CHAPTER IV RESULTS Occurrence of endophytic microorganisms in the roots and shoots of crop plants

Transcription

1 RESULTS

2 CHAPTER IV RESULTS The present study encompasses the isolation and characterization of endophytes of crop plants. The nineteen isolates obtained were analysed for their plant growth promoting potential and the results are presented hereunder Occurrence of endophytic microorganisms in the roots and shoots of crop plants To study the natural occurrence of endophytic microorganisms in the roots and shoots of crop plants, the following crops were collected from the Agricultural Farm, University of Agricultural Sciences, GKVK, Bangalore. The crops selected for the study were Cereal crops Pulse crops : rice (Oryza sativa) and ragi (Eleusine corocana) : cowpea (Vigna ungiculata) and soybean (Glycine max) Oil seed crops : groundnut (Arachis hypogea) and sunflower (Helianthus annus) Vegetables : chilli (Capsicum annuum) and tomato (Lycopersicon esculentum) All the crops selected were found to have endophytes in their shoot and root regions Enumeration of endophytic bacterial population in shoots and roots of crop plants The endophytic bacteria were enumerated and the results are presented in Table1, Fig1 and Plate1. The pulse crops cowpea recorded the highest number of shoot endophytes (5.8 x 10 5 cfu/g). The solanaceous vegetables, tomato and chilli recorded population in the

3 Table 1. Population of endophytic bacteria in the shoots and roots of selected crops. Host group Host plant Shoot (x10 5 cfu/g) Root (x10 5 cfu/g) Cereals Rice 3.5 (5.544) d 4.2 (5.623) c Ragi 2.8 (5.361) e 3.6 (5.477) d Oil seeds Sunflower 1.3 (5.114) g 2.8 (5.591) e Groundnut 2.3 (5.447) f 3.0 (5.556) e Pulses Soybean 5.3 (5.724) b 6.1 (5.785) a Cowpea 5.8 (5.763) a 6.5 (5.813) a Vegetables Chilli 3.9 (5.591) c 4.6 (5.663) c Tomato 4.2 (5.623) c 5.4 (5.732) b SEd CD (0.05) Note: Population count was taken after 72 hours of incubation at 30 0 C. Values in parantheses indicates log transformed values. Different letters in superscripts indicate significantly different values.

4 Population (log value of cfu/g) 6 Population of endophytic bacteria shoot root Rice Ragi Sunflower Groundnut Soybean Cowpea Chilli Tomato Host plant Fig. 1. Population of endophytic bacteria in the shoots and roots of crop plants

5 a)shoots of Tomato b) Shoots of Rice c) Roots of Ragi A. Isolation of endophytes a) Shoots of cowpea b) Shoots of chilli c)roots of soybean B. Enumeration of endophytes Klebsiella sp. Pseudomonas sp. Bacillus sp. C. Colony characters of endophytes Klebsiella sp. Bacillus sp. D. Microscopic characters of endophytes (under 100X) Plate 1. Isolation and characterisation of endophytic bacteria

6 range of 4.2 x 10 5 and 3.9 x 10 5 cfu/g. The cereal crops rice and ragi inhabited 3.5 x 10 5 and 2.8 x 10 5 cfu/g of shoot tissue. The oil seed crops groundnut and sunflower had the least population of endophytes in their shoot region, 2.3 and 1.3 x 10 5 cfu/g. In roots similar trend was recorded for endophytic bacteria. The highest population was detected in the pulse crops, cowpea and soybean (6.5 x 10 5 and 6.1 x 10 5 cfu/g) while the least was in the oilseed crops, groundnut and sunflower (3 x 10 5 and 2.8 x 10 5 cfu/g). The solanaceous vegetables tomato and chilli recorded 5.4 x 10 5 and 4.6 x 10 5 cfu/g root tissue. The cereal crops rice and ragi recorded 3.2 x 10 5 and 2.6 x 10 5 cfu/g. From the above studies those bacterial colonies showing distinct colony morphology and growth in broth was selected for further studies. Nineteen isolates from the above crops were obtained and purified by streak plate technique. They were named according to the host plant and are as follows. A reference culture, Azotobacter chroococcum, AZC20 was used in all the further studies Naming of isolates Host group Host plant Isolates Rice (Oryza sativa) OSE1 OSE2 Cereal crops ECE4 Ragi (Eleusine corocana) ECE5 ECE6 Oil seed crops Groundnut (Arachis hypogea) Sunflower (Helianthus annus) AHE3 HAE7 HAE8

7 Pulse crops Vegetable crops Cowpea (Vigna ungiculata) Soybean (Glycine max) Chilli (Capsicum annuum) Tomato (Lycopersicon esculentum) VUE13 VUE14 GME15 GME16 CAE9 CAE10 CAE11 CAE12 LEE17 LEE18 LEE Morphological characteristics of the endophytic bacterial isolates. The culture characters of the isolates on broth media in the form of surface growth, clouding of broth and sedimentation were studied. Ragi isolates ECE5 and ECE6, the sunflower isolate HAE8, chilli isolate CAE10, soybean isolate GME16, tomato isolates LEE17 and LEE18 and AZC20 showed heavy clouding of the broth. The colony characters in agar media varied from white powdery dotted colonies with irregular margins in the rice isolate OSE1, white flat wrinkled small colonies with serrate margins in groundnut isolate AHE3 and dirty white glistening raised colonies in tomato isolate LEE19. The details of the characters are given in Table 2, Plate Microscopic characteristics of the endophytic bacterial isolates. All the isolates were long rods, rods or short rods and seven of them OSE1, AHE3, ECE4, ECE5, VUE13, VUE14 and GME15 stained

8 Table 2. Morphological characteristics of the endophytic bacterial isolates. Host group Cereals Oilseeds Pulses Vegetables Host plant Isolate Culture characters on broth media Surface Clouding Sediment Colony characters in agar media growth of broth ation White powdery dotted colonies with irregular OSE1 None None Flaky Rice margins OSE2 Ring Slight Flaky Dirty white smooth glistening colonies ECE4 No Slight No White flat wrinkled colonies with irregular margins Ragi ECE5 No Heavy No White smooth glistening raised colonies ECE6 No Heavy No Dirty white slimy glistening colonies Groundnut AHE3 No No No White flat wrinkled small colonies with serrate margins Sunflower HAE7 No No Flaky Yellow raised glistening colonies HAE8 No Heavy No White slimy raised colonies Cowpea VUE13 No Slight Flocculent Pinkish white smooth slimy colonies VUE14 No No Flocculent White flat wrinkled colonies with irregular margins Soybean GME15 Pellicle Slight Flaky White wrinkled colonies with large irregular margins GME16 No Heavy No Slimy white raised colonies CAE9 Pellicle Slight No Pinkish white slimy colonies Chilli CAE10 Pellicle Heavy Scanty Slimy white glistening colonies CAE11 Flaky Slight No Dirty white smooth colonies CAE12 No Slight Granular Dirty white raised colonies with smooth margins LEE17 Pellicle Heavy Flocculent Dirty white slimy colonies Tomato LEE18 Pellicle Heavy No Pinkish white raised slimy colonies LEE19 No Slight No Dirty white glistening raised colonies Reference Azotobacter AZC20 Ring Heavy Flocculent Smooth glistening translucent colonies Note: Observations recorded after 48 hours of incubation at 30 0 C.

9 Gram positive. Other 12 isolates were Gram negative. The tomato isolate LEE17 was curved rods and stained Gram negative. The isolates OSE1, AHE3, ECE4, ECE5, VUE13, VUE14 and GME15 formed endospores in broth media. All the isolates except ragi isolate ECE6 and tomato isolate LEE19 were motile. The results are presented in Table 3, Plate Biochemical characteristics of the endophytic bacterial isolates. For further characterizing the isolates the following biochemical tests were carried out and the results are presented in Table 4. All the isolates tested positive for Catalase, Methyl Red and Citrate utilization tests and negative for indole production and Voges- Proskauer tests. The ragi isolates ECE6, sunflower isolates HAE7 and HAE8, chilli isolates CAE9 and CAE12 and the tomato isolate LEE19 tested negative for oxidase test. All the isolates except the sunflower isolate HAE7, and the chilli isolates CAE9 and CAE12 could reduce nitrate. The ragi isolate ECE6, sun flower isolate HAE8, chilli isolates CAE10 and CAE11, VUE14, and tomato isolates LEE18 and LEE19 did not produce hydrogen sulfide Utilization of carbon sources by the endophytic bacterial isolates The utilization of glucose, sucrose and lactose was studied using the fermentation broth with Durham s tube and the results are presented in Table 5. All the isolates except the chilli isolate CAE11, the soybean isolate GME16 and tomato isolate LEE18 could ferment glucose. OSE2, ECE5, ECE6, HAE8, CAE10, CAE12 and LEE19 produced gas in glucose fermentation broth. ECE4, CAE11, VUE13, GME15, GME16 and LEE18 could not ferment sucrose and eight isolates did not produce gas in sucrose medium. OSE1, AHE3, ECE4, VUE13, VUE14, LEE19 and

10 Table 3. Microscopic characteristics of the endophytic bacterial isolates. Host group Host plant Isolates Gram stain Cell shape Endospore Motility Tentative identification Rice OSE1 + Rods + + Bacillus pasteurii OSE2 - Long Rods - + Citrobacter sp. Cereals ECE4 + Rods + + Bacillus sp. Ragi ECE5 + Rods + + Bacillus pasteurii ECE6 - Rods - - Klebsiella sp. Groundnut AHE3 + Short rods + + Bacillus sp. Oilseeds HAE7 - Cocco-bacilli - + Acinetobacter sp. Sunflower HAE8 - Short rods - - Klebsiella sp. Pulses Cowpea VUE13 + Rods + + Bacillus megaterium VUE14 + Rods + + Bacillus sp. Soybean GME15 + Short rods + + Bacillus sp. GME16 - Rods - + Pseudomonas sp. CAE9 - Rods/ cocci - + Acinetobacter sp. Chilli CAE10 - Long rods - + Citrobacter sp. CAE11 - Short rods - + Pseudomonas sp. Vegetables CAE12 - Rods / cocci - + Acinetobacter sp. LEE17 - Curved rods - + Vibrio sp. Tomato LEE18 - Rods - + Pseudomonas sp. LEE19 - Short rods - - Klebsiella sp. Reference culture Azotobacter AZC20 - Short rods - + Azotobacter chroococcum Note: Tentative identification according to Bergey s Manual of Determinative Bacteriology.

11 Table 4. Biochemical characteristics of the endophytic bacteria Host group Cereals Oilseeds Pulses Vegetables Host plant Isolates Oxidase test Rice Ragi Nitrate reduction H 2S production Catalase Indole production MR test VP test Citrate utilisation OSE OSE ECE ECE ECE Groundnut AHE Sunflower Cowpea Soybean Chilli Tomato HAE HAE VUE VUE GME GME CAE CAE CAE CAE LEE LEE LEE Reference Azotobacter AZC Note: + : positive result - : negative result

12 Table 5. Utilisation of carbon sources by the endophytic bacterial isolates Host group Cereals Oilseeds Pulses Vegetables Host plant Isolates Rice Ragi Glucose Sucrose Lactose Colour Gas Colour Gas Colour Gas OSE OSE ECE ECE ECE Groundnut AHE Sunflower Cowpea Soybean Chilli Tomato HAE HAE VUE VUE GME GME CAE CAE CAE CAE LEE LEE LEE Reference Azotobacter AZC Note: Tested for acid and gas production after 24h of incubation at 30 0 C. + : positive result - : negative result

13 AZC20 fermented lactose, but none of the isolates produced gas in the broth Tentative identification of endophytic bacterial isolates Based on the morphological, microscopic and biochemical characters and their pattern of utilization of carbon sources the endophytic bacterial isolates were tentatively identified according to Bergey s Manual of Determinative Bacteriology. The results are presented in Table 3. The gram positive bacteria were identified as belonging to the genus Bacillus. The rice isolate OSE1 and the ragi isolate ECE5 were tentatively identified as B. pasteurii while cowpea isolate VUE 13 was identified as B. megaterium. Ground nut isolate AHE3, ragi isolate ECE4, cowpea isolate VUE14 and soybean isolate GME15 were Bacillus sp. The Gram negative bacteria belonged to the genera Citrobacter, Klebsiella, Acinetobacter, Pseudomonas and Vibrio. The rice isolate OSE2 and chilli isolate CAE10 were identified as Citrobacter and ragi isolate ECE6, sunflower isolate HAE8 and tomato isolate LEE19 belonged to Klebsiella sp. the sunflower isolate HAE7, and the chilli isolates CAE9 and CAE12 were Acinetobacter while chilli isolate CAE11, soybean isolate GME16 and tomato isolate LEE18 were grouped as Pseudomonas sp. The tomato isolate LEE17 which were curved rods belonged to Vibrio sp. thus it can be concluded that Bacillus and Pseudomonads are the predominant endophytic isolates obtained in this study. The cereals and pulses were dominated by the Bacillus sp.while the gram negative organisms were more in vegetables Enzymatic activities of the endophytic bacterial isolates The enzymatic activity of the endophytic bacterial isolates was studied in relation to gelatinase, amylase, cellulase and pectinases. The results are presented in Table 6. Eight of the endophytic bacteria, the rice isolate OSE2, ragi isolate ECE4, chilli isolate CAE9, CAE10, CAE12,

14 Table 6. Enzymatic activities of the endophytic bacteria. Host group Host plant Isolates Gelatinase Amylase Cellulase Cereals Oilseeds Pulses Vegetables Rice Ragi Pectinase at ph5 Pectinase at ph7 OSE1 L OSE2 L ECE4 L ECE5 L ECE6 L Groundnut AHE3 L Sunflower Cowpea Soybean Chilli Tomato HAE7 L HAE8 L VUE13 L VUE14 L GME15 L GME16 L CAE9 L CAE10 L CAE11 L CAE12 L LEE17 L LEE18 L LEE19 L Reference culture Azotobacter chroococcum AZC20 L Note: L+: Liquefaction of the media L- : No liquefaction of the media +: clear zone around the colonies - : no clear zone

