Endophytic bacterial consortium: powerful antifungal team against menacing Phytopathogens of Apple (Malus Pumila)

Endophytic bacterial consortium: powerful antifungal team against menacing Phytopathogens of Apple (Malus Pumila) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Endophytic bacterial consortium: powerful antifungal team against menacing Phytopathogens of Apple (Malus Pumila) Raouf Ahmad Mir, Mir Khushnawaz Ahmad, Sheema Manzoor This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6210065/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Biological control is a cost effective, natural, and sustainable alternative to chemical pesticides for management of pathogens. The main aim of this study was to investigate new wide spectrum biological agents to develop a microbial formulation against soil borne and air borne Plant pathogens. Almost 100 endophytic strains (Bacterial, fungal and actinomycetes) were screened for their antipathogenic activity against dreadful plant pathogens. Out of 100 strains screened 5 strains (3bacterial and 2 fungal strains) were found to be effective antipathogenic activity against Plant pathogens. The 5 effective strains were identified as Bacillus subtilis, Bacillus megaterium, Pseudomonas species, Trichoderma asperellum and Trichoderma atroviride . All these strains were screened individually as well as in a team of microbial consortium to find out antipathogenic activity. Though all microbes showed promising activity individually against pathogens but bacterial consortium had shown better activity than individual microbes. Compatibility testing had shown that bacterial strains were compatible with each other. The Plant pathogens under test were: Venturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum. Three bacterial strains Bacillus subtillis, Bacillus megaterium and Pseudomonas species produced antifungal compounds to inhibit pathogens where as Trichoderma asperellum and Trichoderma atroviride penetrate pathogenic hyphae and compete for food and produced parasitic inhibition. The cell filtrate of all bacterial strains inhibits the growth of pathogens invitro but extract of fungal strains did not inhibit pathogens invitro. Results proved the efficacy of the individual as well as consortium as biocontrol agents to common soil and air borne pathogens. Biological sciences/Biotechnology Biological sciences/Plant sciences Endophytes Plant Pathogens Consortium Compatibility test Secondary metabolites Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Plant pathogens cause a great threat to agricultural and horticultural production as they lead to great economic loss. Presently the impact has worsened due to the globalization of markets and climatic changes that has increased the infestation of diseases and their rapid spread Beneduzi et al .( 2012). Among the pathogens, soil-borne pathogens like Fusarium oxyporium, Pencillium expansum, Phytophthora infestans, and Phytophthora cactorum are highly active and have a wide host range. Excessive use of conventional pesticides has further decreased soil health and hence makes it favorable for fungal pathogens and develops chemically resistant pathogens Kumar et al. ( 2017 ). Biotic stress mainly due to pathogenic microorganisms gives rise to major crop losses worldwide which are equivalent to nearly 222 billion dollars lost every year Chakraborty and Newton ( 2011 ). To overcome this issue farmers or growers apply a variety of excess chemicals to control these pathogens which are more economic and harmful to the environment as well as human health through the air, water, and soil pollution. Excessive use of chemical pesticides has not only affected soil health but decreased the number of beneficial microbes in soil due to soil pollution. There is a need of the hour to find out an alternative method that is cost-effective, environmentally friendly, equally effective, and sustainable. Biological control of plant pathogens is one such alternative that is being used in agriculture and horticulture. The antagonistic activity of Trichoderma was demonstrated more than eight decades ago Weindling ( 1934 ) and presently it is the most extensively used bio-control agent Mukherjee et al. ( 2012 ). A lot of bio-control microorganisms were isolated by screening several soil samples or plant-associated microorganisms for antagonism against phytopathogens Berg et al. ( 2001 ); Cazorla et al (2017). A large number of bacteria with antifungal activities were isolated from rhizospheric soils; majority of the isolated bacteria were Bacillus and Pseudomonads . Berg et al. ( 2001 ), Cazorla et al ( 2007), Beneduzi et al. ( 2012 ) and Kumar (2012). It is found that many Bacilli genera can promote plant growth Choudhary et al. (2009). Bacteria can promote plant growth through several mechanisms, like improvement in nutrient uptake, activation of systemic resistance, toxicity to pests, and antagonism pathogens Choudhary et al . (2009), Pinchuk et al. (2002), Wang et al . (2007). The antifungal activity of Bacillus strains is due to the synthesis of various antimicrobial peptides, Tan (2010), Falardeau et al. ( 2013 ) secreted enzymes, (Baysal et al. ( 2013 .) proteins, Tan et al. ( 2013 ), and volatile organic compounds (VOCs) Choudhary et al. (2009), Baysal et al. ( 2013 ). The antifungal activity of Bacillus strains makes them an effective bio-control agent (Li et al. 2014 , Baffoni et al. 2015, and Shternshis et al. 2015 ). Fengycin -a cyclic lipopeptide produced by bacillus strains plays an important role in its antifungal activity. (Guo et al. 2014 ). The strains of Bacillus subtilis can vary considerably both phenotypically as well as genotypically and that affects their antagonistic properties. Plants and microorganisms live in interaction mode among each other within nature which can affect plant growth and development, response to any stimuli, and resistance to abiotic and biotic stresses (Nataraja et al. 2019). Endophytes are microorganisms that live in interaction with plants and can be used in biocontrol and induced resistance. Endophytes have an advantage over other bio-control agents since they colonize inside the plant tissues and remain protected from harsh environmental conditions which could otherwise affect their survival. (Latz et al .2018). Thus, endophytes are currently the focus of scientists as a bio-control agent due to their ability to assist plants in growth, development, and resistance. Endophytes have received a lot of attention as both bio-control agents as well as activators of the plant defense against biotic and abiotic stresses. These methods have a positive impact on disease management. Trichoderma species and bacillus species are being used as bio-control agents for decades. This study aimed to directly compare the antagonistic effect of endophytes isolated from resistant apple varieties on apple pathogens invitro by suppressing phytopathogens through the production of cyclic lipopeptides, hydrolytic enzymes, siderophores, parasitic mode, and competition for nutrients. The further aim of this study was to develop a highly effective formulation of an endophytic consortium to control fungal pathogens in Apple trees. MATERIALS AND METHODS Isolation of endophytic microorganisms from Apple tree (Galla): Collection of Plant Material Two year old High density apple trees were sourced from Sher-e-Kashmir University of Agricultural Sciences and Technology Shalimar Sinagar Jammu and Kashmir India- 190025, 0n 9 th of March 2019 under receipt no.503 , and were planted in Hapico Industries Private limited IGC Lassipora Pulwama Jammu and Kashmir India .One year old shoots with leaves from the lowest main branch were sampled from the sourced apple trees (Good growth rate, no deficiency, no disease symptoms, no physical damage, and no insect damage) in spring April-May 2021 when leaves were fully opened. The The plant material was cut and placed in a zip-Lock cover and brought back to the lab. Processing of Plant material All plant parts collected were washed under running tap water three times and then using a 5-step sterilization method Wicaksono et al . (2016), Purushotham et al .