Browsing by Author "Girija, D"

Now showing 1 - 20 of 30

Bioefficacy of native Bacillus thuringiensis isolates from the western ghats of Kerala on pumpkin caterpillar,Diaphania indica (saund.) (Lepidoptera: pyralidae)
(2010) Neema, P.M; Girija, D; Mathew, M.P
Biofertilizer technology for spices:Technical Bulletin 01
(Department of Agricultural Microbiology, College of Agriculture, KAU,Vellanikkara, 2020) Girija, D; Surendra Gopal, K; Santosh Ranjan, Mohanty
Characterization and evaluation of plant growth promoting rhizobacteria from rice soils of Wayanad
(Department of Agricultural Microbiology, College of Agriculture, Vellanikkara, 2021) Wickramasinghe, W R K D W K V; Girija, D
Plant growth promoting rhizobacteria (PGPR) are a group of bacteria that colonize the plant rhizosphere and enhance the growth and yield of plants. The present investigation entitled “Characterization and evaluation of plant growth promoting rhizobacteria from rice soils of Wayanad” was undertaken at the Department of Agricultural Microbiology” during the year 2018-2020, with the objective of isolation, characterization and evaluation of plant growth promoting rhizobacteria from rice soils of Wayanad and formulation of a consortium to improve the growth and yield of rice. Isolation of rhizobacteria with potential plant growth promoting (PGP) activities was attempted from rice rhizosphere soils collected from ten locations in Wayanad district of Kerala. Selective media were used for the isolation of PGPRs including nitrogen fixers, solubilizers of phosphate, K and Zn and fluorescent pseudomonads. A total of 149 isolates obtained on different media were subjected to preliminary screening for growth on selective media, which yielded 32 N-fixers, 16 phosphate solubilizers, four K solubilizers, six Zn solubilizers and two fluorescent pseudomonads. These isolates were evaluated in vitro for PGP activities (production of IAA, NH3, HCN and siderophore) and antagonistic activities against R. solani and X. oryzae. Twenty promising isolates were selected based on their functional efficiency for further characterization using cultural, morphological, biochemical and molecular methods. Four isolates were found to be Gram-positive rods and sixteen isolates were Gram-negative short rods. Eighteen isolates were identified based 16S rRNA gene sequencing and the sequences of all the eighteen isolates deposited in the GenBank of the NCBI. Phylogenetic analysis using MEGA 7 software showed two major clusters and several sub-clusters. A few of the native isolates stood out distinctly from the available accessions in the database, showing that they are genetically diverse. Based on the efficiency of N fixation, P, K and Zn solubilization and other PGP activities, isolates were ranked. Based on ranking, three N-fixers (Bacillus sp. AkNF3, Pseudomonas sp. PkNF4 and Pseudomonas putida KgNF1), three phosphate solubilizers (Bacillus megaterium PkPS1, Acinetobacter schindleri AkPS4 and Achromobacter sp. AvPS1), two K-solubilizers (Microbacterium sp. MvKS1 and Acinetobacter calcoaceticus MvKS3) and two zinc solubilizers (Achromobacter marplatensis ThZnS2 and Cytobacillus kochii PkZnS3) were selected for consortial formulation. Compatibility of ten promising isolates was tested by cross streaking and dual culture methods. Three PGPR based consortia (Consortium 1, 2 and 3) were formulated, each consisting of 5 native isolates (two N-fixers, one each of phosphate, K and Zn solubilizers). These consortia were evaluated in pot culture experiment, along with KAU commercial formulation (PGPR mix-1), at RARS, Ambalavayal, with rice (variety Valichoori) as the test crop. PGPR application was combined with two levels (50% and 75%) of recommended dosage of inorganic fertilizers (RDF). Population of total bacteria, N fixers, P, K and Zn solubilizers was higher in combined application of biofertilizer with inorganic fertilizers than uninoculated treatments and this was indicative of better colonization of native PGPRs in the rice rhizosphere. Growth and yield parameters indicated that application of PGPR consortium with 75% RDF was statistically on par with PoP (KAU) and 100% RDF. Results suggested that 25% inorganic N, P and K can be replaced by using native PGPR consortium without affecting plant