15 cowpea isolate VUE14 and tomato isolate LEE17 were able to produce gelatinase. The enzyme amylase was present in the rice isolate OSE2, groundnut isolate AHE3, ragi isolate ECE4, cowpea isolates VUE13 and VUE14, soybean isolates GME15 and GME16 and tomato isolate LEE18 indicated by the clear zone in starch agar media. All the isolates except the rice isolate OSE2 and sunflower isolates HAE7 and HAE8 were able to produce cellulase. In the case of pectinase very clear zone was not recorded in any of the isolates, however just beneath the colonies clearing of the media was observed indicating that the organism is able to utilize pectin for its growth. The positive result shown in Table 6 indicates this in the case of pectinase. The activity of cellulase was present in the pulse and vegetable isolates while these enzyme activities were less in oilseed isolates Functional properties of the endophytic bacterial isolates Polysaccharide production The polysaccharide production by the endophytic isolates was recorded after 24 hours and 48 hours of incubation in plates at 30 0 C. The plates showing the viscous gelatinous mass of polysaccharide was visually scored and the results are presented in Table 7. High polysaccharide production was recorded in the ragi isolate ECE5, sunflower isolate HAE8 and reference culture AZC20 after 24 hours of incubation. The chilli isolate CAE12 which showed moderate production recorded high production after 48 hours. OSE1, AHE3, ECE6, HAE7, CAE11, GME15 and LEE19 showed slight polysaccharide production after 24 hours and moderate production after 48 hours. The soybean isolate GME16 and tomato isolate LEE18 which did not exhibit any polysaccharide in the media after 24 hours recorded slight production after 48 hours. All the other isolates did not show any polysaccharide

16 Table 7. Polysaccharide production by the endophytic bacteria Host group Host plant Isolates Cereals Oilseeds Pulses Vegetables Reference culture Rice Ragi Polysaccharide production 24 hours 48 hours OSE OSE2 - - ECE4 - - ECE ECE Groundnut AHE Sunflower Cowpea Soybean Chilli Tomato Azotobacter chroococcum HAE HAE VUE VUE GME GME CAE9 - - CAE CAE CAE LEE LEE LEE AZC Note: Polysaccharide production was recorded by visual scoring of the colonies in plates. - : No polysaccharide production. + : Slight polysaccharide production ++ : Moderate polysaccharide production +++ : High polysaccharide production

17 production. Thus the oilseed isolates were found to be good polysaccharide producers Nitrogen fixation ability of the endophytic bacterial isolates The results of the growth of the endophytic bacterial isolates on nitrogen free media - Jensen s medium, N free malate medium and Waksmann 77 medium are recorded in Table 8, Plate 2. In all the N free media tested fourteen isolates out of 19 were able to grow. Eight of these isolates groundnut isolate AHE3, ragi isolates ECE4 and ECE6, sunflower isolate HAE8, chilli isolates CAE10 and CAE11, soybean isolates GME15, GME16 and tomato isolate LEE19 exhibited high growth in the media. OSE1, HAE7, CAE9, CAE12 and LEE18 recorded slight growth while the other five isolates did not grow in the media. It is interesting that the cowpea isolates VUE13 and VUE14 could not grow in N free media Influence of carbon sources on the growth of diazotrophic endophytic bacterial isolates Influence of three carbon sources mannitol, malic acid and yeast extract on the growth of endophytic bacterial isolates was tested and the results are presented in Table 9. There was not much difference among the endophytic isolates with respect to their preference for carbon sources. Both mannitol and malic acid could be utilized by these organisms for their growth in N free media. When Yeast extract was present all the tested endophytic isolates showed moderate to high growth. AHE3, ECE6, CAE10 and GME16 preferred malic acid over mannitol and showed high growth. The isolates ECE5, VUE13, VUE14 and LEE17 did not show any growth in mannitol or malic acid.

18 Table 8. Growth of endophytic bacterial isolates on N free media Host group Cereals Oilseeds Pulses Vegetables Reference Host plant Rice Ragi Isolates Growth on N free media Jensen s medium Waksman 77 medium N free malate medium OSE OSE ECE ECE ECE Groundnut AHE Sunflower Cowpea Soybean Chilli Tomato Azotobacter chroococcum HAE HAE VUE VUE GME GME CAE CAE CAE CAE LEE LEE LEE AZC Note: - : No growth + : Slight growth ++ : Moderate growth +++ : High growth

19 Table 9. Influence of carbon sources on the growth of endophytic bacteria Host group Cereals Oilseeds Pulses Vegetables Reference culture Host plant Rice Ragi Isolates Malic acid Mannitol Mannitol + Yeast Extract Malic acid + Yeast Extract OSE OSE ECE ECE ECE Groundnut AHE Sunflower Cowpea Soybean Chilli Tomato Azotobacter chroococcum HAE HAE VUE VUE GME GME CAE CAE CAE CAE LEE LEE LEE AZC Note: - : No growth + : slight growth ++ : moderate growth +++ : high growth

20 Phosphate solubilization ability of the isolates by plate assay Growth of the endophytic bacterial isolates on Sperber s agar was screened and the results are presented in Table 10, Plate 2. The rice isolate OSE2 and the chilli isolates CAE9, CAE10, CAE11 and CAE12 were not able to form solubilization zone in both media tested. The ragi isolates ECE5 and ECE6, sunflower isolates HAE7 and HAE8, cowpea isolates VUE13 and VUE14 showed more than 4mm zone of solubilisation. AHE3, GME16, LEE17, LEE19 and AZC20 recorded <2mm zone of solubilisation but showed luxuriant growth in the media. Thus the cereal, oilseed and pulse isolates were predominantly phosphate solubilisers Inorganic phosphate solubilization potential of endophytic bacterial isolates The amount of phosphate released in the flasks after incubation was estimated in comparison with a set of uninoculated controls. The results are presented in Table 10, Fig 2, Plate 2. The highest phosphate solubilisation potential was shown by the isolate VUE13 (6.56 ppm) and the least was by LEE19 (0.5ppm). The next best inorganic phosphate solubilisers were the ragi isolate ECE5 (5.5 ppm) sunflower isolates HAE8 and HAE7 (5.14 and 3.96 ppm). The isolates AHE3 and GME16 as well as the tomato isolates LEE17, LEE18 and LEE19 and reference culture had very less solubilisation potential of < 1ppm. The rice isolate OSE2 and the chilli isolates CAE9, CAE10, CAE11, CAE12 were not able to solubilise tricalcium phosphate. All the cereal, pulse and oilseed isolates showed phosphate solubilization potential Intrinsic antibiotic resistance of endophytic bacterial isolates The intrinsic antibiotic resistance was determined against two antibiotics, kanamycin and streptomycin at different concentrations viz.,

21 Table 10. Inorganic phosphate solubilization potential of endophytic bacterial isolates Host group Cereals Oilseeds Pulses Vegetables Reference culture Host plant Rice Ragi Isolates Phosphate solubilization zone on Sperber s medium Phosphate solubilization (ppm)* OSE g OSE ECE f ECE b ECE g Groundnut AHE ij Sunflower Cowpea Soybean Chilli Tomato Azotobacter chroococcum HAE d HAE c VUE a VUE e GME gh GME i CAE CAE CAE CAE LEE h LEE ij LEE j AZC ij SEd 0.09 CD( Note: - : no zone of solubilisation + : <2mm zone of solubilisation ++ : 2 4mm zone +++ : >4mm zone Different letters in superscripts indicate significantly different values. *Estimated after 14 days of incubation with Tricalcium phosphate as Pi source

22 Phosphate (ppm) 7 Phosphate solubilisation by endophytic bacteria Isolates Fig. 2. Inorganic phosphate solubilisation potential of endophytic bacterial isolates

23 A.Growth in Nfree Malic acid medium B.Growth in Waksman77medium VUE13 ECE4 HAE8 ECE5 C. Phosphate solubilisation zone in Sperber s medium Control Blank ECE5 VUE13 HAE8 D. Inorganic Phosphate solubilisation by Molybdenum blue method Plate 2.Functional characteristics of the endophytic bacteria

24 50, 100, 150 and 200 ppm. All the isolates were resistant to 50 ppm and 100 ppm of Streptomycin and Kanamycin and showed high to moderate growth in the agar medium. Thirteen isolates, the rice isolates OSE1 and OSE2, groundnut isolate AHE3, ragi isolate ECE5, chilli isolates CAE9, CAE10, CAE11 and CAE12, cowpea isolates VUE13 and VUE14, soybean isolate GME16, and the tomato isolates LEE18 and LEE19 showed slight growth at 150 ppm streptomycin. ECE4, ECE6, HAE7, HAE8, GME15, LEE17 and the reference culture AZC20 were not resistant to 150 ppm Streptomycin. None of the isolates were resistant to 200ppm Streptomycin. The rice isolates OSE1 and OSE2, ground nut isolate AHE3, ragi isolate ECE5, chilli isolate CAE9, CAE10, CAE11, CAE12, cowpea isolates VUE13 and VUE14 and the soybean isolate GME16 were resistant to 150 ppm Kanamycin and showed slight growth. The cowpea isolate VUE13 and soybean isolate GME16 were resistant to 200 ppm Kanamycin. The cowpea isolates and chilli isolates had intrinsic resistance to 150 ppm of both antibiotics tested. Thus the vegetable isolates were found to be resistant to 150 ppm of streptomycin. The results are recorded in Table Development of antibiotic marker strain of endophyte by UV irradiation To develop a marker strain for the endophytic isolates UV irradiation studies was carried out. The results of the ultraviolet exposure of 200 KV (VUE13) and 200 KG (GME16) as well as wild strains of VUE13 and GME16 are presented in Table 12, Plate 3. When 200 KV was exposed to ultraviolet light its antibiotic resistance increased to 400 ppm Kanamycin, but it did not grow in 500ppm Kanamycin media. 200 KG was found to be resistant upto 500ppm Kanamycin with only slight growth in 500ppm Kanamycin media. From the non exposed plates the

25 Table 11. Intrinsic antibiotic resistance of endophytic bacteria Host group Host plant Isolates Cereals Oilseeds Pulses Vegetables Rice Ragi Streptomycin (ppm) Kanamycin (ppm) OSE OSE ECE ECE ECE Groundnut AHE Sunflower Cowpea Soybean Chilli Tomato HAE HAE VUE VUE GME GME CAE CAE CAE CAE LEE LEE LEE Reference culture Azotobacter chroococcum AZC Note: - No growth + Slight growth ++ Moderate growth +++ High growth

26 Table 12. Effect of UV irradiation on growth of endophytic isolates VUE13 and GME16 Kanamycin in the nutrient agar media VUE KV GME KG exp Non exp exp Non exp exp Non exp exp Non exp 0 ppm ppm ppm ppm ppm ppm Note: 200KV is the VUE13 isolate resistant to 200 ppm Kanamycin 200KG is the GME16 isolate resistant to 200 ppm Kanamycin - No growth + Slight growth ++ Moderate growth +++ High growth Table 13. Establishment of the endophytic marker strain 500KGX on the seeds and shoots of soybean (Glycine max) Parameter Population (cfu/ml) Initial population of 500KGX 16x 10 7 Population of 500KGX in seed after 6 hours incubation 5x10 6 Population of 500KGX in uninoculated seed 0 Population of 500KGX from shoot of inoculated seed after 10 days Population of 500KGX from shoot of uninoculated seed after 10 days 27x Note: Marker strain 500KGX is the GME16 isolate exposed to UV irradiation for 15 min to make it resistant to 500 ppm Kanamycin.

27 500ppm 400ppm 200pp 300ppm A. Growth of 200 KG after irradiation in Kanamycin media 500KGX Control B. Seed bacterisation with 500KGX in soybean SS 500KGX US 500KGX Control C. Isolation of 500KGX from shoots of soybean Plate 3. Development of Antibiotic marker strain 500KGX and its establishment in the seeds and shoots of soybean

28 isolates obtained were resistant to 200ppm Kanamycin only. Thus the isolate 200KG became resistant to 500ppm Kanamycin after exposure to uv light and the colonies which appeared in the 500ppm plates were considered as antibiotic marker strain and were designated as 500KGX Establishment of the endophytic marker strain 500KGX on the seeds and shoots of soybean (Glycine max) The results of the study to determine the population of antibiotic marker strain 500KGX in the seeds and shoots of soybean are presented in Table 13, Plate 3. The initial population of 500KGX in the broth media was 16 x 10 7 cfu/ml. The population of 500KGX after seed bacterisation in surface sterilised soybean seeds was 5 x 10 6 cfu/ seed. The uninoculated seeds did not record any population. 500KGX could be reisolated from the shoots of germinated seedlings and the population was 27 x 10 3 cfu/ml. The uninoculated seedlings did not have any population of 500KGX. Thus, it was confirmed that these isolates were true endophytes as the surface inoculated isolate was reisolated from within the tissue Role of endophytes in plant growth promotion Effect of marker strain 500KGX on seedling growth of tomato The plant growth promotion ability of endophytes was studied by using the antibiotic marker strain 500KGX in tomato and the results are presented in Table 14, Fig 3, Plate 4. The seed bacterisation with 500KGX increased germination percentage to 100 % while there was no significant difference between surface sterilized and non sterilized seeds (80 %). Inoculation with 500KGX increased the shoot length (4.5cm) and root length (5.0cm) significantly and hence these seeds showed the highest vigour index (950). It was followed by non surface sterilized seeds (shoot length- 2.4cm, root length- 3.6cm and vigour index- 480). In the

29 Table 14. Establishment of endophytes and plant growth promotion by 500KGX Treatment Germination (%) Days to 50% germination Shoot length (cm) Root length (cm) Seedling length (cm) Vigour index T1 80 (63.41) b 3.0 b 2.4 b 3.6 b 6.0 b 480 b T2 80 (63.41) b 4.0 c 1.6 c 2.6 c 4.2 c 336 c T3 80 (63.41) b 5.0 d 1.3 c 2.3 d 3.6 c 288 d T4 100 (89.96) a 2.0 a 4.5 a 5.0 a 9.5 a 950 a SEd CD (0.05) Note: Different letters in superscripts indicate significantly different values. Arcsine transformed values are indicated in parantheses. Vigour index = Germination percentage x Seedling length T1: Control Non surface sterilized seeds T2: Surface sterilized seeds on plain agar T3: Surface Sterilised seeds on antibiotic agar T4: 500KGX inoculated SS seeds

