Chitinase Secretion Research Articles

Bioactivites of Penicillium citrinum isolated from a medicinal plant Swertia chirayita

Endophytes associated with plants have the property to produce active biomolecules with their possible applications in agro-industrial sectors. This study provides a project work on analyzing various activities of fungal endophytes isolated from Swertia chirayita of Sikkim Himalayan region. Among several fungal endophytes screened, isolate UTCRF6 was found most active with the secretion of enzymes protease, cellulase, amylase and chitinase, as well as other metabolites Indoleacetic acid and siderophores. This endophyte was found active in restricting the growth of phyto-pathogens, including strains of Fusarium solani, Colletotrichum gloeosporioides, Alternaria alternata, Pestalotiopsis theae and Sclerotinia sclerotiorum. Morphological and molecular studies of this endophytic fungus showed similarity with Penicillium citrinum.Graphic abstract

Multifunctional transcription factor CytR of Vibrio cholerae is important for pathogenesis.

Vibrio cholerae, the Gram-negative facultative pathogen, resides in the aquatic environment and infects humans and causes diarrhoeagenic cholera. Although the environment differs drastically, V. cholerae thrives in both of these conditions aptly and chitinases play a vital role in their persistence and nutrient acquisition. Chitinases also play a role in V. cholerae pathogenesis. Chitinases and its downstream chitin utilization genes are regulated by sensor histidine kinase ChiS, which also plays a significant role in pathogenesis. Recent exploration suggests that CytR, a transcription factor of the LacI family in V. cholerae, also regulates chitinase secretion in environmental conditions. Since chitinases and chitinase regulator ChiS is involved in pathogenesis, CytR might also play a significant role in pathogenicity. However, the role of CytR in pathogenesis is yet to be known. This study explores the regulation of CytR on the activation of ChiS in the presence of mucin and its role in pathogenesis. Therefore, we created a CytR isogenic mutant strain of V. cholerae (CytR¯) and found considerably less β-hexosaminidase enzyme production, which is an indicator of ChiS activity. The CytR¯ strain greatly reduced the expression of chitinases chiA1 and chiA2 in mucin-supplemented media. Electron microscopy showed that the CytR¯ strain was aflagellate. The expression of flagellar-synthesis regulatory genes flrB, flrC and class III flagellar-synthesis genes were reduced in the CytR¯ strain. The isogenic CytR mutant showed less growth compared to the wild-type in mucin-supplemented media as well as demonstrated highly retarded motility and reduced mucin-layer penetration. The CytR mutant revealed decreased adherence to the HT-29 cell line. In animal models, reduced fluid accumulation and colonization were observed during infection with the CytR¯ strain due to reduced expression of ctxB, toxT and tcpA. Collectively these data suggest that CytR plays an important role in V. cholerae pathogenesis.

A holin/peptidoglycan hydrolase‐dependent protein secretion system

Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system that uses a holin membrane protein in tandem with a cell wall-editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall-editing enzymes involved was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS).

Chitinase Production from Locally Isolated Bacillus cereus GS02 from Chitinous Waste Enriched Soil

Background and Objective: Chitin is world’s second most abundant structural carbohydrate in nature and is found as a structural component in the cell wall of fungi and exoskeletons of invertebrates. During senescence, it is degraded by the enzyme, chitinase. In addition, chitinase has been exploited for various commercial applications such as control of insects and fungal pathogens in order to protect the crops, waste management, cosmetics and food industries. Chitinases have been found to be widely distributed in various organisms including viruses, animals, bacteria, fungi, higher plants and insects. In the present study, various soil samples enriched in chitinous waste were screened for the isolation of bacteria capable of producing chitinase.
 Methodology: Chitinase producing bacteria were isolated using serial dilution plating technique onto different agar media. Primary and secondary screening were performed and the isolate producing maximum chitinase was selected for biochemical identification and 16s rRNA sequencing. The secretion of extracellular chitinase by this strain was optimized with respect to pH, temperature, incubation time, substrate concentration, carbon and nitrogen sources and inoculum size. All these components were optimized using OFAT (one factor at a time) approach.
 Results: A total of 29 bacterial isolates were found exhibiting secretion of extracellular chitinase as determined using zones of clearance. Based on the area of zone of clearance, six isolates were selected for secondary screening and the most potent isolate was identified as Bacillus cereus. The maximum chitinase production by this strain was obtained at 37°C and pH 7.0 after 48 hours of incubation. The maximum chitinase secretion was observed on addition of 1% colloidal chitin and 0.05% yeast extract in the medium.

Exploring the Potential for Fungal Antagonism and Cell Wall Attack by Bacillus subtilis natto.

