Isolation and characterization of antagonistic actinomycetes from leaves of neem Azadirachta Indica Neem

work on neem

1. Isolation and characterization of antagonistic endophytic actinomycetes from leaves of Neem (​ Azadirachta indica ​ ) Final Ph.D. Thesis Presentation   By ROOPALI JAIN (Enrolment No. 2019MGU0832)   Under the Guidance of Dr. Adarsh Lalit MANSAROVAR GLOBAL UNIVERSITY SEHORE, MADHYA PRADESH, INDIA

2. Endophytic organisms have received considerable attention after they were found to protect their host against insect, pest, pathogens and even domestic herbivores. Almost all plant species (400,000) harbour one or more endophytic organisms. Studies have been conducted at different parts of the world about the endophytic biodiversity, taxonomy, reproduction, host ecology and their effect host. Currently endophytes are viewed as an outstanding source of bioactive natural products. The term “endophytes” refers to the organisms which throughout or part of its lifecycle invade the tissues of living plants and cause asymptomatic infections. De Bary in 1866 first introduced the term “epiphytes” for the fungi that live on the surface of their host and “endophytes” for those living inside the plant tissue. INTRODUCTION

3. Many endophytic bioactive metabolites possessing a wide variety of biological activities an antibiotic, antitumor, antioxidant, anti-inflammatory etc. have been identified. The aim of the work reported in this paper was to screen the endophytic actinomycetes isolated from leaves of the Neem tree, Azadirachta indica , for activity against common root pathogens like Pythium and Phytophthora and to evaluate their potential as biocontrol agents of a range of antagonistic bacteria and fungi. Azadirachta indica is widely used for the investigation of endophytes and their secondary metabolites, but it remains crucial to further extend the study as it is one of the major traditional medicinal plants used by about 80% of developing nations. Endophytic actinomycetes are now considered as exciting novel sources for obtaining new bioactive compounds and have been reported from several hosts such as tomato, banana, wheat, and maize with promising anti-microbial activity against pathogenic strains(Castillo UF., 2007)

4. Review of literature Azadirachtin is discovered in 1960 firstly and used as insecticide antifeedant, pest growth disruptor and insecticide, recovered from neem seeds. Production of azadiractin is almost 2 to 3% of neem seed (Department of Land Resource Management, 2015; Drugs.com, 2020). There are some other phytoconstituent in this plant viz. nimbin , nimbolinin , nimbiol , nimbidin , sodium nimbinate , salannin and gedunin (Department of Land Resource Management, 2015; Drugs.com, 2020). There are some polyphenolic flavonoids i.e., Quercetin and ß sitosterol, extracted from neem tree leaves and used as antibacterial as well as antifungal agent ( Govindachari et al., 1998; Hossain et al., 2011). Neem extracts have shown good potency against microbes and insects. Limonoid of neem seeds is liable for the toxic effects in pests and insects (Al Akeel et al., 2017; Farjana, Zerin , & Kabir, 2014; Gupta, Ansari, Gupta, & Narwani , 2019; Quelemes et al., 2015).

5. Isolation of endophytic actinomycetes from leaf of azadirachta indica (neem) Screening of recovered endophytic actinomycetes Characterization of potential antagonistic actinomycetes Selection of promising actinomycetes for production of bioactive compounds. Identification of potential isolates by 16s rRNA gene sequencing OBJEC TIVES

6. S tudied samples: Four healthy plants of azadirachta indica A. Juss (neem) were randomly selected from different locations of bhopal . Leaves of each plant were collected in a randomized manner in a sealed sterile bag and brought to laboratory for this study. MATERIALS AND METHODS

7. Methods: Isolation of endophytic fungi: Collected healthy leaves of neem were surface sterilized by following the method of strobel et al. , 1996 with some modification as sequential washings with a solution of 5.3% of hypochlorite followed by 70% ethanol. Leaves were cut into pieces by 1cm long with sterile blade before surface sterilization. Sterilized leaves were then inoculated on SDA supplemented with chloramphenicol (50μg/ml) at room temperature for 7 - 10 days. Mycelia growth was noted after 7 th day of incubation. Observed endophytes were subculture on PDA according to their morphological characteristics and maintained for further studies. Morphological characterization: M orphological characteristics viz. colony growth, presence or absence of aerial mycelium, colony color , presence of wrinkles and furrows, pigment production etc. in reference to Thom and Raper , (1945), Gilman (1957), Ellis (1971, 1976), Ellis and Pamella (1985), Booth (1971), Domsch et al ., (1980) and Barnett, 1992 by lactophenol blue stains under microscope with 40X resolution were performed on all isolated endophytes.

