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Open Access
Plant growth promoting actinobacteria from rhizosphere soils of Black pepper in Wayanad
(Department of Agricultural Microbiology, College of Agriculture, Vellanikkara, 2021) Rineesha Backer, A; KAU; Girija, D
Black pepper (Piper nigrum L.) known as the ‘King of spices’ is one of the important export-oriented spice crops that provides major source of income and employment for rural households in Kerala. Wayanad is one of the main pepper growing tracts in Kerala. However, the production of black pepper in Wayanad has been declining over the past many years, mainly due to the poor soil health status, improper land management and changes in climatic factors. An increasing demand for low-input agriculture has resulted in a greater interest in soil microorganisms which are able to enhance plant health and soil quality. However, actinobacteria have not yet been exploited for this purpose. Hence the present study focused on isolation, screening and characterization of actinobacteria from rhizosphere soils of black pepper in Wayanad and evaluation of their plant growth promoting activity. Rhizosphere soil samples were collected from five different locations of flood affected and non-flood affected black pepper growing areas of Wayanad district. Enumeration of actinobacteria on three different media revealed that the population of actinobacteria ranged from 1.00 x104 cfu to 12.00 x104 cfu g -1 soil in non-flooded soil. Actinobacteria could not be detected in flooded soils, even at a dilution of 10-1 , except in Meppadi. Among the different media, starch casein agar recorded higher population of actinobacteria. A total of 35 isolates were purified and maintained for characterization and screening for plant growth promoting activities. Cultural, morphological and biochemical characters of all 35 isolates of actinobacteria were studied. All the isolates were Gram positive, and they varied in the colony morphology, colour of aerial mycelium, spore chain morphology and pigmentation. Two isolates Vlt-K and Plp-B produced yellow water-soluble pigment and Vlt-I produce purple water insoluble pigment. Screening of all the 35 isolates under in vitro conditions for plant growth promoting (PGP) activities revealed that the isolates were highly variable. Two isolates (Ptd-A and Amb-C) were superior to other isolates in IAA production. Significantly higher N-fixation was noticed in four isolates (Ptd-A, Ptd-E, Ptd-B and Ptr-A). Six isolates solubilized insoluble phosphate to available P, and also reduced the pH of the medium. Reduction in pH by phosphate solubilizers has been reported earlier and this is attributed to the production of organic and inorganic acids. None of the isolates solubilized K or Zn. Three isolates with multiple PGP activities were selected, based on ranking for PGP activities in vitro and identified by 16S rRNA gene sequencing (Ptd-A and Ptr-A as Streptomyces sp. and Ptd-E as Actinobacteria bacterium). These actinobacterial isolates evaluated in-planta for PGP activities with black pepper cuttings (variety Panniyur-1). Treatments also included PGPR mix-1 (KAU commercial formulation) and Organic Package of Practices Recommendations (2017). All the three native isolates exhibited significant increase in shoot length, number of leaves and internode length throughout the growth period from planting to five months. Significantly higher root growth was observed in treatment T2: Ptd-A (Streptomyces sp.), with significantly higher root volume, fresh and dry root weight. Significantly higher plant fresh weight was observed in T2: Ptd-A (Streptomyces sp.) and significantly higher plant dry weight was observed in T2: Ptd-A (Streptomyces sp.) and T3: Ptr-A (Streptomyces sp). The present investigation revealed that native actinobacteria have the potential to improve the growth of black pepper. These isolates may further be evaluated under field conditions before commercialization. Screening for other beneficial traits like disease suppression and drought tolerance may also be carried out. The compatibility of the actinobacteria with other PGP microorganisms may also be evaluated. Actinobacterial spores survive in soil for longer periods and this may be an added advantage. Actinobacterial diversity and the biochemical molecules produced by actinobacteria could be exploited to build up sustainable and eco-friendly agriculture.