30 Vigour index 1000 Vigour index of tomato T1 T2 T3 T4 Treatments Fig. 3. Effect of marker strain 500KGX on seedling growth of tomato T1: Control Non surface sterilized seeds T2: Surface sterilized seeds on plain agar T3: Surface sterilized seeds on antibiotic media T4: 500KGX inoculated SS seeds

31 case of surface sterilized seeds grown on antibiotic agar all the epiphytic and endophytic microorganisms were eliminated and hence showed the least shoot length (1.3cm) and root length (2.3cm) and vigour index (288). Surface sterilized seeds on plain agar recorded vigour index of 336. The surface sterilized seeds grown on antibiotic agar took the more days to 50 % germination (5days) while the 500KGX seeds required only 2 days for 50 % germination Evaluation of endophytic bacteria for seedling growth of paddy The results of the effect of the endophytic isolates on seedling growth of paddy are presented in Table 15, Fig. 4, Plate 4. All the endophytic bacterial isolates tested along with the reference culture Azotobacter chroococcum were found to enhance the seedling growth of paddy in the range of 19.3 % increase over control in rice isolate OSE1 to % increase in the tomato isolate LEE19. The highest seedling length was recorded in the isolate LEE19 (45.60 cm) while the least was in uninoculated control (26.67 cm). Other isolates ECE6, HAE8, CAE10, CAE11, VUE14, GME16 and LEE18 recorded on par values for seedling length (43+ 2 cm). They showed more than 60 % increase over control. The rice isolate OSE1 (19.3 %), groundnut isolate AHE3 (19.4 %), soybean isolate GME15 (28.7 %), sunflower isolate HAE7 (38.7 %), chilli isoate CAE9 (48.7 %) and cowpea isolate VUE13 (49.3 %) recorded less than 50 % increase in seedling growth Production of plant growth regulators by endophytic bacterial isolates The production of plant growth regulators Gibberellic acid (GA), Indole acetic acid (IAA) and cytokinins (Benzyl adenine) by the endophytic bacterial isolates was studied by bioassay as well as spectrophotometric estimation.

32 Table 15. Evaluation of endophytic bacterial isolates on seedling growth of paddy Shoot Root Seedling % increase Host group Host plant Isolates length (cm) length (cm) length (cm) over control Rice OSE jk 9.50 gh j 19.3 OSE e fgh d bcdef 58.1 Cereals ECE fghi de defg 52.5 Ragi ECE de e cdef 56.2 ECE cde de abcde 61.2 Groundnut AHE k g j 19.4 Oilseeds HAE Sunflower f hi 38.7 HAE efghi ab abcd 63.8 Pulses Cowpea VUE hij 16.00d e efgh 49.3 VUE ab f abcd 64.4 Soybean GME ijk g ij 28.7 GME bc de ab 68.8 CAE ijk de fgh 48.7 Chilli CAE fghi a abcd 63.7 CAE cd e abcd 63.7 Vegetables CAE efg cd abcdef 60.0 LEE efghi bc abcd 62.5 Tomato LEE cd d abc 67.5 LEE a f a Reference culture Azotobacter chroococcum AZC k de gh 41.2 Control l 8.33 h k SEd CD (0.05) Note: Observations recorded after 14 days. Each value is the mean of 3 replications with 20 seeds each. Different letters in superscripts indicate significantly different values.

33 Length (cm) Shoot and root length of paddy shoot length root length Isolates Fig. 4. Evaluation of endophytic bacteria on seedling growth of paddy

34 T3 T1 T4 A. Establishment of endophytes and plant growth promotion in tomato Control OSE2 ECE5 ECE6 CAE11 VUE14 GME16 LEE18 LEE19 B. Evaluation of endophytic isolates on seedling growth of paddy Plate 4. Plant growth promotion potential of endophytes

35 Bioassay for Gibberellic Acid (GA) production by starch agar halo test The concentration of GA in the culture filtrate of the endophytic bacterial isolates was determined by the starch agar halo test. This bioassay is based on the principle that GA induces de novo synthesis of amylase in germinating seeds. According to this study the highest GA was recorded in GME16 (9.82 µg) which was followed by ECE6, CAE11, VUE13 and LEE18 which recorded on par values (5.98 µg to 7.22 µg). The least was in HAE7, VUE14, GME15, LEE17, LEE19 and AZC20. All these isolates recorded less than 1.06 µg GA. Results are presented in Table 16, Fig 5, Plate Estimation of Gibberellic Acid (GA) production The spectrophotometric estimation of GA at 254 nm also showed a similar trend and the results are presented in Table 18. The GA concentration in 25 ml of the culture filtrate varied from 9.91 µg in the cowpea isolate GME16 to 1.1 µg in sunflower isolate HAE7. CAE11, VUE13 and LEE18 recorded on par values ( ). The next best isolate was ECE6 (8.74 µg). Azotobacter chroococcum recorded very less GA (1.47 µg). The cereal and oilseed isolates had good GA producing potential Bioassay for IAA production by cucumber root elongation test This bioassay is based on the inhibition of root growth in cucumber by IAA. As the concentration of IAA increases the root elongation of germinating seedlings is inhibited. The results of the bioassay are recorded in Table 17, Fig. 6, Plate 5. Five isolates recorded more than 100 µg of IAA in the culture filtrate. The highest was found in LEE19 ( µg) followed by HAE7 ( µg), OSE1 ( µg),

36 Table 16. Bioassay for Gibberellic Acid (GA) production by endophytic isolates Host group Host plant Isolates Diameter of the zone (mm) GA (µg)* Cereals Rice Ragi OSE c 6.94 b OSE f 5.14 e ECE g 3.62 f ECE h 3.10 g ECE d 5.98 d Groundnut AHE h 3.10 g Oilseeds Pulses Vegetables Sunflower Cowpea Soybean Chilli Tomato HAE k 1.06 h HAE e 6.50b c VUE d 7.18 b VUE l 0.09 i GME j 0.09 i GME a 9.82 a CAE f 5.02d e CAE h 3.10 g CAE b 7.22 b CAE f 5.02 e LEE l 0.09 i LEE b 7.02 b LEE k 0.22 i Reference culture Azotobacter chroococcum AZC i 1.02 h SEd CD (0.05) Note: Bioassay by Starch agar halo test. *Estimated from 25 ml culture filtrate concentrated to 2 ml. Different letters in superscripts indicate significantly different values.

37 GA (µg) 12 Estimation of GA Isolates Fig. 5. Bioassay for Gibberellic Acid (GA) production by endophytes

38 Table 17. Bioassay for IAA production by endophytic bacterial isolates Host group Host plant Isolates Shoot length (cm) IAA (µg)* Cereals Rice Ragi OSE m b OSE f 3.65 h ECE n d ECE h e ECE k c Groundnut AHE j f Oilseeds Pulses Vegetables Sunflower Cowpea Soybean Chilli Tomato HAE l b HAE a 3.09 h VUE f 7.60 h VUE ab 3.09 h GME bc 3.65 h GME cd 3.96 h CAE bc 3.36 h CAE g 8.99 h CAE e 3.96 h CAE h g LEE d 5.97 h LEE i e LEE n a Reference culture Azotobacter chroococcum AZC ab 4.30 h SEd CD (0.05) Note: Bioassay by Cucumber root elongation test. *Estimated from 25 ml culture filtrate concentrated to 2 ml. Different letters in superscripts indicate significantly different values.

39 IAA (µg) 250 Estimation of IAA Isolates Fig. 6. Bioassay for Indole Acetic Acid (IAA) production by endophytes

40 ECE6 ( µg) and ECE4 ( µg). LEE18 recorded µg which was on par with ECE5 (64.32 µg) followed by AHE3 (39.33 µg) and CAE12 (17.33 µg). All other isolates recorded 10 µg IAA. The cereal and oilseed isolates produced IAA than pulses and chilli Estimation of Indole Acetic acid (IAA) production The results of spectrophotometric estimation of IAA are presented in Table 18. Similar trend was found in this also. The IAA concentration in 25 ml of the culture filtrate was the highest in LEE19 ( µg). OSE1 and HAE7 recorded on par values of µg. this was followed by ECE6 ( µg) and ECE4 ( µg). ECE5 and LEE18 recorded on par values ( µg). AHE3 recorded µg and CAE12 produced µg. All other isolates produced 10 µg Bioassay for cytokinin production by cucumber cotyledon greening test The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins and the results of the test are presented in Table 18, Fig.7, Plate 5. Cytokinins accelerate chloroplast differentiation as well as regulate and stimulate chlorophyll (Chl) production in etiolated cucumber cotyledons. The increase in Chlorophyll production is proportional to the concentration of cytokinins and this response provides a sensitive yet rapid bioassay for cytokinins. The cytokinin (benzyl adenine) concentration in 25 ml culture filtrate was highest in the tomato isolate LEE18 (5.816 µg) followed by cowpea isolate GME16 (3.503 µg). The chilli isolate CAE11 produced 3.257µg and the reference culture Azotobacter chroococcum recorded µg. The rice isolates OSE1 and OSE2 produced in the range of and µg respectively. HAE8, CAE12 and LEE17 produced 1.49, and µg cytokinin. All other isolates produced less than 1 µg/ml. The least was in AHE3, ECE4 and ECE5 ( ).

41 Table 18. Estimation of plant growth regulators (GA, IAA, Cytokinins) produced by endophytic bacteria Host group Host Isolates GA (µg)* IAA (µg)* Cytokinin (µg)* Cereals Rice Ragi OSE f b e OSE d 7.53 hi a ECE f d 0.44 l ECE g e 0.53 l ECE e c 7.42 i Groundnut AHE f f 0.63 l Oilseeds Pulses Vegetables Sunflower Cowpea Soybean Chilli Tomato HAE k b 0.76 kl HAE c 4.74 hi f VUE b 7.23 hi 5.89 j VUE i 3.23 i 1.85 k GME h 3.59 i 7.99 i GME a 3.48 i 9.93 h CAE g 4.01 i 8.22 i CAE g h 7.43 i CAE b 4.95 hi c CAE e g g LEE l 6.07 h f LEE b e b LEE m a h Reference culture Azotobacter chroococcum AZC j 4.51 h d SEd CD(.05) Note: Spectrophotometric estimation for GA and IAA. Cucumber cotyledon greening bioassay for cytokinins. *Estimated from 25 ml culture filtrate concentrated to 2 ml. Different letters in superscripts indicate significantly different values.

42 Cytokinin (µg) 7 Estimation of Cytokinins Isolates Fig. 7. Bioassay for cytokinins production by endophytes

43 GME16 LEE18 Control A. Starch agar halo test for GA production Control VUE13 LEE18 AHE3 OSE1 LEE19 HAE7 ECE6 B. Cucumber root elongation test for IAA production LEE18 GME16 Control C. Cucumber cotyledon greening bioassay for cytokinin production Plate 5. Bioassay of plant growth regulators production by endophytic bacterial isolates

44 4.6. Compatibility of endophytic bacterial isolates with beneficial soil microorganisms The compatibility of the endophytic bacterial isolates with Pseudomonas sp., Azotobacter sp., Bacillus subtilis and Bacillus megaterium was tested and the results are presented in Table 19. All the endophytic isolates were found to be compatible with the beneficial soil microorganisms. Both the test organisms grew well in the plate without inhibiting each other Biocontrol efficiency of endophytic isolates against fungal pathogens of vegetables Endophytic bacterial isolates were screened for in vitro growth inhibition of four phytopathogenic fungi Colletotrichum sp., Fusarium oxysporum, Rhizoctonia solani and Pythium aphanidermatum by dual culture method and the results are presented in Table 20, Plate 6. Colletotrichum sp. was inhibited in plate assay by eleven endophytic isolates. The sunflower isolate HAE7 and chilli isolates CAE9 and CAE10 recorded >2mm zone of inhibition while the rice isolates OSE1 and OSE2, groundnut isolate AHE3, ragi isolate ECE4, sunflower isolate HAE8, cowpea isolates VUE13 and VUE14 and soybean isolate GME15 had <2mm zone of inhibition. The other isolates did not inhibit the aerial pathogen. Against Fusarium oxysporum, ten endophytic isolates were found to be antagonistic. OSE1, HAE8, CAE9 and CAE10 showed >2mm zone of inhibition while OSE2, AHE3, HAE7, CAE11, GME16, LEE17 and LEE18 had <2mm zone of inhibition. In the case of Rhizoctonia solani except five isolates, OSE1, ECE5, VUE13, VUE15 and GME16 all others inhibited the pathogen to varying

45 Table 19. Compatibility of endophytic bacterial isolates with beneficial soil bacteria Host group Cereals Oilseeds Pulses Vegetables Rice Ragi Host Isolates Pseudomonas sp. Azotobacter sp. Bacillus megaterium Bacillus subtilis OSE OSE ECE ECE ECE Groundnut AHE Sunflower Cowpea Soybean Chilli Tomato HAE HAE VUE VUE GME GME CAE CAE CAE CAE LEE LEE LEE Reference Azotobacter AZC Note: Compatibility tested by dual culture method. ++ : No inhibition of the test organisms

46 Table 20. Evaluation of endophytic bacterial isolates against fungal plant pathogens Host group Host Isolates Colletotrichum Fusarium Rhizoctonia Pythium Rice OSE OSE Cereals ECE Ragi ECE ECE Groundnut AHE Oilseeds HAE Sunflower HAE Pulses Cowpea VUE VUE Soybean GME GME CAE Chilli CAE CAE Vegetables CAE LEE Tomato LEE LEE Reference culture Azotobacter chroococcum AZC Note: Tested by dual culture method - : no zone of inhibition +: <2mm zone of inhibition ++: >2mm zone of inhibition

47 degree. AHE3, ECE4, HAE7, HAE8, CAE12 and LEE18 showed >2mm zone of inhibition. The root pathogen Pythium aphanidermatum was inhibited by fourteen of the nineteen isolates. ECE4, CAE9, LEE17, LEE18, LEE19 and AZC20 were found to be very effective and exhibited >2mm zone of inhibition in dual culture method Evaluation of endophytic bacterial isolates on growth inhibition of Colletotrichum sp. The endophytic isolates showing positive inhibition of Colletotrichum sp. in plate assay was used for this study. The fungal growth inhibition was determined by mycelial mat weight determination after coinoculation of the pathogen and antagonist in broth and the results are presented in Table 21, Fig.8, Plate 6. The eight cultures which did not inhibit the pathogen in plate assay were not used in this study. The highest mycelial mat dry weight was in uninoculated control (2.39 g). Among the twelve isolates tested there was no significant difference in the dry weight of mycelial mat (0.07 g ) except soybean isolate GME15 and tomato isolate LEE 17. These two isolates did not effectively control the fungus and recorded 1.55 and 2.13 g respectively indicating only % and 10.9 % decrease in dry weight. The other ten isolates could effectively inhibit the growth of Colletotrichum sp. and recorded % to % reduction Evaluation of endophytic bacterial isolates on growth inhibition of Fusarium oxysporum. In the case of Fusarium oxysporum, nine isolates were not tested as they did not show inhibition in plate assay. The results are presented in

48 Table 21. Evaluation of endophytic bacterial isolates on the growth of Colletotrichum sp. Host group Cereals Oilseeds Pulses Vegetables Host Rice Isolates Mycelial mat Wet weight (g) Mycelial mat Dry weight (g) Reduction in wet weight (g) Reduction in dry weight (g) % reduction in dry wt Control 8.28 g 2.39 d OSE b 0.07 a OSE f 0.13 a Ragi ECE a 0.12 a Groundnut AHE e 0.10 a Sunflower Cowpea HAE de 0.10 a HAE e 0.12 a VUE ab 0.12 a VUE cde 0.10 a Soybean GME f 1.55 b Chilli CAE bcd 0.11 a CAE bc 0.13 a Tomato LEE g 2.13 c SEd CD (0.05) Note: Tested in nutrient broth media. Reduction in weight is with reference to control.