growth, yield, plant nutrient content and soil nutrient content. Considering the above parameters, two best consortia (Consortium 2 and Consortium 3) were selected for further field evaluation. Field evaluation was carried out to assess the efficiency of two selected native PGPR consortia at RARS, Ambalavayal. Five treatments included were, consortium 2 + 75% RDF, consortium 3 + 75% RDF, reference biofertilizer PGPR mix-1 + 75% RDF, 100% RDF and farmer’s practice (farm yard manure 5t ha-1 ). Results suggested that root colonization of total bacteria, N fixers, P, K and Zn solubilizers was higher in all treatments of combined application of biofertilizers with 75% inorganic fertilizer than 100% RDF alone. Growth and yield parameters suggested that combined application of Consortium 2 with 75 % RDF was statistically on par with 100% RDF. Therefore, it can be concluded native PGPR strains in consortium 2 (Bacillus sp. strain AkNF3, Pseudomonas putida strain KgNF1, Bacillus megaterium strain PkPS1, Acinetobacter calcoaceticus strain MvKS3 and Cytobacillus kochii PkZnS3) successfully colonized the rice rhizosphere, increased nutrient availability to the plants and produced higher yield. The results also emphasized on the importance of exploiting native, location specific microorganisms as biofertilizer consortium, rather than a common consortium for the entire State. Native PGPR based consortia 2 reduced the 25% of inorganic fertilizer (N, P and K) without affecting the growth and yield of rice. This would be more cost effective and ecofriendly when compared with the use of chemical fertilizers alone. Further multi-locational field trials are required to validate the results before commercialization of this consortium, as a biofertilizer.
Characterization of lepidopteran specific Bacillus thuringiensis isolates using RAPD-PCR
(2010) Jyothi Sara, Jacob; Maicykutty P, Mathew; Girija, D
Characterization of native Bacillus thuringiensis from the western ghats of Kerala
(2010) Neema, P.M; Girija, D; Aparna, C; Firoz, P.M
Characterization of Ralstonia solanacearum (Smith) Yabuuchi et al. causing bacterial wilt in ginger using molecular marker
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2003) Sambasivam, P K; Girija, D
In Kerala, bacterial wilt caused by Ralstonia solanacearum is one of the major constraints in ginger cultivation. Earlier, the pathogen was characterized based on cultural, morphological and biochemical tests. Since these methods are time consuming and involve repeated subculturing and tedious biochemical tests, an attempt was made to characterize the pathogen at molecular level using plasmid profile and RAPD technique. Wilted plant samples were collected from ginger growing tracts of Palakkad, Eranukulam and Wynad districts. The pathogen was isolated on TZC medium, it produced creamy white pink centred fluidal colonies. Stock cultures prepared by suspending single colonies in sterile water were maintained at 4 QC. Pathogenicity of the isolates was established using pseudostem inoculation method. Tomato and brinjal were found to be collateral hosts of the pathogen. Based on hypersensitivity reaction on capsicum the isolates were identified as race 3. This was further confirmed by RAPD assay. The isolates were found to. be Gram negative and showed positive reaction for solubility. in KOH, nitrate reduction, production of catalase and oxidase enzyme, fermentation of glucose. All the isolates utilized lactose, maltose, cellobiose, manitol, sorbitol but not dulcitol and hence, were grouped as biovar III A. Plasmid profile developed for the isolates showed presence of two bands of . . approximately 21 kb size .. Plasmids when transformed to E. coli DH 5a cells, conferred resistance to ampicillin and rifampicin, indicating that the genes encoding resistance to these antibiotics were located on the plasmid. RAPD analysis using 16 primers showed much diversity among the isolates. Primers OPU .13, OPU 17 and OPX 9 showed 100 per cent polymorphism. Dendrogram obtained through cluster analysis showed one major and one sub cluster. All the Palakkad isolates were grouped under a single cluster. The present study indicated possibility of using molecular marker as a tool to detect even slight variability among R. solanacearum isolates infecting ginger.