To develop more ecologically sustainable agricultural practices requires that we reduce our reliance on synthetic chemical pesticides for crop protection. This will likely involve optimized biocontrol approaches – the use of beneficial soil microbes to attack potential plant pathogens to protect plants from diseases. Many bacterial species, including strains of Bacillus subtilis, have been explored for their biocontrol properties, as they can control the growth of harmful fungi, often by disrupting the fungal cell wall. A strain that is not often considered for this particular application is Bacillus subtilis natto, primarily known for fermenting soybeans via cell wall degradation in the Japanese probiotic dish “natto.” Because deconstruction of the fungal cell wall is considered an important biocontrol trait, we were motivated to explore the possible anti-fungal properties of the B. subtilis natto strain. We show that B. subtilis natto can use complex fungal material as a carbon source for growth, and can effectively deconstruct fungal cell walls. We found degradation of fungal cell wall proteins, and showed that growth on a mix of peptides was very strong. We also found that intact fungal cell walls can induce the secretion of chitinases and proteases. Surprisingly, we could show that chitin, the bulk component of the fungal cell wall, does not permit successful growth of the natto strain or induce the secretion of chitinolytic enzymes, although these were produced during exposure to proteins or to complex fungal material. We have further shown that protease secretion is likely a constitutively enabled mechanism for nutrient scavenging by B. subtilis natto, as well as a potent tool for the degradation of fungal cell walls. Overall, our data highlight B. subtilis natto as a promising candidate for biocontrol products, with relevant behaviors that can be optimized by altering growth conditions. Whereas it is common for bacterial biocontrol products to be supplied with chitin or chitosan as a priming polysaccharide, our data indicate that this is not a useful approach with this particular bacterium, which should instead be supplied with either glucose or attenuated fungal material.

Controlling and co-ordinating chitinase secretion in a Serratia marcescens population.

Serratia marcescens is a γ-Proteobacterium and an opportunistic animal and insect pathogen. The bacterium exhibits a complex extracellular protein 'secretome' comprising numerous enzymes, toxins and effector molecules. One component of the secretome is the 'chitinolytic machinery', which is a set of at least four chitinases that allow the use of insoluble extracellular chitin as sole carbon source. Secretion of the chitinases across the outer membrane is governed by the chiWXYZ operon encoding a holin/endopeptidase pair. Expression of the chiWXYZ operon is co-ordinated with the chitinase genes and is also bimodal, as normally only 1% of the population expresses the chitinolytic machinery. In this study, the role of the ChiR protein in chitinase production has been explored. Using live cell imaging and flow cytometry, ChiR was shown to govern the co-ordinated regulation of chiWXYZ with both chiA and chiC. Moreover, overexpression of chiR alone was able to increase the proportion of the cell population expressing chitinase genes to >60 %. In addition, quantitative label-free proteomic analysis of cells overexpressing chiR established that ChiR regulates the entire chitinolytic machinery. The proteomic experiments also revealed a surprising link between the regulation of the chitinolytic machinery and the production of proteins involved in the metabolism of nitrogen compounds such as nitrate and nitrite. The research demonstrates for the first time that ChiR plays a critical role in controlling bimodal gene expression in S. marcescens, and provides new evidence of a clear link between chitin breakdown and nitrogen metabolism.

Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens.

The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high-resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a zinc-containing metalloenzyme, and in vitro assays demonstrate that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp-120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.

Hfq regulates antibacterial antibiotic biosynthesis and extracellular lytic‐enzyme production inLysobacter enzymogenes OH11

Lysobacter enzymogenes is an important biocontrol agent with the ability to produce a variety of lytic enzymes and novel antibiotics. Little is known about their regulatory mechanisms. Understanding these will be helpful for improving biocontrol of crop diseases and potential medical application. In the present study, we generated an hfq (encoding a putative ribonucleic acid chaperone) deletion mutant, and then utilized a new genomic marker-free method to construct an hfq-complemented strain. We showed for the first time that Hfq played a pleiotropic role in regulating the antibacterial antibiotic biosynthesis and extracellular lytic enzyme activity in L. enzymogenes. Mutation of hfq significantly increased the yield of WAP-8294A2 (an antibacterial antibiotic) as well as the transcription of its key biosynthetic gene, waps1. However, inactivation of hfq almost abolished the extracellular chitinase activity and remarkably decreased the activity of both extracellular protease and cellulase in L. enzymogenes. We further showed that the regulation of hfq in extracellular chitinase production was in part through the impairment of the secretion of chitinase A. Collectively, our results reveal the regulatory roles of hfq in antibiotic metabolite and extracellular lytic enzymes in the underexplored genus of Lysobacter.