(2018). Leaf and stem parts were cut into 1 cm portions using a sterile scalpel. Each tissue section was further cut into pieces (1mm thick) with 4 pieces plated on NA, 4 pieces on R2A for endophytic bacterial isolation, and 4 pieces on PDA and SNA (synthetic low agar nutrient) (Berg et al. 2005) for fungal Isolation. The plates were incubated at 25 o C in complete darkness for 4 weeks. A few drops from the final rinse water were placed on R2A and PDA and incubated with inoculated plates at the same temperature and duration in darkness. It was done to check the effectiveness of the surface sterilization process. Isolation and preparation of pure cultures Endophytes were isolated from the incubated plates after 4 weeks. Plates were removed and all isolated colonies were sub-cultured on respective media (Bacteria on NA and Fungi on PDA) to get pure culture for identification and antipathogenic activity Staining and Identification of Endophytes Isolated endophytes were identified by staining method and biochemical tests. Endophytes were stained by conventional methods (Lactophenol cotton blue for endophytic fungi and gram staining for endophytic bacteria) and identified by microscopy based on sporangium structure, spore shape, hyphal structure, hyphal type, (in Fungi) and cell shape and spore type (in bacteria). Biochemical tests such as; catalase test, coagulase test, oxidase test, sugar fermentation test, Indole test, citrate test, and urease test. Screening of Endophytes against Phytopathogens 100 endophytes were isolated and were screened for their antifungal activity against five dreadful plant pathogensviz;( Venturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum ). Out of 100 endophytes, 5 endophytic strains (2 fungal and 3 bacterial) had shown tremendous antipathogenic activity which was further verified by carrying out antifungal activities. The antipathogenic activity of selected 5 endophytic strains The selected endophytic strains (2 fungal and 3 bacterial) were analyzed for their antipathogenic activity against plant pathogens. All selected strains were tested individually as well as in consortium. Testing of Individual endophytic strains against phytopathogens The selected endophytic strains were identified as Bacillus subtilis, Bacillus megaterium, Pseudomonas fluorescens, Trichoderma asperellum, and Trichoderma atroviride . The antipathogenic activity of individual endophytic strains was tested against five plant pathogens viz; ( Venturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum, and Pencillium expansum ). The plant pathogenic fungi were individually grown on PDA plates by placing a disc at the center of the plate and incubated overnight at 25 0 C. The 5 selected endophytic strains were inoculated individually on each pathogenic plate after 12 hours of incubation with pathogens. All plates were incubated at 25 o C for 1 week until the mycelia grew. Testing of endophytic consortium against Phytopathogens Endophytic consortium was formed by combining selected endophytic bacterial strains and used as an endophytic team against Phytopathogens and analyzed the antipathogenic activity of the endophytic team and compare results with individual strains. The plant pathogenic fungi were individually grown on PDA plates by placing a disc at the center of the plate and incubated overnight at 25 o C. The endophytic bacterial strains were inoculated at 3 different locations at the periphery around the central pathogenic disc as an endophytic team. All Plates were incubated at 25 o C for 1 week until the mycelia grew. Compatibility analysis of endophytic strains Cross- streak method was used for the compatibility analysis of endophytic strains. Endophytic bacterial strains were streaked on nutrient plates in such a way that for every single bacterial culture in the center of the plate, other cultures are streaked radiating from the center. The plates were incubated at 35 o C for 48 hours and the zone of inhibition was observed and recorded. Secondary metabolites of endophytes as antagonistic against Phytopathogens All endophytic strains were grown in liquid media for suspension culture to produce secondary metabolites. Bacterial endophytes were grown in ISP medium 4 (g/l) (soluble starch-10, dipotassium phosphate-1, magnesium sulphate-1, sodium chloride-1, ammonium sulphate-2, calcium carbonate-2, ferrous sulphate-1, manganous chloride-1, Zinc sulphate-1) with pH-7. The endophytic bacterial strains were individually inoculated in 150 ml medium (ISP 4) supplemented with optimum carbon and nitrogen source. The cultures were incubated at 35 o C for seven days and the cell-free extract was used for analysis. The cell-free extract was obtained after extraction with ethyl acetate and the bioactive compound was obtained by evaporating the organic layer. The evaporated organic extract was dissolved in 0.5% Tween 80 for analyzing antifungal activity food and the poison method was adopted. The secondary metabolites from endophytic fungi were produced and extracted using the method reported by Vinale et al. (2006). 5 mm diameter plugs from each of the two cultures of Trichoderma isolates ( Trichoderma atroviride and Trichoderma asperellum ) obtained from the margins of actively growing cultures on PDA were separately inoculated into 5 L conical flasks containing 1 L Potato dextrose broth (PDB). The suspension cultures were incubated for 30 days at 25 o C without shaking, after which the fungal mycelium was removed from the broth by vacuum filtration no. 4 filter paper (Whatman). The cell-free extract was obtained after extraction twice with ethyl acetate and a bioactive compound was obtained by evaporating the organic layer. After evaporation of the solvent the organic extract was dissolved in 0.5% Tween 80 for analyzing antifungal activity by food and poison method RESULTS AND DISCUSSION and are very difficult to control. The use of conventional pesticides is posing another threat by causing disease resistance and environmental pollution and also affecting human and animal life. Using endophytes to control plant fungal diseases is a popular topic and has been studied extensively. Endophytes are ubiquitous microbes that inhabit healthy plant tissues without causing disease. Endophytes have been found in every plant examined to date and may be important. Plant-microbe associations play a vital role in the growth and development of plants and may affect agriculture and the environment. Both plants and microbes play their role for the mutual benefit of plants and microbes. Plants and microbes have evolved as symbionts and have modulated their interactions by producing different phytochemicals, hormones, antimicrobial agents, plant growth promoters, and other metabolites. Besides having antimicrobial potential endophytes have gained their popularity as pharmaceutical agents. Endophytes have not only paved their way in medicine, therapeutics, and mining, some novel metabolitesmay be used in sustainable agriculture and plant growth. Endophytes are gaining biotechnological and industrially relevance as a result of their ability to secrete secondary metabolites, serve as bio-control agents, antimicrobial agents, antitumor agents, and immune suppressants, and secrete antiviral compounds and development of natural antioxidants, antidiabetic agents, antibiotics, and insecticidal products (Gouda et al . 2016, Yadav.2018) Recent findings have shown that endophytic fungi can help limit pathogen damage in many crops. (Arnold et al., 2003)Our results support these findings by showing that endophytic consortium isolated from healthy apple leaves and twigs restrict in vitro growth of the six most common and economically important plant pathogens. Isolation of endophytic microorganisms from Apple tree (Galla) Around 100 endophytic strains were isolated from Apple leaves, and twigs. Most of bacterial endophytes isolated belong to genus, Bacillus species Pseudomonas species Rahnella species paenibacillus species Enterobacter species klebsiella species Micrococcus species Ochrobactrum species Sphingomonas species curvibacter spp . And fungal endophytes belong to, Cladosporium species Trichoderma species Fusarium species Pencillium species. Fig 1, Fig 1A. Fig 1B. All isolated endophytes were identified by staining method and biochemical tests. Screening of Endophytes against Phytopathogens Hundred endophytic strains isolated from apple trees were screened against 6 different phytopathogens including Venturia inequalis, Alternaria alternata, Fusarium oxyporium, phytophthora cactorum, Phytophthora infestans, Pencillium expansum. Out of 100 endophytes 5 endophytes (Bacillus subtilis, bacillus megaterium, Pseudomonas fluorescens, Trichoderma asperellum, and Trichoderma atroviride) have shown good antagonistic effect against phytopathogens. These strains were identified, and used for further analysis. Fig 2 Antipathogenic activity of selected 5 endophytic strains The selected endophytic strains (2 fungal and 3 bacterial) were analysed for their antipathogenic activity against plant pathogens. All selected strains were tested individually as well as in consortium. Consortium was found to be better antagonistic as compared to individual strains. Testing of Individual endophytic strains against phytopathogens The selected endophytic strains were identified as Bacillus subtilis, Bacillus megaterium, Pseudomonas fluorescens, , Trichoderma asperellum, and Trichoderma atroviride . The antipathogenic activity of individual endophytic strain was tested against five plant pathogens viz; ( Venturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum ). Antifungal assay. Mycelial discs (6mm) from a 7-day-old pure culture of endophytes ( Bacillus subtilis, Bacillus megaterium, Pseudomonas fluorescens, Trichoderma asperellum, and Trichoderma atroviride. ) were excised and paired individually with pathogenic fungi ( Venturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum ).opposite to each other at a distance of 40 mm apart in a 9 cm petri plate.. Pathogen was inoculated alone on PDA medium as a control. The mycelia growth diameter of both endophyte and pathogen was measured 3, 5 and 7 days after incubation. The inhibition of radial growth of pathogen (PIRG) was evaluated using formula. PIRG=R1−R2/R1×100 Where R1 = diameter (mm) of the pathogen colony in the control; R2 = diameter (mm) of the pathogen in the endophyte-tested plate. The antagonistic activity of the endophyte was also described through visual assessment (Soytong et al) : >75% PIRG = very high antagonistic activity; 61–75% PIRG = high antagonistic activity; 51–60% PIRG = moderate antagonistic activity; <50% PIRG = low antagonistic activity All individual strains were found to be positive antagonistic against phytopathogens. Table 1, fig 3 Table 1.Degree of antagonism of Individual endophtes against six pathogens 7 days after incubation period (DAIP). Treatment pathogen PIRG Antagonistic Activity Bacillus subtilis Venturia inequalis 70 High Bacillus megaterium 62 High Pseudomonas florescens 63 High Trichoderma asperellum 64 High Trichoderma atroviride. 