8. BIOCHEMICAL CHARACTERIZATION: Qualitative and quantative characterization: Amylase: Protease:   Cellulose: Lipase:

9. MOLECULAR CHARACTERIZATION: Isolates were cultivated according to Doyle and Doyle, 1990. 50ml 2% malt extract broth were prepared in 250ml flask and isolated inoculums were inoculated separately. Those were incubated on a 200rpm shaker and 25±2 o C for 7-10 days. Cultivated pure isolates were sent to DNA Labs for DNA sequencing and further molecular identification.

10. FUNCTIONAL CHARACTERIZATION: Phosphate solubilization assay: Production of β-1, 3-glucanase: Siderophore Production Assay: Production of Cellulase:

11.   Qualitative IAA Production Assay Production of IAA was determined by following the protocol of Bric et al. , (1991). Luria Bertani broth (LB) (5 mM L-tryptophan, 0.05% sodium dodecyl sulfate and 1% glycerol) was used for qualitative IAA production in isolated inoculums. Inoculums were inoculated on the media overlaid with an 82-mm-diameter disk of sterile nitrocellulose membrane and incubated inversely at 28 ± 2 o C until the growth was observed. Membrane disc was removed after completion of incubation period from the plate and treated with Salkowski reagent. A red halo zone within the membrane surrounding the colony was seen shows the positive result.

12.   Quantification of IAA Supernatant of isolates were mixed with Salkowsky reagent and incubated at 30 o C for 20 minutes. Absorbance was measured at 535 nm using a spectrophotometer. Quantification was performed as per standard curve with control.

13. Conformation and detection of IAA Confirmation of IAA in crude substance was performed through modified HPTLC (High performance thin layer chromatography) method of Sivagnanam and Gangatharan , 2017. Already defined solvent system Hexane: Ethyl acetate (3.5:1.5) was used. Each extract was applied as band by 4mm in length and 1-l in concentration for leaf using spray technique. Sample were applied twice on pre-coated silica gel 60F254 aluminium sheets (5 x 10 cm) with the help of Linomat 5 applicator connected to CAMAG HPTLC system and programmed through WINCATS. Chromatogram was developed in Twin trough glass chamber 10 x 10 cm saturated with solvent n-Hexane: ethyl acetate (3.5:1.5) for 15 minutes. Plate was then air-dried and viewed in ultraviolet radiation to mid-day light. Visualized chromatograms were scanned by CAMAG HPTLC scanner at 420 nm after spraying with anisaldehyde sulphuric acid. Resulted plot of Rf values were recorded and analyzed by WINCATS.

14. Quantification Phosphate solubilizing ability The isolates were inoculated in Pikovskaya broth medium with Ca 3 (PO 4 ) 2 as phosphate source in a Erlenmeyer flask. That was then incubated at 30 o C for 8 days in rotary shaker. 20 ml culture was filtered with Whatman No. 42 filter paper. Filtrate was then centrifuged for 15 minutes at 1000 rpm. 5.0 ml of supernatant was transferred into test tube. 0.5 ml of concentrated reagents P and concentrated Reagent dye was mixed in that and shaken vigorously. Stand that for 30 minutes to settle. Absorbance was measured at 693 nm using a spectrophotometer. Quantification was performed as per standard curve with control. Pikovskaya broth medium Item Qty glucose 10 g Ca3(PO4)2 5 g (NH4)2 SO4 0,5 g MgSO.2H2O 0,1 g MnSO4 25 mg FeSO4 25mg KCl 0,2 g yeast extract 0,5 g agar 15 g, sterile water Upto 1L

15. Optimization of IAA production by effect of Physical stresses: Different physical parameters i.e. temperature, pH and day of harvesting were imposed in PDA broth media culture. Quantity of IAA production was observed and optimum production was noted.  

16. RESULTS A study of endophytic actinomycetes isolation, screening, characterization and selection of promising actinomycetes from leaf of azadirachta indica (neem) were studied.

17. Images of selected plants and first inoculation

18. Isolate colonised in number of samples collected from different sampling Sampling site Species Chetak Bridge BHMG BHEL Ground Total colonization Cladosporium sp. 3 3 4 10 Nigrospora oryze sp. 3 4 4 11 Streptomyces sp. 2 4 4 10 Acremonium sp. 3 4 4 11 Fusarium sp. 3 4 3 10 Curvularia sp. 3 4 4 11 Total 17 23 23 63

19. Graph of Cladosporium sp. colonised in number of samples collected from different sampling sites Graph of Nigrospora oryzae sp. colonised in number of samples collected from different sampling sites

20. Graph of Streptomyces sp. colonised in number of samples collected from different sampling sites Graph of Acremonium sp. colonised in number of samples collected from different sampling sites

21. Graph of Fusarium sp. colonised in number of samples collected from different sampling sites Graph of Curvularia sp. colonised in number of samples collected from different sampling sites

