenotypic analysis, and RNA sequencing. The ability of the AMPK complex mutants (including ΔAosnf1, ΔAogal83, and ΔAosnf4) to utilize a nonfermentable carbon source (glycerol) was reduced, and the spore yields and trap formation were remarkably decreased. Moreover, AMPK plays an important role in regulating stress response and nematode predation efficiency. Transcriptomic profiling between the wild-type strain and ΔAosnf1 showed that differentially expressed genes were enriched for peroxisome, endocytosis, fatty acid degradation, and multilipid metabolism (sphingolipid, ether lipid, glycerolipid, and glycerophospholipid). Meanwhile, a reduced lipid droplet accumulation in ΔAosnf1, ΔAogal83, and ΔAosnf4 mutants was observed, and more vacuoles appeared in the mycelia of the ΔAosnf1 mutant. These results highlight the important regulatory role of AMPK in the utilization of carbon sources and lipid metabolism, as well as providing novel insights into the regulatory mechanisms of the mycelia development, conidiation, and trap formation of nematode-trapping (NT) fungi.IMPORTANCE NT fungi are widely distributed in various ecosystems and are important factors in the control of nematode populations in nature; their trophic mycelia can form unique infectious devices (traps) for capturing nematodes. Arthrobotrys oligospora is a representative NT fungi which can develop complex three-dimensional networks (adhesive networks) for nematode predation. Here, we demonstrated that AMPK plays an important role in the glycerol utilization, conidiation, trap formation, and nematode predation of A. oligospora, which was further confirmed by transcriptomic analysis of the wild-type and mutant strains. In particular, our analysis indicated that AMPK is required for lipid metabolism, which is primarily associated with energy regulation and is essential for trap formation. Therefore, this study extends the functional study of AMPK in NT fungi and helps to elucidate the molecular mechanism of the regulation of trap development, as well as laying the foundation for the development of efficient nematode biocontrol agents.

Chitosan-harvested polyhydroxybutyrate-rich Zobellella denitrificans ZD1 as a multifunction feed for aquaculture

The security of the aquaculture industry is jeopardized by antibiotics overuse and increasing demand for aquaculture feeds, which remain heavily derived from capture fisheries. Biopolymer poly(3-hydroxybutyrate) (PHB) and some microorganisms are promising replacements for antibiotics and aquaculture feed, respectively. Therefore, we investigated the potential of using chitosan-harvested PHB-rich Zobellella denitrificans ZD1 (designated as CP-ZD1 hereafter) as a multifunction feed for brine shrimp Artemia (model species). Both PHB and chitosan and their intermediates (3-hydroxybutyrate and chitosan oligosaccharides) showed antimicrobial properties toward three known aquaculture pathogens (Vibrio campbellii, Aeromonas hydrophila, and Streptococcus agalactiae) and non-pathogens (non-pathogenic Escherichia coli, Bacillus megaterium, and Rhodococcus jostii RHA1). Low doses of 3-hydroxybutyrate and chitosan oligosaccharides mixtures effectively suppressed the growth of the pathogens. When supplied with CP-ZD1, the starved Artemia showed prolonged survival and a healthy gut microbiome, suggesting that CP-ZD1 can serve as a feed/energy source. Up to 80% enhanced survival and immune response was observed in pathogenic V. campbellii-challenged Artemia when fed with CP-ZD1. Overall, the results showed that CP-ZD1 could serve as an efficient biocontrol agent, a feed/energy source, and an immunostimulant in support of sustainable aquaculture.