77 very high Bacillus subtilis Alternaria alternata 72 high Bacillus megaterium 60 moderate Pseudomonas florescens 64 high Trichoderma asperellum 73 high Trichoderma atroviride. 75 very high Bacillus subtilis Fusarium oxyporium 66 high Bacillus megaterium 55 moderate Pseudomonas florescens 50 low Trichoderma asperellum 60 moderate Trichoderma atroviride. 73 very high Bacillus subtilis Phytophthora infestan 65 high Bacillus megaterium 55 moderate Pseudomonas florescens 51 moderate Trichoderma asperellum 62 high Trichoderma atroviride. 73 high Bacillus subtilis Phytophthora cactorum 62 moderate Bacillus megaterium 50 low Pseudomonas florescens 40 low Trichoderma asperellum 65 high Trichoderma atroviride. 69 high Bacillus subtilis Pencillium expansum 70 high Bacillus megaterium 60 moderate Pseudomonas florescens 73 high Trichoderma asperellum 70 high Trichoderma atroviride. 75 very high Values are mean of triplicate and presented as a percentage Visual assessment on antagonistic activity of the endophyte (Soytong et al) >75% PIRG = very high antagonistic activity; 61–75% PIRG = high antagonistic activity; 51–60% PIRG = moderate antagonistic activity; <50% PIRG = low antagonistic activity. Testing of bacterial endophytic consortium against Phytopathogens An endophytic consortium was formed by combining three selected endophytic strains Bacillus subtilis, Bacillus megaterium, and Pseudomonas fluorescens, as an endophytic team against Phytopathogens and analyzing the antipathogenic activity of the endophytic team and comparing results with individual strains. The hyphal growth of pathogenic fungi was restricted by the tested organisms. The endophytic consortium limited the mycelia growth of pathogenic fungi at 3, 5, and 7 days after incubation. The pathogenic fungi grow fully inside the control plate while the treated plates show limited growth after 7 days of incubation. The consortium of endophytes has shown better antagonistic properties against the pathogen as compared to when used individually. Table 2, Fig 4 Table 2: Degree of antagonism of endophtytic team against six pathogens after 3,5and 7 days after incubation period (DAIP) Mycelial growth in mm Microbial strains 3DAIP 5DAIP 7DAIP Bacillus subtilis 20 40 60 Bacillus megaterium 18 30 40 Pseudomonas florescens 20 23 30 Venturia inequalis 25 60 90 Bacillus subtilis 45 55 65 Bacillus megaterium 25 28 30 Pseudomonas florescens 22 35 40 Alternaria alternata 45 65 90 Bacillus subtilis 20 29 50 Bacillus megaterium 22 28 30 Pseudomonas florescens 15 26 35 Fusarium oxyporium 30 70 90 Bacillus subtilis 20 33 40 Bacillus megaterium 18 27 30 Pseudomonas florescens 15 19 20 Phytophthora infestan 38 60 90 Bacillus subtilis 38 45 55 Bacillus megaterium 25 27 30 Pseudomonas florescens 22 26 30 Phytophthora cactorum 25 55 90 Bacillus subtilis 33 40 60 Bacillus megaterium 19 22 25 Pseudomonas florescens 9 10 10 Pencillium expansum 35 70 90 Indicated values are the mean of triplicate. Compatibility analysis of endophytic strains: Cross- streak method was used for the compatibility analysis of endophytic strains. Endophytic bacterial strains were streaked on nutrient plates in such a way that for every single bacterial culture in the center of the plate, other cultures are streaked radiating from the center. The plates were incubated at 35 o C for 48 hours and the zone of inhibition was observed and recorded. Fig 5 Secondary metabolites of endophytes as antagonistic against Phytopathogens: All endophytic strains were grown in liquid media for suspension culture to produce secondary metabolites. Bacterial endophytes were grown in ISP medium 4 supplemented with optimum carbon and nitrogen source. The cultures were incubated at 35oC for seven days and the cell-free extract was used for analysis. The secondary metabolites from endophytic fungi were produced and extracted using the method reported by (Vinale et al . 2006). 5 mm diameter plugs from each of the two cultures of Trichoderma isolates ( Trichoderma atroviride and Trichoderma asperellum ) and obtained from the margins of actively growing cultures on PDA were separately inoculated into 5 L conical flasks containing 1 L Potato dextrose broth (PDB). The suspension cultures were incubated for 30 days at 25oC without shaking, after which the fungal mycelium was removed from the broth by vacuum filtration (Whatman 4). The cell-free extract was obtained after extraction with ethyl acetate in both methods and bioactive compounds were obtained by evaporating the organic layer. After evaporation of the solvent, the organic extract was dissolved in 0.5% Tween 80 for analyzing antifungal activity by food and poison method. The cell-free extract of endophytic fungi was not so effective in controlling the growth of phytopathogens in invitro conditions but is inhibiting the growth by parasitic mode and competition for nutrients. Instead, the cell-free extract of endophytic bacteria was effective enough to inhibit the growth of phytopathogens in invitro hence favors the role of antifungal compounds produced by endophytic bacteria. Fig 6 Biocontrol of plant pathogens is the need of the hour and is a viable approach in plant disease management. Endophytic microbes ( Bacillus subtilis, Bacillus megaterium, pseudomonas species Trichoderma asperellum and Trichoderma atroviride ) isolated from apple leaves and twigs inhibited the growth of( Venturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum ) by antibiosis. Our observations on the potential of endophytes as a possible antifungal agents imply that such organisms can be considered as management options against pathogens in apple. Our findings reveal that endophytic bacterial consortium shows better results in controlling the fungal pathogens as compared to individual strains. Thus, further investigation must be conducted to identify the inhibitory compounds produced by the endophytes, and determine the efficacy level of those compounds and appropriate methods of application. Declarations Author Contribution Mr.Raouf Ahmad Mir and Mir Khushnawaz Ahmad prepared the manuscript, did all research work , wrote the main manuscript , Mr Khushnawaz prepared figures and tables , Miss Sheema Manzoor reviewed the manuscript Acknowledgement The authors are very much indebted to the Department of Biotechnology, Hapico Industries Private Limited IGC SIDCO Lassipoora for the financial support of the project References Arnold et al., 2003; Evans et al., 2003; Mejı´a et al., 2003; Holmes et al., 2004, 2006; Rubini et al., 2005; Tondje et al., 2006; Pierre Roger Tonje, IRAD, Cameroon; personal communication Baffoni, L., Gaggia, F., Dalanaj, N., Prodi, A., Nipoti, P., Pisi, A., Biavati, B. and Di Gioia, D. (2015) Microbial Inoculants for the Biocontrol of Fusarium spp. in Du rum Wheat. BMC Microbiology,15, 242. https://doi.org/10.1186/s12866-015-0573-7 Baysal, O., Lai, D., Xu, H., Siragusa, M., Casiskan, M., Carimi, F., Silva, J.A.T. and Tor, M. (2013) A Proteomic Approach Provides New Insights into the Control of Soil-Borne Plant Pathogens by Bacillus Species. PloS One, 8, e53182. https://doi.org/10.1371/journal.pone.0053182 Bell, D.K.; Wells, D.H.; Markham, R.C. In vitro antagonism of Trichoderma against six fungal plant pathogens. Phytopathology 1982, 72, 379–382. DOI: 10.1094/Phyto-72-379. Beneduzi, A., Ambrosini, A. and Passaglia, L.M. (2012) Plant Growth-Promoting Rhizobacteria (PGPR): The Potential as Antagonists and Biocontrol Agents. Genet ics and Molecular Biology, 35, 1044-1051. https://doi.org/10.1590/S1415-47572012000600020 Berg, G., Fritze, A., Roskot, N. and Smalla, K. (2001) Evaluation of Potential Bio-control Rhizobacteria from Different Host Plants of Verticillium dahliae Kleb. Journal of Applied Microbiology, 91, 963-971. https://doi.org/10.1046/j.1365-2672.2001.01462.x Cazorla, F.M., Romero, D., Perez-Garcia, A., Lugtenberg, B.J., Vicente, A.D. and Bloemberg, G. (2007) Isolation and Characterization Of antagonistic Bacillus subti lis Strains from the Avocado Rhizoplane Displaying Biocontrol Activity. Journal of Applied Microbiology, 103, 1950-1959. https://doi.org/10.1111/j.1365-2672.2007.03433.x Chakraborty, S., and Newton, A. C. (2011). Climate change, plant diseases and food security: an overview. Plant Pathol. 60, 2–14. doi: 10.1111/j.1365-3059.2010.02411.x Choudhary, D.K. and Johri, B.N. (2009) Interactions of Bacillus spp. and Plants— with Special Reference to Induced Systemic Resistance (ISR). Microbiological Re search, 164, 493-513. https://doi.org/10.1016/j.micres.2008.08.007 Falardeau, J., Wise, C., Novitsky, L. and Avis, T.J. (2013) Ecological and Mechanis tic Insights into the Direct and Indirect Antimicrobial Properties of Bacillus subtilis lipopeptides on Plant Pathogens. Journal of Chemical Ecology, 39, 869-878. https://doi.org/10.1007/s10886-013-0319-7 Gouda, S., Das, G., Sen, S. K., Shin, H.-S., and Patra, J. K. (2016). Endophytes: a treasure house of bioactive compounds of medicinal importance. Front. Microbiol. 7:1538. doi:10.3389/fmicb.2016.01538 Guo, Q., Dong, W., Li, S., Lu, X., Wang, P., Zhang, X., Wang, Y. and Ma, P. (2014) Fengycin Produced by Bacillus subtilis NCD-2 Plays a Major Role in Biocontrol of Cotton Seedling Damping-Off Disease. Microbiological Research, 169, 533-540. https://doi.org/10.1016/j.micres.2013.12.001 Kumar G, A Maharshi, J Patel, A Mukherjee, HB Singh and BK Sharma. 