22. Isolates Chetak Bridge BHMG BHEL Ground Total colonization Cladosporium sp. 100.00 75.00 100.00 91.67 Nigrospora oryzae sp. 100.00 100.00 100.00 100.00 Streptomyces sp. 66.67 100.00 100.00 88.89 Acremonium sp. 100.00 100.00 100.00 100.00 Fusarium sp. 100.00 100.00 75.00 91.67 Curvularia sp. 100.00 100.00 100.00 100.00 Total 94.44 95.83 95.83 95.37 Colony Frequency Percentage (CF%) of isolates in collected samples

23. Graph of Colony Frequency Percentage (CF%) of Cladosporium sp. in collected samples Graph of Colony Frequency Percentage (CF%) of Nigrospora oryzae sp. in collected samples

24. Graph of Colony Frequency Percentage (CF%) of Streptomyces sp. in collected samples Graph of Colony Frequency Percentage (CF%) of Acremonium sp. in collected samples

25. Graph of Colony Frequency Percentage (CF%) of Fusarium sp. in collected samples Graph of Colony Frequency Percentage (CF%) of Curvularia sp. in collected samples

26. FREQUENCY CLASS OF ISOLATES IN COLLECTED SAMPLES This was studied as per method of Raunker’s class and presented in Table 4.3. As per this study all the isolates comes under frequency class of ‘E’ only. Total Colonized frequency percentage (CF%) and Frequency class of identified fungal species from collected samples   Isolates Number of samples colonized (A) CF (%) (C) (A/N) x 100 Frequency class(F)* Cladosporium sp. 10 90.91 E Nigrospora oryze sp. 11 100.00 E Streptomyces sp. 10 90.91 E Acremonium sp. 11 100.00 E Fusarium sp. 10 90.91 E Curvularia sp. 11 100.00 E Where N = 11. * Class Frequency % = A: 0 - 20; B: 21 – 40; C: 41 – 60; D: 61 – 80; E: 81 – 100  

27. MORPHOLOGICAL CHARACTERIZATION: There were six different morphological distinguished actinomycetes seen in each samples. Identification of these actinomycetes was done as per classification of Domsch et al., 1980. They were coded as A, B, C for sampling station; 1, 2, 3, 4 for samples; i , ii, iii, iv, v, vi for isolates. So, isolate number one of plant A and sample 1 was coded as A1.i. The best sub-cultured isolates are presented in Table 4.4. Table 4.4: Morphological characterization of isolated actinomycetes Isolate code Endophyte (Morphological basis) Colour of mycelia Pigmentation Spore arrangement A3.i Cladosporium oxysporum Olive brown Dark brown Un-branched A1.ii Nigrospora oryzae sp. White Black Un-branched B3.iii Streptomyces sp. White Whitish green Branched filamentous C3.iv Acremonium implicatum White Not appeared Single celled B2.v Fusarium solani White Whitish pink Sickle shaped B4.vi Curvularia pallescens . Blackish brown Greenish brown Branched septa

28.   Cladosporium oxysporum : 600 to 650μm long and 4-5μm wide colony of isolate were seen. Spores were arranged in upward branched chain and fragile. 7 to 9 chained septa were seen. Straight and slit bend conidiophores were arranged in oval shape. Colony colour of this isolate was brown to black with olive brown powdery conidia were seen. Asexual reproduction with sporulation was seen. This was reported slow growing. Morphologically this was a species of Cladosporium oxysporum . Well develop plate and their microscopic image is presented in Figure 4.14. Cladosporium sp .

29. Nigrospora oryzae Colony of this isolate was seen 6 to 9cm in diameter. Spores were arranged in smooth branched, hyaline and septet. Conidiophores aggregated in black sporodochia, and were extensively flexuous or straight branched on cluster of hyphae. Conidia were abundantly, globose, black shiny and smooth. Cluster of perithecia grown in irregular row. Colony was white to grey in colour, marginal circular and grows rapidly. Asexual reproduction with sporulation was seen. Morphologically this was a species of Nigrospora oryzae . Well develop plate and their microscopic image is presented in Figure 4.15. Nigrospora oryzae sp.

30. Streptomyces sp. Isolate was reported 1 to 2 μm in diameter. This was gram positive and having vegetative hyphae with branched mycelium. Spores are arranged in chain with smooth mouth and globose shaped. Colour of mycelia was reported white with whitish green pigment. Conidiophores aggregated in black sporodochia, and were extensively flexuous or straight branched on cluster of hyphae. Sexual reproduction was seen in this isolate. Morphologically this was a species of Streptomyces. Well develop plate and their microscopic image is presented in Figure 4.16. Streptomyces sp .