A holin and an endopeptidase are essential for chitinolytic protein secretion in Serratia marcescens.

Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria.

Unlocking the secrets of chitinase secretion

Researchers discover a new mechanism of protein secretion in a pathogenic bacterium.

Heterologous expression of VHb can improve the yield and quality of biocontrol fungus Paecilomyces lilacinus, during submerged fermentation

Paecilomyces lilacinus is an egg-parasitic fungus which is effective against plant-parasitic nematodes and it has been successfully commercialized for the control of many plant-parasitic nematodes. However, during the large-scale industrial fermentation process of the filamentous fungus, the dissolved oxygen supply is a limiting factor, which influences yield, product quality and production cost. To solve this problem, we intended to heterologously express VHb in P. lilacinus ACSS. After optimizing the vgb gene, we fused it with a selection marker gene nptII, a promoter PgpdA and a terminator TtrpC. The complete expression cassette PgpdA-nptII-vgb-TtrpC was transferred into P. lilacinus ACSS by Agrobacterium tumefaciens-mediated transformation. Consequently, we successfully screened an applicable fungus strain PNVT8 which efficiently expressed VHb. The submerged fermentation experiments demonstrated that the expression of VHb not only increased the production traits of P. lilacinus such as biomass and spore production, but also improved the beneficial product quality and application value, due to the secretion of more protease and chitinase. It can be speculated that the recombinant strain harboring vgb gene will have a growth advantage over the original strain under anaerobic conditions in soil and therefore will possess higher biocontrol efficiency against plant-parasitic nematodes.

Characterisation of shell disease syndrome in the brown crab, Cancer pagurus, in a Discrete Irish Sea Fishery

Shell disease is the progressive degradation of a crustacean’s exoskeleton through the secretion of chitinases and other enzymes from a suite of microorganisms. It affects a number of crustacean fisheries worldwide, through increased pre-capture mortality and rendering many individuals unsalable or with a reduced market value. The European edible crab (also known as the brown crab), Cancer pagurus Linnaeus, 1758, is highly susceptible to shell disease, but while intertidal populations have been comprehensively assessed, there has been little attention from a commercial fishery perspective. This study assessed the prevalence (% of catch infected) and severity (% cover of lesions on the carapace) of shell disease in the fishery around the Isle of Man for C. pagurus, in the Irish Sea. Shell disease was assessed in relation to sex and size, and the pattern of infection on ventral and dorsal surfaces described. A total of 2361 individuals was sampled between June and August 2012. Almost 25% of crabs were infected to some degree and of those infected an average of just over 2% of the carapace was covered by lesions. The proportion of individuals infected and the severity of infections was found to increase with size and was greater in males and those exhibiting limb losses. Differences in the pattern of lesions on the carapace between sexes suggest behavioural differences between genders can result in different patterns of individual infection. Both prevalence and severity of the disease varied considerably around the Isle of Man. Based on assessment of eight different fishing grounds, prevalence varied from as low as 15% of the population to as high as 35%, while the severity of infection varied between 0.8% to almost 3%. These findings not only suggest that shell disease syndrome could be causing a significant economic loss in the fishery, but also that environmental factors may be driving the condition. However, further investigation is required to fully elucidate the environmental drivers of this syndrome.

REGULATION OF AMYLASE, CELLULASE AND CHITINASE SECRETION IN THE DIGESTIVE TRACT OF THE TWO‐SPOTTED FIELD CRICKET, Gryllus bimaculatus

The secretion of amylase and cellulase in Gryllus bimaculatus is determined by increased food intake, whereby shortly after molting food consumption increases. About half of the standing amylase concentration (activity) in the endothelial cells can be secreted within 30 min. The peak of amylase and cellulase secretion that occurs in the photophase is related to the feeding peak in the previous scotophase. The secretion of chitinase on the other hand is primarily controlled by the molting cycle. Only amylase secretion was affected by calcium in the incubation medium, suggesting an apocrine release mechanism. Refeeding experiments (after 5 days without food) suggest that the release of amylase in response to a nutrient in the lumen (glucose) is not due to simple stimulation of exocytosis, but rather a stimulation of synthesis.