2017. Trichoderma: A potential fungal antagonist to control plant diseases. SATSA Mukhapatra- Annual Technical Issue 21: 206-21 https://www.researchgate.net/publication/314946122 Kumar, P., Dubey, R.C. and Maheshwari, D.K. (2012) Bacillus Strains Isolated from Rhizosphere Showed Plant Growth Promoting and Antagonistic Activity against Phytopathogens. Microbiological Research, 167, 493-499. https://doi.org/10.1016/j.micres.2012.05.002 Kim, P.I., Ryu, J., Kim, Y.H. and Chi, Y.T. (2010) Production of Biosurfactant Li popeptides Iturin A., Fengycin, and Surfactin A from Bacillus subtilis CMB32 for Control of Colletotrichum gloeosporioides. Journal of Microbiology and Biotech nology, 20, 138-145. doi: 10.4014/jmb.0905.05007 Latz, M.A.C.; Jensen, B.; Collinge, D.B.; Jørgensen, H.J.L. Endophytic Fungi as Biocontrol Agents: Elucidating Mechanisms in Disease Suppression. Plant Ecol. Divers. 2018, 11, 555–567. https://doi.org/10.1080/17550874.2018.1534146 Li, X.Y., Yang, J.J., Mao, Z.C., Ho, H.H., Wu, Y.X. and He, Y.Q. (2014) Enhance ment of Biocontrol Activities and Cyclic Lipopeptides Production by Chemical Mutagenesis of Bacillus subtilis XF-1, a Biocontrol Agent of Plasmodiophora bras sicae and Fusarium solani. Indian Journal of Microbiology, 54, 476-479. https://doi.org/10.1007/s12088-014-0471-y Mukherjee M, PK Mukherjee, BA Horwitz, C Zachow, G Berg and S Zeilinger. 2012. Trichoderma- Plant-Pathogen interactions: Advances in genetics of biological control. Indian J. Microbiol. 52(4): 522-529. doi: 10.1007/s12088-012-0308-5 Nataraja, K.N.; Suryanarayanan, T.S.; Shaanker, R.U.; Senthil-Kumar, M.; Oelmüller, R. Plant–Microbe Interaction: Prospects for Crop Improvement and Management. Plant Physiol. Rep. 2019, 24, 461–462. DOI 10.1007/s40502-019-00494-4 Pinchuk, I.V., Bressollier, P., Sorokulova, I.B., Verneuil, B. and Urdaci, M.C. (2002) Amicoumacin Antibiotic Production and Genetic Diversity of Bacillus subtilis Strains Isolated from Different Habitats. Research in Microbiology, 153, 269-276. https://doi.org/10.1016/S0923-2508(02)01320-7 Purushotham, N., Jones, E., Monk, J. and Ridgway, H. (2018) Community structure of endophytic Actinobacteria in a New Zealand native medicinal plant Pseudowintera colorata (Horopito) and their influence on plant growth. Microb Ecol 76, 729–740 DOI: 10.1007/s00248-018-1153-9 Shternshis, M.V., Belyaev, A.A., Matchenko, N.S., Shpatova, T.V. and Lelyak, A.A. (2015) Possibility of Biological Control of Primocane Fruiting Raspberry Disease Caused by Fusarium sambucinum. Environmental Science and Pollution Research, 22, 15656-15662. https://doi.org/10.1007/s11356-015-4763-5 Soytong, K. Identification of of Chaetomium in the Philippines and Screening for Their Biocontrol Properties against Seedborne Fungi of Rice. Ph.D. Thesis, University of the Philippines Los Baños, Laguna, Philippines, 1988. Tan, Z., Lin, B. and Zhang, R. (2013) A Novel Antifungal Protein of Bacillus subtilis B25. SpringerPlus, 2, 543. https://doi.org/10.1186/2193-1801-2-543 Wang, S., Wu, H., Qiao, J., Ma, L., Liu, J., Xia, Y. and Gao, X. (2009) Molecular Mechanism of Plant Growth Promotion and Induced Systemic Resistance to To bacco Mosaic Virus by Bacillus spp. Journal of Microbiology and Biotechnology, 19, 1250-1258. https://doi.org/10.4014/jmb.0901.008 Weindling R. 1934. Studies on a lethal principle effective in the parasitic action of Trichoderma lingorum on Rhizoctonia solani and other soil fungi. J. Phytopathol. 24: 1153-1159. Wicaksono, W.A., Jones, E.E., Monk, J. and Ridgway, H.J. (2016) The bacterial signature of Leptospermum scoparium (M anuka) reveals core and accessory communities with bioactive properties. PLoS ONE 11, e0163717. https://doi.org/10.1371/journal.pone.0163717 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6210065","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":439019530,"identity":"6c575327-a227-4e03-8d8a-9baaf3e2fcd6","order_by":0,"name":"Raouf Ahmad Mir","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACZijN3t4DJA2gvARitPCcOUOsFhjguZFDpLt023kfMFfU2CT2SL49+LmgwE6Ogf3wAYaHO3BrMTvMbsB45lhaYo90XrL0DINkYwaetASGxDP4tLAxMDawHU7cL51jIM1jcCCxQYLHgCGxjZCWf4eBDjtj/Jt4LY1tQC0SPGbE23KwsS/NuIcnx8wa5Bc2oF8O4NVy/hjjw4ZvNrI97GeMbxf8sZPjZz988OFPPFpA4ACMAY5WNmQRgoCZsJJRMApGwSgYiQAAE7lHancksB8AAAAASUVORK5CYII=","orcid":"","institution":"Hapico Industries Private Limited IGC Lassipoora Pulwama","correspondingAuthor":true,"prefix":"","firstName":"Raouf","middleName":"Ahmad","lastName":"Mir","suffix":""},{"id":439019531,"identity":"293198d0-3f92-415b-a9de-ff725e4126e8","order_by":1,"name":"Mir Khushnawaz Ahmad","email":"","orcid":"","institution":"Hapico Industries Private Limited IGC Lassipoora Pulwama","correspondingAuthor":false,"prefix":"","firstName":"Mir","middleName":"Khushnawaz","lastName":"Ahmad","suffix":""},{"id":439019532,"identity":"a1e66b19-6371-4a29-bbe8-f0b32ae0eb50","order_by":2,"name":"Sheema Manzoor","email":"","orcid":"","institution":"Hapico Industries Private Limited IGC Lassipoora Pulwama","correspondingAuthor":false,"prefix":"","firstName":"Sheema","middleName":"","lastName":"Manzoor","suffix":""}],"badges":[],"createdAt":"2025-03-12 08:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6210065/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6210065/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80630693,"identity":"dd929396-6042-4fa5-9dfc-faa2df4f83b6","added_by":"auto","created_at":"2025-04-15 11:41:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72295,"visible":true,"origin":"","legend":"\u003cp\u003eIsolation of endophytes from apple bark and leaves. A. Bacterial Endophytes. B. Fungal Endophytes.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6210065/v1/00ff99d59d1ed612ed78772b.jpg"},{"id":80630695,"identity":"1c7fd5cc-5ec4-439c-b716-226f1ec32371","added_by":"auto","created_at":"2025-04-15 11:41:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":136340,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of Endophytes against Phytopathogens\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6210065/v1/d508e91e0f7625cffa029976.jpg"},{"id":80630694,"identity":"e205df1d-6ca3-4c70-8379-f69d8714be3a","added_by":"auto","created_at":"2025-04-15 11:41:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108239,"visible":true,"origin":"","legend":"\u003cp\u003eAntifungal activity of individual endophytic strains against pathogens.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6210065/v1/baca47df9f8053056ab4b01b.jpg"},{"id":80630698,"identity":"27c6c9cf-c8f1-40ef-bd5f-2cfc097af114","added_by":"auto","created_at":"2025-04-15 11:41:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":161448,"visible":true,"origin":"","legend":"\u003cp\u003eTesting of bacterial endophytic consortium against Phytopathogens\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6210065/v1/1dfbb006d1a3253bf0df9252.jpg"},{"id":80631659,"identity":"dc2f81cf-c23b-4a17-9c51-d6831a2537c9","added_by":"auto","created_at":"2025-04-15 11:49:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":82872,"visible":true,"origin":"","legend":"\u003cp\u003eCompatibility analysis of endophytic strains\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6210065/v1/ddb0576858bd5df361439bca.jpg"},{"id":80633287,"identity":"906b31c0-e0a4-443d-abd2-f2dffb60874d","added_by":"auto","created_at":"2025-04-15 11:57:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119436,"visible":true,"origin":"","legend":"\u003cp\u003eSee image for figure legend.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6210065/v1/85d9cff0ca014ab013c176ca.jpg"},{"id":90855435,"identity":"571917e1-a317-49d2-8d4f-4299fe142679","added_by":"auto","created_at":"2025-09-09 04:46:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1596482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6210065/v1/c66246c6-e32f-4086-b210-350bde53c156.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Endophytic bacterial consortium: powerful antifungal team against menacing Phytopathogens of Apple (Malus Pumila)","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePlant pathogens cause a great threat to agricultural and horticultural production as they lead to great economic loss. Presently the impact has worsened due to the globalization of markets and climatic changes that has increased the infestation of diseases and their rapid spread Beneduzi \u003cem\u003eet al\u003c/em\u003e.