Bacterial endophytes: Potential bioagents against plant pathologens

Endophytic bacteria: potential bioagents against plant pathologens.
Special thanks to sagar, niteen, Mahendra, santhosh, sangamesh.

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2. 8/6/2024 2 So how do we get MORE FOOD to feed increasing population?

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4. Destruction of other beneficial organisms Pesticide residue Indiscriminate use Development of resistant strains in plant pathogens 8/6/2024 Dept. of PAT 4

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6. UNIVERSITY OF HORTICULTURAL SCIENCES BAGALKOT 06-08-2024 Dept. of PAT 6 COLLEGE OF HORTICULTURE, BAGALKOT SEMINAR – II Endophytic bacteria: Potential biocontrol agents against plant pathogens Name of the Student : Manohar Gowda B P ID. No. : UHS 22 PGM1 531 Degree Programme : Sr. M.Sc. Department : Plant Pathology 1.Introduction 2.History 3.Mode of entry 4.Plant growth promotion 5. Case studies 6. Limitations 7. Conclusion

7. 1.Introduction 2.History 3.Mode of entry 4.Plant growth promotion 5. Case studies 6. Limitations 7. Conclusion Flow of seminar

8. 8 Endophytes (Bacon and White, 2020)

9. 8/6/2024 9 HISTORY

10. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Heinrich Friedrich – first to describe the endophytes as “ Entophytae ” Heinrich Friedrich HISTORY Heinrich Friedrich Anton De Bary Anton De Bary

11. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Anton De Bary De Bary coined the term “ Endophyte ” HISTORY Anton De Bary

12. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Marie Louis Victor Galippe reported occurrence of bacteria and fungi interior tissues of vegetables. HISTORY Orlando Petrini Orlando Petrini

13. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Orlando Petrini defined endophytes as “all organisms inhabiting plant organs that at some part of time in their life cycle and can colonize internal plant tissues without causing apparent harm to their host” Orlando Petrini HISTORY Orlando Petrini

14. Endophyte Diversity Prokarytotic Endophytes Glomeromycota , Ascomycota, Basidiomycota and Zygomycota. 23 Phyla ; Archaea – 2, Bacteria 21 It is largely represented by a few phylum: Firmicutes, Bacteroidetes, Proteobactria and Actinobacteria Hardoim et al ., 2015 14 Eukaryotic Endophytes

15. Mode of entry Active entry It occurs through the release of cell wall digesting enzymes like cellulose, glucanase etc . Passive entry Reinhold- Hurek et al ., 1993

16. Rhizosphere as a bacterial reservoir The rhizosphere is the soil region around plant roots, acting as a reservoir for various bacteria due to the various mutual association. Based on the beneficial activity of the bacteria the plants which allows to the bacteria in several mechanisms Vaishnav et al ., 1993

17. 8/6/2024 17 Interaction between endophyte and host plant Root Exudates Chemical Signals Selective Attraction Physical Barriers Cell wall Cell membrane Microbial competition Beneficial Microbes Microbial Consortia Root Architecture Root Structure Root zones Vaishnav et al ., 1993

18. 8/6/2024 18 Symbiotic relationship

19. Comparison between rhizosphere bacteria and endophytic bacteria Feature Rhizosphere Bacteria Endophytic Bacteria Location Soil around plant roots Inside plant tissues Interaction External interaction with plant roots Internal relationship within plant tissues Diversity More diverse and numerous Less diverse and fewer Population Density 10 6 to 10 9 cells/gram of soil 10 4 to 10 8 cells/gram of root tissue Role Nutrient cycling, disease suppression, growth promotion Growth promotion, stress resistance, nutrient acquisition Environmental Influence Soil type, pH, moisture, root exudates Host plant species Common Genera Pseudomonas, Bacillus, Azospirillum , Rhizobium Bacillus, Burkholderia , Pseudomonas, Stenotrophomonas

20. 06-08-2024 Dept. of PAT 20 Transmission Horizontal   Vertical Transmission of endophytes INSECT SOIL WIND VEGITATIVE PROPAGATION SEEDS

21. Colonization of endophytes Bacterial endophytes most often occupy intercellular spaces in the plant, most likely because these areas have an abundance of carbohydrates, amino acids, and inorganic nutrients.