Chemical control of stack burn disease of rice
(Kerala Agricultural University, 1993) Girija, D; Vasavan, M G; Rema Devi, L; Sheela Paul, T; Santhakumari, P
Cloning of genes encoding insecticidal proteins (cry/vip genes ) of Bacillus thuringiensis from Western Ghats of Kerala
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Neema, P M; Girija, D
The study entitled ‘Cloning of genes encoding insecticidal proteins (cry/vip genes) of Bacillus thuringiensis from Western Ghats of Kerala’ was carried out in the Molecular Biology Laboratory of the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2005- 2007. The crystal protein genes (cry/vip) of B. thuringiensis possess insecticidal activity against larvae of insect orders Lepidoptera, Diptera and Coleoptera. In the present study, an attempt was made to isolate and clone cry genes of B. thuringiensis from the Western Ghats of Kerala. Bacillus thuringiensis strains were isolated from soil samples collected from different locations of the Western Ghats of Kerala. The pure colonies obtained were stab inoculated and stored under refrigerated conditions. Variability among the isolates were studied by various cultural, morphological and biochemical tests. The insecticidal activity of the isolates was determined by bioassay against the major lepidopteran pest of cucurbitaceous vegetables, the pumpkin caterpillar. The information on cry1A gene sequences of different species of Bacillus thuringiensis available in the public domain NCBI was collected and subjected to multiple sequence alignment to detect conserved boxes of the gene among species. Based on the data, one pair of gene specific primer was designed for amplification of partial cry1A gene fragment of about 800bp in B. thuringiensis isolates. Total DNA was isolated from the B. thuringiensis strains of Western Ghats of Kerala. Profiling of cry1 and cry4 genes of bacterial isolates were done using universal primers for cry1 and cry4. Amplification was obtained with cry1 gene for seven isolates and with cry4 gene primer for two isolates. The amplicons obtained with universal cry1 primer from two isolates and with cry4 primer from one isolate were used for cloning. The amplicons obtained with cry1 and cry4 primers were eluted, cloned in pGEMT vector and transformed into competent cells. High level of recombination was observed on blue-white screening. Recombination of the insert was confirmed by PCR of the plasmid isolated from white colonies. The cloned fragments were sequenced. The amplicon obtained with cry4 primer in the second isolate was sequenced after purifying the PCR product. The cry1ky5 and cry1em11 sequences when subjected to Blast search revealed significant levels of homology with cry1 genes reported from other B. thuringiensis strains deposited in the public domain. The cry4em10 sequence when subjected to Blast search, showed high level of similarity with cry4 genes from B. thuringiensis. The cry4ky1 sequence showed similarity with cry genes of different species of B. thuringiensis. The sequences were also subjected to various sequence analysis using bioinformatics tools which include ORF finder, SOPMA, GENSCAN, AASTAT and TCAG tools of Biology Workbench and Interproscan. PCR of all the isolates found positive in cry1 gene profiling, was done with cry1A primer designed during this study. Amplification was obtained with cry1A primer in two isolates. The amplicon obtained in one isolate was subjected to sequencing after purifying the PCR product. The cry1Aky3 sequence showed similarity with other cry genes of Bacillus thuringiensis present in the NCBI databank. 1500 bp long variable region of cry1 was amplified in two isolates using specific primers. Future research works should be focused on the isolation of B. thuringiensis from completely undisturbed ecological niches. Novelty of cry genes can be detected by restriction digestion of the genes. Characterization of novel full-length cry genes and its expression in transgenic crops will help to develop resistant varieties thereby reducing insecticide applications and resistance development in insect pest populations.
Detection of Ralstonia solanacearum race 3 causing bacterial wilt of solanaceous vegetables in Kerala, using random amplified polymorphic DNA (RAPD) analysis
(Kerala Agricultural University, Vellanikara, 2003) Deepa, James; Girija, D; Sally K, Mathew; Nazeem, P A; Babu, T D; Sukumara Varma, A
Nine strains of Ralstonia solanacearum (Smith) Yabuuchi et al. isolated from bacterial wilt affected plants of brinjal, chilli and tomato in three different agroclimatic zones of Kerala were compared based on the utilization of carbohydrates, hypersensitivity reaction on capsicum leaves and RAPD analysis. Among these, six isolates were grouped into Biovar III and three, into Biovar IIIA. The isolates belonged to Races 1 and 3. RAPD analysis with 10 decamer primers revealed a high degree of polymorphism among the isolates. The primer OPF 8 yielded a unique band of 1.45 kb size for Race 3. This could be considered as a marker for rapid identification of Race 3 isolates of R. solanacearum.