( 2012). Among the pathogens, soil-borne pathogens like \u003cem\u003eFusarium oxyporium, Pencillium expansum, Phytophthora infestans, and Phytophthora cactorum\u003c/em\u003e are highly active and have a wide host range. Excessive use of conventional pesticides has further decreased soil health and hence makes it favorable for fungal pathogens and develops chemically resistant pathogens Kumar et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiotic stress mainly due to pathogenic microorganisms gives rise to major crop losses worldwide which are equivalent to nearly 222\u0026nbsp;billion dollars lost every year Chakraborty and Newton (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). To overcome this issue farmers or growers apply a variety of excess chemicals to control these pathogens which are more economic and harmful to the environment as well as human health through the air, water, and soil pollution. Excessive use of chemical pesticides has not only affected soil health but decreased the number of beneficial microbes in soil due to soil pollution. There is a need of the hour to find out an alternative method that is cost-effective, environmentally friendly, equally effective, and sustainable. Biological control of plant pathogens is one such alternative that is being used in agriculture and horticulture. The antagonistic activity of \u003cem\u003eTrichoderma\u003c/em\u003e was demonstrated more than eight decades ago Weindling (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1934\u003c/span\u003e) and presently it is the most extensively used bio-control agent Mukherjee et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A lot of bio-control microorganisms were isolated by screening several soil samples or plant-associated microorganisms for antagonism against phytopathogens Berg et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2001\u003c/span\u003e); Cazorla \u003cem\u003eet al\u003c/em\u003e (2017). A large number of bacteria with antifungal activities were isolated from rhizospheric soils; majority of the isolated bacteria were \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonads\u003c/em\u003e. Berg et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), Cazorla \u003cem\u003eet al\u003c/em\u003e( 2007), Beneduzi et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and Kumar (2012). It is found that many \u003cem\u003eBacilli\u003c/em\u003e genera can promote plant growth Choudhary \u003cem\u003eet al.\u003c/em\u003e (2009). Bacteria can promote plant growth through several mechanisms, like improvement in nutrient uptake, activation of systemic resistance, toxicity to pests, and antagonism pathogens Choudhary \u003cem\u003eet al\u003c/em\u003e. (2009), Pinchuk \u003cem\u003eet al.\u003c/em\u003e(2002), Wang \u003cem\u003eet al\u003c/em\u003e. (2007). The antifungal activity of \u003cem\u003eBacillus\u003c/em\u003e strains is due to the synthesis of various antimicrobial peptides, Tan (2010), Falardeau et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) secreted enzymes, (Baysal et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e.) proteins, Tan et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and volatile organic compounds (VOCs) Choudhary \u003cem\u003eet al.\u003c/em\u003e (2009), Baysal et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The antifungal activity of Bacillus strains makes them an effective bio-control agent (Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Baffoni \u003cem\u003eet al.\u003c/em\u003e 2015, and Shternshis et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Fengycin -a cyclic lipopeptide produced by bacillus strains plays an important role in its antifungal activity. (Guo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The strains of \u003cem\u003eBacillus subtilis\u003c/em\u003e can vary considerably both phenotypically as well as genotypically and that affects their antagonistic properties.\u003c/p\u003e \u003cp\u003ePlants and microorganisms live in interaction mode among each other within nature which can affect plant growth and development, response to any stimuli, and resistance to abiotic and biotic stresses (Nataraja \u003cem\u003eet al.\u003c/em\u003e2019). Endophytes are microorganisms that live in interaction with plants and can be used in biocontrol and induced resistance. Endophytes have an advantage over other bio-control agents since they colonize inside the plant tissues and remain protected from harsh environmental conditions which could otherwise affect their survival. (Latz \u003cem\u003eet al\u003c/em\u003e.2018). Thus, endophytes are currently the focus of scientists as a bio-control agent due to their ability to assist plants in growth, development, and resistance. Endophytes have received a lot of attention as both bio-control agents as well as activators of the plant defense against biotic and abiotic stresses. These methods have a positive impact on disease management. \u003cem\u003eTrichoderma\u003c/em\u003e species and \u003cem\u003ebacillus\u003c/em\u003e species are being used as bio-control agents for decades. This study aimed to directly compare the antagonistic effect of endophytes isolated from resistant apple varieties on apple pathogens invitro by suppressing phytopathogens through the production of cyclic lipopeptides, hydrolytic enzymes, siderophores, parasitic mode, and competition for nutrients. The further aim of this study was to develop a highly effective formulation of an endophytic consortium to control fungal pathogens in Apple trees.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eIsolation of endophytic microorganisms from Apple tree (Galla):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of Plant Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo year old High density apple trees were sourced from Sher-e-Kashmir University of Agricultural Sciences and Technology Shalimar Sinagar \u0026nbsp; Jammu and Kashmir India- 190025, \u0026nbsp; 0n 9\u003csup\u003eth\u003c/sup\u003e of March 2019 under receipt no.503 , and were planted in Hapico Industries Private limited IGC Lassipora Pulwama Jammu and Kashmir India .One year old shoots with leaves from the lowest main branch were sampled from the sourced \u0026nbsp;apple trees (Good growth rate, no deficiency, no disease symptoms, no physical damage, and no insect damage) in spring April-May 2021 when leaves were fully opened. \u0026nbsp;The The plant material was cut and placed in a zip-Lock cover and brought back to the lab.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcessing of Plant material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll plant parts collected were washed under running tap water three times and then using a 5-step sterilization method Wicaksono \u003cem\u003eet al\u003c/em\u003e. (2016), Purushotham \u003cem\u003eet al\u003c/em\u003e.(2018). Leaf and stem parts were cut into 1 cm portions using a sterile scalpel. Each tissue section was further cut into pieces (1mm thick) with 4 pieces plated on NA, 4 pieces on R2A for endophytic bacterial isolation, and 4 pieces on PDA and SNA (synthetic low agar nutrient) (Berg et al. 2005) for fungal Isolation. The plates were incubated at 25\u003csup\u003eo\u003c/sup\u003eC in complete darkness for 4 weeks. A few drops from the final rinse water were placed on R2A and PDA and incubated with inoculated plates at the same temperature and duration in darkness. It was done to check the effectiveness of the surface sterilization process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and preparation of pure cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndophytes were isolated from the incubated plates after 4 weeks. Plates were removed and all isolated colonies were sub-cultured on respective media (Bacteria on NA and Fungi on PDA) to get pure culture for identification \u0026nbsp;and antipathogenic activity\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStaining and Identification of Endophytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolated endophytes were identified by staining method and biochemical tests. Endophytes were stained by conventional methods (Lactophenol cotton blue for endophytic fungi and gram staining for endophytic bacteria) and identified by microscopy based on sporangium structure, spore shape, hyphal structure, hyphal type, (in Fungi) and cell shape and spore type (in bacteria). Biochemical tests such as; catalase test, coagulase test, oxidase test, sugar fermentation test, Indole test, citrate test, and urease test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening of Endophytes against Phytopathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e100 endophytes were isolated and were screened for their antifungal activity against five dreadful plant pathogensviz;(\u003cem\u003eVenturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Pencillium expansum\u003c/em\u003e). Out of 100 endophytes, 5 endophytic strains (2 fungal and 3 bacterial) had shown tremendous antipathogenic activity which was further verified by carrying out antifungal activities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe antipathogenic activity of selected 5 endophytic strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selected endophytic strains (2 fungal and 3 bacterial) were analyzed for their antipathogenic activity against plant pathogens. All selected strains were tested individually as well as in consortium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTesting of Individual endophytic strains against phytopathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selected endophytic strains were identified as \u003cem\u003eBacillus subtilis, Bacillus megaterium, Pseudomonas fluorescens, Trichoderma asperellum,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Trichoderma atroviride\u003c/em\u003e. The antipathogenic activity of individual endophytic strains was tested against five plant pathogens viz; (\u003cem\u003eVenturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Pencillium expansum\u003c/em\u003e). The plant pathogenic fungi were individually grown on PDA plates by placing a disc at the center of the plate and incubated overnight at 25\u003csup\u003e0\u003c/sup\u003eC. The 5 selected endophytic strains were inoculated individually on each pathogenic plate after 12 hours of incubation with pathogens. All plates were incubated at 25\u003csup\u003eo\u003c/sup\u003eC for 1 week until the mycelia grew.