Morphological and molecular identification of the entomopathogenic fungus Purpureocillium lilacinum and its virulence against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) larvae and pupae

BackgroundThe tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is a serious pest of solanaceous plant species, mainly Solanum lycopersicum L. The entomopathogenic fungi, an alternative to chemical insecticides, proved to be an efficient biocontrol agent in reducing pest population density. In the present study, an entomopathogenic fungus, Purpureocillium lilacinum isolated from soil was identified based on the morphological and molecular characteristics and its pathogenicity was tested against target pest.ResultsThe micromorphological characters showed variations in growth pattern, shape and colour on different cultural media. For molecular analysis, a phylogenetic tree based on ITS/LSU and ITS/β-tubulin (benA) gene regions was constructed which revealed the isolate (FC18) as P. lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones and Samson (Family: Ophiocordycipitaceae). Further, the pathogenicity of P. lilacinum was tested using different spore concentrations (1 × 108, 1 × 107, 1 × 106, 1 × 105 and 1 × 104 spores/ml) on larval and pupal stages of T. absoluta, which showed a dose-dependent mortality. At the highest concentration (1 × 108 spores/ml), the mean mortality of 92.99, 83.05, 72.0% of second, third and fourth instar was observed, respectively. Pupal mortality also showed significant differences at different spore concentrations.ConclusionObtained results showed that the virulence of the indigenous strain of P. lilacinum on T. absoluta can be utilized in the field suppression of the pest as a potent biocontrol agent.

Egg Predation by Phytoseiid Predatory Mites: Is There Intraguild Predation Towards Predatory Bug Eggs?

Phytoseiid predatory mites are efficient biocontrol agents of important thrips pests, such as the western flower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae). Until recently, it was believed that first instars, and to a lesser extent second instars, were the most vulnerable developmental stages of thrips to be attacked by phytoseiids. However, recent evidence showed that some phytoseiids can detect and prey upon thrips eggs inserted in the leaf tissue. As phytoseiid predatory mites often co-occur with other beneficial insects, such as mirid and anthocorid predatory bugs which also insert their eggs inside leaf material, this raises the question whether phytoseiid predatory mites may also feed on predatory bug eggs. Here we first tested the potential of Amblyseius swirskii Athias-Henriot, Transeius montdorensis Schicha, and Amblydromalus limonicus Garman and McGregor (Acari: Phytoseiidae) to kill eggs of F. occidentalis in leaf tissue. Secondly, we tested whether those phytoseiids were capable of killing eggs of Orius laevigatus Fieber (Hemiptera: Anthocoridae), Macrolophus pygmaeus Rambur and Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), three biocontrol agents that also insert their eggs inside plant tissue. Our results showed that A. swirskii and A. limonicus could kill thrips eggs, whereas T. montdorensis could not. Furthermore, we show that the presence of phytoseiid predatory mites does not affect the hatch rate of predatory bugs that insert their eggs inside leaves.

Identification of two leafminer parasitoids (Hymenoptera: Eulophidae),Neochrysocharis formosaandProacriassp. from Australia, with both showing thelytoky and infection byRickettsia

AbstractLiriomyza huidobrensis,L. sativaeandL. trifoliiare polyphagous agromyzid leafminers that have recently arrived in Australia, posing a threat to Australian vegetable and ornamental crops. Adventive and endemic hymenopteran parasitoids of agromyzid leafminers already present in Australia should assist in the management of these invasive agromyzid species.Neochrysocharis formosa(Hymenoptera: Eulophidae: Entedoninae) is an idiobiont endoparasitoid commonly attackingLiriomyzaspp. in many countries, but it has not been formally identified in Australia. This study provides the first confirmed Australian record ofN. formosaas well as an unidentifiedProacriasspecies, another entedonine species. Females of both species were reared from several adventive and endemic agromyzid leafminers in southern Australia. Laboratory cultures of both species established the presence of thelytokous reproduction. DNA barcodes (5′ COI, 3′ COI, ITS1, ITS2, 28S) were used to delineate species boundaries, with the 5′ end of the mitochondrial COI sequences pointing to multiple cryptic lineages amongN. formosa. Additionally, 16S rRNA sequencing indicated that both species were infected with aRickettsiabacterium, which is related to theRickettsiapresent in thelytokous populations ofN. formosain Japan and China. These findings expand records for parasitoids attacking leafminers in Australia and highlight the potential for an endosymbiont to produce thelytokous strains that could provide for more efficient biocontrol agents for augmentative release.