Open Access
Comparative assessment of climate resilient capacity of two indigenous goat breeds based on changes in both phenotypic and genotypic traits
(Academy of Climate Change Education and Research, Vellanikkara, 2020) Reshma Nair, M R; KAU; Sejian, V
Open Access
Exopolysaccharide producing bacteria from soil based nesting structures of insects
(Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2022-03-14) Sruthi, Suresh; KAU; Chitra, N
The study entitled ―Exoploysaccharide producing bacteria from soil based nesting structures of insects‖ was conducted during the year 2019-2021 in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram with the objective to isolate and characterize exopolysaccharide producing bacteria from mud wasp and termitaria and the ability of the best two isolates in soil aggregate stability study. Thirty three bacterial isolates were obtained from different termitaria and mud dauber wasp nest. Among them colonies of fifteen isolates had slimy mucoidal appearance and were identified as exopolysaccharide producers. The exopolysaccharide production of the isolates ranged from 25.78 µg mL-1 (Klebsiella pneumoniae KWP23) to 2.58 µg mL-1 . The total carbohydrate content in exopolysaccharide was maximum in bacterial isolate Paenibacillus polymyxa KTM17 (14.48 µg mL-1 ). The best five isolates were selected for molecular characterization by 16s rRNA sequencing and identified as Priestia aryabhattai TWP12, Priestia aryabhattai TWP13, Klebsiella pneumoniae KWP23, Priestia megaterium KTM4, Paenibacillus polymyxa KTM17. They were screened for phosphate, potassium and silicate solubilization. Klebsiella pneumoniae KWP23 showed solubilization zone for phosphate (4 mm) and potassium (1 mm). None of the isolates chosen were silicate solubilizers. Two bacterial isolates with maximum EPS production viz. Paenibacillus polymyxa KTM17 and Klebsiella pneumoniae KWP23 were selected for the further studies. Effect of carbon source and carbon: nitrogen ratio on exopolysaccharide production was studied. Glucose was found to be the best carbon source for EPS production in Klebsiella pneumoniae KWP23 at C: N ratio of 10:1. In Paenibacillus polymyxa KTM17, highest EPS production was recorded when sucrose was used as carbon source at a C: N ratio of 50:1. The effect of temperature on EPS production of the isolates was evaluated. Increase in incubation temperature resulted in a corresponding increase in EPS production in case of Klebsiella pneumoniae KWP23. The highest EPS 3 production by Klebsiella pneumoniae KWP23 was (23.00 µg mL-1 ) at 50oC and Paenibacillus polymyxa KTM17 at 30oC (31.50 µg mL-1 ). The effect of salinity on EPS production by Klebsiella pneumoniae KWP23 and Paenibacillus polymyxa KTM17 isolates was also assessed. For Klebsiella pneumoniae KWP23, the EPS production was maximum without NaCl (18.0 µg mL-1 ) and for Paenibacillus polymyxa KTM17, the EPS production was maximum at 6% (1 M) NaCl (26.25 µg mL-1 ). Klebsiella pneumoniae KWP23 and Paenibacillus polymyxa KTM17 were further subjected to dual culture plate assay to study the biocontrol potential of the isolates. Paenibacillus polymyxa KTM17 showed a clear inhibition zone on dual culture assay with Rhizoctonia solani, but Klebsiella pneumoniae KWP23 did not inhibit the growth of the fungus. Both Klebsiella pneumoniae KWP23 and Paenibacillus polymyxa KTM17 inhibited the growth of Helminthosporium sp. The potential of the isolates to form soil aggregates and its aggregate stability was studied. The mean weight diameter of dry aggregates obtained was highest for the soil amended with Paenibacillus polymyxa KTM17 (1.068 mm). The study on wet aggregate stability on water immersed revealed that the best soil aggregate stability was given by the isolate Paenibacillus polymyxa KTM17. The stability of the aggregate lasted for more than 24 hours when immersed in water whereas that in the aggregate kept as control disintegrated immediately on pouring water. The effect of agitation and water immersion on the aggregate stability was also analyzed. On oscillation of the aggregates in water the aggregates in control soil showed maximum turbidity (0.254 nm). The dry weight of the disintegrated soil was minimum in the soil aggregate amended with Paenibacillus polymyxa KTM17 (0.014 g). Based on the results of the present study, it can be concluded that among the selected isolates Klebsiella pneumoniae KWP23 had maximum production of EPS. Both Klebsiella pneumoniae KWP23 and Paenibacillus polymyxa KTM17 has biocontrol potential against plant pathogenic fungi. Paenibacillus polymyxa KTM17 is a potential isolate which can effect soil aggregate stability in dry and wet condition.