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTesting of endophytic consortium against Phytopathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndophytic consortium was formed by combining selected endophytic bacterial strains and used as an endophytic team against Phytopathogens and analyzed the antipathogenic activity of the endophytic team and compare results with individual strains. The plant pathogenic fungi were individually grown on PDA plates by placing a disc at the center of the plate and incubated overnight at 25\u003csup\u003eo\u003c/sup\u003eC. The endophytic bacterial strains were inoculated at 3 different locations at the periphery around the central pathogenic disc as an endophytic team. All Plates were incubated at 25\u003csup\u003eo\u003c/sup\u003eC for 1 week until the mycelia grew.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompatibility analysis of endophytic strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCross- streak method was used for the compatibility analysis of endophytic strains. Endophytic bacterial strains were streaked on nutrient plates in such a way that for every single bacterial culture in the center of the plate, other cultures are streaked radiating from the center. The plates were incubated at 35\u003csup\u003eo\u003c/sup\u003eC for 48 hours and the zone of inhibition was observed and recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecondary metabolites of endophytes as antagonistic against Phytopathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll endophytic strains were grown in liquid media for suspension culture to produce secondary metabolites. Bacterial endophytes were grown in ISP medium 4 (g/l) (soluble starch-10, dipotassium phosphate-1, magnesium sulphate-1, sodium chloride-1, ammonium sulphate-2, calcium carbonate-2, ferrous sulphate-1, manganous chloride-1, Zinc sulphate-1) with pH-7. The endophytic bacterial strains were individually inoculated in 150 ml medium (ISP 4) supplemented with optimum carbon and nitrogen source. The cultures were incubated at 35\u003csup\u003eo\u003c/sup\u003eC for seven days and the cell-free extract was used for analysis. The cell-free extract was obtained after extraction with ethyl acetate and the bioactive compound was obtained by evaporating the organic layer. The evaporated organic extract was dissolved in 0.5% Tween 80 for analyzing antifungal activity food and the poison method was adopted. The secondary metabolites from endophytic fungi were produced and extracted using the method reported by Vinale et al. (2006). 5 mm diameter plugs from each of the two cultures of \u003cem\u003eTrichoderma\u003c/em\u003e isolates (\u003cem\u003eTrichoderma atroviride and Trichoderma asperellum\u003c/em\u003e) obtained from the margins of actively growing cultures on PDA were separately inoculated into 5 L conical flasks containing 1 L Potato dextrose broth (PDB). The suspension cultures were incubated for 30 days at 25\u003csup\u003eo\u003c/sup\u003eC without shaking, after which the fungal mycelium was removed from the broth by vacuum filtration no. 4 filter paper (Whatman). The cell-free extract was obtained after extraction twice with ethyl acetate and a bioactive compound was obtained by evaporating the organic layer. After evaporation of the solvent the organic extract was dissolved in 0.5% Tween 80 for analyzing antifungal activity by food and poison method\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eand are very difficult to control. The use of conventional pesticides is posing another threat by causing disease resistance and environmental pollution and also affecting human and animal life. Using endophytes to control plant fungal diseases is a popular topic and has been studied extensively.\u003c/p\u003e\n\u003cp\u003eEndophytes are ubiquitous microbes that inhabit healthy plant tissues without causing disease. Endophytes have been found in every plant examined to date and may be important. Plant-microbe associations play a vital role in the growth and development of plants and may affect agriculture and the environment. Both plants and microbes play their role for the mutual benefit of plants and microbes. Plants and microbes have evolved as symbionts and have modulated their interactions by producing different phytochemicals, hormones, antimicrobial agents, plant growth promoters, and other metabolites. Besides having antimicrobial potential endophytes have gained their popularity as pharmaceutical agents. Endophytes have not only paved their way in medicine, therapeutics, and mining, some novel metabolitesmay be used in sustainable agriculture and plant growth. Endophytes are gaining biotechnological and industrially relevance as a result of their ability to secrete secondary metabolites, serve as bio-control agents, antimicrobial agents, antitumor agents, and immune suppressants, and secrete antiviral compounds and development of natural antioxidants, antidiabetic agents, antibiotics, and insecticidal products (Gouda \u003cem\u003eet al\u003c/em\u003e. 2016, Yadav.2018)\u003c/p\u003e\n\u003cp\u003eRecent findings have shown that endophytic fungi can help limit pathogen damage in many crops. (Arnold et al., 2003)Our results support these findings by showing that endophytic consortium isolated from healthy apple leaves and twigs restrict in vitro growth of the six most common and economically important plant pathogens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of endophytic microorganisms from Apple tree (Galla)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAround 100 endophytic strains were isolated from Apple leaves, and twigs. Most of bacterial endophytes isolated belong to genus, Bacillus species Pseudomonas species \u003cem\u003eRahnella\u003c/em\u003e species \u003cem\u003epaenibacillus\u003c/em\u003e species Enterobacter species \u003cem\u003eklebsiella\u003c/em\u003e species \u003cem\u003eMicrococcus\u003c/em\u003e species \u003cem\u003eOchrobactrum\u003c/em\u003e species \u003cem\u003eSphingomonas\u003c/em\u003e species \u003cem\u003ecurvibacter spp\u003c/em\u003e. And fungal endophytes belong to, \u003cem\u003eCladosporium\u0026nbsp;\u003c/em\u003especies \u003cem\u003eTrichoderma\u003c/em\u003e species \u003cem\u003eFusarium\u0026nbsp;\u003c/em\u003especies \u003cem\u003ePencillium\u0026nbsp;\u003c/em\u003especies. Fig 1, Fig 1A. Fig 1B. All isolated endophytes were identified by staining method and biochemical tests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening of Endophytes against Phytopathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHundred endophytic strains isolated from apple trees were screened against 6 different phytopathogens including \u003cem\u003eVenturia inequalis, Alternaria alternata, Fusarium oxyporium, phytophthora cactorum, Phytophthora infestans, Pencillium expansum.\u003c/em\u003e Out of 100 endophytes 5 endophytes \u003cem\u003e(Bacillus subtilis, bacillus megaterium, Pseudomonas fluorescens, Trichoderma asperellum, and Trichoderma atroviride)\u003c/em\u003e have shown good antagonistic effect against phytopathogens. These strains were identified, and used for further analysis. Fig 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntipathogenic activity of selected 5 endophytic strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selected endophytic strains (2 fungal and 3 bacterial) were analysed for their antipathogenic activity against plant pathogens. All selected strains were tested individually as well as in consortium. Consortium was found to be better antagonistic as compared to individual strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTesting of Individual endophytic strains against phytopathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selected endophytic strains were identified as \u003cem\u003eBacillus subtilis, Bacillus megaterium, Pseudomonas fluorescens, \u0026nbsp;, Trichoderma asperellum, and Trichoderma atroviride\u003c/em\u003e. The antipathogenic activity of individual endophytic strain was tested against five plant pathogens viz; (\u003cem\u003eVenturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntifungal assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMycelial discs (6mm) from a 7-day-old pure culture of endophytes ( \u0026nbsp;\u003cem\u003eBacillus subtilis, Bacillus megaterium, Pseudomonas fluorescens, Trichoderma asperellum, and Trichoderma atroviride.\u003c/em\u003e ) were excised and paired individually with pathogenic fungi (\u003cem\u003eVenturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum\u003c/em\u003e).opposite to each other at a distance of 40 mm apart in a 9 cm petri plate.. Pathogen was inoculated alone on PDA medium as a control. The mycelia growth diameter of both endophyte and pathogen was measured 3, 5 and 7 days after incubation. The inhibition of radial growth of pathogen (PIRG) was evaluated using formula.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePIRG=R1\u0026minus;R2/R1\u0026times;100\u003c/p\u003e\n\u003cp\u003eWhere R1 = diameter (mm) of the pathogen colony in the control; R2 = diameter (mm) of the pathogen in the endophyte-tested plate.\u003c/p\u003e\n\u003cp\u003eThe antagonistic activity of the endophyte was also described through visual assessment (Soytong et al) : \u0026gt;75% PIRG = very high antagonistic activity; 61\u0026ndash;75% PIRG = high antagonistic activity; 51\u0026ndash;60% PIRG = moderate antagonistic activity; \u0026lt;50% PIRG = low antagonistic activity\u003c/p\u003e\n\u003cp\u003eAll individual strains were found to be positive antagonistic against phytopathogens. \u0026nbsp; Table 1, fig 3\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"458\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 458px;\"\u003e\n \u003cp\u003eTable 1.Degree of antagonism of Individual endophtes against six pathogens 7 days after incubation period (DAIP).