Effect of Azospirillum inoculation on seedling vigour in rice
(Kerala Agricultural University, 1994) Girija, D; Varadarajan Nair, P; Reena Mathew
Evaluation of bacillus thuringiensis isolates against diaphania indica (saund.) (Lepidoptera:pyralidae)
(Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2017) Janish Rose Jacob; Girija, D
Chemical pesticides provide significant benefit by controlling pests of agricultural crops. However, their use has increased at an alarming rate, along with proportionate increase in their adverse effect on the environment. In recent years, several insects have developed resistance towards a few pesticides due to their indiscriminate use. Several microbial agents have been used as an alternative for chemical pesticides and Bacillus thuringiensis is one among them. B. thuringiensis is a sporulating, Gram positive, facultative anaerobic soil bacterium, capable of synthesizing δ-endotoxins or Cry proteins during sporulation. Due to the insecticidal properties of these proteins, the bacterium is used against insect species of the orders Lepidoptera, Coleoptera and Diptera. More than 500 isolates of B. thuringiensis obtained from the Western Ghats of Kerala, in a DBT funded project, are being maintained in the repository of the Department of Agricultural Microbiology, College of Horticulture, Thrissur. Several microbial inoculants such as Pseudomonas fluorescens, Trichoderma viride, Beauvaria bassiana, Lecanicillium lecanii have been developed by Kerala Agricultural University but there is no formulation of B. thuringiensis. A study was conducted to develop a commercial formulation of B. thuringiensis and to evaluate its bio-efficacy against the pumpkin caterpillar, Diaphania indica (Saund.). Twenty native isolates with cent per cent mortality in previous studies were selected from the repository of the Department of Agricultural Microbiology. Morphological, biochemical and molecular characterisation of these native B. thuringiensis were carried out. The isolates showed only very slight variations in their cultural characteristics. Colonies appeared circular, creamy white with entire to undulate margin and flat elevation. The isolates showed positive reaction to starch and esculin hydrolysis, sucrose fermentation test and negative to Voges-Proskauer test. The isolates were screened for the presence of lepidopteran specific insecticidal genes, using PCR. Among the 20 native B. thuringiensis isolates, seven isolates yielded cry1 gene amplicons. None of the isolates produced cry2 and cry9 amplicons. The cry1 amplicons were further sequenced and when subjected to Blastn analysis showed homology towards cry1, cry1A, cry1Aa and cry1Ac. All the isolates have shown identity in the range of 93-96 per cent to the known cry1 genes. Thus, the presence of cry1 gene was confirmed. Based on the abundance of crystal protein and cry1 gene, three native isolates (KAU-11, KAU-474 and KAU-2189) were further selected for laboratory bioassay against the lepidopteran pest, D. indica. Among the native isolates, KAU-2189 showed highest per cent morality and was further selected for liquid formulation studies. Suitability of three media (soy flour broth, coconut water broth and T3 broth) was assessed based on the population and spore count at 0 h, 72 h and 96 h after inoculation. Coconut water yielded higher population than the standard medium (T3 broth). Spore count of B. thuringiensis in coconut water and T3 were statistically on par. Thus, coconut water served as the best among the tested substrates for B. thuringiensis production and this was further used for the liquid formulation. Bio-efficacy of the liquid formulation was evaluated in pot culture experiment against D. indica using little gourd (Coccinia indica) as the test crop. Btk (ABTEC), a commercial formulation was used as standard. The higher per cent mortality was recorded for the formulations containing KAU-2189 in coconut water broth and HD-1 in coconut water broth and both were statistically on par with each other. Minimum leaf damage was also recorded in treatments with these formulations and was statistically on par with each other. Shelf life studies of liquid formulations indicated that both population and spore count decreased from fourth month onwards. The study revealed that the native isolates have the potential to be developed into a biopesticide. Coconut water could be used as an ingredient for low cost liquid formulation. Further evaluation under field conditions is required to confirm the efficiency of KAU-2189 as a biopesticide.