22. Endophytic association can either be :- 8/6/2024 Dept of Plant Pathology 22 Commensalism Mutualism

23. Source of nutrients Place of survival Protected environment Nutrient Supply Reduced Competition Enhanced Survival and Dissemination Benefits - Endophytes

24. Improving Nutrient Acquisition Improved Plant Health Improved Yield and Quality Disease Resistance Improved Stress Tolerance Enhanced Growth and Development 8/6/2024 Dept. of FSC 24 Benefits – Host plant Datta et al ., 2012

25. 8/6/2024 25 Isolation of endophytic bacteria Direct tissue isolation method Vacuum extraction method Tissue Maceration method Cryo-Maceration method Tissue lyophilization method Garner (1982)

26. 8/6/2024 26 Direct tissue isolation Garner (1982)

27. 8/6/2024 27 Plant origin Endophytic bacteria References Fruit crops Citrus Bacillus cereus , B . lentus , B . Pumilis , B . subtilis , B . megaterium Araujo et al. (2001) Grapes Comamonas sp. Bell et al. (1995) Pseudomonas cichori , Xanthomonas sp. Moraxella bovis , Enterobacter sp. West et al. (2010) Apple Micrococcus luteus , B . subtilis & P . aeruginosa Miliute et al. (2016) Avocado Bacillus sp. Prasad and Dagar (2014) Indian fig tree P . viridiflava , Streptomyces sp. & Serratia marcescens Abdallah et al. (2016) List of endophytic bacteria isolated from major horticultural crops

28. 8/6/2024 28 Plant origin Endophytic bacteria References Vegetable crops Onion Bacillus spp , Pseudomonas spp & Burkholderia phytofirmans Weilharter et al. (2011) Potato Variovorax paradoxus , Bacillu spp , Peudomonas spp & Chrysiobacterium sp Han et al. (2011) common bean Bacillus thuringiensis ,  Micrococcus luteus Costa et al. (2011) Soyabean B . megaterium Smita and Dipak (2015) Cucumber Bacillus spp. Raupach and Kloepper (1998) Pea B . Pumilus Chaudhary et al. (2009) Plantation and spices crops Black pepper Bacillus sp., Pseudomonas sp Aravind et al. (2009) Coconut B . Subtilis & Pseudomonas spp Salo et al . (2020) Turmeric B . Pumilis and B . turingiensis Kumar et al. (2016) Cacao Bacillus sp. Melnick et al . (2008)

29. 8/6/2024 29 Methods for in vitro evaluation of antimicrobial activity of endophytes Dual c ulture assay Agar well d iffusion assay Disk diffusion a ssay Spot Inoculation assay Volatile organic c ompounds a ssay

30. 8/6/2024 30 In vitro evaluation of antagonistic potential of endophytic bacteria Against pathogenic fungi The antagonistic ability of endophytic bacteria against fungal pathogen can be done by dual culture technique

31. 8/6/2024 31 Against pathogenic bacteria The antagonistic ability of endophytic bacteria against pathogenic bacteria can be done by Disk diffusion method. The percent inhibition of pathogens by endophytic bacteria can be calculated using formula

32. In Vitro Efficacy of Bacterial Endophytes against Pythium sp. Causing Soft Rot of Ginger in Meghalaya Meshanki Bamon*, Dipali Majumder, Dwipendra Thakuria and Thangaswami Rajesh Objective: To evaluate the efficacy of bacterial endophytes in managing rhizome rot of ginger caused by Pythium spp. in vitro condition Published: 2018 Department of Plant Pathology, College of Post-Graduate Studies, Central Agricultural University (Imphal), Umiam-793103, Meghalaya, India 8/6/2024 32 Case study - 1

33. 8/6/2024 33 Materials and method Bamon et al ., 2018

34. 8/6/2024 34 Fig. 1: In vitro evaluation of endophytic bacteria against phythium sp. Bamon et al . (2018)