Open Access
Isolation and characterization of beneficial rhizosphere microorganisms from ragi grown in Attappady hill tract of Kerala
(Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2021) Gayathri, M; KAU; Chitra, N
The study entitled “Isolation and characterization of beneficial rhizosphere microorganisms from ragi grown in Attappady hill tract of Kerala” was conducted during the year 2019-2021 in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram with the objective to isolate nitrogen fixing and phosphate solubilizing bacteria from soil and root samples collected from Attapady and select efficient isolates through in vitro screening process and assess the plant growth promotion activity by roll towel assay. Thirty six nitrogen fixing bacteria and eight phosphate solubilizing bacteria were isolated from root and soil samples from ragi grown in Attappady region. The isolates were designated as ATY1-ATY36 for NFB isolates and PSB1-PSB8 for PSB isolates. Twenty isolates were selected after eliminating isolates with similarities in colony morphological characters, in order to avoid repetition among the isolates obtained from the same sample. Both NFB and PSB isolates were characterized by morphology and biochemical tests. All selected isolates were subjected to plant growth promotion activity studies. The Indole Acetic Acid (IAA) production showed a wide range of variation from 101.22 µg mL-1 to 3.26 µg mL-1 . Gibberellic Acid (GA) production of all the twenty isolates were done and the results ranged between 10.07 µg mL-1 and 2.18 µg mL-1 . Maximum IAA and GA production was recorded in isolate ATY10. Extracellular ammonia production of the bacterial isolates ranged between 176.53±5.07 μmol mL−1 and 75.59 μmol mL−1. The nitrogen content of NFB isolates ranged between 30.75 µg mL-1 and 9 µg mL-1 . The isolate ATY33 had maximum nitrogen content. The selected PSB isolates were subjected to plate assay and broth assay using Pikovskaya’s medium. The isolate PSB1 recorded maximum zone of solubilization (18.23 mm), phosphate solubilizing index (2.82) and quantification of phosphate solubilization (53.41 mg L-1 ). The bacterial isolates were screened based on weighted average ranking. In NFB, ATY10, ATY34 and ATY35 were selected for further studies and in PSB, the isolates PSB1, PSB3 and PSB4 were selected. The selected isolates were 164 identified as Pantoea agglomerans ATY10, Rhizobium sp. ATY34, Ensifer adhaerens ATY35, Burkholderia territorii PSB1, Burkholderia cepacia PSB3 and Aeromonas hydrophila PSB4 by 16s rRNA sequencing. Effect of these isolates were assessed for plant growth promotion in vitro in ragi seeds using roll towel assay. Maximum germination percentage was recorded by Aeromonas hydrophila PSB4 (81.24 per cent) and the minimum number of days taken for germinationwas recorded by Pantoea agglomerans ATY10 treated seeds. Also, the treatment Pantoea agglomerans ATY10 recorded maximum shoot length (3.31 cm), root length (7.76 cm), seedling length (11.08 cm), seedling vigour index (876.62), root dry weight (0.443 mg) and root shoot ratio (0.50). The treatment Burkholderia territorii PSB1 recorded maximum root fresh weight (3.762 mg) and shoot dry weight (0.877 mg). The six isolates were subjected to assess the multiple traits such as phosphorus, potassium and silicate solubilizing capacity, siderophore production and antifungal activity. Burkholderia territorii PSB1 showed maximum zone of inhibition against Rhizoctonia solani (3.16 mm) and Sclerotium rolfsii (0.58 mm). The maximum antagonistic activity against Fusarium sp. and Helminthosporium sp. was recorded by Burkholderia cepacia PSB3 with 4.13 mm and 5.66 mm of zone of inhibition respectively. Burkholderia territorii PSB1 and Burkholderia cepacia PSB3 solubilized potassium alumino silicate in agar also, with clearance zone of 11.00 mm and 12.66 mm (in diameter), respectively. None of the isolates solubilized magnesium trisilicate in Bunt and Rovira medium. The siderophore production was observed in Aeromonas hydrophila PSB4 isolate in Chrome Azurol S (CAS) agar medium plates. Based on the results of the present study, it can be concluded that Pantoea agglomerans ATY10 is the superior isolate among NFB isolates for plant growth promotion. Also, the isolate Burkholderia territorii PSB1 is best among PSB isolates for plant growth promotion and biocontrol activity.
Open Access
Development of encapsulated formulation of PGPR mix-I and IRS evaluation
(Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2020) Ayisha, Y L; KAU; Meenakumari, K S