31. Acremonium implicatum Diameter of single colony mycelia of this isolate was seen 12-20mm after 10 day of incubation. Conidia were hyaline, cylindrical and arranged at top of the phialide apex. Colour of the mycelia was white without pigmentation. Colony colour was white grey, slow growing and moist powdery structure. Spore arrangement was single celled and doing sexual reproduction. Morphologically this was a species of Acremonium implicatum . Well develop plate and their microscopic image is presented in Figure 4.17. Acremonium sp.

32. Fusarium solani Diameter of mycelia of this isolate was seen 60-70mm in after 7 day of incubation. Colony was white cottony with violet colour centre. Aerial hyphae and lateral conidiophores were seen. Conidiophores were elongated and thin, that have capacity to produce conidia. A pair of oval hyaline and smooth conidia seen in this isolate. Sexual reproduction was reported. Spores were arranged in sickle shaped. Morphologically this was a species of Fusarium solani . Well develop plate and their microscopic image is presented in Figure 4.18 . Fusarium sp.

33. Curvularia pallescens Conidiophores were between 50-500µm in diameter with four celled cylindrical conidia and pale brown in colour. C entrally located septum colony was seen in this isolate. Colony was fast growing and brown to blackish brown in colour with greenish brown pigmentation . Conidiophores were erected, septate, sympodial conidia and branch septa spore arrangement reported. Sexual reproduction with sporulation was reported. Mycelium was septet. Morphologically this was a species of Curvularia pallescens . Well develop plate and their microscopic image is presented in Figure 4.19. Curvularia sp.

34. QUALITATIVE ENZYME ACTIVITIES Quantitative enzyme activities for Amylase, Protease, Cellulase and Lipase were performed and presented in table 2. Qualitative enzyme activities characterization of isolated actinomycetes Isolate code Name of Endophyte (Morphological basis) Amylase Protease Cellulase Lipase A3.i Cladosporium sp. - + + + A1.ii Nigrospora oryze sp. - - - + B3.iii Streptomyces sp. + + + - C3.iv Acremonium sp. + - + + B2.v Fusarium sp. + - - + B4.vi Curvularia sp. - + - -

35. Amylase (U/ml) Amylase (U/ml) was reported nil in isolates of Cladosporium sp., Nigrospora oryze sp. and Curvularia sp. whereas isolates of Streptomyces sp., Acremonium sp. and Fusarium sp. reported 2.40, 2.10 and 2.75 respectively. Graph of Quantitative Amylase (U/ml) enzyme activities of isolated actinomycetes is presented in Figure 4.20. Quantitative Amylase (U/ml) enzyme activities of isolated actinomycetes

36. Quantitative enzyme activities characterization of isolated actinomycetes. Isolate code Name of Endophyte (Morphological basis) Amylase (U/ml) Protease (U/ml) Cellulase (U/ml) Lipase (U/ml) A3.i Cladosporium sp. 0.00 1.25 0.26 0.17 A1.ii Nigrospora oryze sp. 0.00 0.00 0.00 0.23 B3.iii Streptomyces sp. 2.40 1.71 0.47 0.00 C3.iv Acremonium sp. 2.10 0.00 0.43 0.11 B2.v Fusarium sp. 2.75 0.00 0.00 0.06 B4.vi Curvularia sp. 0.00 1.20 0.00 0.00

37. Protease (U/ml) Protease (U/ml) was reported nil in isolates of Cladosporium sp ., Streptomyces sp. and Curvularia sp. whereas isolates of Streptomyces sp., Acremonium sp. and Fusarium sp. reported 1.25, 1.71 and 1.20 respectively. Graph of Quantitative Amylase (U/ml) enzyme activities of isolated actinomycetes is presented in Figure 4.21. Quantitative Protease (U/ml) enzyme activities of isolated actinomycetes.  

38. Cellulase (U/ml) Cellulase (U/ml) was reported nil in isolates of Cladosporium sp . , Fusarium sp. and Curvularia sp. whereas isolates of Nigrospora oryze sp., Streptomyces sp. and Acremonium sp. reported 0.26, 0.47 and 0.43 respectively. Graph of Quantitative Amylase (U/ml) enzyme activities of isolated actinomycetes is presented in Figure 4.22. Quantitative Cellulase (U/ml) enzyme activities of isolated actinomycetes.  

39. Lipase (U/ml) Lipase (U/ml) was reported nil in isolates of Nigrospora oryze sp. and Curvularia sp. whereas isolates of Cladosporium sp . , Streptomyces sp., Acremonium sp. and Fusarium sp. reported 0.17, 0.23, 0.11 and 0.06 respectively. Graph of Quantitative Amylase (U/ml) enzyme activities of isolated actinomycetes is presented in Figure 4.23. Figure 4.23: Quantitative Lipase (U/ml) enzyme activities of isolated actinomycetes.