35. 8/6/2024 35 Bamon et al . (2018) In vitro evaluation found that four isolates were highly effective as it gave more than 70% inhibition in growth of Pythium sp. Fig. 2: T he per cent inhibition of bacterial endophytes against Pythium sp. in in vitro evaluation

36. Endophytic bacteria could serve as potential antagonist to prevent the infection of plant pathogens. In vitro evaluation of 4 bacterial endophytes revealed that all isolates were effectively inhibit the growth of Pythium spp. Inference 8/6/2024 36

37. 8/6/2024 37 Role of endophytes in plant growth promotion Direct growth promotion Indirect growth promotion

38. 8/6/2024 38 Direct growth promotion Biological nitrogen fixation Inside the plant tissues, the endophytic bacteria convert atmospheric nitrogen (N₂) into ammonia (NH₃) through the action of the nitrogenase enzyme. This process requires a significant amount of energy, which the bacteria obtain from the plant in the form of carbohydrates and other organic compounds.

39. 8/6/2024 39 Phosphate solubilization Siderophores synthesis The most powerful strains of phosphate Solubilizers are Pseudomonas , Bacillus .

40. 8/6/2024 40 Phytohormone (IAA) production Indole acetic acid enhances cell division and differentiation which eventually increases the root length and root hair, so this helps in absorbing more nutrients which stimulate plant growth.

41. 8/6/2024 41 Indirect growth promotion Biocontrol agents plant pathogens They mainly control or reduce the incidence and severity of the plant diseases. Pseudomonas and Bacilli species are the predominant bacterial biological control agents.

42. 8/6/2024 42 Quorum Quenching Endophytes can interfere with the signals of harmful bacteria use to coordinate actions like producing toxins. This disruption helps protect plants from diseases, offering a natural and eco-friendly method of disease control. Quorum quenching is a process which disrupts bacterial communication by blocking their signals, preventing them from coordinating harmful actions, which helps control bacterial infections. Fekete et al . (2010)

43. 8/6/2024 43 Secondary metabolites It plays a vital role in the functioning of plants as the defence compound or as the signaling molecule during ecological interactions and environmental stresses. It acts as – antimicrobial, anti-cancerous, phytohormone and its precursor, vitamin (B12), bioprotectants Some examples of secondary metabolites are: Alkaloids, steroids, terpenoids and phenols

44. Isolation, identification and characterization of endophytic bacteria antagonistic to Phytophthora palmivora causing black pod of cocoa in Malaysia Wael Alsultan & Ganesan Vadamalai & Ahmad Khairulmazmi & Halimi Mohd Saud & Abdullah M. Al-Sadi & Osamah Rashed & Ahmad Kamil Mohd Jaaffar & Abbas Nasehi Objective: To study biological control activities of endophytic bacteria isolated from cocoa against Phytophthora palmivora and also evaluate different secondary metabolites. Published: 2020 Department of Plant Protection UniversitiPutra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia 8/6/2024 44 Case study - 2

45. Materials and method Isolation of bacterial endophytes from Cocoa In vitro evaluation of endophytes against pathogen Phytophthora palmivora Identification of endophytic bacteria Detection of Secondary metabolites using GCMS Dept. of PAT 8/6/2024 45 Alsultan et al . (2020)

46. 8/6/2024 46 State Locations Total Number of Isolates Leaf Branch Pod Perak Teluk Intan Beruas Manjung Hilir Perak Perak Tengah 12 11 10 Pahang Raub Triang Damak Jerantut Jengka 5 4 5 Sabah Madai Gudang Tingkat 9 3 3 Malacca Jasin 5 4 3 Johor Batu Pahat Muar 4 5 3 Selangor Sabak Bernam 3 1 1 Sarawak Kuching 8 4 Total 38 36 29 Table 1: Isolates of endophytic bacteria present on cocoa tissues (pods, branches, leaves) from various locations in Malaysia Alsultan et al . (2021)