49 Table 22, Fig.8, Plate 6. Of the eleven isolates tested highest inhibition was recorded in OSE2 (97.26 %). The dry weight of the mycelia mat was only 0.14 g as compared to the 5.25 g in uninoculated control. The least inhibition was 54 % exhibited by OSE1 where the mat dry weight was 2.39g. More than 80 % inhibition was recorded in seven isolates OSE2, AHE3 (80.98 %), HAE7 (94.3 %), CAE9 (91.47 %), CAE10 (96.38 %), GME16 (87.91 %) and LEE17 (86.96 %) Evaluation of endophytic bacterial isolates on growth inhibition of Rhizoctonia solani. The results of the growth inhibition of Rhizoctonia solani are presented in Table 23, Fig 8, Plate 6. All the isolates except OSE1, ECE5, VUE13, GME15 and GME16 were tested for growth inhibition against Rhizoctonia sp. and they inhibited the mycelial mat in the range of 5.5 to 85 %. The highest inhibition was recorded in AHE3 (0.04 g) as against the least in uninoculated control (0.29 g), % reduction in dry weight. This was followed by CAE9 and CAE12 recording and % inhibition respectively. VUE14 and LEE19 exhibited 75 % reduction in dry weight. Tomato isolates LEE17 and LEE18 could inhibit Rhizoctonia sp. to 60 %. OSE2 and CAE10 recorded 0.28 g and 0.27 g dry weight showing only 5.5 and 5.84 % inhibition Evaluation of endophytic bacterial isolates on growth inhibition of Pythium aphanidermatum. The results of the growth inhibition of endophytic bacteria on Pythium aphanidermatum are presented in Table 24, Fig. 8, Plate 6. CAE10 and VUE13 showed highest inhibition of Pythium sp. (97.53 % and %) followed by LEE19 (90.12 %). LEE17 and GME15 were inhibiting the pathogen to around 85 % which was found to be on par. OSE1 recorded 9.88 % inhibition and CAE %. Azotobacter chroococcum recorded the least inhibition of 6 % with a dry weight of

50 Table 22. Evaluation of endophytic bacterial isolates on the growth of Fusarium oxysporum. Host group Cereals Oilseeds Host Rice Isolates Mycelial mat wet weight (g) Mycelial mat dry weight (g) Reduction in wet weight (g) Reduction in dry weight (g) % reduction in dry weight Control 8.98 h 5.25 j OSE f 2.39 i OSE c 0.14 a Groundnut AHE f 1.00 e Sunflower HAE b 0.30 b HAE d 2.04 g Pulses Soybean GME e 0.64 d Vegetables Chilli Tomato CAE c 0.45 c CAE a 0.19 ab CAE g 2.17 h LEE g 0.69 d LEE d 1.89 f SEd CD (0.05) Note: Tested in nutrient broth media. Reduction in weight is with reference to control.

51 Table 23. Evaluation of endophytic bacterial isolates on the growth of Rhizoctonia solani. Host group Cereals Oilseeds Host Isolates Mycelial mat wet weight (g) Mycelial mat dry weight (g) Reduction in wet wt (g) Reduction in dry wt (g) % reduction in dry wt Control 5.09 o 0.29 h Rice OSE l 0.28 g Ragi ECE b 0.12 d ECE m 0.21 f Groundnut AHE a 0.04 a Sunflower HAE k 0.20 i HAE n 0.19 hi Pulses Cowpea VUE f 0.07 cd Vegetables Chilli Tomato CAE h 0.05 ab CAE i 0.27 i CAE d 0.15 e CAE de 0.06 b LEE c 0.11 d LEE ef 0.12 d LEE g 0.07 c Reference Azotobacter AZC j 0.16 e SEd CD (0.05) Note: Tested in nutrient broth media. Reduction in weight is with reference to control.

52 Table 24. Evaluation of endophytic bacterial isolates on the growth of Pythium aphanidermatum. Host group Cereals Host Rice Isolates Mycelial mat wet weight (g) Mycelial mat dry weight (g) Reduction in wet wt (g) Reduction in dry wt (g) % reduction in dry wt Control 7.54 l k OSE g i OSE i d Ragi ECE j h Oilseeds Groundnut AHE f g Pulses Vegetables Cowpea VUE k a Soybean GME e c Chilli Tomato CAE h i CAE a a CAE b f LEE d bc LEE b e LEE a b Reference Azotobacter AZC c j SEd CD (0.05) Note: Tested in nutrient broth media. Reduction in weight is with reference to control.

53 % reduction in mat dry weight % reduction in mat dry weight % reduction in mat dry weight % reduction in mat dry weight Colletotrichum sp Fusarium sp Rhizoctonia sp Pythium sp. Isolates Fig. 8. Interaction of endophyte with fungal plant pathogens

54 A. Inhibition of Rhizoctonia sp.by plate assay B. Inhibition of Colletotrichum sp. C. Inhibition of Fusarium sp. D. Inhibition of Rhizoctonia sp. E. Inhibition of Pythium sp. Control AHE3 HAE8 OSE2 CAE10 LEE18 LEE19 F. Plant infection study Plate 6. Interaction of endophytic isolates with fungal plant pathogens

55 0.076 g while uninoculated control recorded 0.81 g dry mycelial mat weight Plant infection study The effects of seed bacterisation of tomato with endophytic bacterial isolates on the control of damping off disease by root pathogens are presented in the Table 25, Fig. 9, Plate 6. In the case of Fusarium damping off., the highest control of the disease was recorded in OSE1 and LEE % and % respectively followed by CAE11 while the least was recorded in OSE2 (21.67 %). The uninoculated control had the minimum seedlings of %. In Rhizoctonia damping off the uninoculated control recorded 28 % while the highest control was recorded in rice isolate OSE2 and chilli isolate CAE10 (70 %). The chilli isolates CAE9 and CAE11 could control only upto % incidence. The highest control of Pythium damping off was shown by the rice isolate OSE1 and ground nut isolate AHE3 (60 %) while the least of 33.3 % control was exhibited by the chilli isolate CAE10 and tomato isolate LEE Amplification of nitrogenase gene (nifh) by PCR The expected nifh gene fragments (360 bp) were amplified by PCR in the tomato isolate LEE19 and in the positive strain Azotobacter chroococcum, but not in the blank. The results of gel electrophoresis of nifh gene PCR products is presented in Plate 7.

56 LEE19 AZC20 Ladder bp Plate 7. Amplification of nifh gene in LEE19

57 Table 25. Effect of seed treatment of endophytic bacterial isolates on the control of damping off disease in tomato Host group Cereals Host Rice Isolates Fusarium Rhizoctonia Pythium Control j k i OSE a a OSE i a f Groundnut AHE g i a Oilseeds Sunflower HAE h e 0.00 HAE d f 0.00 Soybean GME f CAE g j b Chilli CAE h b h Vegetables CAE c g 0.00 Tomato LEE e e e LEE b e c Note: The number of healthy seedlings was recorded after 14 days. SEd CD (0.05)

58 % control of damping off 80 Control of tomato damping off by endophytic bacterial isolates Fusarium sp. Rhizoctonia sp. Pythium sp Control OSE1 OSE2 AHE3 HAE7 HAE8 CAE9 CAE10 CAE11 CAE12 LEE17 LEE18 LEE19 AZC20 Treatments Fig. 9. Effect of seed treatment of endophyte on the control of damping off disease in tomato

59 4.9. Application of endophytes Application of endophytes in Nursery technology Effect of endophytic isolates on the rooting and establishment of cuttings of the ornamental plant, Hibiscus rosasinensis. The effect of the endophytic isolates viz. ECE6, HAE7, LEE18, LEE19 and the consortia of the above four was studied in the green house experiment and the results are presented in Tables 26, 27 and Effect of endophytic bacterial isolates on sprouting percentage of Hibiscus rosasinensis The number of cuttings with fresh sprouts was recorded at 15 th day, 30 th day, 60 th day and 90 th day of planting and expressed as sprouting percentage in Table 26, Fig. 10, Plate 8. On 15 th day, the maximum sprouting of 75 % was recorded in the chemical treatment (IBA) followed by the commercial formulation (62.5 %). Among the endophytic treatments LEE18 showed the best sprouting percentage (50 %) followed by LEE19 which was on par with uninoculated control (33.30 %). ECE6, HAE7 and the consortia recorded the least sprouting (12.5 %). However by 90 th day the endophytic treated plants showed better sprouting (75 %) than the commercial formulation Quicroot (62.5 %) and 100 % sprouting was recorded in IBA treatment. The treatment with the consortia showed 50 % sprouting. Control had only 33.3 % sprouting. Similar trend was noted on the 60 th day and 90 th day of planting.

60 Table 26. Effect of endophytic bacterial isolates on sprouting per cent of Hibiscus rosasinensis Treatments Sprouting per cent 15 th day 30 th day 60 th day 90 th day T1 Uninoculated control (35.23) d (35.23) e (35.23) e (35.23) e T2 Commercial formulation (Quicroot dip for 30s) T3 Chemical control (IBA 2500ppm for 30s) (52.18) b (52.18) c (52.18) c (52.18) c (59.18) a (89.96) a (89.96) a (89.96) a T4 Ragi isolate ECE (20.69) e (59.97) b (59.97) b (59.97) b T5 Sunflower isolate HAE (20.69) e (44.98) d (44.98) d (44.98) d T6 Tomato isolate LEE (44.98) c (59.97) b (59.97) b (59.97) b T7 Tomato isolate LEE (35.23) d (59.97) b (59.97) b (59.97) b T8 Consortium of ECE6, HAE7, LEE18 and LEE (20.69) e (44.98) d (44.98) d (44.98) d SEd CD(.05) Note: Arcsine transformed values are indicated in parantheses. Different letters in superscripts indicate significantly different values. Sprouting per cent = No of cuttings with fresh sprouts x 100 No of cuttings planted

61 Effect of endophytic bacterial isolates on shoot parameters of Hibiscus rosasinensis cuttings The effect of the endophytic bacteria on the shoot parameters like shoot length, no of sprouts/ cutting and the no of leaves / cuttings are presented in Table 27, Fig. 10, Plate 8. IBA treatment recorded the highest shoot length of 43.5cm and the least was in uninoculated control (22 cm). The treatment with HAE7, LEE18, LEE19 as well as Quicroot dip recorded on par values ( cm). Consortia recorded shoot length of 26 cm. IBA treatment as well as LEE19 recorded 13 sprouts per cutting while the least number of sprouts was in uninoculated control and consortia (4.5 and 5 respectively). 12 sprouts were there in LEE18 and 9 sprouts in ECE6 and HAE7. The Quicroot dip had only 6 sprouts/ cutting. In the case of number of leaves also IBA treatment had the highest number (168) and the commercial formulation Quicroot recorded only 48 leaves/ cutting. Among the endophytic isolates LEE19 had 152 leaves followed by LEE18 (149). The next best was ECE6 which had 94 leaves and the consortia recorded 58 leaves. The least number of leaves was in uninoculated control (35leaves) Effect of endophytic bacterial isolates on root parameters of Hibiscus rosasinensis cuttings The results of the effect of the bacterial endophytes on rooting parameters like rooting percentage, root length, fresh weight and dry weight of roots recorded after 90 days of planting are presented in Table 28, Fig. 10, Plate 8.

62 Table 27. Effect of endophytic bacterial isolates on shoot parameters of Hibiscus rosasinensis cuttings after 90 days of planting. Treatments Shoot length (cm) No of sprouts/ cutting No of leaves/ cutting T1 Uninoculated control 22.0 g 4.5 f 35.0 g T2 Commercial formulation (Quicroot dip for 30s) T3 Chemical control (IBA 2500ppm for 30s) 37.8 c 6.0 d 48.0 f 43.5 a 13.0 a a T4 ECE e 9.0 c c T5 HAE b 9.0 c 94.0 d T6 LEE bc 12.0 b b T7 LEE c 13.0 a b T8 Consortium of ECE6, HAE7, LEE18 and LEE f 5.0 e 58.0 e SEd CD (0.05) Note: Shoot length was recorded from the base to the tip of the topmost leaf. All values are the mean of three replications. Different letters in superscripts indicate significantly different values.