40. FUNCTIONAL CHARACTERIZATION: Phosphate solubilisation assay, production of β-1, 3-glucanase, siderophore production assay and production of cellulase of functional characteristics were studied and reported in Table 4.7. All reported isolates were given positive results against performed functional characteristics . Functional characterization of isolated actinomycetes Isolate code Name of Endophyte Phosphate solubilization assay Production of β-1, 3-glucanase Siderophore Production Assay Production of Cellulase A3.i Cladosporium sp. + + + + A1.ii Nigrospora oryze sp. + + + + B3.iii Streptomyces sp. + + + + C3.iv Acremonium sp. + + + + B2.v Fusarium sp. + + + + B4.vi Curvularia sp. + + + +

41. CONFIRMATION OF IAA PRODUCTION: Productions of Indole-3-acetic acid in isolates were determined. Quantification was done by spectrophotometric method and confirmation of presence of Indole-3-acetic acid in isolate extract was confirmed by HPTLC. Quantification of IAA Observes absorbance and concentration of Indole-3-acetic are presented in Table 4.8 and graph in Figure 4.23. Table 4.8 Quantification of IAA in isolated actinomycetes. Isolate code Name of Endophyte Absorbance (λ) Concentration (mg/l) A3.i Cladosporium sp. 0.096 1.337 A1.ii Nigrospora oryze sp. 0.086 1.112 B3.iii Streptomyces sp. 0.134 1.937 C3.iv Acremonium sp. 0.123 1.807 B2.v Fusarium sp. 0.079 1.014 B4.vi Curvularia sp. 0.083 1.001 Concentration (mg/l) of IAA in isolates of Neem

42. Confirmation of IAA through HPTLC Presence of IAA in isolates was further confirmed by following the High performance thin layer chromatography. HTLC plate with sample and standard is showing in Figure 5. Bands of standard and samples as M - IAA Standard, S1 to S6 – isolated samples as A3.i = Cladosporium sp., A1.ii = Nigrospora oryze sp . , B3.iii = Streptomyces sp., C3.iv = Acremonium sp., B2.v = Fusarium sp., B4.vi = Curvularia sp. Plot of HPTLC scanning are presented in

43. HPTLC graph plot  

44. CONFIRMATION OF PHOSPHATE SOLUBILIZING ABILITY: Productions of phosphate solubilize in isolates were determined. Quantification was done by spectrophotometric method. Quantification of phosphate solubilizer Readings of absorbance and concentration of phosphate solubilizer are presented in Table 4.10 and graph in Figure 4.25. Quantification of phosphate solubilizer in isolated actinomycetes Isolate code Name of Actinomycetes Absorbance (λ) Concentration (mg/l) A3.i Cladosporium sp. 0.423 1.973 A1.ii Nigrospora oryze sp. 0.614 2.974 B3.iii Streptomyces sp. 0.496 2.327 C3.iv Acremonium sp. 0.752 3.636 B2.v Fusarium sp. 0.614 2.942 B4.vi Curvularia sp. 0.732 3.555 Concentration (mg/l) of phosphate solubilizer.

45. MOLECULAR CHARACTERIZATION: Partial sequencing of isolates was performed to identify the species and found that the species reported as per morphological, biochemical and functional confirms the molecular identification of isolates. Prominent species reported in this study were Cladosporium, Nigrospora oryzae , Streptomyces, Acremonium, Fusarium and Curvularia . All these species were producing good quatity of IAA and also able to solubilized the phosphate. Sequences of these isolates were aligned and submit to NCBI for further studies. Accession numbers of such isolates are presented in Table 4.11. Complete Genebank details are presented in Figure 4.26 to 4.31. Isolate code Name of Actinomycetes Accession No. A3.i Cladosporium sp. OR492471 A1.ii Nigrospora oryze sp. OR492604 B3.iii Streptomyces sp. OR492606 C3.iv Acremonium sp. OR492470 B2.v Fusarium sp. OR492602 B4.vi Curvularia sp. SUB13800117 Table 4.11 NCBI accession numbers of such isolates

46. EFFECT OF PHYSIOLOGICAL STRESS ON IAA PRODUCTION: A study for optimisation of IAA production by isolates was performed with different physical stress conditions viz. temperature, pH and days of harvesting. EFFECT OF TEMPERATURE ON PRODUCTION OF IAA BY DIFFERENT ISOLATES All isolates were incubated at four different temperatures i.e. 30, 35, 40 and 45 o C for 8 days. Harvesting of IAA was done on 8 th day and concentration of IAA was calculated with the help of spectroscopy. Obtained results are presented in Table 4.12. Lowest production of IAA was reported in isolate of Curvularia with 0.654mg/l at 35 o C whereas highest production reported in isolates of Acremonium . Gradually decreased production reported in species of Streptomyces , Cladosporium , Nigrospora oryzae and Fusarium.