Shielding of Photosynthetic Apparatus by Consortia of Bacterial Endophytes in Tomato Plants Suffering From Fusarium Wilt

Fusarium oxysporum is one of the most damaging plant pathogens causing Fusarium wilt in many plants leading to serious economic loss. The fungus colonizes the xylem, which leads to resistance in water flow in the plant thereby affecting the rate of photosynthesis. The present study focuses on the selection of bacterial endophytes isolated from tomato plants and evaluating their potential to antagonize Fusarium oxysporum in tomato in vivo. The results obtained indicated that two endophytic isolates, namely Pseudomonas fluorescens BUMD5 and Bacillus velezensis BUMD9, could act as efficient biocontrol agents (BCAs) as they inhibited the growth of pathogen by 67.2 and 69.1%, respectively, in vitro. Both the isolates were found to produce hydrolytic enzymes chitinase and protease. They also produced siderophore and hydrogen cyanide (HCN). The consortia of both the isolates significantly reduced the infection percentage by about 67% and a 3-fold decrease in disease severity was observed as compared to pathogen control. The treatment of infected plants with these potent isolates was also beneficial in improving the overall photosynthetic performance index (PI). Thus, plants treated with consortia of these isolates exhibited better overall plant growth despite being infected by the pathogen.

Bacillus subtilis suppresses the charcoal rot disease by inducing defence responses and physiological attributes in soybean.

Endophytes can induce the defence responses and modulates physiological attributes in host plants during pathogen attacks. In the present study, 127 bacterial endophytes (BEs) were isolated from different parts of healthy soybean plant. Among them, two BEs (M-2 and M-4) resulted a significant antagonistic property against Macrophomina phaseolina, causes charcoal rot disease in soybean. The antagonistic potential was evaluated through dual culture plate assay, where M-4 expressed higher antifungal activity than M-2 against M. phaseolina. The M-4 produces cell wall degrading enzymes viz. cellulase (145.71 ± 1.34 μgmL-1), chitinase (0.168 ± 0.0009 unitmL-1) and β,1-3 endoglucanase (162.14 ± 2.5 μgmL-1), which helps in cell wall disintegration of pathogens. Additionally, M-4 also can produce siderophores, indole-3-acetic acid (IAA) (17.03 ± 1.10 μgmL-1) and had a phosphate solubilization potential (19.89 ± 0.26 μgmL-1). Further, GC-MS profiling of M-4 has been carried out to demonstrate the production of lipophilic secondary metabolites which efficiently suppress the M. phaseolina defensive compounds under co-culture conditions. Bio-efficacy study of M-4 strain shown a significant reduction in disease incidence around 60 and 80% in resistant and susceptible varieties of soybean, respectively. The inoculation of M-4 potentially enhances the physiological attributes and triggers various defence responsive enzymes viz. superoxide dismutase (SOD), phenol peroxidase (PPO), peroxidase (PO) and catalase (CAT). The histopathological study also confirmed that M-4 can reduce the persistence of microsclerotia in root and shoot tissue. Conclusively, M-4 revealed as an efficient biocontrol agent that can uses multifaceted measures for charcoal rot disease management, by suppress the M. phaseolina infection and enhance the physiological attributes of soybean.

The gut commensal bacterium Enterococcus faecalis LX10 contributes to defending against Nosema bombycis infection in Bombyx mori.