Exploration of native mineral phosphate solubilizing microorganisms as biofertilizer for the acidic soils of Kerala
(Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2016) Saranya, K S; Girija, D
A study was undertaken on ‘Exploration of native mineral phosphate solubilizing microorganisms as biofertilizer for the acidic soils of Kerala’. The main objective was to exploit native microorganisms with mineral phosphate solubilization and plant growth promoting activities for the acidic soils of Kerala.
Functional diversity of beneficial microorganisms from the rhizosphere of black pepper in Wayanad
(Department of agricultural microbiology, College of Horticulture,Vellanikkara, 2015) Athira, P S; Girija, D
Black pepper (Piper nigrum L.) is a perennial, woody and flowering climber belonging to family Piperaceae. It is one of the important spice crops which provides major source of income and employment for rural households in Kerala. Wayanad dominated in pepper farming in the state about 20 years ago. Annual production of pepper was 40,000 tonnes in the mid-1980s, which comprised about half of India’s total pepper production. But recently, the production has declined drastically due to the infestation of pests and diseases. Foot rot caused by Phytophthora capsici and yellowing of black pepper are the major diseases devastating most of the plantations in Wayanad. However, some of the plants in the disease affected areas remain healthy which could be due to inherent activity of native rhizosphere microflora. The present study focused on assessing the functional diversity of beneficial microorganisms which could possibly be exploited for the benefit of plant growth. Four healthy gardens, four gardens each affected by foot rot and yellowing were selected for sample collection. Rhizosphere soil samples were collected from five healthy vines in each garden. Population of beneficial microbes in the rhizosphere soils of healthy and disease affected gardens were compared. In general, rhizosphere soil from healthy gardens recorded higher population of bacteria, fungi, phosphate solubilizers and fluorescent pseudomonads. A total of 207 isolates (including 112 bacteria, 32 actinomycetes and 63 fungi) were purified and maintained to study their plant growth promoting and antagonistic activities. Maximum IAA production (292.50 μg ml-1) was recorded by HPLBC-6 followed by HABC-3 (46.43 μg ml-1). The isolate HPLPSB-3 was the most efficient P solubiliser (162.7 μg ml-1) followed by HPLF-5 (161.3 μg ml-1). The isolate YPTN- 3 fixed maximum amount of nitrogen (46.92 mg of N g-1 of sucrose) followed by HVKN-6 (32.62 mg of N g-1 of sucrose). From the invitroexperiment, two most promising isolates each of IAA producers, phosphate solubilizers and nitrogen fixers were selected for preliminary screening for growth promotion on blackpepper cuttings. The isolate HPLPSB-3 (P solubiliser) recorded maximum sprouting, vine length, number of leaves, number of roots and roots fresh weight underinplanta screening for plant growth promotion. However, maximum root length was observed in HPLBC-6 (IAA producer). All the isolates were screened in vitro for their antagonistic activity against foot rot pathogen Phytophthora capsici. Among the bacteria, isolate HPLPSB-6 recorded maximum inhibition (69.27 %) of the pathogen. Among the actinomycetes, HVZACT-1 recorded maximum mycelial inhibiton of 66.66 %. Among the fungal isolates screened, maximum inhibition (75.17 %) was recorded by the isolate FPRF-3. The three most promising PGPM selected from preliminary in planta screening and three antagonists from in vitro screening were further tested for their efficiency in controlling foot rot disease in blackpepper nursery. Minimum disease incidence (6.23%) and severity (4.00 %) were observed in isolate FPRF-3. This was followed by actinomycete HVZACT-1 with disease incidence of 13.20 % and severity of 8.00 %. Maximum disease incidence and severity were observed in control with pathogen alone. In addition to biocontrol activity, FPRF-3 also improved plant growth parameters such as length of vine, number of leaves and roots. The selected growth promoting isolates HPLPSB-3, HPLBC-6 and YPTN-3 were identified as Acinetobacter grimontii, Providencia sp. and Paenibacillus sp. The three selected antagonists HPLPSB-6, HVZACT-1 and FPRF-3 were identified as Paenibacillus polymyxa, Streptomyces termitum and Trichoderma viride respectively. Based on in planta evaluation, Acinetobacter grimontii was considered as the best PGPM and Trichoderma viridethe most promosing antagonist against P. capsici. These isolates could be further exploited for improving the growth and managing foot rot disease, after validation under field conditions. The compatibility of PGPM with antagonists and chemical fungicides may also be evaluated.This is the first report of antagonistic activity of the actinomycete S. termitum against P. capsici causing foot rot disease in blackpepper.