The study entitled “Development of encapsulated formulation of PGPR mix-I and its evaluation” was conducted during 2018-2020, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to develop calcium alginate based encapsulated formulation of PGPR mix-I and its evaluation for slow release and biodegradation. The component cultures of PGPR mix-I were procured from the Department of Agricultural Microbiology, College of Agriculture, Vellayani for standardization of protocol for preparation of calcium alginate based encapsulated bead formulation of PGPR mix-I. Encapsulated bead formulation of PGPR mix-I was prepared by standard procedures. An experiment was carried out to standardize the protocol for preparation of calcium alginate based encapsulated formulation of PGPR mix–I in completely randomized design with different treatments such as 10% Standard starch, 15% Standard starch, 10% Wheat flour, 15% Wheat flour, 10% Talc, 15% Talc and control treatment as 2% Sodium alginate alone in three replications. Consistent viable count was recorded in encapsulated formulation amended with 10% Standard starch. It exhibited maximum viable count of each of the component cultures of PGPR mix-I as a result of three month population study. A significant decline of total viable population in control treatment was observed in each month compared to encapsulated formulation amended with 10% Standard starch. Based on the population study, encapsulated formulation of PGPR mix-I 10% Standard starch amended was adjudged as the best combination of filler material and hence the shelf life studies of the same had to be continued at monthly intervals at room temperature and refrigerated conditions for six months by serial dilution and plate count method. Significant viable count was recorded in encapsulated beads stored at room temperature condition throughout the shelf life study. The moisture content of beads were also monitored during standardization and shelf life study. During standardization study, moisture content of PGPR mix-I encapsulated beads of each treatment was monitored for a period of three months at monthly intervals at room temperature and it showed a significant variation among treatments in each month. A reduction in moisture content of beads was observed from first month to the end of sixth month in all treatments. Beads amended with 10% Standard starch showed a moisture content of 13.37%, 12.07%, 11.72% and 11.45% after 24 hours of drying, first, second and third month respectively. During shelf life study, 10% Standard starch combination at refrigerated condition showed moisture content in the range of 12.83% to 11.45% while at room temperature the same has recorded values in the range of 12.07% to 10.70%. Evaluation of rate of release of immobilized bacteria from encapsulated beads was determined as per the procedure described by Bashan (1986) and the number of released bacteria was determined by the plate count method in respective selective medium. The higher cfu of component cultures of PGPR mix-I was observed after gentle shaking at 32⁰C for 24hours (T1) in75ml of sterile saline solution. Evaluation of biodegradation of encapsulated beads was studied at weekly intervals in sterile and non-sterile soil with PGPR mix-I inoculated and non-inoculated beads with three replications each (Bashan, 1986). Both the sets were observed weekly for their rate of biodegradation. As per biodegradation scale values like 0, ˃0-0.5, ˃0.5-1, ˃1-2, ˃2-2.5 or 3 was assigned according to the degree of visible degradation which indicates no visible degradation,onset of degradation, slight visible degradation on bead edges, one-half to three-fourth of the beads degraded, 90% of beads become mushy, full degradation (beads are disintegrated into small pieces or not found in the nylon bag) repectively (Bashan, 1986). The PGPR mix-I inoculated beads with bacteria in non-sterile soil showed highest scale of biodegradation throughout the biodegradation study (mean value 1.34) and beads without bacteria in sterile soil showed the lowest scale (mean value 0.52). Kruskal-Wallis rank sum test was done and there was a significant difference between treatments and so multiple comparison was done using Dunn test. During all the four weeks of biodegradation study, treatment T1 (beads with PGPR mix-I in non sterile soil) recorded the highest biodegradation and T4 (beads without PGPR mix-I in sterile soil) recorded the least biodegradation. Treatments T2 (beads with PGPR mix-I in sterile soil) and T3 (beads without PGPR mix-I in non sterile) were on par with both the treatments T1 and T4 in all the four weeks. Treatment wise evaluation of biodegradation of beads was done with Kruskal-Wallis rank sum test and gives a chi-squared value of 46.205 with df = 15 and p-value = 4.932e-05. There was a significant difference between treatments and so multiple comparison was done using Dunn test. Treatment T4 (beads with bacteria in non sterile soil during fourth week) showed significantly different from treatment T13 (beads without bacteria in sterile soil during first week). In the present investigation, calcium alginate based encapsulated beads of PGPR mix-I amended with 10% Standard starch exhibited maximum viable count of component cultures of PGPR mix-I throughout the three months period of standardization study. In terms of evaluation of shelf life and moisture retention during storage, beads stored under room temperature condition was found to be better. The rate of release of component cultures of PGPR mix-I from the encapsulated formulation was more during the first 24-48 hours. Biodegradation studies of encapsulated beads of PGPR mix-I revealed that the beads inoculated with PGPR mix-I in non sterile soil showed highest biodegradation throughout the period of investigation.
Open Access
Field evaluation of abiotic stress tolerant strains of Trichoderma harzianum and Pseudomonas fluorescens for Phytophthora disease management in black paper (Piper nigrum L.)
(Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2020) Rima, K R.; KAU; Surendra Gopal, K
Black pepper (Piper nigrum L.), known as “the king of spices” is an important spice commodity of commerce and trade in India since pre-historic period. It is highly sensitive to abiotic stresses like climate changes. The abiotic stresses in plants can be overcome by the use of beneficial microorganisms. However, the microorganisms themselves are vulnerable to abiotic and biotic stresses. Therefore, abiotic stress tolerant strains of beneficial microorganisms have to be developed for black pepper to overcome changes due to micro-climatic variables and soil parameters. In an earlier study in the Department of Agricultural Microbiology, College of Horticulture, native abiotic stress tolerant isolates of Trichoderma harzianum (CKT isolate) and Pseudomonas fluorescens (PAP isolate) were identified for growth promotion and disease management in black pepper (Piper nigrum L.) under pot culture studies. As the performance of these strains varies under field conditions, a study on “Field evaluation of abiotic stress tolerant strains of Trichoderma harzianum and Pseudomonas fluorescens for Phytophthora disease management in black pepper (Piper nigrum L.)” was undertaken with an objective to study the effect of micro- climatic and soil parameters on abiotic stress tolerant strains under field conditions. Before field evaluation, pot culture experiment was conducted to evaluate the efficiency of the abiotic stress tolerant Trichoderma harzianum (CKT) and Pseudomonas fluorescens (PAP) for growth promotion and Phytophthora disease management in black pepper during April – October, 2017. T. harzianum (CKT) was the most promising isolate for growth promotion and disease management based on biometric (number of leaves, plant height, number of lateral branches, number of nodes, intermodal length), biological and disease management characters under pot culture studies. It was found that population of Trichoderma harzianum (CKT), Pseudomonas fluorescens (PAP), Trichoderma viride (KAU reference culture) and Pseudomonas fluorescens (KAU reference culture) was negatively correlated with soil temperature and positively correlated with soil moisture. There was no significant correlation between the population of microbes and soil pH and soil respiration. Field evaluation revealed that Trichoderma harzianum (CKT) + KAU POP was the most promising treatment for growth promotion under field conditions based on biometric and biological characters. The population of Trichoderma harzianum (CKT), Pseudomonas fluorescens (PAP), Trichoderma viride (KAU reference culture) and Pseudomonas fluorescens (KAU reference culture) were negatively correlated with soil temperature and positively correlated with soil moisture content. However, soil pH and soil respiration did not have any correlation with the population of Trichoderma sp. and Pseudomonas fluorescens. Micro-climatic and soil parameters did not show any effect on the production of ammonia, HCN and siderophore but had significant effect on production of IAA in Trichoderma sp. and Pseudomonas fluorescens. Soil temperature (26.8 to 30.6 °C), soil moisture content (14 to 17.6 %), soil pH (5.7 to 6.1) and soil respiration (8.1 to 9.8 mg CO2 g-1 day-1) were found to be the optimum micro-climatic and soil parameters for IAA production. Trichoderma harzianum (CKT) was the most promising isolate for growth promotion under field conditions. However, multilocational field trials at different agro- ecological zones of Kerala need to be conducted before commercialization of Trichoderma harzianum (CKT).
Open Access
Isolation and in vitro screening of silicate solubilizing bacteria from paddy rhizosphere
(Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2020) Akhila P, Subash.; KAU; Meenakumari, K S
The study entitled “Isolation and in vitro screening of silicate solubilizing bacteria from paddy rhizosphere”, was conducted during 2018-2020, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective of isolation and in vitro screening of bacteria which are capable of solubilizing insoluble form of silicate. Bacteria capable of solubilizing silicates were isolated from the rhizosphere soils collected from different upland and low land paddy fields by serial dilution and plate count method using Bunt and Rovira medium supplemented with 0.25 per cent magnesium trisilicate. Based on the clear halo zone formed around the bacterial colonies on solid media, they were identified as Silicate Solubilizing Bacteria (SSB). Twenty seven isolates of bacteria capable of solubilizing insoluble form of silicate (magnesium trisilicate) were obtained from different locations and were allotted code numbers from SSB 1 to SSB 27. These isolates were subjected to plate and broth assay in Bunt and Rovira medium supplemented with 0.25 per cent magnesium trisilicate. After three days of incubation of test plates at room temperature, all the twenty seven isolates solubilized magnesium trisilicate and produced clearing zone around the bacterial colonies on solid media. The size of clearance zone ranged from 3 mm to 13 mm in plates. The maximum clearance zone of 13 mm was recorded with the isolate SSB 14 which was significantly superior to all other isolates. In broth culture, SSB 20 showed the highest silicate solubilization of 94.65 mg L-1. Based on plate as well as broth assay of all the twenty seven isolates obtained, five isolates viz., SSB 3, SSB 14, SSB 18, SSB 20 and SSB 22 which showed the maximum clearance zone in plate and silicate solubilization in broth were selected as superior