BACKGROUNDMicrosporidia, a group of obligate intracellular fungal‐related parasites, have been used as efficient biocontrol agents for agriculture and forestry pests due to their host specificity and transovarial transmission. They mainly infect insect pests through the intestinal tract, but the interactions between microsporidia and the gut microbiota of the host have not been well demonstrated.RESULTSBased on the microsporidia–Bombyx mori model, we report that the susceptibility of silkworms to exposure to the microsporidium Nosema bombycis was both dose and time dependent. Comparative analyses of the silkworm gut microbiome revealed substantially increased abundance of Enterococcus belonging to Firmicutes after N. bombycis infection. Furthermore, a bacterial strain (LX10) was obtained from the gut of B. mori and identified as Enterococcus faecalis based on 16S rRNA sequence analysis. E. faecalis LX10 reduced the N. bombycis spore germination rate and the infection efficiency in vitro and in vivo, as confirmed by bioassay tests and histopathological analyses. In addition, after simultaneous oral feeding with E. faecalis LX10 and N. bombycis, gene (Akirin, Cecropin A, Mesh, Ssk, DUOX and NOS) expression, hydrogen peroxide and nitric oxide levels, and glutathione S‐transferase (GST) activity showed different degrees of recovery and correction compared with those under N. bombycis infection alone. Finally, the enterococcin LX protein was identified from sterile LX10 fermentation liquid based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis.CONCLUSIONAltogether, the results revealed that E. faecalis LX10 with anti‐N. bombycis activity might play an important role in protecting silkworms from microsporidia. Removal of these specific commensal bacteria with antibiotics and utilization of transgenic symbiotic systems may effectively improve the biocontrol value of microsporidia. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Antagonistic efficacy of <i>Trichoderma</i> isolates against soil-borne plant pathogens, <i>Pythium aphanidermatum</i> and <i>Rhizoctonia solani</i>

<p class="Default"><em>Trichoderma</em> spp. are long been recognized as efficient fungal biocontrol agents for the control of plant disease and for their ability to increase plant growth and development. Management of soil borne diseases has become very much important since it causes high crop yield losses. The present study was carried out to isolate <em>Trichoderma</em> spp. from soil samples collected from different locations of Kerala and to test their in vitro efficacy against soil borne pathogens viz., <em>Pythium aphanidermatum</em> and <em>Rhizoctonia solani</em>. The <em>Trichoderma</em> spp. was isolated on Trichoderma Selective Medium (TSM) and observed that the isolates differed in radial growth and colony characters such as colony colour, texture and sporulation. In vitro studies revealed the potential of <em>Trichoderma</em> isolates against soil borne pathogens. Isolates TRPN3 and TRPN7 exhibited no sporulation and white mycelial colour. Isolates which completed their growth at four days after inoculation include TRKR1, TRPN3, TRPN7, TRPN10 and TRPN18. Biocontrol activities against different pathogens resulted in inhibition of pathogens. Maximum inhibition percentage was observed by the isolates TRPN7, TRPN15 and TRKR2 against both the pathogens. The maximum inhibition exhibited against both the pathogens is due to the antagonistic property displayed by the isolates.</p>

Scanning Electron Microscopic Analysis of Antennal Sensilla and Tissue-Expression Profiles of Chemosensory Protein Genes in Ophraella communa (Coleoptera: Chrysomelidae).

Simple SummaryLeaf beetle Ophraella communa is considered an effective biocontrol agent against the common ragweed Ambrosia artemisiifolia. However, there are no studies that assess the antennal sensilla and chemosensory proteins expression profiles of O. communa. In this study, the types and morphology of sensilla on the antennae were examined by scanning electron microscopy (SEM). The results showed that there are six types of sensilla (sensilla trichodea, sensilla chaetica, sensilla basiconica, sensilla styloconica, sensilla coeloconica, and Böhm bristles) distributed on the antennae. We also found that the expression levels of five chemosensory proteins in male and female antennae were higher than those in other tissues by quantitative real-time polymerase chain reaction. Our results lay the foundation for interpreting the olfactory functions of adult O. communa.Ophraella communa is an efficient biocontrol agent used against the invasive weed Ambrosia artemisiifolia. It is an herbivorous insect that feeds on specific plants; the olfactory functions of this insects plays an important role in their search for host plants. There are no reports on O. communa sensilla types, morphology, or chemosensory protein (CSP) genes. In this study, we observed the external structure and distribution of antennal sensilla in adult O. communa antennae by scanning electron microscopy; moreover, we cloned 11 CSPs (CSP1–CSP11) and elucidated their tissue-expression profiles using quantitative real-time polymerase chain reaction. Six types of sensilla were identified: sensilla trichodea (including two subtypes), sensilla chaetica, sensilla basiconica (including two subtypes), sensilla styloconica, sensilla coeloconica, and Böhm bristles. Both male and female antennae had all six types of sensilla, and no sexual dimorphism was noted in sensillar types or distribution. We also found that the expression levels of CSP2, CSP3, CSP4, CSP6, and CSP7 in male and female antennae were higher than those in other tissues, which suggests that these five CSPs may be related to olfactory function in O. communa. Ultimately, our results lay the foundation for interpreting the olfactory functions of adult O. communa.