Evaluation of the Resistance and Differential Induction of Chitinases in Tomato in Response to Inoculation with Fusarium oxysporum f. sp. lycopersici

Evaluation of the Resistance and Differential Induction of Chitinases in Tomato in Response to Inoculation with Fusarium oxysporum f. sp.lycopersici Fusarium oxysporum f. sp. lycopersici (FOL) causes Fusarium wilt in tomato, a disease that significantly affects its production. This project investigated the response of different tomato genotypes to Fusarium wilt, aiming at selecting sources of resistance, as well as identifying the differential chitinase secretion during infection between resistant and susceptible cultivars. Results show that the BHRS cultivar proved to be resistant to the disease and there was increased chitinolytic activity in the roots of this genotype six days after inoculation (dai).

Influences of Medium Composition on Mycelial Growth, Sporulation and Chitinase Secretion of Metarhizium anisopliae HY‐2

Metarhizium anisopliae HY‐2, an entomopathogenic fungus isolated from pest cadaver in South Korea, is applicable as a potent bio‐pesticide because of its insecticidal activity to Adoretus tenuimaculatus. Here we report this strain varied in its ability to grow under different nutritional and environmental factors. Temperature at 28°C and pH from 6 to 7 were obtained the best‐fit environmental requirements. Insect pupa and tryptone were found the optimal nitrogen sources for both mycelial growth and sporulation. The strain reached the most suitable effects at the carbon‐to‐nitrogen ratio from 10:1 to 20:1 when the carbon concentration was 4 g/ml provided by glucose and nitrogen supplied by tryptone. Additionally, strain HY‐2 produced an extracellular chitinase at a relatively high level (21.1 mU/ml) when cultivated for 20 days on a medium supplemented with colloidal chitin (1%) and insect pupa (0.5%), indicating that synthesis of chitinase is subject to induction by certain substrate.

Chitinase effects on immune cell response in neuromyelitis optica and multiple sclerosis

Background: Recent studies conducted in arthritis, asthma, and inflammatory bowel disease suggest that chitinases are important in inflammatory processes and tissue remodeling. Objective: To investigate the role of chitinases in multiple sclerosis (MS) and neuromyelitis optica (NMO). Methods: Levels of chitotriosidase, acid mammalian chitinase (AMCase), and chitinase 3-like-1 (CHI3L1) were measured using ELISA, in cerebrospinal fluid (CSF) and in serum from 24 patients with relapsing remitting (RR) MS, 24 patients with secondary progressive (SP) MS, 12 patients with NMO, 24 patients with other inflammatory neurological diseases (OIND), and 24 healthy controls (HCs). The number of anti-MOG cytokine-secreting cells was studied using ELISPOT. Eotaxins, MCP-1, RANTES, and IL-8 were assessed using ELISA. Cell transmigration was determined using an in vitro blood–brain barrier (BBB) model, in the presence and absence of chitinases. Results: CSF chitinase levels were significantly increased in patients with RRMS and NMO compared with HCs and patients with SPMS and OIND. In contrast, no significant differences were detected in serum chitinase levels between groups. Chitinase CSF levels showed correlation with anti-MOG IL-13-producing cells, and eotaxin levels. In vitro experiments showed macrophage chitinase secretion was significantly increased by IL-13, but not by IL-5, IL-6, IL-12, or IFN-γ. Moreover, chitinases enhanced IL-8, RANTES, MCP-1, and eotaxin production, increasing migratory capacity in eosinophils, T cells, and macrophages across an in vitro BBB model. Conclusions: Chitinases increased in the CSF from patients with NMO in response to IL-13. These enhanced levels could contribute to central nervous system inflammation by increasing immune cell migration across the BBB.

Flavobacterium johnsoniae sprB Is Part of an Operon Spanning the Additional Gliding Motility Genes sprC , sprD , and sprF

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the gliding motor that propels cell surface adhesins, such as the 669-kDa SprB. A novel protein secretion apparatus called the Por secretion system (PorSS) is required for assembly of SprB on the cell surface. Genetic and molecular analyses revealed that sprB is part of a seven-gene operon spanning 29.3 kbp of DNA. In addition to sprB, three other genes of this operon (sprC, sprD, and sprF) are involved in gliding. Mutations in sprB, sprC, sprD, and sprF resulted in cells that failed to form spreading colonies on agar but that exhibited some motility on glass in wet mounts. SprF exhibits some similarity to Porphyromonas gingivalis PorP, which is required for secretion of gingipain protease virulence factors via the P. gingivalis PorSS. F. johnsoniae sprF mutants produced SprB protein but were defective in localization of SprB to the cell surface, suggesting a role for SprF in secretion of SprB. The F. johnsoniae PorSS is involved in secretion of extracellular chitinase in addition to its role in secretion of SprB. SprF was not needed for chitinase secretion and may be specifically required for SprB secretion by the PorSS. Cells with nonpolar mutations in sprC or sprD produced and secreted SprB and propelled it rapidly along the cell surface. Multiple paralogs of sprB, sprC, sprD, and sprF are present in the genome, which may explain why mutations in sprB, sprC, sprD, and sprF do not result in complete loss of motility and suggests the possibility that semiredundant SprB-like adhesins may allow movement of cells over different surfaces.