isolates. All the isolates obtained were subjected to plate and broth assay for phosphate solubilization in Pikovskaya’s medium and potassium solubilization in Aleksandrov medium. Among them, fourteen isolates showed phosphate solubilization in plates and the clearance zone ranged from 0.87 mm to 5.50 mm. The maximum clearance zone of 5.50 mm was recorded with the isolate SSB 22 which was on par with SSB 23 with 5 mm clearance zone in plate and highest solubilization of 41.92 mg L-1 in broth was shown by SSB 22 which was significantly superior to all other isolates. Twelve isolates showed potassium solubilization in plates and the clearance zone ranged from 2.25 mm to 5.50 mm. Maximum clearance zone of 5.50 mm was recorded with the isolate SSB 8 which was significantly superior to all other isolates. The highest potassium solubilization of 37.50 mg L-1 in broth was observed with isolates SSB 1, SSB 2, SSB 7, SSB 8, SSB 13, SSB 18, SSB 21 and SSB 22 which were found to be statistically on par. Acid production by the five superior SSB isolates, SSB 3, SSB 14, SSB 18, SSB 20 and SSB 22 was detected as a yellow halo around the bacterial colonies in bromophenol blue amended Bunt and Rovira medium. All the five superior isolates tested showed positive results for acid production. The antagonistic activity of the five superior SSB isolates were assessed against major pathogens of paddy viz., Rhizoctonia solani, Magnaporthe grisea, Helminthosporium oryzae and Xanthomonas oryzae pv. oryzae following dual culture method. Out of the five isolates tested, three isolates (SSB 18, SSB 20 and SSB 22) inhibited Rhizoctonia solani. Three isolates (SSB 3, SSB 18 and SSB 22) showed antagonism against Magnaporthe grisea and four isolates (SSB 3, SSB 18, SSB 20 and SSB 22) inhibited Helminthosporium oryzae. The bacterial pathogen, Xanthomonas oryzae pv. oryzae was inhibited by three isolates (SSB 18, SSB 20 and SSB 22). Among all the five isolates tested against different phytopathogens, SSB 18 was found superior with the maximum zone of inhibition of 9.65 mm, 14.45 mm, 10.80 mm and 11.50 mm against Rhizoctonia solani, Magnaporthe grisea, Helminthosporium oryzae and Xanthomonas oryzae pv. oryzae respectively. The five superior isolates were characterized based on morphological and biochemical characters. The results revealed that all the isolates were rod shaped, Gram positive endospore formers. Based on the results of present study, it can be concluded that SSB 3, SSB 14, SSB 18, SSB 20 and SSB 22 are the superior silicate solubilizing bacterial isolates. Among them, SSB 18 showed the highest antagonistic activity against major pathogens of paddy.
Open Access
Development of encapsulated formulation of PGPR mix-I and its evaluation
(Department of Agricultural Microbiology, College of Agriculture, Vellayani, 2020) Ayisha, Y L; KAU; Meenakumari, K S
The study entitled “Development of encapsulated formulation of PGPR mix-I and its evaluation” was conducted during 2018-2020, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to develop calcium alginate based encapsulated formulation of PGPR mix-I and its evaluation for slow release and biodegradation. The component cultures of PGPR mix-I were procured from the Department of Agricultural Microbiology, College of Agriculture, Vellayani for standardization of protocol for preparation of calcium alginate based encapsulated bead formulation of PGPR mix-I. Encapsulated bead formulation of PGPR mix-I was prepared by standard procedures. An experiment was carried out to standardize the protocol for preparation of calcium alginate based encapsulated formulation of PGPR mix–I in completely randomized design with different treatments such as 10% Standard starch, 15% Standard starch, 10% Wheat flour, 15% Wheat flour, 10% Talc, 15% Talc and control treatment as 2% Sodium alginate alone in three replications. Consistent viable count was recorded in encapsulated formulation amended with 10% Standard starch. It exhibited maximum viable count of each of the component cultures of PGPR mix-I as a result of three month population study. A significant decline of total viable population in control treatment was observed in each month compared to encapsulated formulation amended with 10% Standard starch. Based on the population study, encapsulated formulation of PGPR mix-I 10% Standard starch amended was adjudged as the best combination of filler material and hence the shelf life studies of the same had to be continued at monthly intervals at room temperature and refrigerated conditions for six months by serial dilution and plate count method. Significant viable count was recorded in encapsulated beads stored at room temperature condition throughout the shelf life study. The moisture content of beads were also monitored during standardization and shelf life study. During standardization study, moisture content of PGPR mix-I encapsulated beads of each treatment was monitored for a period of three months at monthly intervals at room temperature and it showed a significant variation among treatments in each month. A reduction in moisture content of beads was observed from first month to the end of sixth month in all treatments. Beads amended with 10% Standard starch showed a moisture content of 13.37%, 12.07%, 11.72% and 11.45% after 24 hours of drying, first, second and third month respectively. During shelf life study, 10% Standard starch combination at refrigerated condition showed moisture content in the range of 12.83% to 11.45% while at room temperature the same has recorded values in the range of 12.07% to 10.70%. Evaluation of rate of release of immobilized