Short communication: The preference and functional response of Sycanus aurantiacus (Hemiptera: Heteroptera: Reduviidae) on three prey types in laboratory conditions

Abstract. Yuliadhi KA, Supartha IW, Wijaya IN, Pudjianto, Nurmansyah A, Susila IW, Yudha IKW, Utama IWEK, Wiradana PA. 2021. Short communication: The preference and functional response of Sycanus aurantiacus (Hemiptera: Heteroptera: Reduviidae) on three prey types in laboratory conditions. Biodiversitas 22: 5662-5667. Sycanus aurantiacus Ishikawa et Okajima (Hemiptera: Heteroptera) is a valuable predatory insect that can be cultivated in biological pest control programs, particularly in vegetable crops. The research aimed to investigate the preferences and functional responses of the predator Sycanus aurantiacus to three different prey species in the laboratory conditions, included Plutella xylostella (Linnaeus), Crocidolomia pavonana (Fabricius), and Tenebrio molitor (Linnaeus). The preference experiment was carried out by monitoring the prey on which S. aurantiacus preyed the most. The functional responsiveness of S. aurantiacus was determined by measuring the time it took to detect and prey on various kinds of prey, as well as the number of prey that were successfully preyed upon when the quantity of prey was increased. The predatory S. aurantiacus preferred P. xylostella over C. pavonana and T. molitor. S. aurantiacus functional response to three kinds of prey, including type II, is shown. In P. xylostella, the instantaneous prey search rate (a) was 0.2/hour, with a handling time (Th) of 1 hour 40 minutes/prey, which was quicker than the other two kinds of prey. As a result of our findings, the predator S. aurantiacus can be maintained in the lab and has the potential to be developed as an efficient biocontrol agent, particularly against the cabbage leaf caterpillar P. xylostella.

Bacillus velezensis: A Treasure House of Bioactive Compounds of Medicinal, Biocontrol and Environmental Importance

Bacillus velezensis gram-positive bacterium, is frequently isolated from diverse niches mainly soil, water, plant roots, and fermented foods. B. velezensis is ubiquitous, non-pathogenic and endospore forming. Being frequently isolated from diverse plant holobionts it is considered host adapted microorganism and recognized of high economic importance given its ability to promote plant growth under diverse biotic and abiotic stress conditions. Additionally, the species suppress many plant diseases, including bacterial, oomycete, and fungal diseases. It is also able after plant host root colonization to induce unique physiological situation of host plant called primed state. Primed host plants are able to respond more rapidly and/or effectively to biotic or abiotic stress. Moreover, B. velezenis have the ability to resist diverse environmental stresses and help host plants to cope with, including metal and xenobiotic stresses. Within species B. velezensis strains have unique abilities allowing them to adopt different life styles. Strain level abilities knowledge is warranted and could be inferred using the ever-expanding new genomes list available in genomes databases. Pangenome analysis and subsequent identification of core, accessory and unique genomes is actually of paramount importance to decipher species full metabolic capacities and fitness across diverse environmental conditions shaping its life style. Despite the crucial importance of the pan genome, its assessment among large number of strains remains sparse and systematic studies still needed. Extensive knowledge of the pan genome is needed to translate genome sequencing efforts into developing more efficient biocontrol agents and bio-fertilizers. In this study, a genome survey of B. velezensis allowed us to (a) highlight B. velezensis species boundaries and show that Bacillus suffers taxonomic imprecision that blurs the debate over species pangenome; (b) identify drivers of their successful acquisition of specific life styles and colonization of new niches; (c) describe strategies they use to promote plant growth and development; (d) reveal the unlocked strain specific orphan secondary metabolite gene clusters (biosynthetic clusters with corresponding metabolites unknown) that product identification is still awaiting to amend our knowledge of their putative role in suppression of pathogens and plant growth promotion, and (e) to describe a dynamic pangenome with a secondary metabolite rich accessory genome.