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003epathogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003ePIRG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eAntagonistic Activity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cem\u003eVenturia inequalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Trichoderma asperellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma atroviride.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003every high\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlternaria alternata\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Trichoderma asperellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma atroviride.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003every high\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cem\u003eFusarium oxyporium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Trichoderma asperellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma atroviride.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003every high\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhytophthora infestan\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Trichoderma asperellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma atroviride.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhytophthora cactorum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Trichoderma asperellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma atroviride.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Pencillium expansum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003emoderate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Trichoderma asperellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma atroviride.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003every high\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues are mean of triplicate and presented as a percentage\u003c/p\u003e\n\u003cp\u003eVisual assessment on antagonistic activity of the endophyte (Soytong et al) \u0026gt;75% PIRG = very high antagonistic activity; 61\u0026ndash;75% PIRG = high antagonistic activity; 51\u0026ndash;60% PIRG = moderate antagonistic activity; \u0026lt;50% PIRG = low antagonistic activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTesting of bacterial endophytic consortium against Phytopathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn endophytic consortium was formed by combining three selected endophytic strains \u003cem\u003eBacillus subtilis, Bacillus megaterium, and Pseudomonas fluorescens,\u0026nbsp;\u003c/em\u003eas an endophytic team against Phytopathogens and analyzing the antipathogenic activity of the endophytic team and comparing results with individual strains.\u003c/p\u003e\n\u003cp\u003eThe hyphal growth of pathogenic fungi was restricted by the tested organisms. The endophytic consortium limited the mycelia growth of pathogenic fungi at 3, 5, and 7 days after incubation. The pathogenic fungi grow fully inside the control plate while the treated plates show limited growth after 7 days of incubation. The consortium of endophytes has shown better antagonistic properties against the pathogen as compared to when used individually. Table 2, Fig 4\u003c/p\u003e\n\u003cp\u003eTable 2: Degree of antagonism of endophtytic team against six pathogens after 3,5and 7 days after incubation period (DAIP)\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"574\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 405px;\"\u003e\n \u003cp\u003eMycelial growth in mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003eMicrobial strains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3DAIP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e5DAIP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e7DAIP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eVenturia inequalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eAlternaria alternata\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eFusarium oxyporium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhytophthora infestan\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003ePhytophthora cactorum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas florescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Pencillium expansum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIndicated values are the mean of triplicate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompatibility analysis of endophytic strains:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCross- streak method was used for the compatibility analysis of endophytic strains. Endophytic bacterial strains were streaked on nutrient plates in such a way that for every single bacterial culture in the center of the plate, other cultures are streaked radiating from the center. The plates were incubated at 35\u003csup\u003eo\u003c/sup\u003eC for 48 hours and the zone of inhibition was observed and recorded. Fig 5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecondary metabolites of endophytes as antagonistic against Phytopathogens:\u003c/strong\u003e All endophytic strains were grown in liquid media for suspension culture to produce secondary metabolites. Bacterial endophytes were grown in ISP medium 4 supplemented with optimum carbon and nitrogen source. The cultures were incubated at 35oC for seven days and the cell-free extract was used for analysis. The secondary metabolites from endophytic fungi were produced and extracted using the method reported by (Vinale \u003cem\u003eet al\u003c/em\u003e. 2006). 5 mm diameter plugs from each of the two cultures of Trichoderma isolates (\u003cem\u003eTrichoderma atroviride and Trichoderma asperellum\u003c/em\u003e) and obtained from the margins of actively growing cultures on PDA were separately inoculated into 5 L conical flasks containing 1 L Potato dextrose broth (PDB). The suspension cultures were incubated for 30 days at 25oC without shaking, after which the fungal mycelium was removed from the broth by vacuum filtration (Whatman 4). The cell-free extract was obtained after extraction with ethyl acetate in both methods and bioactive compounds were obtained by evaporating the organic layer. After evaporation of the solvent, the organic extract was dissolved in 0.5% Tween 80 for analyzing antifungal activity by food and poison method. The cell-free extract of endophytic fungi was not so effective in controlling the growth of phytopathogens in invitro conditions but is inhibiting the growth by parasitic mode and competition for nutrients. Instead, the cell-free extract of endophytic bacteria was effective enough to inhibit the growth of phytopathogens in invitro hence favors the role of antifungal compounds produced by endophytic bacteria. Fig 6\u003c/p\u003e\n\u003cp\u003eBiocontrol of plant pathogens is the need of the hour and is a viable approach in plant disease management. Endophytic microbes (\u003cem\u003eBacillus subtilis, Bacillus megaterium, pseudomonas species Trichoderma asperellum and Trichoderma atroviride\u003c/em\u003e) isolated from apple leaves and twigs inhibited the growth of( \u003cem\u003eVenturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum\u003c/em\u003e) by antibiosis. Our observations on the potential of endophytes as a possible antifungal agents imply that such organisms can be considered as management options against pathogens in apple. Our findings reveal that endophytic bacterial consortium shows better results in controlling the fungal pathogens as compared to individual strains. Thus, further investigation must be conducted to identify the inhibitory compounds produced by the endophytes, and determine the efficacy level of those compounds and appropriate methods of application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMr.Raouf Ahmad Mir and Mir Khushnawaz Ahmad prepared the manuscript, did all research work , wrote the main manuscript , Mr Khushnawaz prepared figures and tables , Miss Sheema Manzoor reviewed the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors are very much indebted to the Department of Biotechnology, Hapico Industries Private Limited IGC SIDCO Lassipoora for the financial support of the project\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArnold et al., 2003; Evans et al., 2003; Mejı\u0026acute;a et al., 2003; Holmes et al., 2004, 2006; Rubini et al., 2005; Tondje et al., 2006; Pierre Roger Tonje, IRAD, Cameroon; personal communication Baffoni, L., Gaggia, F., Dalanaj, N., Prodi, A., Nipoti, P., Pisi, A., Biavati, B. and Di Gioia, D. (2015) Microbial Inoculants for the Biocontrol of Fusarium spp. in Du rum Wheat. BMC Microbiology,15, 242. https://doi.org/10.1186/s12866-015-0573-7\u003c/li\u003e\n\u003cli\u003eBaysal, O., Lai, D., Xu, H., Siragusa, M., Casiskan, M., Carimi, F., Silva, J.A.T. and Tor, M. (2013) A Proteomic Approach Provides New Insights into the Control of Soil-Borne Plant Pathogens by Bacillus Species. PloS One, 8, e53182. https://doi.org/10.1371/journal.pone.0053182\u003c/li\u003e\n\u003cli\u003eBell, D.K.; Wells, D.H.; Markham, R.C. In vitro antagonism of Trichoderma against six fungal plant pathogens. Phytopathology 1982, 72, 379\u0026ndash;382. DOI: 10.1094/Phyto-72-379.