47. Table 4.12 Production of IAA on different temperature Isolate code   Temp.( o C ) Name of Actinomycetes Concentration (mg/l) 30 35 40 45 A3.i Cladosporium sp. 1.012 1.476 1.123 0.654 A1.ii Nigrospora oryzae sp. 0.635 0.905 0.792 0.251 B3.iii Streptomyces sp. 1.562 2.111 1.632 0.901 C3.iv Acremonium sp. 1.654 2.571 1.741 0.913 B2.v Fusarium sp. 0.517 0.873 0.568 0.163 B4.vi Curvularia sp. 0.465 0.651 0.493 0.114

48. Production of IAA by Cladosporium isolates Highest production IAA by isolate of Cladosporium species was given in broth incubated at 35 o C with 1.476mg/l. Production of IAA was reported 1.123mg/l in broth incubated at 40 o C and slight less production reported at 30 o C with 1.012. Decreased or very less IAA produced at 45 o C with 0.654mg/l. Production of IAA by Cladosporium isolates at different temperature

49. Production of IAA by Nigrospora oryzae isolates Highest production IAA by isolate of Nigrospora oryzae species was given in broth incubated at 35 o C with 0.905 mg/l. Production of IAA was reported 0.792 mg/l in broth incubated at 40 o C and slight less production reported at 30 o C with 0.635 mg/l. Decreased or very less IAA produced at 45 o C with 0.251 mg/l. Production of IAA by Nigrospora oryzae isolates at different temperature .  

50.   Production of IAA by Streptomyces isolates Highest production IAA by isolate of Streptomyces species was given in broth incubated at 35 o C with 2.111mg/l. Production of IAA was reported 1.632mg/l in broth incubated at 40 o C and slight less production reported at 30 o C with 1.562mg/l. Decreased or very less IAA produced at 45 o C with 0.901mg/l. Production of IAA by Streptomyces isolates at different temperature .

51. Production of IAA by Acremonium isolates Highest production IAA by isolate of Acremonium species was given in broth incubated at 35 o C with 2.571mg/l. Production of IAA was reported 1.741mg/l in broth incubated at 40 o C and slight less production reported at 30 o C with 1.654mg/l. Decreased or very less IAA produced at 45 o C with 0.913mg/l. Production of IAA by Acremonium isolates at different temperature.  

52. Production of IAA by Fusarium isolates Highest production IAA by isolate of Fusarium species was given in broth incubated at 35 o C with 0.873mg/l. Production of IAA was reported 0.568mg/l in broth incubated at 40 o C and slight less production reported at 30 o C with 0.517mg/l. Decreased or very less IAA produced at 45 o C with 0.163mg/l. Production of IAA by Fusarium isolates at different temperature.

53. Production of IAA by Curvularia isolates Highest production IAA by isolate of Curvularia species was given in broth incubated at 35 o C with 0.651mg/l. Production of IAA was reported 0.493mg/l in broth incubated at 40 o C and slight less production reported at 30 o C with 0.465mg/l. Decreased or very less IAA produced at 45 o C with 0.114mg/l. Figure 4.37: Production of IAA by Curvularia isolates at different temperature .

54. EFFECT OF pH ON PRODUCTION OF IAA BY DIFFERENT ISOLATES All isolates were incubated at four different pH i.e. 5.5, 6.5, 7.5 and 8.5units for 8 days. Harvesting of IAA was done on 8 th day and concentration of IAA was calculated with the help of spectroscopy. Obtained results are presented in Table 4.13. Lowest production of IAA was reported in isolate of Curvularia broth of pH 7.5units with 0.406mg/l whereas highest production reported in isolates of Acremonium . Gradually decreased production reported in species of Streptomyces , Cladosporium , Nigrospora oryzae and Fusarium. Table 4.13 Production of IAA on different pH Isolate code pH (Unit) Name of Actinomycetes Concentration (mg/l) 5.5 6.5 7.5 8.5 A3.i Cladosporium sp. 0.236 1.021 1.452 0.965 A1.ii Nigrospora oryzae sp. 0.124 0.785 0.914 0.423 B3.iii Streptomyces sp. 0.521 1.223 2.121 1.002 C3.iv Acremonium sp. 0.593 1.314 2.543 1.018 B2.v Fusarium sp. 0.206 0.421 0.880 0.498 B4.vi Curvularia sp. 0.195 0.406 0.674 0.412

55. Production of IAA by Cladosporium isolates Highest production IAA by isolate of Cladosporium species was given in broth of pH 7.5units incubated for 8 days with 1.452mg/l. Production of IAA was reported 1.021mg/l in broth of pH 6.5units and slight less production reported in broth of pH 8.5units with 0.965mg/l. Decreased or very less IAA produced in broth of pH 5.5 units with 0.236mg/l. Production of IAA by Cladosporium isolates in broth of different pH.  