Genetic transformation of chilli (Capsicum annuum L.) with osmotin gene
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Resmi Henry, T; Girija, D
The study entitled ‘genetic transformation of chilli (Capsicum annuum L.) with osmotin gene’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, from December 2003 to September 2005. The study was undertaken to standardize in vitro regeneration and Agrobacterium mediated transformation of chilli with osmotin gene. Different explants, media and hormonal combinations (auxin and cytokinin) were tried in order to standardize in vitro regeneration in chilli var.Ujwala. The best explant for in vitro regeneration was hypocotyl. Only the buds originated from hypocotyl explants showed elongation. Shoot buds formed from cotyledon and leaf segments did not elongate in any of the media tested. Based on the percentage regeneration in cultures and no. of shoot-buds per explant, MS medium containing the hormonal combination; BA (5.0 mg l-1) + IAA (0.3 mg l-1) was found optimum. The regenerated plantlets were transferred to pots for acclimatization so that they can sustain and survive in the natural conditions. The hardened plantlets were planted out. Agrobacterium mediated transformation protocol was optimized considering all the factors for successful transformation. Optimum inhibitory concentration of selectable marker (Kanamycin: 100mg l-1) was established. The antibiotic cefotaxime (200 mg l-1) was selected for killing the bacteria. Agrobacterium strain EHA 105 harbouring the gus reporter gene was used for the standardization of transformation. Hypocotyl explants of chilli were co-cultivated with Agrobacterium strain (EHA 105). The inoculum density-0.1 OD600 nm, infection time-5minutes and co-cultivation period-2 days were found optimum based on GUS assay and survival rate 60 days after co-cultivation. The hypocotyl explants of chilli were co-cultivated with Agrobacterium strain (GV2260) harbouring osmotin gene in the plasmid pGV2260 tagged with 35S CaMV promoter. The transformed explants were regenerated on the selection medium optimized for regeneration of chilli. However, none of the transformed buds were elongated in the elongation medium containing selection agent. Hence the transformed plants were transferred to elongation medium containing no antibiotics. Even in this medium, no elongation of shoots was observed even after 60 days. So, further refinement of transformation protocol using an optimal selectable marker is needed for the production of transgenic chilli. After selection of the transformants, the putative transgenics were characterized employing molecular biology techniques viz. PCR-utilizing the gene specific primers of nptII. The presence of transgene was confirmed in the transformed plants.
Hairy root induction in adapathiyan (Holostemma ada-kodien K. Schum.)
(Kerala Agricultural University, Vellanikara, 2001) Karmarkar, S H; Keshavachandran, R; Nazeem, P A; Girija, D
Holostemma ada-kodien, commonly known as adapathiyan is a laticeferous climber belonging to the family Asclepiadaceae. The root tubers of the plant are useful to cure various ailments of eye and many other human diseases. Due to the indiscriminate collection of root tubers, the plant population in the natural habitats has declined drastically and consequently it has been listed out as vulnerable and rare in the FRLHT red list of medicinal plants. The present study reports the hairy root induction in Holostemma useful in conservation of the plant and also to explore possibilities for in vitro production of the active chemicals in Holostemma, which would be a good alternative to meet its ever-increasing demand. The procedure for induction of hairy roots is given in detail.