bacteria from encapsulated beads was determined as per the procedure described by Bashan (1986) and the number of released bacteria was determined by the plate count method in respective selective medium. The higher cfu of component cultures of PGPR mix-I was observed after gentle shaking at 32⁰C for 24hours (T1) in75ml of sterile saline solution. Evaluation of biodegradation of encapsulated beads was studied at weekly intervals in sterile and non-sterile soil with PGPR mix-I inoculated and non-inoculated beads with three replications each (Bashan, 1986). Both the sets were observed weekly for their rate of biodegradation. As per biodegradation scale values like 0, ˃0-0.5, ˃0.5-1, ˃1-2, ˃2-2.5 or 3 was assigned according to the degree of visible degradation which indicates no visible degradation,onset of degradation, slight visible degradation on bead edges, one-half to three-fourth of the beads degraded, 90% of beads become mushy, full degradation (beads are disintegrated into small pieces or not found in the nylon bag) repectively (Bashan, 1986). The PGPR mix-I inoculated beads with bacteria in non-sterile soil showed highest scale of biodegradation throughout the biodegradation study (mean value 1.34) and beads without bacteria in sterile soil showed the lowest scale (mean value 0.52). Kruskal-Wallis rank sum test was done and there was a significant difference between treatments and so multiple comparison was done using Dunn test. During all the four weeks of biodegradation study, treatment T1 (beads with PGPR mix-I in non sterile soil) recorded the highest biodegradation and T4 (beads without PGPR mix-I in sterile soil) recorded the least biodegradation. Treatments T2 (beads with PGPR mix-I in sterile soil) and T3 (beads without PGPR mix-I in non sterile) were on par with both the treatments T1 and T4 in all the four weeks. Treatment wise evaluation of biodegradation of beads was done with Kruskal-Wallis rank sum test and gives a chi-squared value of 46.205 with df = 15 and p-value = 4.932e-05. There was a significant difference between treatments and so multiple comparison was done using Dunn test. Treatment T4 (beads with bacteria in non sterile soil during fourth week) showed significantly different from treatment T13 (beads without bacteria in sterile soil during first week). In the present investigation, calcium alginate based encapsulated beads of PGPR mix-I amended with 10% Standard starch exhibited maximum viable count of component cultures of PGPR mix-I throughout the three months period of standardization study. In terms of evaluation of shelf life and moisture retention during storage, beads stored under room temperature condition was found to be better. The rate of release of component cultures of PGPR mix-I from the encapsulated formulation was more during the first 24-48 hours. Biodegradation studies of encapsulated beads of PGPR mix-I revealed that the beads inoculated with PGPR mix-I in non sterile soil showed highest biodegradation throughout the period of investigation.
Open Access
Plant associated endospore forming bacteria from amaranthus as growth promoters and biocontrol agents against rhizoctonia leaf blight
(Department of Agricultural Microbiology, college of Agriculture, Vellayani, 2020) Yashwasini, M S; KAU; Anith, K N
The study entitled “Plant associated endospore forming bacteria from amaranthus as growth promoters and biocontrol agents against Rhizoctonia leaf blight”, was conducted during 2018-2020, in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective of isolation and characterization of endospore forming bacteria from endorhizosphere and phyllosphere of Amaranthus and evaluate their effect on growth promotion and disease suppression. Endospore forming bacteria from endorhizosphere and phyllosphere were isolated from healthy roots and leaves of red amaranthus variety Arun, green amaranthus var. CO1 and wild relative (Amaranthus viridis) respectively by enrichment method on NA medium. A total of eight endospore forming bacterial isolates were obtained each from endorhizosphere and phyllosphere of the variety Arun, variety CO-1 and wild relative (Amaranthus viridis). All the isolates were identified as Bacillus spp. based on morphological, biochemical and molecular characterization. These isolates designated as AR1, AR2, AR3, GR1, GR2, GR3, WR1, WR2, AL1, AL2, AL3, GL1, GL2, GL3, WL1 and WL2 were identified as Bacillus sp., Bacillus siamensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus amyloliquefaciens, Bacillus amyloliquefaciens, Bacillus sp., Bacillus amyloliquefaciens, Bacillus sp., Bacillus subtilis, Bacillus sp., Bacillus amyloliquefaciens, Bacillus sp., Bacillus sp., Bacillus subtilis and Bacillus amyloliquefaciens respectively based on 16S rRNA sequence. Indole Acetic Acid production by the different endospore forming endorhizosphere bacterial isolates ranged from 4.09 to 9.73 μg mL-1 of culture filtrate. The isolate AR3 produced the maximum IAA of 9.73 μg mL-1 of culture filtrate. In roll towel assay significant increase in germination percentage, seedling shoot length, root length and seedling vigour index compared to control was observed when seeds were treated with the endospore forming endorhizosphere bacterial isolates. Isolate WR1 showed the maximum germination (43.33%) whereas control recorded a germination of 30%. Isolate WR1 showed maximum seedling vigour index (223.66) compared to the control (136.53). A portray experiment was also conducted to study the influence of endospore forming endorhizosphere bacterial isolates on plant growth parameters of amaranthus in the nursery stage. Highest root length