63 Table 28. Effect of endophytic bacterial isolates on root parameters of Hibiscus rosasinensis cuttings. Treatments Rooting (%) Root length (cm) Fresh weight of roots (g) Dry weight of roots (g) T1 Uninoculated control 60 (50.75) c 9.06 g f d T2 Commercial formulation (Quicroot dip for 30s) 80 (63.41) b b b ab T3 Chemical control (IBA 2500ppm for 30s) 100 (89.96) a a a a T4 ECE6 80 (63.41) b 30.2 d c bc T5 HAE7 80 (63.41) b e d c T6 LEE18 80 (63.41) b d c c T7 LEE (89.96) a c c b T8 Consortium of ECE6, HAE7, LEE18 and LEE19 60 (50.75) c f d c SEd CD (0.05) Note: Arcsine transformed values are indicated in parantheses. Different letters in superscripts indicate significantly different values. Rooting per cent = No of cuttings with roots x 100 No of cuttings planted

64 Treatment details T1 Uninoculated control T2 Commercial formulation (Quicroot dip for 30s) T3 Chemical control (IBA 2500ppm for 30s) T4 Ragi isolate ECE6 T5 Sunflower isolate HAE7 T6 Tomato isolate LEE18 T7 Tomato isolate LEE19 T8 Consortium of ECE6, HAE7, LEE18 and LEE19

65 Dry weight of roots (g) No of leaves per cutting Per cent Sprouting and Rooting in Hibiscus rosasinensis Sprouting % Rooting % T1 T2 T3 T4 T5 T6 T7 T8 Treatments Number of leaves T1 T2 T3 T4 T5 T6 T7 T8 Treatments Root biomass T1 T2 T3 T4 T5 T6 T7 T8 Treatments Fig. 10. Effect of endophytic bacteria on rooting and establishment of Hibiscus rosasinensis

66 Treatment details T1 Uninoculated control T2 Commercial formulation (Quicroot dip for 30s) T3 Chemical control (IBA 2500ppm for 30s) T4 Ragi isolate ECE6 T5 Sunflower isolate HAE7 T6 Tomato isolate LEE18 T7 Tomato isolate LEE19 T8 Consortium of ECE6, HAE7, LEE18 and LEE19

67 A. Control B. IBA treatment C. LEE19 treatment T1 T2 T3 T4 T5 T6 T7 T8 E. Rooting of cuttings due to endophyte treatment T3 T6 T1 T1 F. Control F. IBA treatment G. LEE19 treatment Plate 8. Effect of endophytes on rooting and establishment of Hibiscus rosasinensis.

68 100 % rooting was observed in IBA treatment as well as LEE19 inoculation. The commercial formulation Quicroot and the other test endophytes showed 80 % rooting. The least rooting of 60 % was recorded in uninoculated control and combination of endophytic isolates. There was significant difference in the average root length of the cuttings. The average root length varied from 9cm in uninoculated control to 48.5cm in IBA treatment. The second best in root length was the commercial formulation Quicroot (37.75 cm). Among the endophytic isolates the longest root was recorded in LEE19 (35.4 cm). LEE18 (31.5 cm) and ECE6 (30.2 cm) recorded on par values. The fresh weight of roots was highest in IBA treatment (0.504 g) followed by commercial formulation Quicroot (0.453 g) and the least was in uninoculated control g. LEE19, LEE18 and ECE6 recorded on par root fresh weight (0.3 g) and HAE7 and combination showed on par values (0.2 g). The dry weight of roots also showed significant difference between treatments. It was highest in IBA treatment (0.105 g) and least in uninoculated control (0.019 g). Among the endophytic treatments the best was found to be LEE19 (0.084 g) and the least was in combination (0.035 g) Effect of endophytes on growth and establishment of tissue culture banana var. Nanjangudu rasabale Effect of endophytes on growth parameters of Tissue culture banana The effect of endophytes on growth of tissue culture banana var. Nanjangudu Rasabale was recorded at two stages, first at the time of primary hardening and then at the time of secondary hardening. The

69 following growth parameters root length (cm), pseudostem height (cm) and pseudostem girth (cm) were recorded and the results are presented in Table 29, Fig. 11, Plate 9. After the primary hardening of the tissue culture plantlets, there was significant difference between the treatments in the case of root length. The highest root length was recorded in LEE18 treatment (24 cm) while the least was in uninoculated control (5.58 cm). LEE19 and GME16 recorded on par values of and cm respectively. The combination of the isolates recorded slightly lesser root length (17.03 cm). Similar trend was observed after secondary hardening also. The best treatment was LEE18 which recorded cm. The uninoculated control had the least root length (8.20 cm). GME16 and LEE 19 did not have much difference in root length (31 cm) and the combination recorded 28 cm. After primary hardening the pseudostem length was highest in LEE18 (19.2 cm) followed by GME16 (18 cm) and LEE19 (15.37 cm). The least pseudostem length was in uninoculated control (10.37 cm). After secondary hardening the length of pseudostem recorded similar trend. GME16 and LEE18 recorded highest length (30.00 cm and cm). LEE19 recorded cm and combination showed cm. The least was in uninoculated control (20.33 cm). Thus the tissue culture plantlet length was increased due to the treatment with LEE18 which recorded 43.2 cm after primary hardening and cm after secondary hardening in comparison with control (15.95 cm and cm) This was followed by GME16 (35.33 cm and cm), LEE19 (36.4 cm and cm) and combination (34.27 cm and cm).

70 Table 29. Effect of endophytes on growth parameters of Tissue culture banana var. Nanjangudu Rasabale Root length (cm) Shoot length(cm) Plantlet length(cm) Shoot girth (cm) Treatment I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) Control 5.58 d c d b c c 2.85 d 5.00 b GME b ab ab a b a 4.12 b 7.00 a LEE a a a a a a 4.50 a 7.30 a LEE b ab c b b ab 3.93 bc 5.53 b Consortium c b 14.2 c b b b 3.70 c 5.05 b SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

71 After primary hardening of the tissue culture plantlets there was significant difference among the treatments in the case of pseudostem girth. LEE18 showed the highest (4.5 cm) while uninoculated control recorded the least (2.85 cm). GME16, LEE19 and consortium recorded on par values of ( ) cm. After secondary hardening there was no significant difference in shoot girth among LEE19, consortium and uninoculated control (5+0.89). LEE18 recorded the highest shoot girth of 7.30 cm which was on par with GME16 (7.00 cm) Effect of endophytes on leaf parameters of tissue culture banana The leaf parameters like length of leaf, breadth of leaf and leaf area index was recorded and the results are presented in Table 30, Fig. 11. The length and breadth of leaf and the leaf area index showed significant difference among the treatments. However with respect to the number of leaves LEE18 had 7 leaves at primary hardening and 8 leaves after secondary hardening stage. The least was 5 leaves in uninoculated control which increased to 7 leaves at secondary hardening stage. All the endophytic treatments GME16, LEE18 and LEE19 recorded 8 leaves after secondary hardening. The length of the leaf varied from cm in LEE18 and GME16 to 6.57 cm in uninoculated control. LEE19 and combination recorded on par values (8 cm) after primary hardening. After secondary hardening similar trend was shown with GME16 and LEE18 having the lengthiest leaf (24 cm and 23.5 cm). The least was in uninoculated control (18.5 cm). The leaf width did not show much significant difference and the best was in LEE18 (4.73 cm) GME16 (3.70 cm) and while LEE19 (3.17 cm), combination (2.63 cm) and control (1.70 cm) were on par after

72 Table 30. Effect of endophytes on leaf parameters of Tissue culture banana var. Nanjangudu Rasabale No of Leaves Width of top leaf(cm) Length of top leaf (mm) Leaf area (cm 2 ) Treatment I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) Control 5.00 c 7.00 b 1.70 e 8.00 b 6.57 c bc e d GME b 8.00 a 3.70 b a a a b a LEE a 8.00 a 4.73 a a a b a b LEE b 8.00 a 3.17 c 9.00 b 8.47 b bc c c Consortium 6.00 b 7.00 b 2.63 d 8.50 b 8.03 b c d d SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

73 primary hardening. After secondary hardening also similar trend was followed. The leaf area index showed significant difference among treatments after primary hardening. LEE18 recorded maximum leaf area (270.97) followed by GME16 (187.01) and LEE19 (128.88). The least was in control (44.68). After secondary hardening the leaf area index was maximum in GME16 ( ) followed by LEE18 (1654.4). Combination (833) and uninoculated control (828) were on par Effect of endophytes on biomass of Tissue culture banana The effect of endophytes on the biomass production of tissue culture banana was recorded after secondary hardening and the results are presented in Table 31, Fig. 11, Plate 9. The shoot weight recorded significant difference after primary hardening as well as secondary hardening. LEE18 recorded highest shoot weight (12.32 g and g) which was on par with GME16 (11.30g and 20.15g) followed by LEE 19 (9.30 g and g). The least shoot biomass was in uninoculated control 3.84 g and 6.38 g respectively. The root weight increased substantially due to endophytes and the best treatment was found to be LEE18 which recorded highest root weight both after primary hardening and secondary hardening (15.00 g and g). This was followed by GME 16 which was on par with LEE18 after primary hardening (14.25 g) and g after secondary hardening. LEE19 recorded 16.6 g and 21.8 g respectively at the two stages and the least was in uninoculated control (2.42 g and 4.37 g). Similar trend was noted in plant biomass with the highest biomass being recorded in LEE18 at both stages g and g respectively

74 Table 31. Effect of endophytes on biomass of Tissue culture banana var. Nanjangudu Rasabale Shoot fresh weight (g) Root fresh weight (g) Plant fresh weight (g) Treatment I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) I 0 hardening (After 45 days in cups) 2 0 hardening (After 45 days in polybags) Control 3.84 c 6.38 d 2.42 d 4.37 e 6.26 d d GME a a a b a a LEE a a a a a a LEE b b b c b b Consortium 8.15 b c 7.83 c 8.35 d c c SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

75 Fresh weight (g) Leaf area Length (cm) Plantlet length in Tissue culture banana Pri.hardening Sec. hardening Control GME16 LEE18 LEE19 Combination Treatments Leaf area Pri.hardening Sec. hardening Control GME16 LEE18 LEE19 Combination Treatments Plant biomass Pri.hardening Sec. hardening Control GME16 LEE18 LEE19 Combination Treatments Fig. 11. Effect of endophytes on growth and establishment of Tissue culture banana

76 Treatment details T1 Uninoculated control T2 Soybean isolate GME16 T3 Tomato isolate LEE18 T4 Tomato isolate LEE19 T5 Consortium of ECE6, HAE7, LEE18 and LEE Hardening: Primary hardening in cups for 45 days 2 0 Hardening: Secondary hardening in polybags for 45 days

77 LEE18 Control LEE18 Control Growth in bottles Rooting 1 0 Hardening Mist chamber for 1 0 Hardening 2 0 Hardening General view of Tissue culture banana in polybags Control GME16 LEE18 LEE19 Consortium Comparison of growth LEE18 Control Rooting of Tissue Culture Banana Plate 9. Effect of endophytes on growth and establishment of Tissue culture banana var. Nanjangudu Rasabale.

78 which was on par with GME16 (25.55 g and g). This was followed by LEE19 inoculation (21.80 g and g). Consortia of endophytes gave less biomass (15.98 g and g) but more than uninoculated control (6.26 g and g) Applications of endophytes in the field Comparison of the effect of plant growth promoting endophytic bacterial isolates on the seedling growth of different varieties of aerobic rice. The effect of plant growth promoting endophytic isolates on the seedling growth of four varieties of aerobic rice; MAS 145, MAS 946, MAS 99 and MAS 26 was studied. The seedling growth parameters like germination percentage, seedling length and vigour index was recorded and the results are presented in Table 32, Fig. 12. The germination percentage showed significant difference due to seed bacterisation with endophytic isolates compared to uninoculated control in all the varieties tested. There was 100 % germination in all varieties due to the cowpea isolate GME16 and tomato isolates LEE18 treatment and LEE19 recorded on par values at 90 % in MAS99 and MAS26. The consortia recorded slightly lesser germination in MAS 145, MAS 99 and MAS 26 (80 %, 80 % and 70 %) respectively but 100 % in MAS946. The least germination was in uninoculated control with only 70 % germination in all varieties except MAS946 which showed 90 % germination. The seedling length also showed a similar trend with the cowpea isolate GME16 being the best isolate. The highest seedling length was in MAS26 (15.3cm) with GME16 treatment followed by LEE18 and LEE19 which recorded on par values for MAS26. Uninoculated seedlings

79 Table 32. Comparison of the effect of plant growth promoting endophytic bacteria on the seedling growth of different varieties of aerobic rice Germination (%) Seedling length (cm) Vigour index Treatment MAS 145 MAS 946 MAS 99 MAS 26 MAS 145 MAS 946 MAS 99 MAS 26 MAS 145 MAS 946 MAS 99 MAS 26 GME (89.96) a (89.96) a (89.96) a 100 (89.96) a 11.1 a 11.2 a 12.6 a 15.3 a 910 a 1120 a 1134 a 1530 a LEE (89.96) a 100 (89.96) a 100 (89.96) a 100 (89.96) a 9.1 b 8.8 c 10.5 c 12.3 b 888 ab 880 c 1050 b 1230 b LEE (89.96) a 100 (89.96) a 90 (71.54) ab 90 (71.54) ab 8.7 c 9.3 b 11.5 b 12.2 b 870 b 930 b 920 c 1098 c Consortium 80 (63.41) b 100 (89.96) a 80 (63.41) bc 70 (56.77) b 7.0 d 7.8 d 9.5 d 11.6 c 700 c 780 d 760 d 812 d Control 70 (56.77) bc 90 (71.54) b 70 (56.77) c 70 (56.77) b 7.5 e 5.4 e 6.1 e 8.4 d 525 d 486 e 427 e 588 e SEd CD (0.05) Note: Observations recorded after 14 days. Each value is the mean of three replications. Different letters in superscripts indicate significantly different values. Arcsine transformed values are indicated in parantheses.