56.   Production of IAA by Nigrospora oryzae isolates Highest production IAA by isolate of Nigrospora oryzae species was given in broth of pH 7.5units incubated for 8 days with 0.914 mg/l. Production of IAA was reported 0.785 mg/l in broth of pH 6.5units and slight less production reported in broth of pH 8.5units with 0.423 mg/l. Decreased or very less IAA produced in broth of pH 5.5 units with 0.124 mg/l. Production of IAA by Nigrospora oryzae isolates in broth of different pH.  

57. Production of IAA by Streptomyces isolates Highest production IAA by isolate of Streptomyces species was given in broth of pH 7.5units incubated for 8 days with 2.121 mg/l. Production of IAA was reported 1.223 mg/l in broth of pH 6.5units and slight less production reported in broth of pH 8.5units with 1.002 mg/l. Decreased or very less IAA produced in broth of pH 5.5 units with 0.521 mg/l. Production of IAA by Streptomyces isolates in broth of different pH.  

58. Production of IAA by Acremonium isolates Highest production IAA by isolate of Acremonium species was given in broth of pH 7.5units incubated for 8 days with 2.543mg/l. Production of IAA was reported 1.314mg/l in broth of pH 6.5units and slight less production reported in broth of pH 8.5units with 1.018mg/l. Decreased or very less IAA produced in broth of pH 5.5 units with 0.593mg/l. Production of IAA by Acremonium isolates in broth of different pH.

59. Production of IAA by Fusarium isolates Highest production IAA by isolate of Fusarium species was given in broth of pH 7.5units incubated for 8 days with 0.880mg/l. Production of IAA was reported 0.498mg/l in broth of pH 8.5units and slight less production reported in broth of pH 6.5units with 0.421mg/l. Decreased or very less IAA produced in broth of pH 5.5 units with 0.206mg/l. Production of IAA by Fusarium isolates in broth of different pH.    

60. Production of IAA by Curvularia isolates Highest production IAA by isolate of Curvularia species was given in broth of pH 7.5units incubated for 8 days with 0.674mg/l. Production of IAA was reported 0.412mg/l in broth of pH 8.5units and slight less production reported in broth of pH 6.5units with 0.406mg/l. Decreased or very less IAA produced in broth of pH 5.5 units with 0.195mg/l. Production of IAA by Curvularia isolates in broth of different pH.

61. EFFECT OF HARVESTING DAY ON PRODUCTION OF IAA BY DIFFERENT ISOLATES All isolates were incubated in a same media but harvest at 5 th , 10 th , 15 th , 20 th , 25 th , and 30 th day. Production of IAA was calculated with the help of spectroscopy. Obtained results are presented in Table 4.14. Lowest production of IAA was reported in isolate of Curvularia broth on 20 th day of harvesting with 0.710mg/l whereas highest production reported in isolates of Acremonium with 2.498mg/l gradually decreased production reported in species of Streptomyces , Cladosporium , Nigrospora oryzae and Fusarium. Isolate code   No. of Days Name of Actinomycetes Concentration (mg/l) 5 10 15 20 25 30 A3.i Cladosporium sp. 0.121 0.229 1.109 1.426 1.410 1.253 A1.ii Nigrospora oryze sp. 0.082 0.102 0.814 0.964 0.950 0.912 B3.iii Streptomyces sp. 0.223 0.787 1.964 2.224 2.156 1.946 C3.iv Acremonium sp. 0.229 0.864 2.012 2.498 2.254 1.984 B2.v Fusarium sp. 0.078 0.219 0.843 0.912 0.897 0.756 B4.vi Curvularia sp. 0.041 0.246 0.632 0.710 0.685 0.597 Production of IAA on different harvesting day

62. Production of IAA by Cladosporium isolates Highest production IAA by isolate of Cladosporium species was given at 20 th day of harvesting with 1.426mg/l. Lowest production of IAA was reported 0.121mg/l on 5 th day of harvesting. 1.410, 1.253, 1.109 and 0.229mg/l IAA production was reported in broth harvested on 25 th , 30 th , 15 th and 10 th day respectively. Production of IAA by Cladosporium isolates on different harvesting day.  

63. Production of IAA by Nigrospora oryzae isolates Highest production IAA by isolate of Nigrospora oryzae species was given at 20 th day of harvesting with 0.964 mg/l. Lowest production of IAA was reported 0.082mg/l on 5 th day of harvesting. 0.950 , 0.912 , 0.814 and 0.102 mg/l IAA production was reported in broth harvested on 25 th , 30 th , 15 th and 10 th day respectively. Production of IAA by Nigrospora oryzae isolates on different harvesting day.