was observed in the seedlings treated with isolate GR1 (4.76 cm) whereas the control recorded 3.91 cm. Shoot length was the maximum in seeds treated with WR1 (6.34 cm) whereas control recorded a shoot length of 5.59 cm. Maximum germination percentage of 100 was obtained in seeds treated with the isolate WR2. Significant influence of the bacterial isolates on seedling vigour index was observed wherein the isolate WR2 recorded maximum of 884.50. Highest root fresh weight was recorded in the plants treated with isolate GR1 (8.12 mg/plant) and the lowest root fresh weight of 3.56 mg/plant was recorded in control plants. It was found that the highest root dry weight was found in plants treated with isolate WR1 (2.99 mg/plant) as against the control (2.46 mg/plant). Dual culture plate assay was carried out on Potato Dextrose Agar medium to check the ability of endospore forming phyllopshere bacterial isolates to inhibit Rhizoctonia solani causing foliar blight disease. All the isolates inhibited the mycelial growth of Rhizoctonia solani, of which GL3 and GL1 exhibited the maximum and minimum zone of inhibition (ZOI) of 8.73 and 1.00 mm respectively. All the endospore forming phyllopshere bacterial isolates tested for their indirect antagonism inhibited Rhizoctonia solani mycelial growth, of which isolate GL3 showed the maximum ZOI of 2.88 mm and the minimum was recorded by the isolate GL1 (0.50 mm). In detached leaf assay, significant difference was noticed in the lesion size appeared on the detached leaves treated with endospore forming phyllosphere bacterial isolates and water (control) on 3rd and 4th day after pathogen inoculation. Control exhibited maximum lesion area of 3.49 and 12.23 cm2 and the minimum lesion area of 0.42 and 3.11 cm2 was developed on the leaves treated with isolate WL2 on 3rd and 4th day after pathogen inoculation respectively. Based on in vitro studies on plant growth promotion and antagonistic activity against R. solani the endospore forming endorhizosphere bacterial isolates AR1, AR2, GR1, GR2, WR1 and WR2 and endospore forming phyllosphere bacterial isolates - AL1, AL3, GL2, GL3, WL1 and WL2 were selected for further pot culture study. A pot culture experiment was conducted to evaluate the efficacy of selected endospore forming endorhizosphere bacteria for their plant growth promoting activity in Amaranthus (var. Arun). Endospore forming endorhizosphere bacteria inoculated plants showed significant increase in biometric parameters such as number of leaves, number of branches, plant height and dry matter production compared to control. Plants treated with Bacillus amyloliquefaciens GR1 exhibited the maximum shoot fresh weight of 54.30 g/plant, whereas the control plants showed the minimum of 40.24 g/plant. Shoot dry matter production was maximum with plants treated with the isolate AR2 (6.74 g/plant) and the control plants recorded 4.76 g/plant. Bacillus amyloliquefaciens GR1 treated plants produced maximum root fresh weight of 13.64 g/plant, whereas the control plants had root fresh weight of 9.88 g/plant. Maximum root dry weight was exhibited by plants treated with Bacillus amyloliquefaciens GR1 (1.98 g/plant), lowest was produced by control plants with 1.37 g/plant. Suppression of Rhizoctonia leaf blight in the variety Arun treated with selected endospore forming phyllosphere bacterial isolates was studied by challenge inoculation with the pathogen on intact leaves. The bacterial suspension containing bacterial cells of concentration 107 cfu ml-1 was sprayed on to the intact leaves of plants one week prior to pathogen inoculation. On 3rd day after pathogen inoculation, plants treated with Bacillus sp GL3 showed the minimum percent disease index of 11.80 with 32.92% disease 11.80 with 32.92% disease 11.80 with 32.92% disease 11.80 with 32.92% disease 11.80 with 32.92% disease 11.80 with 32.92% disease 11.80 with 32.92% disease 11.80 with 32.92% disease suppression over the pathogen co suppression over the pathogen co suppression over the pathogen co suppression over the pathogen co suppression over the pathogen co suppression over the pathogen co suppression over the pathogen co suppression over the pathogen co suppression over the pathogen cosuppression over the pathogen control, which was on par with the ntrol, which was on par with the ntrol, which was on par with the ntrol, which was on par with the ntrol, which was on par with the ntrol, which was on par with the ntrol, which was on par with the ntrol, which was on par with the ntrol, which was on par with the ntrol, which was on par with the Bacillus sp AL3 with with 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. 11.81 percent disease index. No symptom development was observed in absolute control. On 5th day after pathogen inoculation, Bacillus sp AL3 treated plants showed the minimum percent disease index of 18 .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the .75 with 44.51% disease suppression over the pathogen inoculated control. pathogen inoculated control. pathogen inoculated control. pathogen inoculated control. pathogen inoculated control. pathogen inoculated control. The present study revealed that seed bacterization of amaranthus plants with endospore forming endorhizosphere and foliar spray of phyllosphere bacterial isolates improved plant growth and suppressed Rhizoctonia solani leaf blight incidence respectively and helps in better establishment of plants. Bacillus amyloliquefaciens GR1 and Bacillus sp AL3 were selected as the best isolates for plant growth promotion and disease suppression respectively.

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