47. 8/6/2024 47 In vitro evaluation of endophytes against pathogen Phytophthora palmivora Fig. 3: Inhibition of growth of P. palmivora in dual culture test medium by AS1 (a), AS2(b) and control (c) Alsultan et al . (2021) AS1 AS2 CONTROL - P. palmivora

48. 8/6/2024 48 Molecular identification of endophytic bacteria Table 2: Identification of endophytic bacterial isolates using 16S rDNA gene sequencing Isolates Isolation source Origin Species Accession Number AS1 Branch Perak Pseudomonas aeruginosa MH800529 AS2 Branch Perak Chryseobacterium proteolyticum MH800530 Alsultan et al . (2021)

49. 8/6/2024 49 Bacterial isolates Phosphate solubilization Siderophores HCN Production Ammonia production P. aeruginosa + + + + C. proteolyticum + - - + Table 3: In-vitro plant growth promotional activities of the two endophytic bacteria Alsultan et al . (2021)

50. 8/6/2024 50 Different inhibitory effects towards P. palmivora in Culture filtrate test Fig. 4: Different inhibitory effects towards P. palmivora in culture filtrate test AS1(a), AS2 (b) and control (c) a b c AS1 AS2 CONTROL - P. palmivora Alsultan et al . (2021)

51. 8/6/2024 51 Table 4: Chemical composition of effective entophytic bacteria by GC-MS Alsultan et al . (2021)

52. 8/6/2024 52 Table 5 : Antimicrobial substances produced by two effective endophytes Sl. No Compounds Biological activity Antifungal activity References 1. Eicosane Antifungal, Cytotoxic and Antitumor Sphaerotheca fuliginea and Botrytis cinerea on cucumber (Zhang et al. 2013) Hsouna et al .(2011), Belakhdar et al .(2015) 2. Hexatriacontane Antifungal, Anti-bacterial, Anti-cancer, nematicide and pesticidal compound Penicillium expansum , Pythium ultiumu and Fusarium solani (Hashem et al. 2016) Umaiyambigai et al . (2017) 3. Tetratetracontane Anti-cancerous activity Fusarium wilt of banana Singaria et al . (2015) Alsultan et al . (2021)

53. Among 103 endophytes two are effectively inhibited the growth of Phytophthora palmivora and the GCMS analysis of this isolates revealed that the secondary metabolites produced by this isolate having anti-microbial properties this is responsible for pathogen inhibition. This study is the first report on the antagonistic activity of endophytic bacterial isolates against the black pod pathogen Inference 8/6/2024 53

54. Isolation and characterization endophytic bacteria as biological control of fungus Colletotrichum sp. on onion plants ( Allium cepa L .) A Murtado, N R Mubarik* and A Tjahjoleksono Objective: To isolate endophytic bacteria from onion and test the ability of bacteria to inhibit the growth of the pathogenic fungus Colletotrichum sp. Published: 2020 Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Jalan Agatis - IPB Dramaga , Bogor 16680, Indonesia 8/6/2024 54 Case study - 3

55. 8/6/2024 55 Materials and method Murtado et al . (2020)

56. 8/6/2024 56 Table 6: Number of endophytic bacteria isolated from different parts on onion plants Sample collection Sample location of onion plant Total Healthy onion plant Diseased onion plant Root 12 6 18 Stem 4 5 9 Leaf 5 8 3 Total 21 19 40 Murtado et al . (2020)

57. 8/6/2024 57 Table 7: Potential isolates of endophytic bacteria that have inhibited the growth of pathogenic fungi SL No. Isolate code Sampel origin Fungus hyphae growth length Percentage of inhibition (%) R1 (cm) R2(cm) 1 BBP4.1 Stem 4.5 3.0 33.3 2 DBS5.2 Leaf 4.5 3.0 33.3 3 DBP4.2 Leaf 4.5 3.0 33.3 4 ABP4.2 Root 4.5 2.4 46.7 5 DBP4.2 Leaf 4.5 2.3 48.9 6 ABP5.1 Root 4.5 2.2 49.1 7 ABP4.1 Root 4.5 1.8 60.0 8 DBP4.1 Leaf 4.5 1.8 60.0 9 ABS4.1 Root 4.5 1.7 62.2 10 ABP5.1 Root 4.5 1.4 68.9 11 ABP5.2 Root 4.5 1.3 71.1 12 BBP5.2 Stem 4.5 1.2 73.3 Murtado et al . (2020)