80 Vigour index Vigour index of aerobic rice varieties MAS 145 MAS 946 MAS 99 MAS GME16 LEE18 LEE19 Combination Control Treatments Fig. 12. Comparison of the effect of plant growth promoting endophytes on the seedling growth of different varieties of aerobic rice

81 recorded the least seedling length in all varieties ranging from 5.4cm in MAS946 to 8.4cm in MAS26. The vigour index of aerobic rice increased substantially due to endophyte inoculation and the cowpea isolate which the cowpea GME16 was found to be the best isolate in the case of all varieties with values ranging from 910 in MAS 145 to 1530 in MAS26. The next best isolate was LEE18 which recorded 888 in MAS145, 880 in MAS946, 1050 in MAS99 and 1230 in MAS26. This was on par with LEE19 in MAS145 and MAS946. Consortia of endophytes did not record more vigour index than individual inoculation and the values ranged from 700 in MAS145 to 812 in MAS26. The uninoculated control recorded the least vigour index in all varieties Pot culture studies to evaluate the plant growth promotion potential of endophytic bacteria on crop plants Effect of endophytic bacteria on seedling vigour of tomato var. Vaibhav. The germination percentage, seedling length and vigour index of tomato var. Vaibhav was recorded after 14 days of sowing and the results are presented in Table 33, Fig 14. The germination percentage increased due to seed bacterisation with endophyte to 100 % compared to uninoculated treatments where it was only 80 %. The treatment with the consortia resulted in a decrease in germination to 90 % than individual inoculation which recorded 100 %. In the case of seedling length T6 with the tomato isolate LEE18 and NPK recorded the best result (15.6 cm). 75 % NPK+LEE18 recorded 13.4 cm which was on par with T3 (13.3 cm), T4 (12.8cm), T5 (13.3 cm),

82 Table 33. Effect of endophytic isolates on seedling growth of tomato var. Vaibhav. Treatments Germination per cent Seedling length (cm) Vigour index T1- Control 80(63.41) c 8.4 j 672 k T2 NPK 80(63.41) c 10.2 i 816 j T3 3 org+ spray 90(71.54) b 13.3b bc 1197 de T4 NPK + 3org + spray 90(71.54) b 12.8 bcd 1152 f T5 - NPK + GME16 + spray 100(89.96) a 13.3b bc 1330 b T6 - NPK + LEE18 + spray 100(89.96) a 15.6 a 1560 a T7 - NPK + LEE19 + spray 100(89.96) a 12.3 cdef 1230 c T8 50% NPK + 3org + spray 90(71.54) b 13.2 bc 1188 e T9 50% NPK + GME16 + spray 100(89.96) a 10.3 hi 1030 i T10 50% NPK + LEE18 + spray 100(89.96) a 12.4 bcde 1240 c T11 50% NPK + LEE19 + spray 100(89.96) a 12.1 def 1210 d T12 75% NPK + 3org + spray 90(71.54) b 11.3 fgh 1130 g T13-75% NPK + GME16 + spray 100(89.96) a 11.6 efg 1160 f T14-75% NPK + LEE18 + spray 100(89.96) a 13.4 b 1340 b T15-75% NPK + LEE19 + spray 100(89.96) a 10.6 ghi 1060 h SEd CD (0.05) Note: Observations recorded after 14 days. Each value is the mean of three replications. Different letters in superscripts indicate significantly different values. Arcsine transformed values are indicated in parantheses.

83 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

84 Plant height (cm) 140 Plant height of tomato 30DAS 60DAS 90DAS 120DAS T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Fig. 13. Effect of endophytic isolates on the plant height of tomato var. Vaibhav

85 T8 (13.2 cm) and T10 (12.4 cm). The NPK control recorded 10.2 cm which was significantly lower than all inoculated treatments. Absolute control recorded the least seedling length (8.4 cm). Similar trend was noted in the case of vigour index. The best treatment was the treatment with NPK+ LEE18 which recorded a vigour index of 1560 followed by NPK+GME16 (1330) on par with 75 %NPK+LEE18 (1340). NPK+LEE19 (1230) and NPK+Consortia (1240) recorded on par values. The consortia of endophytes alone recorded 1197 which was on par with T11 (1210) and T8 (1188) while the NPK control recorded lower vigour index (816) and the least was in absolute control (672) Effect of endophytes on seedling parameters of tomato var. Vaibhav The shoot length, root length and seedling length were measured after 30 days at the time of transplanting and the results are presented in Table 34. The shoot length showed substantial increase due to the inoculation with endophytes and the highest was recorded in the treatment with the tomato isolate LEE18 (23.33 cm) with full NPK dose. This was followed by NPK+LEE19 (22.50 cm) and 75 % NPK+LEE18 (22.33cm), both treatments involving the tomato isolates LEE19 and LEE18 respectively. NPK+GME16 recorded 21.67cm and 75% NPK+LEE19 recorded 21 cm. The treatment with the consortia of endophytes alone recorded cm, while the NPK control showed 20.50cm and the least was in the absolute control cm. In the case of root length also treatment with NPK+ LEE18 recorded cm root length which was the highest followed by 75 % NPK+LEE18 (18.33 cm) and NPK+LEE19 (17.67 cm). 75 %NPK+LEE19

86 Table 34. Effect of endophytes on seedling parameters of tomato var. Vaibhav Treatments Shoot length (cm) Root length (cm) Seedling length (cm) T1- Control i i l T2 NPK ef g h T3 3 org+ spray j f k T4 NPK + 3org + spray f g f T5 - NPK + GME16 + spray c e d T6 - NPK + LEE18 + spray a a a T7 - NPK + LEE19 + spray b c c T8 50% NPK + 3org + spray i g i T9 50% NPK + GME16 + spray g g i T10 50% NPK + LEE18 + spray e f g T11 50% NPK + LEE19 + spray ef g j T12 75% NPK + 3org + spray h h h T13-75% NPK + GME16 + spray g g e T14-75% NPK + LEE18 + spray b b b T15-75% NPK + LEE19 + spray d d d SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

87 recorded 17cm and NPK+GME cm. The treatment with the consortia of endophytes alone recorded cm while NPK control recorded cm. The least was in the absolute control 12 cm. Similar trend was noted in seedling length with NPK+ LEE18 being the best treatment (44.00 cm) followed by 75 % NPK+LEE18 (40.83 cm) and NPK+LEE19 (38.17 cm). The next one was NPK+GME16 (37.83 cm) which was on par with 75 % NPK+LEE19 (37.66 cm) followed by 75 % NPK+GME16 (37.00 cm). The treatment with the consortia of endophytes alone recorded cm while NPK control recorded slightly higher seedling length (34.83 cm). The least was recorded in absolute control which recorded cm Effect of endophytic isolates on the plant height of tomato var. Vaibhav The plant height was recorded at monthly intervals viz. 30 days, 60 days, 90 days and 120 days and the results are presented in Table 35, Fig 13, Plate 10. At 30 days of sowing the highest plant height was recorded in the treatment with NPK+ LEE18 (23.33 cm) followed by NPK+LEE19 (922.5 cm) and 75 % NPK+LEE18 (22.33 cm) which were on par with each other. NPK+GME16 recorded cm and 75 % NPK+LEE19 recorded 21 cm after 30 days of sowing. NPK control recorded cm while the treatment with consortia recorded 16.33cm which was slightly better than absolute control (15.67 cm). After 60 days also same trend was followed with the highest plant height in treatment with NPK+ LEE18 (68 cm) followed by NPK+LEE19 (66.50 cm) and NPK+GME16 (65 cm) which was on par with 75 % NPK+LEE18 (64.50 cm). NPK control recorded the next best plant height of 63cm while the absolute control showed the least plant height of 45

88 Table 35. Effect of endophytic isolates on the plant height of tomato var. Vaibhav Plant height (cm) Treatments 30 days 60 days 90 days 120 days T1- control k m j k T2 NPK ef d c f T3 3 org+ spray j k i j T4 NPK + 3org + spray f f fg hi T5 - NPK + GME16 + spray c c a d T6 - NPK + LEE18 + spray a a a a T7 - NPK + LEE19 + spray b b b c T8 50% NPK + 3org + spray ef k e hi T9 50% NPK + GME16 + spray i l h g T10 50% NPK + LEE18 + spray e 58.67g 90.33c e T11 50% NPK + LEE19 + spray g i d g T12 75% NPK + 3org + spray h j ef h T13-75% NPK + GME16 + spray g h g i T14-75% NPK + LEE18 + spray b c b ab T15-75% NPK + LEE19 + spray d e c d SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

89 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

90 Yield (g) Dry weight (g) Vigour index Vigour index of tomato T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Dry matter production T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Yield T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Fig. 14. Effect of endophytic isolates on growth and yield of tomato var. Vaibhav

91 cm. The treatment with consortia alone, recorded 51.5 cm which was on par with T8 which involves the consortia along with 50 % NPK (52 cm). After 90 days there was significant difference in the plant height due to the treatment with endophytes. The highest was recorded in treatment with NPK+ LEE18 (98 cm) and NPK+GME16 (97.33 cm) followed by NPK+LEE19 (92.5 cm) and 75 % NPK+LEE18 (92 cm). NPK control, T15 and T10 were found to be on par recording cm and cm respectively cm was recorded in the treatment with consortia alone while the absolute control recorded the least plant height of 60 cm. At harvest (120days of sowing) the highest plant height was recorded in treatment with NPK+ LEE18 ( cm) which was found to be on par with 75 % NPK+LEE18 ( cm). NPK+LEE19 was the next best treatment with cm plant height. This was followed by 75 % NPK+LEE19 ( cm) which was on par with NPK control ( cm). Absolute control recorded 65 cm only while treatment with consortia alone recorded cm. Thus the treatment with LEE18 with full dose of NPK was found to be the best in case of plant height of tomato at all intervals Effect of endophytic isolates on the number of leaves and branches of tomato var. Vaibhav The growth parameters like no of leaves and no of branches per plant were recorded after 60days, 90 days and at harvest (120 days) of planting and the results are presented in Table 36. The no of leaves significantly increased due to the treatment with endophytes and the best treatment was found to be treatment with NPK+ LEE18. This treatment with the tomato isolate, LEE18 and full dose of NPK recorded 18.67, and leaves at 60, 90 and 120days after

92 Table 36. Effect of endophytic isolates on the number of leaves and branches of tomato var. Vaibhav Treatment No of leaves/ plant No of branches / plant 60days 90days 120 days 60days 90days 120days T1- Control 8.00 l m k f 2.33 f T2 NPK c d f c 3.67 c T3 3 org+ spray k j j f 3.00 e T4 NPK + 3org + spray i h g d 3.67 c T5 - NPK + GME16 + spray e c d c 4.33 b T6 - NPK + LEE18 + spray a a a a 4.50 a T7 - NPK + LEE19 + spray d b b b 4.33 b T8 50% NPK + 3org + spray l l h f 2.33 f T9 50% NPK + GME16 + spray j i e e 3.00 e T10 50% NPK + LEE18 + spray h e g e 3.33 d T11 50% NPK + LEE19 + spray g g d e 3.33 d T12 75% NPK + 3org + spray i k i d 3.00 e T13-75% NPK + GME16 + spray h g j c 3.67 c T14-75% NPK + LEE18 + spray b c c b 4.33 b T15-75% NPK + LEE19 + spray f f c c 4.33 b SEd NS CD (0.05) NS Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

93 sowing respectively. The least was recorded in absolute control (8, and 21 leaves). After 60 days of sowing the second best was 75 % NPK+LEE18 which recorded leaves followed by NPK control (16.67). NPK + LEE19 recorded while 50 % NPK + LEE18 recorded 16 leaves. The treatment with consortia alone had only 13 leaves. However after 90 days of sowing the NPK+LEE19 (26.33) was found to be the second best followed by 75 % NPK+LEE18 (24.33) and NPK+GME16 (25.5). NPK control recorded 24.67cm while the treatment with consortia was only Absolute control recorded leaves. At harvest after treatment with NPK+ LEE18 the next best treatment was NPK+LEE19 with 28 leaves. This was followed by 75 % NPK+LEE18 and 75 % NPK+LEE19 recording on par values (27 leaves). The NPK control recorded leaves. The treatment with consortia recorded 22 leaves which were slightly higher than the absolute control (21 leaves). The number of branches did not show any significant difference after 60 days of sowing as almost all the treatments recorded only 1 branch. After 90 days of sowing the branches increased and the highest number of 4.00 was recorded in treatment with NPK+ LEE18. This was followed by NPK+LEE19 and 75 % NPK+LEE18 which were on par (3.67). NPK control, T5, T13 and T15 recorded 3.33 branches. Treatment with consortia as well as absolute control recorded 2.33 branches. After 120 days also the same trend was noted with more number of branches in treatment with NPK+ LEE18 (4.5). This was followed by T5, T7, T14 and T15 (4.33). The treatment with consortia recorded 3 branches while the absolute control had 2.33 branches.

94 Effect of endophytic isolates on dry matter production of tomato var. Vaibhav The shoot dry weight, root dry weight and the drymatter per plant was recorded after harvest and the results are presented in Table 37, Fig 14, Plate 10. The shoot dry weight varied from g in absolute control to 88.1 g in LEE18 with full NPK dose. LEE18 with 75 % NPK recorded 87 g. Full dose of NPK with LEE19 (86.14 g) was found to be on par with full dose of NPK and GME16 (85.38 g). 75 % NPK+LEE19 was found to be on par with NPK control both recording 85 g shoot weight. The treatment with microbial consortia recorded slightly more shoot weight (74.70 g) than absolute control. Similar trend was noted in root dry weight also with treatment with NPK+ LEE18 recording the highest of 70 g. Full NPK with LEE19 and GME16 recorded on par values of 64 g which was on par with 75 % NPK with LEE % NPK with LEE19 was the next best (62.5 g) which was on par with NPK control (62.89 g) followed by 75 % NPK with GME16 (60.33 g). The treatment with the consortia alone recorded g which was slightly higher than the absolute control which recorded g. The plant drymatter production was highest in the treatment with LEE18 and full dose of NPK which recorded g. Full NPK with GME16 and full NPK with LEE19 recorded on par values (149 g and150 g). This was on par with 75 % NPK with LEE18 treatment ( g). 75 % NPK with LEE19 recorded 147.5g drymatter which was on par with full NPK with consortia of endophytes ( g). NPK control recorded g while absolute control recorded only g drymatter. The treatment with consortia alone performed better and recorded g.