English

Biocontrol agents are the potential microbes and used for the control of aerial and soil-borne pathogens present in all crops. An attempt was made on identification, morphological and molecular characterization of Lasiodiplodia theobromae, a pathogen causing disease in nuts and leaves of coconut. A virulent isolate Lasiodiplodia theobromae L26 was selected based on the growth parameters and pycnidiospore production. Three efficient biocontrol agents (BCAs) namely Trichoderma asperellum, Bacillus subtilis and Streptomyces rochei, were selected for in vitro studies. Among these, T. asperellum showed a significantly higher percentage of inhibition (81%) in dual culture assay against L26. The inhibition was also confirmed in light microscopic observation, the mycelium of L26 was distorted, lysis of cell wall during the interaction. Volatile organic compounds (VOCs) emitted from BCAs inhibited the fungal growth of L26 by 59.61–47.03% in sealed plate method. Solid-phase microextraction GC-MS analysis revealed numerous new VOCs compounds emitted from the BCAs, whereas the dominant compound was identified as peptaibols, 2,4-di-tert-butylphenol, 2-piperidinone. The strength of peaks of these compounds augmented during the interaction of BCAs with L26, the peak intensity for terpenoids was the predominant class, followed by phenols and heterocyclic organic compound. Crude metabolite (75 μL) of each antagonist tested through agar well method against L26 and showed a complete inhibition. This study demonstrated the ability of BCAs to produce volatile and nonvolatile antifungal compounds, showing that there could a major mechanism involved in and that will be responsible for the successful inhibition of L26 under in vitro. In future combination of these three strains as commercial formulation may be a better management practices for leaf blight and malformation of nuts in coconut. © 2021 Friends Science Publishers

Identifying and detecting Entomopathogenic fungi using Surface-enhanced Raman spectroscopy / Identificação e detecção de fungos entomopatogênicos utilizando Superficie-enhanced Raman espalhamento

In the natural ecosystem, fungal entomopathogens are the most efficient biocontrol agents against insect pests. In this study we offer an alternative for conventional fungal diagnostic, Surface-enhanced Raman spectroscopy (SERS) technique combine with principal component analysis (PCA) for detection and identification three entomopathogenic fungi, namely, IBCB 66 Beauveria bassiana, IBCB 130 Isaria fumosorosea, and IBCB 425 Metarhizium anisopliae. Using a simple preparation approach, highly active silver nanoparticles suitable for detecting complex biomolecules were produced for application in the SERS technique. Entomopathogens fungi produced highly enhanced and reproducible Raman signals based on their biochemical composition due to the high density of hot spots at the confluence of silver nano-aggregates, allowing the three entomopathogens species to be differentiated in the SERS spectrum fingerprint region, 550-1700 cm-1. The SERS method, along with PCA analysis, accounted for over 99 % of total variance and allowed for very high probability discrimination between the three entomopathogens, allowing taxonomic affiliation to be determined in a short period of time. These findings suggest that the SERS methodology can be used to develop a new, fast, accurate, and cost-effective diagnostic method for fungal entomopathogens.

Los aislados de hongos del género Trichoderma inducen la resistencia al marchitamiento del guisante causada por Fusarium oxysporum f. sp. pisi a través de la inhibición competitiva

Four different Trichoderma species were isolated from the soil rhizosphere of pea field from selected areas of Punjab, Pakistan. Based on morphological and cultural characteristics species were recognized as T. harzianum, T, viridae, T. hamatum and T. koningii. The isolated species were screened for the production of extracellular hydrolytic enzymes. The strains were found positive for chitinase and glucanase activities.  Fusarium oxysporum f. sp. pisi was isolated from roots of wilted pea plants. In-vitro biocontrol potential of the isolated Trichoderma species was assayed by dual culture technique against Fusarium oxysporum f. sp pisi. The maximum growth inhibition was observed by T. viridae (76.42%) followed by T. harzianum (74.29%), T. koningii (71.43%) and T. hamatum (69.64%), after 7 days of incubation. All four isolated species of Trichoderma were used in confrontational assay against Fusarium oxysporum pisi (FOP). A competition of Trichoderma sp. v/s FOP was evident, all Trichoderma strains showed strong antagonistic activity that was clearly observed on dual culture agar plates and further confirmed under scanning electron microscopy (SEM). Pot experiments also confirmed a very strong competitive inhibition of FOP evidently due to presence of glucanase and chitinases in Trichoderma spp. The primary screening and basic findings of present work will be helpful to obtain an efficient and novel biocontrol agent for further experimental trials on pea plants and may also enhance the chance of using Trichoderma species in integrated disease management (IDM) programs as an effective biological agent against several phyto-pathogens.