64. Production of IAA by Streptomyces isolates Highest production IAA by isolate of Streptomyces species was given at 20 th day of harvesting with 2.224mg/l. Lowest production of IAA was reported 0.223mg/l on 5 th day of harvesting. 2.156, 1.964, 1.946 and 0.787mg/l IAA production was reported in broth harvested on 25 th , 30 th , 15 th and 10 th day respectively. Production of IAA by Streptomyces isolates on different harvesting day.

65. Production of IAA by Acremonium isolates Highest production IAA by isolate of Acremonium species was given at 20 th day of harvesting with 2.498mg/l. Lowest production of IAA was reported 0.229mg/l on 5 th day of harvesting. 2.254, 2.012, 1.984 and 0.864mg/l IAA production was reported in broth harvested on 25 th , 30 th , 15 th and 10 th day respectively. Production of IAA by Acremonium isolates on different harvesting day.

66. Production of IAA by Fusarium isolates Highest production IAA by isolate of Fusarium species was given at 20 th day of harvesting with 0.912mg/l. Lowest production of IAA was reported 0.078 on 5 th day of harvesting. 0.897, 0.843, 0.756 and 0.219mg/l IAA production was reported in broth harvested on 25 th , 30 th , 15 th and 10 th day respectively. Production of IAA by Fusarium isolates on different harvesting day.  

67. Production of IAA by Curvularia isolates Highest production IAA by isolate of Curvularia species was given at 20 th day of harvesting with 0.710mg/l. Lowest production of IAA was reported 0.041mg/l on 5 th day of harvesting. 0.685, 0.632, 0.597 and 0.246mg/l IAA production was reported in broth harvested on 25 th , 30 th , 15 th and 10 th day respectively. Production of IAA by Curvularia isolates on different harvesting day.

68. Total six actinomycetes were isolated from neem plant leaves of three different locations of Bhopal viz. Chetak Bridge, BHMG ( Barkheda Haat and Market Ground), BHEL Ground. Samples were coded as A for Chetak Bridge, B for BHMG and C for BHEL Ground. The collected neem leaves sample bare the colonization of different microbial species. Species of Cladosporium, Nigrospora oryze , Streptomyces, Acremonium, Fusarium and Curvularia are reported in this study. Total 63 colonies were isolated and did that pure. All reported specis are prone to produce IAA and optimize their production on 35 o C temperature, 7.5pH and 20 th day of harvesting. These strains can be used for production of commercialized IAA. This can helps in elongation as well as in cell division, to form root hair and to make short root length. This is also helps to increase the nutrient absorption capability of plant. IAA producing actinobacteria are also act as a plant growth promoter.     CONCLUSIONS

69. Discovery of novel antimicrobial compounds: Further research could lead to the discovery of new antibiotics, antifungals, and anticancer agents. Development of biopesticides: Endophytic actinomycetes from Neem leaves could be used as biopesticides, reducing chemical usage in agriculture. Improvement of Neem-based products: Characterization of endophytic actinomycetes could lead to enhanced Neem-based products for agriculture, medicine, and cosmetics. Exploration of Neem-endophyte synergy: Research could focus on understanding the symbiotic relationship between Neem and its endophytic actinomycetes, leading to new insights into plant defense mechanisms. Bioprospecting for novel enzymes: Endophytic actinomycetes from Neem leaves could be a source of novel enzymes for industrial applications. Development of targeted drug delivery systems: Endophytic actinomycetes could be used as vectors for targeted drug delivery, exploiting their ability to colonize specific plant tissues. Development of novel biofertilizers: Endophytic actinomycetes from Neem leaves could be used as biofertilizers, promoting plant growth and soil health. Exploration of Neem-endophyte-based solutions for environmental remediation: Research could investigate the potential of endophytic actinomycetes from Neem leaves for bioremediation of pollutants. FUTURE SCOPES

70. ACKNOWLEDGEMENTS I owe a deep sense of gratitude Prof. Arun Kumar Pandey, Hon’ble Vice Chancellor, Mansarovar Global University Sehore (M.P.) for his valuable support. A special thanks to the Dr. Pushpendra Tiwari Dean faculty of Biotechnology Mansarovar Global University Sehore (M.P.) for providing me with all the necessary facilities for the research. It is a genuine pleasure to express my deep sense of thank and gratitude to my supervisor Dr. Adarsh Lalit Department of Biotechnology Mansarovar Global University Sehore (M.P.). To my family, thank you for encouraging me in all of my pursuits and inspiring me to follow my dreams.

71. THANK YOU