95 Table 37. Effect of endophytic isolates on dry matter production of tomato var. Vaibhav Treatment Shoot dry weight (g) Root dry weight (g) Plant dry weight (g) T1- control k k k T2 NPK d c cd T3 3 org+ spray i j j T4 NPK + 3org + spray d d de T5 - NPK + GME16 + spray c b bc T6 - NPK + LEE18 + spray a a a T7 - NPK + LEE19 + spray c b b T8 50% NPK + 3org + spray j h j T9 50% NPK + GME16 + spray i g i T10 50% NPK + LEE18 + spray h f h T11 50% NPK + LEE19 + spray g i h T12 75% NPK + 3org + spray g e g T13-75% NPK + GME16 + spray f d f T14-75% NPK + LEE18 + spray b b b T15-75% NPK + LEE19 + spray d c d SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

96 Effect of endophytic isolates on yield of tomato var. Vaibhav The yield parameters like number of flowering clusters per plant, no of fruits per plant, individual fruit weight and yield per plant were recorded and the results are presented in Table 38, Fig 14, Plate 10. There was significant increase in the number of flowering clusters due to the treatment with endophytes. The highest number was obtained in treatment with NPK+ LEE18 (16) followed by NPK+LEE19 (14.67). NPK control, 75 % NPK+LEE18 and 75 % NPK+LEE19 recorded on par values (14, and 14 respectively). 75 % NPK+consortia and NPK+GME16 were on par while treatment with consortia alone recorded 8.67 flowers only. The absolute control had the least number of flowers (8.12). The highest number of fruits per plant was also recorded in treatment with NPK+ LEE18 (15.33) followed by NPK+LEE19 (15.00). T14 and T5 had 14 fruits which were on par with NPK control (13.85). T13 and T15 were on par with 13 fruits while the treatment with consortia recorded only 9 fruits and the absolute control had 7.33 fruits. The individual fruit weight did not show much difference among the treatments. NPK control, T4, T5, T6, T7, T12, T13, T14 and T15 recorded on par values (43+3.6). Absolute control (35.33 g) and treatment with consortia were found to be on par (37.50 g). T8, T9, T10, and T11 also were on par with this (38+3.6). The yield per plant showed significant increase due to the inoculation with endophytes. The treatment with the tomato isolate LEE18 with full dose of fertilizers showed the highest yield of g. The other tomato isolate LEE19 along with full dose of NPK was the next one with 675g. With 75 % NPK dose and the LEE18 isolate showed

97 Table 38. Effect of endophytic isolates on yield parameters of tomato var. Vaibhav Treatment No of flowering clusters/ plant No of fruits/ plant Individual fruit weight (g) Yield/ plant (g) T1- control 8.12 j 7.33 j c m T2 NPK c c ab e T3 3 org+ spray 8.67 i 9.00 i c l T4 NPK + 3org + spray e e ab f T5 - NPK + GME16 + spray d c a d T6 - NPK + LEE18 + spray a a a a T7 - NPK + LEE19 + spray b b a b T8 50% NPK + 3org + spray g 9.67 h c k T9 50% NPK + GME16 + spray g g c j T10 50% NPK + LEE18 + spray f e bc h T11 50% NPK + LEE19 + spray h f bc i T12 75% NPK + 3org + spray g f a g T13-75% NPK + GME16 + spray d d a f T14-75% NPK + LEE18 + spray c c a c T15-75% NPK + LEE19 + spray c d a f SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

98 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

99 Plant height (cm) Plant height of paddy 30DAS 60DAS 90DAS T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Fig.15. Effect of endophytic isolates on the plant height of paddy var. MAS99

100 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

101 A. General view of the pot culture experiment T2 T1 T6 T1 T2 At flowering stage (45 DAS) At fruiting stage (75DAS) B. Comparison of treatments T6 T1- Control T2 NPK T6 NPK + LEE18 T14-75% NPK+ LEE18 C. Yield of tomato Plate 10. Effect of endophytes on growth and yield of Tomato var. Vaibhav

102 significantly higher yield of 650g which was more than the yield recorded in the treatment with GME16 and full NPK dose (639 g). NPK control recorded 609 g while the absolute control had only 258 g. The treatment with the consortia alone also significantly increased the yield to g Effect of endophytic isolates on seedling vigour of paddy MAS99. The germination percentage, seedling length and vigour index of paddy was recorded 14 days after sowing and the results are presented in Table 39, Fig. 16. The germination percentage did not record significant difference among endophyte treatments. The absolute control, NPK control, and the treatments with consortia recorded 90 % germination while all other treatments recorded 100 %. In the case of seedling length, GME16+NPK recorded highest (19.33 cm). The treatment with NPK+ LEE18 and 75 %NPK+GME16 recorded on par seedling length (19 cm) followed by NPK+LEE19 (17.67 cm) and T9 (17.33 cm). 75 % NPK+LEE19 recorded cm which was on par with NPK control (16.37 cm). The treatment with consortia recorded 14.67cm and absolute control recorded cm. Seedling vigour recorded significant difference among the treatments and the maximum was recorded in NPK+GME16 (1933) followed by NPK+ LEE18 and 75 % NPK+GME16 (1900). This was followed by NPK+LEE19 (1767). 75 % NPK with LEE18 was on par with 50 % NPK and GME16 (1733). This was followed by 75 % NPK with LEE19 (1633). NPK control recorded while the treatment with consortia showed The least was in absolute control

103 Table 39. Effect of endophytic isolates on seedling growth of paddy var. MAS99 Treatment Germination (%) Seedling length (cm) Vigour index T1- Control 90(71.54) b j k T2 NPK 90(71.54) b e h T3 3 org+ spray 90(71.54) b g j T4 NPK + 3org + spray 90(71.54) b f 1395 i T5 - NPK + GME16 + spray 100(89.96) a a 1933 a T6 - NPK + LEE18 + spray 100(89.96) a b 1900 b T7 - NPK + LEE19 + spray 100(89.96) a c 1767 c T8 50% NPK + 3org + spray 90(71.54) b h 1260 j T9 50% NPK + GME16 + spray 100(89.96) a d 1733 d T10 50% NPK + LEE18 + spray 100(89.96) a f 1500 g T11 50% NPK + LEE19 + spray 100(89.96) a f 1533 f T12 75% NPK + 3org + spray 90(71.54) b g j T13-75% NPK + GME16 + spray 100(89.96) a b 1900 b T14-75% NPK + LEE18 + spray 100(89.96) a d 1733 d T15-75% NPK + LEE19 + spray 100(89.96) a e 1633 e SEd CD (0.05) Note: Observations recorded after 14 days. Each value is the mean of three replications. Different letters in superscripts indicate significantly different values. Arcsine transformed values are indicated in parantheses.

104 Effect of endophytic isolates on plant height of paddy var. MAS 99 The effect of endophytic isolates on plant height was recorded after 30 days, 60days and 90days of sowing and the results are presented in Table 40, Fig. 15, Plate 11. At all intervals the highest plant height was recorded in NPK+GME16 (44.00 cm, cm, cm) followed by treatment with NPK+ LEE18 (43.33 cm, cm, cm). NPK+LEE19 recorded cm, cm and cm respectively for 30 days, 60 days and 90 days. NPK control recorded (39.67 cm, cm and cm). The least plant height was in absolute control (31.34 cm, cm and cm). The treatment with consortia recorded cm, cm and cm at 30, 60 and 90 days of sowing Effect of endophytic isolates on number of leaves and tillers of paddy var. MAS 99 The effect of endophytic isolates on no of leaves and no of tillers of paddy is presented in Table 41. The number of leaves per plant was recorded at three intervals, 30, 60 and 90 days and results are presented in Table 43. The best treatment for the number of leaves was found to be NPK+GME16 (19.33, and 34.00) followed by NPK+ LEE18 (16.67, and 31.67) which was on par with 75 % NPK+GME16 (16.33, and 33.00). This was followed by 75 % NPK+LEE18 which recorded 15, 30 and leaves. The next good treatment was NPK+LEE19 which recorded 14.50, and leaves. The NPK control recorded 13.67, and 29 leaves while absolute control had only 8, 12 and 18 leaves respectively.

105 Table 40. Effect of endophytic isolates on plant height of paddy var. MAS99 Treatment Plant height (cm) 30 days 60 days 90 days T1- Control l l k T2 NPK e e d T3 3 org+ spray j k i T4 NPK + 3org + spray f c h T5 - NPK + GME16 + spray a a a T6 - NPK + LEE18 + spray b b b T7 - NPK + LEE19 + spray c d c T8 50% NPK + 3org + spray j j j T9 50% NPK + GME16 + spray g g h T10 50% NPK + LEE18 + spray i i i T11 50% NPK + LEE19 + spray f h h T12 75% NPK + 3org + spray e hi g T13-75% NPK + GME16 + spray d e e T14-75% NPK + LEE18 + spray i f f T15-75% NPK + LEE19 + spray h g f SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

106 Table 41. Effect of endophytic isolates on number of leaves and tillers of paddy var. MAS99 Treatment No of leaves/ plant No of tillers/ plant 30 days 60 days 90 days 30days 60days 90days T1- Control 8.00 k l l 2.00 g 3.00 f 3.00 h T2 NPK f i e 4.00 c 5.33 d 6.33 e T3 3 org+ spray 9.67 l k k 3.00 f 5.00 e 5.00 g T4 NPK + 3org + spray g e g 3.33 e 5.33 d 6.00 f T5 - NPK + GME16 + spray a a a 4.67 a 8.00 a 9.00 a T6 - NPK + LEE18 + spray c c c 4.33 b 6.00 c 7.33 b T7 - NPK + LEE19 + spray d d d 4.00 c 5.33 d 6.33 e T8 50% NPK + 3org + spray f j ij 3.33 e 6.00 c 6.00 f T9 50% NPK + GME16 + spray b i h 3.67 d 5.33 d 6.33 e T10 50% NPK + LEE18 + spray j g hi 3.33 e 5.00 e 6.00 f T11 50% NPK + LEE19 + spray i h jk 3.33 e 5.00 e 6.00 f T12 75% NPK + 3org + spray h j hi 3.67 d 5.00 e 5.00 g T13-75% NPK + GME16 + spray c b b 4.00 c 6.33 b 7.00 c T14-75% NPK + LEE18 + spray e e d 3.00 f 6.00 c 6.67 d T15-75% NPK + LEE19 + spray d f f 4.00 c 6.05 c 6.00 f SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

107 In the number of tillers per plant there was not much difference and after 30 days of planting the number varied from 3 in absolute control to 4 in the treatments. The best treatment was 4.67 recorded in NPK+GME16. By 60 days the number increased to 8 in this. Absolute control had only 3 tillers per plant after 60 days. After 90 days the number slightly increased to 9 in T5 but remained the same in absolute control. In NPK control the number of tillers was 4 after 30days which was on par with NPK+LEE19, 75 % NPK+GME16 and 75 % NPK+LEE18. It increased to 5.33 in NPK control and NPK+LEE19 after 60 days and 6.33 after 90 days. In 75 % NPK+GME16 and 75 % NPK+LEE18 it increased to 6.33 after 60 days and 7 and 6.67 respectively after 90 days. In the treatment with consortia alone the number of tillers was 3 after 30 days, and 5 after 60 and 90 days Effect of endophytic isolates on dry matter production of paddy MAS99 The shoot dry weight, root dry weight and plant dry matter was recorded at harvest and the results are presented in Table 42, Fig16, Plate 11. There was significant difference among the treatments in shoot dry weight and the highest was recorded in NPK+GME16 (932 g) while the uninoculated control recorded only 12.40g. The treatment with NPK and LEE18 recorded followed by the treatment with 75 % NPK and GME16 (29.10 g) which was on par with NPK treatment (29.3 g). The treatment with consortia alone recorded16.95 g. Similar trend was noted in root dry weight as well. T5 was the best treatment with 30 g roots. 75 % NPK with GME16 also recorded g. The NPK treatment recorded g while the consortia had 18 g. The least was in uninoculated control with a root mass of only 13 g.

108 Table 42. Effect of endophytic isolates on dry matter production of paddy var. MAS99 Treatment Shoot dry weight (g) Root dry weight (g) Plant dry matter (g) T1- control n k j T2 NPK c d c T3 3 org+ spray m j i T4 NPK + 3org + spray h f e T5 - NPK + GME16 + spray a a a T6 - NPK + LEE18 + spray b c b T7 - NPK + LEE19 + spray d c c T8 50% NPK + 3org + spray l i h T9 50% NPK + GME16 + spray i g f T10 50% NPK + LEE18 + spray j g f T11 50% NPK + LEE19 + spray k h g T12 75% NPK + 3org + spray g f e T13-75% NPK + GME16 + spray cd b b T14-75% NPK + LEE18 + spray e e d T15-75% NPK + LEE19 + spray f de d SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

109 The plant dry matter recorded the highest in GME16 + full dose of NPK (62 g). Full NPK with LEE18 and 75 % NPK with GME16 recorded the same dry matter of g. NPK control recorded 57 g while uninoculated control had only 25.4 g dry matter. The consortia recorded g dry matter Effect of endophytic isolates on yield parameters of paddy MAS99 The yield parameters of paddy like number of panicle, panicle, number of grains / panicle, panicle weight and yield / plant were recorded at harvest and the results are presented in Table 43, Fig. 16, Plate 11. The number of panicle per plant recorded significant difference among the treatments and the best was recorded in NPK+GME16 (7.67). The treatment with NPK+ LEE18 recorded 6.33 followed by NPK+LEE19 which had 5 panicles per plant. It was on par with 75 %NPK+GME16 (4.89). This was followed by 75 % NPK+LEE18 and T4 (4.33). NPK control had 4 panicles which were on par with T12 and T15 (4 panicles/ plant). T9, T10 and T11 had 3.67 panicles. The treatment with consortia had 3 panicles which were on par with T8. Absolute control recorded 2.33 panicle. There was no significant difference among the treatments in panicle length which varied from 22.00cm in absolute control to 23.00cm in endophyte treatments. The number of grains per panicle also did not record any significant difference with the least number of 98 in absolute control and maximum of 106 grains in the treatment with NPK+ GME16. The panicle weight did not record any significant difference among treatments. It varied slightly from 2.55g in NPK+GME16 to 2.35g in absolute control.

