Browsing by Author "Sreeja, S J"

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    Bioefficacy of endophytic actinomycetes on plant growth promotion and management of bacterial wilt in tomato.
    (Department of Plant Pathology, College of Horticulture, Vellanikkara, 2011) Sreeja, S J; Surendra Gopal, K
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    Endophytic bacillus consortium formulation for plant growth promotion and management of chilli anthracnose
    (Department of Plant Pathology, College of Agriculture, Vellayani, 2025) Renjini, R S.; Sreeja, S J
    The present study entitled “Endophytic Bacillus consortium formulation for plant growth promotion and management of chilli anthracnose” was carried out at the College of Agriculture, Vellayani (2022–2024) to develop and evaluate effective formulations of endophytic Bacillus consortium for improving plant growth and managing anthracnose disease in chilli (Capsicum annuum L.). Thirteen compatible endophytic Bacillus isolates, obtained through previous studies conducted at the Department of Plant Pathology were assessed for their antagonistic activity against Colletotrichum capsici, the causal agent of chilli anthracnose. Among the isolates tested, CFLE3, WR2, WL2, and WR1 displayed strong antagonistic activity in both dual culture and culture filtrate assays. The roll towel method employed to evaluate seed germination and seedling vigour indicated that biopriming with CFLE3, WR1, WR2, and WL2 significantly improved seed germination rates, seedling growth, and biomass accumulation, while treatments with PNRE5, GR2, and NAT001 performed poorly and were comparable to the untreated control in all growth parameters. Based on their dual efficacy in antagonism and plant growth promotion, ten promising isolates viz., PNRE4, PNSE5, AR2, GR1, WR1, WR2, WL2, CBSE5, CBRE5, and CFLE3, were combined to formulate a microbial consortium. This consortium was developed into both solid and liquid bioformulations using talc, plain nutrient broth (NB), and nutrient broth amended with additives such as 2% glycerol (NB-GLY), 2% polyvinylpyrrolidone (NB-PVP), and 15 mM trehalose (NB-TREH). The population dynamics of the endophytic Bacillus consortium in different formulations were monitored over six months. While initial populations were comparable across treatments, NB-GLY maintained the highest bacterial counts throughout the storage period, followed by NB-TREH. In contrast, talc and unamended NB showed marked population decline. The in vitro efficacy of the formulations was assessed over six months using detached leaf and fruit assays, as 124 well as pro tray studies. Over the six-month evaluation period, the NB-TREH and NB-GLY formulations consistently demonstrated effective suppression of lesion progression in both detached chilli leaves and fruits artificially inoculated with Colletotrichum capsici. Seed priming with NB-TREH and NB-GLY also led to consistent improvements in all seedling growth parameters, including faster germination, higher germination percentages, increased shoot and root lengths, and enhanced fresh and dry biomass. Based on their superior performance, NB-TREH and NB-GLY were selected for in vivo evaluation of formulation of endophytic Bacillus consortium for promoting plant growth and managing anthracnose in chilli. In pot culture studies, the liquid Bacillus formulations NB-GLY and NB-TREH consistently suppressed anthracnose in chilli leaves, showing significantly reduced lesion progression (0.26–2.23 cm) and disease severity (18.12–65.62) compared to untreated and singly amended treatments. Similarly, in fruit assays, NB-GLY and NB-TREH effectively limited lesion development from 0.47 to 2.76 cm and maintained lower fruit rot severity (20.20–72.70) across all observation periods. These two bioformulations performed on par with or better than the chemical fungicide, while glycerol, trehalose alone, and the inoculated control showed poor disease suppression and complete tissue rotting by 10–15 DAI. Subsequent pot culture experiment for plant growth promotion, validated these findings, showing significant enhancement in plant growth and yield with NB-GLY and NBTREH formulations. NB-GLY-treated plants attained the greatest height (46.60 cm), earliest flowering (30.80 days), and longest shoots and roots (49.78 cm and 26.25 cm). Biomass accumulation was also highest in NB-GLY, with shoot and root fresh weights of 49.63 g and 21.28 g, and dry weights of 13.30 g and 5.70 g, respectively. Yield data further reinforced the superior performance of these formulations, with NB-TREH and NB-GLY yielding the highest fruit numbers (24.49 and 23.00) and fruit yield per plant (290.57 g and 294.98 g). Endophytic re-isolation from harvested chilli seeds confirmed the persistence of the endophytic Bacillus, with NB-GLY supporting the highest Bacillus population (1.14 × 10⁷ cfu g⁻¹), indicating successful colonization and 125 long-term survival of the inoculated bacteria. In conclusion, liquid formulation of endophytic Bacillus consortium amended with 2 % glycerol (NB-GLY) proved to be effective, eco-friendly alternatives to chemical fungicides by supporting microbial viability, suppressing anthracnose, promoting growth, and enhancing yield in chilli.