58. Fig. 5: Results antagonistic test of six potential bacterial against Colletotrichum sp. Control (A), bacterial isolates BBP5.2 (B), ABP5.2 (C), ABS4.1 (D), DBP4.1 (E), ABP4.1 (F) and ABP5.1 (G) 8/6/2024 58 Murtado et al . (2020)

59. 8/6/2024 59 Hypersensitivity tests on tobacco plants Fig. 6: Results hypersensitivity test of potential endophytic bacteria on tobacco leaves for 48 hours. The negative results of four isolates selected ABS4.1, ABP5.1, BBP5.2, and ABP5.2 (A), positive control of Xanthomonas oryzae (B) and negative control of NB media and sterile aquades (C). Murtado et al . (2020)

60. 8/6/2024 60 Hemolysis test Fig. 7: Results of hemolysis test from four isolate of endophytic bacteria for 24 hours incubation. The isolates selected ABS4.1 (A), ABP5.1 (B), ABP5.2 (C) and BBP5.2 (D) Murtado et al . (2020)

61. 40 endophytic bacterial isolates were obtained from healthy and diseased onion plants. Six isolates effectively inhibited the growth of Colletotrichum fungi, with one isolate, BBP5.2, showing the greatest inhibition and being non-pathogenic to plants and humans. Inference 8/6/2024 61

62. 8/6/2024 62 Isolation and identification of endophytic bacteria from potato tissues and their effects as biological control agent against bacterial wilt Objectives : To isolate and identify endophytic bacteria from potato plant tissues to investigate their antagonistic effects against Ralstonia solanacearum. Kambiz Bahmani a , Nader Hasanzadeh a , Behrouz Harighi b , Alireza Marefat c Department of Plant Protection, Faculty of Agricultural Sciences and Food Industries, Science and Research Branch, Islamic Azad University, Tehran, Iran Case study - 4 Published: 2021

63. 8/6/2024 63 Materials and method Bahmani et al. (2021)

64. Isolation of endophytic bacteria Bahmani et al. (2021) 8/6/2024 64 Sampling areas (cities) Number of farms sampled in each area Number of endophytic bacteria isolated in each area Number of endophytic bacteria isolated from each part of potato plants in different areas Leaf (L) Stem (S) Root (R) Tuber (T) Sanandaj 2 25 8 6 10 1 Dehgolan 9 53 3 16 22 12 Qorveh 9 46 20 12 14 - Kamyaran 5 27 - 12 7 8 Mariwan 5 17 - 11 6 - Saqqez 12 64 13 25 17 9 Divandarreh 4 4 - 1 3 - Total 236 44 83 79 30 Table 8: Number of endophytic bacteria on potato plants isolated from different locations

65. Table 9: In vitro inhibition of growth of R. solanacearum by antagonistic endophytic bacteria from potato through the production of siderophore, hydrogen cyanide and protease 8/6/2024 65 Strains Mean diameter of inhibition zone (mm) a Siderophore production test Protease production test Hydrogen cyanide production test Bp91 11.2c±0.17 + + - Bp1 11.5c±0.05 + + - Bp49 11.1c±0.44 + + - Bl17 15.2b±0.14 + + - Pa86 16.3a±0.06 + - - Psb101 17.6a±0.28 + + - Ps169 17.4a±0.12 + + - Ps52 15.5b±0.26 + + + Ch54 8.5e±0.20 + + - Chl98 8.5e±0.34 + + - Mi41 9.5d±0.28 + + - R. Solanacearum + Sterile distilled water - + + - a Values in the same column with the same letter(s) are not significant different as by DMRT test (P = 0.05). B + = positive, - = negative Bahmani et al. (2021)