110 Table 43. Effect of endophytic isolates on yield parameters of paddy vvar. MAS99 Treatment No of panicle /plant Panicle length (cm) No of grains /panicle Panicle weight (g) Yield/ plant (g) T1- Control 2.33 h h T2 NPK 4.00 e de T3 3 org+ spray 3.00 g g T4 NPK + 3org + spray 4.33 d de T5 - NPK + GME16 + spray 7.67 a a T6 - NPK + LEE18 + spray 6.33 b b T7 - NPK + LEE19 + spray 5.00 c c T8 50% NPK + 3org + spray 3.00 g g T9 50% NPK + GME16 + spray 3.67 f f T10 50% NPK + LEE18 + spray 3.67 f f T11 50% NPK + LEE19 + spray 3.60 f f T12 75% NPK + 3org + spray 4.00 e e T13-75% NPK + GME16 + spray 4.89 c c T14-75% NPK + LEE18 + spray 4.33 d d T15-75% NPK + LEE19 + spray 4.00 e de SEd 0.05 NS NS NS 0.45 CD (0.05) 0.11 NS NS NS 0.92 Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

111 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

112 Yield (g) Dry matter (g) Vigour index 2500 Vigour index of paddy T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Dry matter production T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Yield T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Fig. 16. Effect of endophytic isolates on growth and yield of paddy var. MAS99

113 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

114 A. General view of the pot culture experiment T2 T5 T1 B. Comparison of treatments T1-Control T2 NPK T5 NPK+GME16 C. Yield of Paddy Plate11. Effect of endophytes on growth and yield of Paddy var. MAS99

115 However the yield per plant recorded significant difference among treatments. The best treatment for improving the yield of paddy was found to be NPK+GME16 which recorded g. This was followed by treatment with NPK+ LEE18 (15.83 g). NPK+LEE19 had yielded g which was on par with T13 (12.32 g). NPK control, T4, T14 and T15 recorded on par yield of 10 g while the treatment with 50 % NPK and endophytes recorded on par yield of 8 g. The treatment with consortia alone was on par with the treatment with 50 % NPK (7 g). Absolute control recorded the least yield of 5.48 g Effect of endophytic isolates on seedling vigour of cowpea var. KBC 2 The effect of endophytic isolates on germination percentage, seedling length and vigour index was recorded after 14 days of sowing and the results are presented in Table 44, Fig. 18. The germination percentage recorded for individual inoculation of seeds was 100 % while the treatments with consortia as well as uninoculated treatments recorded 90 % germination. The seedling length significantly differed from each other and the highest was found in NPK+GME16 (42.67cm) followed by 75 %NPK+GME16 (41.67cm). T4, T6 and T14 recorded on par values of 40cm. The treatment with consortia alone as well as T7 and T13 recorded on par values of 39cm. The NPK control recorded very less seedling length of 32.11cm and the absolute control only 23.67cm. The vigour index recorded significant difference among the treatments and highest was in NPK+GME16 (4267) followed by 75 % NPK+GME16 (4167). The treatment with NPK+ LEE18 and 75 %NPK+ LEE18 recorded on par values (4033 and 4025). NPK+LEE19 recorded 3950 and T13 recorded The treatment with consortia alone

116 Table 44. Effect of endophytic isolates on seedling vigour of cowpea var. KBC 2 Treatment Germination (%) Seedling length (cm) Vigour index T1- Control 90(71.54) b l T2 NPK 90(71.54) b j k T3 3 org+ spray 90(71.54) b d i T4 NPK + 3org + spray 90(71.54) b c h T5 - NPK + GME16 + spray 100(89.96) a a 4267 a T6 - NPK + LEE18 + spray 100(89.96) a c 4033 c T7 - NPK + LEE19 + spray 100(89.96) a d 3950 d T8 50% NPK + 3org + spray 90(71.54) b i j T9 50% NPK + GME16 + spray 100(89.96) a e 3833 f T10 50% NPK + LEE18 + spray 100(89.96) a f 3767 g T11 50% NPK + LEE19 + spray 100(89.96) a h 3633 h T12 75% NPK + 3org + spray 90(71.54) b d i T13-75% NPK + GME16 + spray 100(89.96) a b 4167 b T14-75% NPK + LEE18 + spray 100(89.96) a c 4025 c T15-75% NPK + LEE19 + spray 100(89.96) a g 3900 e SEd CD (0.05) Note: Arcsine transformed values are indicated in parantheses. Each value is the mean of three replications. The same alphabets on superscripts indicate on par values.

117 recorded 3539 while NPK control had only The absolute control recorded the least of Effect of endophytic isolates on plant height of cowpea var. KBC 2 The plant height was recorded at 30, 60, 90 and 120 days of planting and the results are presented in Table 45, Table 17, Plate 12. There was significant difference among the treatments in plant height. The best plant height was recorded at all intervals in NPK+GME16 (32.00 cm, cm, cm and cm). This was followed by NPK+ LEE18 (30.00 cm, cm, cm and cm). 75 % NPK+GME16 recorded cm after 30 days, cm after 60days, cm after 90 days and cm after 120days of sowing. NPK control recorded cm, cm, 185 cm and cm at harvest. The least plant height was in absolute control (13.45 cm, 62 cm, 105 cm and cm) Effect of endophytic isolates on the number of leaves and branches of cowpea var. KBC2 The effect of endophytes on number of leaves per plant and no of branches per plant was recorded 60 days, 90 days and at harvest (120 days) after sowing and the results are presented in Table 46. There was significant difference among the treatments in the case of number of leaves and the best treatment was found to be NPK+GME16 (20.67, and 45.00). This was followed by NPK+ LEE18 (16.33, and 42.00) which was on par with the NPK control which recorded 15.50, and leaves. NPK+LEE19 was the next which recorded after 60days, after 90days and after 120 days which was on par with T13 (15.5, and 39). The treatment with consortia

118 Table 45. Effect of endophytic isolates on plant height of cowpea var. KBC 2 Treatment Plant height (cm) 30 days 60 days 90 days 120days T1- control j k k m T2 NPK fg e 185 e f T3 3 org+ spray i j j l T4 NPK + 3org + spray c f f d T5 - NPK + GME16 + spray a a a a T6 - NPK + LEE18 + spray b b a b T7 - NPK + LEE19 + spray e d c b T8 50% NPK + 3org + spray h i 145 g k T9 50% NPK + GME16 + spray de g i h T10 50% NPK + LEE18 + spray f hi i i T11 50% NPK + LEE19 + spray fg h h k T12 75% NPK + 3org + spray g h f j T13-75% NPK + GME16 + spray b c b c T14-75% NPK + LEE18 + spray de d d e T15-75% NPK + LEE19 + spray d g e g SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

119 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

120 Plant height (cm) 400 Plant height of cowpea 30DAS 60DAS 90DAS 120DAS T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Fig. 17. Effect of endophytic isolates on the plant height of cowpea var. KBC 2

121 Table 46. Effect of endophytic isolates on number of leaves and branches of cowpea var. KBC2 Treatment No of leaves/ plant No of branches/ plant 60days 90days 120 days 60days 90days 120days T1- control 6.00 k j k f 2.33 f T2 NPK c a b f 3.00 e T3 3 org+ spray 9.00 j i j d 2.33 f T4 NPK + 3org + spray e b b e 3.00 e T5 - NPK + GME16 + spray a a a a 5.00 a T6 - NPK + LEE18 + spray b b b c 4.00 b T7 - NPK + LEE19 + spray d c c c 3.33 d T8 50% NPK + 3org + spray g g j f 3.00 e T9 50% NPK + GME16 + spray f f f d 3.00 e T10 50% NPK + LEE18 + spray h h g f 2.33 f T11 50% NPK + LEE19 + spray i j i f 2.33 f T12 75% NPK + 3org + spray f e h f 3.33 d T13-75% NPK + GME16 + spray c d c b 3.67 c T14-75% NPK + LEE18 + spray e d e c 3.33 d T15-75% NPK + LEE19 + spray f e d e 3.00 e SEd NS CD (0.05) NS Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

122 alone had very less number of leaves per plant (9.00, and 18.33) while the least was recorded in absolute control (6.00, and12.67). The number branches was found to be non significant after 60days of sowing with only 1 branch in the treatments. After 90 days NPK+GME16 had 4 branches while NPK+LEE18, NPK+LEE19 and 75 % NPK+LEE18 had 3 branches. Absolute control, NPK control, as well as the treatment with consortia alone recorded 2 branches. After 120 days also the same trend was followed with the highest number of branches (5) in NPK+GME16 followed by NPK+ LEE18 (4) and 75 % NPK+GME16 (3.67). NPK control, T4, T8, T9 and T15 had 3 branches while absolute control had only 2.33 branches at harvest Effect of endophytic isolates on dry matter production of cowpea KBC2 The shoot dry weight, root dry weight and plant drymatter was recorded at harvest and the results are presented in Table 47, Fig. 18, Plate 12. There was significant increase in shoot dry weight due to endophyte treatment and the highest was recorded in NPK+GME16 (90.33 g). This was followed by NPK+ LEE18 (86 g) which was on par with 75 % NPK+GME16 (84.3 g). 75 % NPK + LEE18 recorded 79 g and T7 recorded 75 g shoot weight. NPK control recorded 62 g. The treatment with consortia recorded 47 g which was on par with absolute control (46 g). Similar trend was followed in root weight also. The best treatments were 75 % NPK+GME16 and NPK+GME16 (54 g) which was on par with 75 % NPK+LEE18 (52 g). 51g was recorded in NPK+ LEE18 and 50 g in NPK+LEE19. The treatment with consortia alone recorded 42

123 Table 47. Effect of endophytic isolates on dry matter production of cowpea var. KBC2 Treatment Shoot dry weight (g) Root dry weight (g) Plant dry weight (g) T1- Control l m m T2 NPK g g h T3 3 org+ spray kl k l T4 NPK + 3org + spray f j g T5 - NPK + GME16 + spray a ab a T6 - NPK + LEE18 + spray b c bc T7 - NPK + LEE19 + spray d d e T8 50% NPK + 3org + spray j l k T9 50% NPK + GME16 + spray i e i T10 50% NPK + LEE18 + spray i h j T11 50% NPK + LEE19 + spray k i k T12 75% NPK + 3org + spray h f h T13-75% NPK + GME16 + spray b a b T14-75% NPK + LEE18 + spray c b d T15-75% NPK + LEE19 + spray e d f SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

124 g root dry weight. NPK control recorded 46 g, while absolute control had only 38 g. The plant dry matter production was significantly different among the treatments with NPK+GME16 being the best, which recorded g. This was followed by 75 % NPK+GME16 (138 g) and NPK+ LEE18 (137 g) which were on par. 75 % NPK+LEE18 recorded 132 g and T7 had 125 g plant drymatter. The treatment with the consortia alone recorded 89 g while NPK control had g. The least dry matter production was in absolute control which recorded only g Effect of endophytic isolates on yield parameters of cowpea KBC2 The yield parameters like number of flowering clusters/plant, number of fruits/ plant, fruit length, pod weight, number of seeds/pod and yield/ plant were recorded at harvest and the results are presented in Table 48, Fig. 18, Plate 12. The highest number of flowering clusters was recorded in NPK+GME16 (5.67) followed by 75 % NPK+GME16 (5.33). The treatment with NPK+ LEE18, NPK+LEE19 and 75 % NPK+LEE18 recorded on par values (5) followed by 75 % NPK+LEE19 (4.67). NPK control was on par with T4 and T10 with 4.33 flowering clusters. The treatment with the consortia alone recorded 4 flowering clusters. Absolute control had only 3.67 flowers. There was significant difference in the number of fruits per plant and the maximum number of fruits was in NPK+GME16 (15) followed by 75 % NPK+GME16 (14.50). The treatment with NPK+ LEE18 had fruits followed by 75 % NPK+LEE18 with 13 fruits. T5 had fruits and T7 12 fruits. The NPK control recorded fruits while absolute

125 Table 48. Effect of endophytic isolates on yield of cowpea var.kbc2 Treatment No of flowering clusters/plant No of fruits/ plant Fruit length (cm) Pod weight (g) No of seeds/pod Yield/ plant (g) T1- Control 3.67 h 6.89 j l i i l T2 NPK 4.33 f d f g e d T3 3 org+ spray 4.00 g 8.50 i i i g k T4 NPK + 3org + spray 4.33 f f g e e e T5 - NPK + GME16 + spray 5.67 a a a a a a T6 - NPK + LEE18 + spray 5.00 c c c c b b T7 - NPK + LEE19 + spray 5.00 c f b d a e T8 50% NPK + 3org + spray 4.00 g h k h h i T9 50% NPK + GME16 + spray 4.50 e f h e f g T10 50% NPK + LEE18 + spray 4.33 f h j f g i T11 50% NPK + LEE19 + spray 4.00 g h l h h j T12 75% NPK + 3org + spray 4.00 g g e gh d h T13-75% NPK + GME16 + spray 5.33 b b d 4.50 d c d T14-75% NPK + LEE18 + spray 5.00 c d e e d d T15-75% NPK + LEE19 + spray 4.67 d e e e d d SEd CD (0.05) Note: Each value is the mean of three replications. Different letters in superscripts indicate significantly different values.

126 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

127 Yield (g) Dry matter (g) Vigour index Vigour index of cowpea T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Dry matter production T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Yield T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Treatments Fig. 18. Effect of endophytic isolates on growth and yield of cowpea var. KBC 2

128 Treatment details T1- Control T2 NPK T3 3 org + spray T4 NPK + 3org + spray T5 - NPK + GME16 + spray T6 - NPK + LEE18 + spray T7 - NPK + LEE19 + spray T8 50% NPK + 3org + spray T9 50% NPK + GME16 + spray T10 50% NPK + LEE18 + spray T11 50% NPK + LEE19 + spray T12 75% NPK + 3org + spray T13-75% NPK + GME16 + spray T14-75% NPK + LEE18 + spray T15-75% NPK + LEE19 + spray

129 A. General view of the pot culture experiment T5 T4 T3 T2 T1 T5 T2 B. Comparison of growth T1 T2 T5 C. Drymatter production at harvest Plate 12. Effect of endophytes on the growth and yield of cowpea var. KBC2