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    Endophytic bacteria for plant growth promotion and management of web blight of cowpea
    (Department of Plant Pathology, College of Agriculture , Vellayani, 2021-11-29) Siva, M.; Sreeja, S J
    The study entitled “Endophytic bacteria for plant growth promotion and management of web blight of cowpea” was conducted in Department of Plant Pathology, College of Agriculture, Vellayani during 2019-2021 with the objective to evaluate endophytic bacteria inplant growth promotion and management of web blight of cowpea. Endophytic bacteria were isolated from healthy root, stem and leaves of fodder cowpea var. Aiswarya (22 isolates) and bush cowpea var. Bhagyalakshmi (16 isolates) from Instructional farm, COA Vellayani and farmer’s field at Naruvamoodu respectively. The isolates were purified and maintained for further studies. The pathogen Rhizoctonia solani causing web blight of cowpea was isolated from infected plant samples and pathogenicity tests were carried out. The isolates of endophytic bacteria were screened against R.solani by testing their antagonistic properties adopting dual culture technique. The results revealed that isolate CBRE5 (9.33 mm) showed maximum zone of inhibition against R. solani which was on par with CBRE1 (8.83 mm) followed by CBSE5 (8.33 mm). The mycelial growth inhibition of R. solani was recorded maximum (48.52 %) by the isolate CBRE5 followed by CBRE1 (42.22 %) which was on par with CBSE5 (41.85 %). The culture filtrate obtained from the isolates CFLE3 and CBRE5 recorded maximum zone of inhibition against R. solani. Based on the results of direct and indirect antagonism of endophytic bacteria, 20 isolates were selected as promising ones for biopriming of cowpea seeds. Surface sterilized cowpea seeds were bio-primed with selected bacterial endophytes at 108 cfu/ ml using roll towel assay. The cowpea seedlings treated with isolate CFLE3 recorded the maximum seedling vigour index – I and II (4087.33 and 7.66 respectively) which was on par with CBRE5 (3598.00 and 6.73 respectively) followed by CBSE5 (3452.33 and 6.70 respectively). Based on the results of roll towel assay the isolates CFLE3, CBRE5 and CBSE5 were selected as the best three bacterial endophytes for further studies. The promising endophytic bacterial isolates were tentatively identified as Bacillus through morphological, cultural and biochemical characters. The identities of the species of these isolates were further confirmed through 16S rRNA analyses as Bacillus subtilis(CFLE3), B. amyloliquefaciens (CBRE5) and B. velezensis (CBSE5). The compatibility studiesamong the best three bacterial endophytes following disc diffusion method and cross streak plate assay indicated that the isolates were compatible among themselves. In pot culture experiment, seed biopriming for 4 h along with foliar application (20 and 40 DAS) of B. velezensis CBRE5recorded minimum percent disease index (PDI) (32.33) on 14 DAI which was on par with the treatment with B. amyloliquefaciens CBSE5 (PDI- 39.33) followed by treatment combination with B. amyloliquefaciens CBSE5 and B. velezensis CBRE5 (PDI – 41.33). In vivo evaluation of best three endophytic bacterial isolates and their combinations for plant growth promotion indicated that combined seed biopriming for 4 h along with foliar application of B. amyloliquefaciens CBSE5 and B. velezensis CBRE5 (20 and 40 DAS) recorded maximum values for all growth parameters viz. shoot length (47.40 cm), root length (40.93 cm), shoot fresh weight (139.16 g), root fresh weight (49.33 g), shoot dry weight (27.83 g), root dry weight (6.33 g), pod yield (236.66 g) and number of pods per plant (36). However, the plants treated with isolate B. amyloliquefaciens CBSE5 exhibited early flowering (33 DAS). The best three endophytic bacterial isolates were evaluated for their plant growth promoting traits such as production of indole acetic acid (IAA), ammonia, phosphate, siderophore and HCN. B. amyloliquefaciens CBSE5 recorded highest IAA (3.54 μg/ml) and siderophore (zone of colouration – 6 mm) production. Whereas, the isolate B. velezensis CBRE5 showed highest ammonia (177.29 μmol/ml) and phosphate (zone solubilization - 2mm) production. Thus, the present study indicated that seed biopriming for 4 h along with foliar application (20 and 40 DAS) of B. velezensis CBRE5 was most effective treatment for the management of web blight of cowpea whereas combined seed biopriming for 4 h along with foliar application (20 and 40 DAS) of B. amyloliquefaciens CBSE5 and B. velezensis CBRE5 was the best treatment in plant growth promotion in vivo which could be further validated under field conditions for ecofriendly and sustainable safe – to – eat crop production.