66. Bahmani et al. (2021) 8/6/2024 66 Isolate code Origin The part of the potato plant from which endophytic bacteria have been isolated GeneBank Accession number Species Leaf Stem Root Tuber S1L2(Bp91) Sanandaj + - - - MN326732 Bacillus pumilus Sq1BS1(Bp1) Saqqez - + - - MN326725 Bacillus pumilus Q1R1(Bp49) Qoeveh - - + - MN326728 Bacillus pumilus Q3L1(Bl17) Qoeveh + - - - MN326726 Bacillus licheniformis D2L1(Pa86) Dehgolan + - - - MN326731 Paenibacillus peoriae D7R2(Psb101) Dehgolan - - + - MN326734 Pseudomonas brassicacearum Sq6S1(Ps169) Saqqez - + - - MN326735 Pseudomonas brassicacearum Q8R1(Ps52) Qoeveh - - + - MN326729 Pseudomonas putida Q5L4(Ch54) Qoeveh + - - - MN326730 Chrysiobacterium indologenes D9S1(Chl98) Dehgolan - + - - MN326733 Chrysiobacterium lathyri Q7S2(Mi41) Qoeveh - + - - MN326727 Microbacterium phyllospaerae Table 9: Identification of endophytic bacterial isolates using 16S rDNA gene sequencing

67. Table 10: Effect of antagonistic endophytic bacteria on disease biocontrol and reduction of disease symptoms in greenhouse condition. Bahmani et al. (2021) 8/6/2024 67 Treatment Disease incidence (%) Bp91+ R. Solanacearum 48.06d a ±0.27 Bp1+ R. Solanacearum 48.11d±0.12 Bp49+ R. Solanacearum 48.24d±0.27 Bl17+ R. Solanacearum 44.14c±0.43 Pa86+ R. Solanacearum 41.81b±0.32 Psb101+ R. Solanacearum 33.07a±0.38 Ps169+ R. Solanacearum 33.82a±0.39 Ps52+ R. Solanacearum 34.29b±0.35 Ch54+ R. Solanacearum 52.72e±0.37 Chl98+ R. Solanacearum 52.30e±0.09 Mi41+ R. Solanacearum 75.22g±0.28 R. Solanacearum - a Values in the same column with the same letter(s) are not significant different as by DMRT test (P = 0.05).

68. Table 11: The effect of antagonistic endophytic bacteria on growth promotion and increase of potato biomass in greenhouse conditions Fig. 8: The seedlings treated with antagonistic endophytic bacteria show drastically wilt disease reduction and plant growth promotion in greenhouse conditions 8/6/2024 68

69. 11 different endophytic bacteria isolated from the potato were able to control the disease under both in vitro and in vivo conditions and they also had a significant effect on the growth mass of the treated plants Inference 8/6/2024 69

70. Department of Agricultural and Environmental Sciences, College of Agriculture, Human and Natural Sciences, Tennessee State University, Nashville, TN, USA Evaluation of Selected Bacterial Endophytes for Biocontrol Potential against Phytophthora Blight of Bell Pepper ( Capsicum annuum L .) Irabor A and Mmbaga MT* Objective: To explore the potential of bacterial endophytes as biological control agents for managing Phytophthora blight in bell pepper and impact of these endophytes on plant growth and compatibility with the fungicide mefenoxam. Published: 2017 8/6/2024 70 Case study - 5

71. 8/6/2024 71 Materials and method Location: College of Agriculture, Human and Natural Sciences, Tennessee State University, Nashville, TN, USA Isolation and confirmation the pathogen In vitro evaluation of bacterial endophytes against P. capsica Effects of selected bacterial endophytes on Phytophthora blight severity in greenhouse Effects of bacterial endophytes on plant growth and yield of bell pepper Sensitivity of selected bacterial endophytes and P. capsici isolate to mefenoxam Irabor and Mmbaga . ( 2017)