The deletion of chiMaD1, a horizontally acquired chitinase of Metarhizium anisopliae, led to higher virulence towards the cattle tick (Rhipicephalus microplus).

The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions.

Proteomic analyses reveal mechanisms of action of biocontrol agents on ochratoxin A repression in Penicillium nordicum

Penicillium nordicum is among the main ochratoxin A (OTA) producers in dry-cured fermented sausages. Recent strategies to prevent its development on the surface of these products include the substitution of synthetic antifungals, such as potassium sorbate, by natural compounds. However, the mechanisms of action of the latter are still unclear. The Proteomic analysis is a useful tool to elucidate the basis of the antifungal compound effects on moulds. Therefore, the aim of this study was to evaluate the efficiency of biocontrol agents on the OTA repression in P. nordicum and its relation to proteome changes. Seven different combinations of rosemary leaves, rosemary essential oil, acorn shell extract and the yeast Debaryomyces hansenii have been tested as biocontrol agents against two ochratoxigenic P. nordicum strains on a dry-cured fermented sausage-based medium. The rosemary essential oil and the combination of rosemary leaves with D. hansenii provoked significant inhibition of OTA production in both moulds (p ≤ 0.05). The acorn shell extract and D. hansenii individually decreased OTA only in one of the mould strains. The Proteomic analyses revealed that rosemary essential oil reduced the abundance of proteins involved in the polyketide synthase enoylreductase domain in both moulds, which would explain the OTA reduction. However, the combination of rosemary leaves with D. hansenii showed inter-strain differences, reducing the abundance of proteins related to the cell wall integrity and to the purine pathway, depending on the tested mould. In conclusion, rosemary essential oil and the combination of rosemary leaves with D. hansenii can be proposed as efficient biocontrol agents against ochratoxigenic P. nordicum on dry-cured fermented sausages, although some inter-strain differences at proteome level were found regarding the mechanism of action involved in the reported OTA repression.

Could Gnomoniopsis castaneae Be Used as a Biological Control Agent against Insect Pests?

Gnomoniopsis castaneae is the cause of the chestnut brown rot but has been also regarded as an important mortality factor for the chestnut gall wasp Dryocosmus kuriphilus. The question to whether G. castaneae could serve as a natural biocontrol agent against insect pests is investigated in the present study. We used three serious insect pests as experimental model insects: Plodia interpuctella and Trogoderma granarium, which are important pests of stored products, and Myzus persicae, a cosmopolitan, serious pest of annual and perennial crop plants. Although chemical pesticides represent effective control means, they are also related to several environmental and health risks. In search for alternative pest management methods, scientific interest has been focused, inter alia, on the use of entomopathogenic fungi. While Isaria fumosorosea has long been recognized as an effective control agent against several pests, G.castaneae has been very little studied. The present study examined whether and to what extent G. castaneae and I. fumosorosea exhibit insecticidal activity against fourth-instar larvae of P. interpunctella and T. granarium and adults of M. persicae. Mortality was examined in interrelation with dosage and time exposure intervals. Both fungi exhibited pesticidal action. However, G. castaneae induced noteworthy mortality only at very high doses. In general, we concluded that G. castaneae failed to cause high insect pathogenicity at normal doses and may not be an efficient biocontrol agent compared with other entomopathogens. On the other hand, our study reiterates the pathogenic potential of I. fumosorosea. More studies are needed to further our insight into the potential of EF species as a component of IPM.