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    Management of fusarium wilt of vegetable cowpea with Consortia of Endophytic Bacilli
    (Department of Plant Pathology, College of Agriculture, Vellayani, 2024) Anuvinda, A R.; Sreeja, S J
    The study entitled “Management of Fusarium wilt of vegetable cowpea with consortia of endophytic Bacilli” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2021-2023 with the objective to evaluate the consortia of endophytic Bacilli for the management of Fusarium wilt of vegetable cowpea. Twenty endophytic Bacillus isolates, sourced from prior studies conducted at the Department of Plant Pathology and the Department of Agricultural Microbiology, were used for the current study. The antagonistic potential of all endophytic Bacillus isolates against Fusarium oxysporum was evaluated through both dual culture plate assay and culture filtrate assay. Out of the 20 isolates, 14 exhibited antagonistic activity against the pathogen. The isolate WL1 stood out, displaying maximum inhibition zone of 7.00 mm against Fusarium oxysporum. Meanwhile, the isolate GR1 achieved the maximum mycelial growth inhibition (30.62%) against F. oxysporum. Culture filtrate of the isolates PCSE8, PCSE10, PNRE4, AR2, AR1, GR1, GR2 and CFLE3 also had inhibitory action against F. oxysporum, indicating antagonism by diffusible metabolites. The findings from direct and indirect antagonism indicated that isolates PNRE5, NAT001, WR2, WR1, VLY17, and PNSE5 did not exhibit antagonistic properties against the wilt pathogen in cowpea. The endophytic Bacillus isolates underwent assessment for various plant growth promoting (PGP) traits, including nitrogen fixation, phosphate solubilization, potassium solubilization, IAA and gibberellic acid production, siderophore production, ACC deaminase activity, ammonia and HCN production, as well as the release of volatile organic compounds. Among the 20 isolates, ten demonstrated nitrogen-fixing capabilities, while 15 exhibited phosphorus solubilizing activity. None of the isolates displayed potassium solubilization, as evidenced by the absence of a clearance zone in Aleksandrov medium. All tested bacterial isolates produced IAA, ranging from 14.64 μg mL⁻¹ to 0.08 μg mL⁻¹. However, none of the isolates demonstrated gibberellic acid (GA) production. Regarding ACC deaminase activity, nine isolates were able to produce α ketobutyrate within the range of 1.35 to 8.303 α-KB μmol. With the exception of four isolates, all tested ones produced siderophore, with the maximum production recorded for CBRE5 at 68.92 μg/ml. Among the 20 isolates, 12 exhibited ammonia production within the range of 5.18 to 0.234 μmol/ml and 11 isolates produced HCN. All isolates tested positive for the production of volatile organic compounds, with PCSE10 demonstrating the maximum mycelial growth inhibition (46.93%) against Fusarium sp. due to the volatile compounds emitted. In the protray growth experiment, there was a significant improvement in all the growth parameters of the seedlings treated with endophytic Bacillus isolates PNRE4, NAT001, AR1, WR2, VLY17, WL1 and PNSE5. Seedling vigour index I values ranged from 3444.80 (AR1) to 1191.00 (GL3) while seedling vigour index II ranged from 92.32 (AR1) to 30.00 (GL3). The compatibility studies among the endophytic Bacillus isolates following disc diffusion method and spot on lawn method indicated that isolates PNRE5, PNRE4, NAT001, AR2, GR1, GR2, WR2, WR1, WL2, PNSE5, CBRE5, CBSE5 and CFLE3 were compatible among themselves. Based on the results of in vitro experiments, eight compatible antagonistic endophytic Bacillus isolates viz., PNRE4, AR2, GR1, GR2, WL2, CBRE5, CBSE5 and CFLE3 with superior plant growth promoting traits and seedling vigour index were pooled together to form Consortium I. All the 20 endophytic Bacillus isolates selected for the study were pooled together to form Consortium II. Seed biopriming for four hours along with soil application of Consortium 1 thrice (@ 20, 40 and 60 DAS) recorded only 33.33% wilt incidence while, the similar mode of application of Consortium II recorded 50 % disease incidence in vivo. The results of field evaluation revealed that seed biopriming for four hours along with soil application of Consortium 1 thrice (@ 20, 40 and 60 DAS) recorded 28% wilt incidence while, the similar mode of application of Consortium II recorded 32 % wilt incidence. No significant difference between Consortium I and II was observed on days taken for flowering, number of pods/plants, pod yield/plant, shoot fresh and dry weight except root fresh and dry weight. The present study emphasizes the importance of assessing the compatibility of strains while formulating a microbial consortium. Therefore, it can be inferred that inoculating cowpea plants with a consortium of compatible antagonistic endophytic Bacillus isolates possessing superior plant growth-promoting traits and seedling vigor index substantially mitigated the occurrence of Fusarium wilt in cowpea. Additionally, it facilitated the improved establishment of plants by enhancing overall plant growth