72. Isolates Endophytes Plant Source B17B Serratia marcescens Dogwood stem E Enterobacter sp Snap bean root IMC8 Bacillus thuringiensis Dogwood stem Y B. thuringiensis Dogwood stem Ps B. vallismortis Papaya stem Psl B. amyloliquefaciens Papaya stem Prt B. subtilis Papaya root Table 12: Bacterial endophytes and their sources 8/6/2024 72 Irabor and Mmbaga . ( 2017)

73. Treatments (bacteria) Mycelial growth (cm) % Growth Inhibition B17B 1.84 g 51.59 g E 1.28 d 66.27 d IMC8 1.43 e 62.50 e Y 1.49 f 60.69 f Ps 0.99 b 73.92 b Psl 0.88 a 76.78 a Prt 1.07 c 71.86 c Control 3.81 h 0.00 h Table 13: Effect of bacterial endophytes on the mycelial growth of Phytophthora capsici in Dual culture assay. 8/6/2024 73 Irabor and Mmbaga . ( 2017)

74. Table 14: Sensitivity of Bacillus isolates Ps, Psl and Prt different levels of mefenoxam Treatment (µg ml -1 ) BCA concentration at OD600 Ps Psl Prt 0.83 0.82 0.72 10 0.72 0.76 0.59 100 0.79 0.86 0.78 1000 0.67 0.64 0.65 BCA, biological control agents; OD600, optical density measured at 600 nm Sensitivity of Bacillus isolates Ps, Psl and Prt to mefenoxam at 0 ppm, 10 ppm, 100 ppm, and 1000 ppm showed that all isolates tested were tolerant to mefenoxam even at concentrations higher than the manufacturer’s recommended rates. 8/6/2024 74 Irabor and Mmbaga . ( 2017)

75. The isolates tested have biological control potential against phytophthora blight and may be useful in strategies integrating biological control agents with chemical control of Phytophthora diseases. Inference 8/6/2024 75

76. 8/6/2024 76 Formulations of Endophytic Microbes for Field Applications Characteristics of a good carrier material It should be easily sterilized, chemically and physically uniform as possible, having high water-holding capacity and suitable for many microbes It should be reasonably priced, easily manufactured and mixed by existing industry It should allow addition of nutrients and adjustment of pH It should be nontoxic, biodegradable, non-polluting and have sufficient shelf life Selection of formulation is very crucial as it can determine the success or failure of endophytic microbes. Formulation typically consists of an active ingredient either as microbe(s) or as a product of microbe(s) in a suitable carrier material (sterile or non-sterile) with additives.

77. 8/6/2024 77 Different Carrier materials used in the formulation Types of Formulations Liquid-Based Formulations Talc-Based Formulation Sawdust-Based Formulation Fly Ash-Based Formulation Encapsulation-Based Formulation - Encapsulation of microbial cells in polymeric gel (alginate or gluten) Peat, talc, charcoal, cellulose powder, farmyard manure, vermin-compost and compost, lignite, bagasse and press mud

78. 8/6/2024 78 Endophytes Shelf life Type of formulation reference Bacillus subtilis Up to 1 year Talc powders Trivedi et al . (2005) Ochrobactrum anthropi Up to 9 months Talc powders Chakraborty et al . (2009) Bacillus amyloliquefaciens Up to 9 months Sawdust Chakraborty et al . (2013) Serratia marcescens Up to 9 months Talc powder Bacillus Pumilus Up to 9 months Rice husk Different formulations of endophytic bacteria and their shelf life

79. 8/6/2024 79 Biopesticide Mass-production Units in India Mishra et al. (2020)

80. 8/6/2024 80 Type of biopesticide distribution Per cent distribution of bacterial biopesticide Mishra et al. (2020)

81. 8/6/2024 81 State-wise consumption of biopesticide formulations during 2014-20 in India Crop-wise consumption of biopesticide formulations during 2014-20 in India Murali et al. (2023)