\u003c/li\u003e\n\u003cli\u003eBeneduzi, A., Ambrosini, A. and Passaglia, L.M. (2012) Plant Growth-Promoting Rhizobacteria (PGPR): The Potential as Antagonists and Biocontrol Agents. Genet ics and Molecular Biology, 35, 1044-1051. https://doi.org/10.1590/S1415-47572012000600020\u003c/li\u003e\n\u003cli\u003eBerg, G., Fritze, A., Roskot, N. and Smalla, K. (2001) Evaluation of Potential Bio-control Rhizobacteria from Different Host Plants of Verticillium dahliae Kleb. Journal of Applied Microbiology, 91, 963-971. https://doi.org/10.1046/j.1365-2672.2001.01462.x\u003c/li\u003e\n\u003cli\u003eCazorla, F.M., Romero, D., Perez-Garcia, A., Lugtenberg, B.J., Vicente, A.D. and Bloemberg, G. (2007) Isolation and Characterization Of antagonistic Bacillus subti lis Strains from the Avocado Rhizoplane Displaying Biocontrol Activity. Journal of Applied Microbiology, 103, 1950-1959. https://doi.org/10.1111/j.1365-2672.2007.03433.x\u003c/li\u003e\n\u003cli\u003eChakraborty, S., and Newton, A. C. (2011). Climate change, plant diseases and food security: an overview. Plant Pathol. 60, 2\u0026ndash;14. doi: 10.1111/j.1365-3059.2010.02411.x\u003c/li\u003e\n\u003cli\u003eChoudhary, D.K. and Johri, B.N. (2009) Interactions of Bacillus spp. and Plants\u0026mdash; with Special Reference to Induced Systemic Resistance (ISR). Microbiological Re search, 164, 493-513. https://doi.org/10.1016/j.micres.2008.08.007\u003c/li\u003e\n\u003cli\u003eFalardeau, J., Wise, C., Novitsky, L. and Avis, T.J. (2013) Ecological and Mechanis tic Insights into the Direct and Indirect Antimicrobial Properties of Bacillus subtilis lipopeptides on Plant Pathogens. Journal of Chemical Ecology, 39, 869-878. https://doi.org/10.1007/s10886-013-0319-7\u003c/li\u003e\n\u003cli\u003eGouda, S., Das, G., Sen, S. K., Shin, H.-S., and Patra, J. K. (2016). Endophytes: a treasure house of bioactive compounds of medicinal importance. Front. Microbiol. 7:1538. doi:10.3389/fmicb.2016.01538\u003c/li\u003e\n\u003cli\u003eGuo, Q., Dong, W., Li, S., Lu, X., Wang, P., Zhang, X., Wang, Y. and Ma, P. (2014) Fengycin Produced by Bacillus subtilis NCD-2 Plays a Major Role in Biocontrol of Cotton Seedling Damping-Off Disease. Microbiological Research, 169, 533-540. https://doi.org/10.1016/j.micres.2013.12.001\u003c/li\u003e\n\u003cli\u003eKumar G, A Maharshi, J Patel, A Mukherjee, HB Singh and BK Sharma. 2017. Trichoderma: A potential fungal antagonist to control plant diseases. SATSA Mukhapatra- Annual Technical Issue 21: 206-21 https://www.researchgate.net/publication/314946122\u003c/li\u003e\n\u003cli\u003eKumar, P., Dubey, R.C. and Maheshwari, D.K. (2012) Bacillus Strains Isolated from Rhizosphere Showed Plant Growth Promoting and Antagonistic Activity against Phytopathogens. Microbiological Research, 167, 493-499. https://doi.org/10.1016/j.micres.2012.05.002\u003c/li\u003e\n\u003cli\u003eKim, P.I., Ryu, J., Kim, Y.H. and Chi, Y.T. (2010) Production of Biosurfactant Li popeptides Iturin A., Fengycin, and Surfactin A from Bacillus subtilis CMB32 for Control of Colletotrichum gloeosporioides. Journal of Microbiology and Biotech nology, 20, 138-145. doi: 10.4014/jmb.0905.05007\u003c/li\u003e\n\u003cli\u003eLatz, M.A.C.; Jensen, B.; Collinge, D.B.; J\u0026oslash;rgensen, H.J.L. Endophytic Fungi as Biocontrol Agents: Elucidating Mechanisms in Disease Suppression. Plant Ecol. Divers. 2018, 11, 555\u0026ndash;567. https://doi.org/10.1080/17550874.2018.1534146\u003c/li\u003e\n\u003cli\u003eLi, X.Y., Yang, J.J., Mao, Z.C., Ho, H.H., Wu, Y.X. and He, Y.Q. (2014) Enhance ment of Biocontrol Activities and Cyclic Lipopeptides Production by Chemical Mutagenesis of Bacillus subtilis XF-1, a Biocontrol Agent of Plasmodiophora bras sicae and Fusarium solani. Indian Journal of Microbiology, 54, 476-479. https://doi.org/10.1007/s12088-014-0471-y\u003c/li\u003e\n\u003cli\u003eMukherjee M, PK Mukherjee, BA Horwitz, C Zachow, G Berg and S Zeilinger. 2012. Trichoderma- Plant-Pathogen interactions: Advances in genetics of biological control. Indian J. Microbiol. 52(4): 522-529. doi: 10.1007/s12088-012-0308-5\u003c/li\u003e\n\u003cli\u003eNataraja, K.N.; Suryanarayanan, T.S.; Shaanker, R.U.; Senthil-Kumar, M.; Oelm\u0026uuml;ller, R. Plant\u0026ndash;Microbe Interaction: Prospects for Crop Improvement and Management. Plant Physiol. Rep. 2019, 24, 461\u0026ndash;462. DOI 10.1007/s40502-019-00494-4\u003c/li\u003e\n\u003cli\u003ePinchuk, I.V., Bressollier, P., Sorokulova, I.B., Verneuil, B. and Urdaci, M.C. (2002) Amicoumacin Antibiotic Production and Genetic Diversity of Bacillus subtilis Strains Isolated from Different Habitats. Research in Microbiology, 153, 269-276. https://doi.org/10.1016/S0923-2508(02)01320-7\u003c/li\u003e\n\u003cli\u003ePurushotham, N., Jones, E., Monk, J. and Ridgway, H. (2018) Community structure of endophytic Actinobacteria in a New Zealand native medicinal plant Pseudowintera colorata (Horopito) and their influence on plant growth. Microb Ecol 76, 729\u0026ndash;740 DOI: 10.1007/s00248-018-1153-9\u003c/li\u003e\n\u003cli\u003eShternshis, M.V., Belyaev, A.A., Matchenko, N.S., Shpatova, T.V. and Lelyak, A.A. (2015) Possibility of Biological Control of Primocane Fruiting Raspberry Disease Caused by Fusarium sambucinum. Environmental Science and Pollution Research, 22, 15656-15662. https://doi.org/10.1007/s11356-015-4763-5\u003c/li\u003e\n\u003cli\u003eSoytong, K. Identification of of Chaetomium in the Philippines and Screening for Their Biocontrol Properties against Seedborne Fungi of Rice. Ph.D. Thesis, University of the Philippines Los Ba\u0026ntilde;os, Laguna, Philippines, 1988.\u003c/li\u003e\n\u003cli\u003eTan, Z., Lin, B. and Zhang, R. (2013) A Novel Antifungal Protein of Bacillus subtilis B25. SpringerPlus, 2, 543. https://doi.org/10.1186/2193-1801-2-543\u003c/li\u003e\n\u003cli\u003eWang, S., Wu, H., Qiao, J., Ma, L., Liu, J., Xia, Y. and Gao, X. (2009) Molecular Mechanism of Plant Growth Promotion and Induced Systemic Resistance to To bacco Mosaic Virus by Bacillus spp. Journal of Microbiology and Biotechnology, 19, 1250-1258. https://doi.org/10.4014/jmb.0901.008\u003c/li\u003e\n\u003cli\u003eWeindling R. 1934. Studies on a lethal principle effective in the parasitic action of Trichoderma lingorum on Rhizoctonia solani and other soil fungi. J. Phytopathol. 24: 1153-1159.\u003c/li\u003e\n\u003cli\u003eWicaksono, W.A., Jones, E.E., Monk, J. and Ridgway, H.J. (2016) The bacterial signature of Leptospermum scoparium (M anuka) reveals core and accessory communities with bioactive properties. PLoS ONE 11, e0163717. https://doi.org/10.1371/journal.pone.0163717\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Endophytes, Plant Pathogens, Consortium, Compatibility test, Secondary metabolites","lastPublishedDoi":"10.21203/rs.3.rs-6210065/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6210065/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiological control is a cost effective, natural, and sustainable alternative to chemical pesticides for management of pathogens. The main aim of this study was to investigate new wide spectrum biological agents to develop a microbial formulation against soil borne and air borne Plant pathogens. Almost 100 endophytic strains (Bacterial, fungal and actinomycetes) were screened for their antipathogenic activity against dreadful plant pathogens. Out of 100 strains screened 5 strains (3bacterial and 2 fungal strains) were found to be effective antipathogenic activity against Plant pathogens. The 5 effective strains were identified as \u003cem\u003eBacillus subtilis, Bacillus megaterium, Pseudomonas species, Trichoderma asperellum\u003c/em\u003e and \u003cem\u003eTrichoderma atroviride\u003c/em\u003e. All these strains were screened individually as well as in a team of microbial consortium to find out antipathogenic activity. Though all microbes showed promising activity individually against pathogens but bacterial consortium had shown better activity than individual microbes. Compatibility testing had shown that bacterial strains were compatible with each other. The Plant pathogens under test were: \u003cem\u003eVenturia inequalis, Alternaria alternata, Fusarium oxyporium, Phytophthora infestans, Phytophthora cactorum and Pencillium expansum.\u003c/em\u003e Three bacterial strains \u003cem\u003eBacillus subtillis, Bacillus megaterium\u003c/em\u003e and \u003cem\u003ePseudomonas species\u003c/em\u003e produced antifungal compounds to inhibit pathogens where as \u003cem\u003eTrichoderma asperellum\u003c/em\u003e and \u003cem\u003eTrichoderma atroviride\u003c/em\u003e penetrate pathogenic hyphae and compete for food and produced parasitic inhibition. The cell filtrate of all bacterial strains inhibits the growth of pathogens invitro but extract of fungal strains did not inhibit pathogens invitro. Results proved the efficacy of the individual as well as consortium as biocontrol agents to common soil and air borne pathogens.\u003c/p\u003e","manuscriptTitle":"Endophytic bacterial consortium: powerful antifungal team against menacing Phytopathogens of Apple (Malus Pumila)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-15 11:41:31","doi":"10.21203/rs.3.rs-6210065/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fc3cbcf1-6bea-4207-9100-05a60c902ed0","owner":[],"postedDate":"April 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46740537,"name":"Biological sciences/Biotechnology"},{"id":46740538,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-09-09T04:38:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-15 11:41:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6210065","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6210065","identity":"rs-6210065","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}