Optimization of Culture Media for Enhanced Chitinase Production from a Novel Strain of Stenotrophomonas maltophilia Using Response Surface Methodology

Chitinase is one of the most important mycolytic enzymes with industrial significance. This enzyme is produced by a number of organisms including bacteria. In this study we describe optimization of media components with increased production of chitinase for selected bacteria Stenotrophomonas maltophilia isolated from the soil. Different components of the defined media responsible for influencing chitinase secretion by the bacterial isolate were screened using Plackett-Burman experimental design and were further optimized by Box-Behnken factorial design of response surface methodology (RSM) in liquid culture. Maximum chitinase production was predicted in medium containing chitin 4.94 g/l, maltose 5.56 g/l, yeast extract 0.62 g/l, KH2PO4 1.33 g/l and MgSO4.7H2O 0.65 g/l using Response surface plots and point prediction tool of DESIGN EXPERT 7.1.6 (Statease, USA) software.

Development of embryo-like structures in the suspension cultures of flax coincides with secretion of chitinase-like proteins

Flax suspension cultures have been established from the callus induced from the cotyledons, hypocotyls, and immature zygotic embryos (iZE). The growth of flax suspension culture (expressed as a sedimented cell volume) was compared in both conditioned (by liquid from embryogenic Pinus nigra suspension culture) and non-conditioned media. Conditioning of media significantly increased the growth of the cell lines of hypocotyl and iZE origin; however, it had no promotive effect on embryogenic response of these flax liquid cultures. Formation of embryo-like structures (ELS), confirmed also histologically, has only been found in the cell line derived from iZE and cultivated in non-conditioned MS medium supplemented with 1 mg l−1 2,4-D. The process of ELS formation in this cell line was accompanied by the expression of the protein(s) with chitinolytic activity and molecular weight approximately of 25 kDa. The relationship between the formation of ELS and secretion of chitinase(s) is discussed.

Biological control of phytopathogenic fungi of vanilla through lytic action of Trichoderma species and Pseudomonas fluorescens

Trichoderma, soil-borne filamentous fungi, are capable of parasitising several plant pathogenic fungi. Secretion of lytic enzymes, mainly glucanases, proteases and chitinases, is considered the most crucial step of the mycoparasitic process. The lytic enzymes degrade the cell walls of the pathogenic fungi, enabling Trichoderma to utilise both their cell walls and cellular contents for nutrition. Twenty-one strains of Trichoderma spp. were isolated from vanilla rhizosphere and suppressive soils from different regions of Tamil Nadu. The antagonistic effect of Trichoderma was studied towards a range of phytopathogenic fungi; Fusarium oxysporum f.sp. vanillae, P. meadii and Colletotrichum vanillae in vanilla. Coculture of the phytopathogens and Trichodema under laboratory conditions clearly showed dominance of the Trichoderma species. In all cases, Trichoderma overgrew the phytopathogens and subsequently developed a conidial lawn over the surface. The percent inhibition of pathogen growth by isolates T1 and T17 were significantly high by 49.5 and 48.2% respectively. In these studies the lytic action of the pathogen was clearly apparent and the inhibition of growth appears directly related to its ability to hydrolyse the cell walls of the tested microorganisms. Protease, chitinase and β-1,3-glucanase activities were determined in the extracellular fluid of Trichoderma. The data imply that the nature of Trichoderma antagonism is based on mycoparasitism (lysis) and appears to optimalize with contact between the mycelia. Six of them (Tricho1, Tricho6, Tricho10, Tricho12, Tricho15 and Tricho17) were prioritized for further evaluation to utilize different substrates by chitinase production. T. viride isolates T1 and T17 produced more extracellular protease, chitinase and β-1,3 glucanase, which cause disruption of the cell wall and membrane structure of pathogen. The increase in mycelial weight, inhibition behaviour and production of extracellular enzymes by these isolates suggest that the isolates can be used as biological control agents in vanilla plantations. In vanilla field evaluation with microbial consortia involving P. fluorescens strains and Trichoderma isolates, the combinations viz., Pf1 + FP7 + chitin (soil and foliar), Pf1 + Tricho1 + chitin (soil) + Pf1 + chitin (foliar) and Pf1 + Tricho17 + chitin (soil) + Pf1 + chitin (foliar) performed well in reducing the disease incidence.