82. 8/6/2024 82 Name of Company Organism Trade name Target biotic stress Kan biosys Pvt. Ltd., Pune Pseudomonas florescens Sudo 0.5% WP Late leaf spot of groundnut Deepa Farm inputs Private Limited Trichoderma viride Bio-plus tricho Soil and seed borne diseases Biotech International Ltd., New Delhi Trichoderma viride BIODERMA WP/AS Root rot, stem rot, damping off, fusarium wilt and verticillium wilt, all types of leaf spot, leaf & blight Ampelomyces quisqualis ARMOUR WP/AS Powdery Mildew in pulses, vegetables, fruits & ornamental crops International Panaacea Ltd., New Delhi Trichoderma harzianum Bioharz 2% AS Fusarium, Charcoal rot, Black scurf, Karnal bunt, Silver leaf of plum & peach, Rhizoctonia, Pythium, Sclerolims , Verticillium, Alternaria Bannari Amman Sugars Ltd., Tamil Nadu Bacillus subtilis LEAF CARE Fungal diseases List of microbial biopesticide formulations registered in India

83. 8/6/2024 83 Kera probio Talc formulation of Bacillus megaterium Talc based formulation of Pseudomonas putida . Cocoa probio Registered biopesticides for growth promotion

84. 8/6/2024 84 Limitations Limited Efficacy Host Specificity Environmental Factors Complex Interactions Cost and Scalability Long-term Effects

85. 8/6/2024 85 CONCLUSION All plants have beneficial bacteria inside them called endophytes, which help protect against diseases and environmental stress. By using these bacteria, we can reduce the need for chemical pesticides, making farming healthier and more environmentally friendly. These bacteria hold great potential for creating a greener and stronger agricultural future .

86. 8/6/2024 86 The future belongs to nations who have grains not guns - Dr. M S Swaminathan

87. 8/6/2024 87 Before spray… Think about pray…

88. 8/6/2024 88 Honor the invisible champions of Greener world

89. 8/6/2024 89 Vacuum extraction method Surface sterilize the collected samples Cut into small pieces and placed Vacuum chamber Due to pressure by the vacuum pump The extract were collected in a sterile container Plating on nutrient agar (NA) medium Incubation at 28 ° C for 72 hrs Homogenisation / Trituration method • Surface-sterilization of the healthy plant sample. • Sample is homogenised with 12.5 mM potassium phosphate buffer. • Serial dilution in same buffer • Plating on nutrient agar (NA) medium • Incubation at 28 ° C for 72 hrs Bell et al. (1995) Petrini (1986)

90. Fig. 9: Pathogenicity test of Phytophthora capsici isolate on bell pepper Disease severity of 0-5 scale = no visible symptoms 1 = Slightly wilted with brownish lesions beginning on the stem 2 = Stem lesions extending to cotyledons & 30% of plant diseased 3 = Stem lesions extending to petioles and 50% of the plant diseased 4 = Petioles collapse and 80% of the plant diseased 5 = Entire plant dead 8/6/2024 90 Irabor and Mmbaga . ( 2017)

91. Fig. 8: Effects of selected bacterial endophytes on Phytophthora blight severity in greenhouse 8/6/2024 91 Irabor and Mmbaga . ( 2017)

92. Fig. 9: Effects of bacterial endophytes on plant growth promotion activity in bell peppers Treatment Chlorophyll content Root length (cm) Shoot length (cm) Plant fresh weight (g) Plant dry weight (g) Number of fruits/ plant Fruit weight (g)/plant Control 7.24 32.28 30.45 63.18 7.78 0.75 7.48 Ps 9.41 30.92 40.98 108.58 8.50 2.20 48.14 Psl 5.98 28.33 31.00 90.08 9.03 2.00 22.55 Prt 8.96 30.73 32.02 110.90 8.75 2.00 41.72 All three Bacillus isolates promoted plant growth in shoot height, fruit number and fruit weight with Ps and Prt being slightly better than Psl 8/6/2024 92 Irabor and Mmbaga . ( 2017)

93. 8/6/2024 93 Future Research Directions Future research should focus on understanding and manipulating the interactions between endophytic bacteria and different environmental conditions to enhance their adaptability and efficacy as biocontrol agents across diverse ecosystems and also Appropriate formulation and delivery methods need to be developed and tested for agronomic use