Metagenomic analysis of bacterial diversity in the rhizosphere of arecanut palms affected by yellowing in Wayanad
(Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2017) mahesh Mohan; Girija, D
Metagenomic analysis of bacterial diversity in the rice rhizosphere of kole lands of Thrissur
(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2021) Athira Krishnan, L R; Girija, D
Kole wetlands of Kerala are a complex ecological system and are known for the higher productivity of rice. The Kole lands remain submerged under flood water for about six months in a year and this seasonal alteration gives it both terrestrial and water related properties which determine the ecosystem structure. Though several studies have been conducted for exploring the diversity of fishes, birds, flora, butterflies, etc., in Kole lands, no systematic studies have been made on rhizosphere microbial diversity. This study was intended to analyze the bacterial diversity in the rice rhizosphere ecosystem of Kole lands of Thrissur. Rhizosphere soil samples were collected from three locations of Kole lands of Thrissur viz. Puzhakkal (Pzk), Mullassery (Mls) and Cherpu (Chr) and analyzed for physico-chemical and biological properties. Culturable microflora was enumerated using serial dilution plate method for bacteria, fungi, actinomycetes, N- fixers, P, K and Zn-solubilizers. Twenty four predominant bacterial isolates were purified and screened for PGP activities including production of IAA and ammonia and phosphate solubilization. The bacterial diversity of the rhizosphere samples was analyzed by metagenomic library construction and sequencing of V3-V4 regions of the 16S rRNA gene, using Next Generation Sequencing (NGS) technology. The sequences thus obtained were analyzed for the Operational Taxonomic Units (OTUs) using MEGAN and MG- RAST server. The analysis of physico-chemical parameters showed a comparatively low pH in all the samples. An extreme low pH can reduce the availability of major and secondary nutrients in the soil. The sample Pzk showed higher content of organic C. Culturable microflora and microbial biomass C analysis also showed a slight increase in the sample Pzk. The soil organic C content and microbial biomass C are reported to be positively correlated. The microbial biomass C is the measure of the weight of the organisms present.The predominant bacterial phyla in the rice rhizosphere of Kole lands of Thrissur included Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, Bacteroidetes and Nitrospirae. The bacterial population was found higher in the sample Puzhakkal and comparatively lower in the sample Chr. Phylum Proteobacteria was found to be the most predominant bacterial phylum in Pzk while, Chloroflexi was more predominant in Mls and Chr. The classes Acidobacteria and Ktedonobacteria were found dominant in the samples Mls and Chr and the Pzk sample was dominated by Acidobacteria and Deltaproteobacteria. The phylum level bacterial diversity was found highest in the sample Chr with 21 phyla while the genus level bacterial diversity was highest in the sample Mls. The abundance of genera Desulfobacca, Thermoanaerobaculum, Thioalkalispira, Anaerolinea, Ktedonobacter, Gemmatimonas, Puedolabrys, Sulfuricurvum, Syntrophobacter, Haliangium, Geobacter and Syntrophorhabdus was observed in the Kole land rice rhizosphere samples. Many of these genera are involved in geo-cycling of nutrients like Fe, S and Mn and a few are used in waste water treatment. The species- level bacterial diversity was found to be highest in the sample Mls as indicated by the Chao1 and observed species indices. The predominant archaeal phyla in the rice rhizosphere of Kole lands of Thrissur included Euryarchaeota, Crenarchaeota and Thaumarchaeota. Archaea are still an under- detected and little-studied part of the soil, so their full influence on global biogeochemical cycles remains largely unexplored. This study has thrown light on the diversity of bacterial and archaebacterial communities in the peculiar ecosystem of Kole lands of Thrissur. Many of the biofertilizer organisms like Azospirillum, Paenibacillus, Cellulosimicrobium and biocontrol agents like Bacillus and Pseudomonas could be detected, which could be cultured and used as potential acid tolerant biofertilizers and PGPR. Many of the ‘Unclassified’ genera could be novel bacteria and more research is needed to identify their taxonomic position and functional role in the ecosystem.
Metagenomic approach to assess diversity of bacterial community in saline pokkali habitats of kerala
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2012) Sarveshwar Sah; Girija, D
Metagenomics to assess bacterial diversity in the soil as influenced by organic and chemical inputs
(Center for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Ashwini, S P; Girija, D
Microbial inoculants for enhancing degradation of biosolid waste in aerobic composting
(Department of Agricultural Microbiology, College of Horticulture, Vellanikkara, 2019) Shilpa, P; Girija, D
Solid waste management is a major challenge throughout the world, especially in urban areas, due to the rapid growth of population along with urbanization. Earlier, centralized management of biosolid waste was being practiced. However, due to problems in transportation and segregation, management at source is being promoted. Aerobic composting has been practiced from time immemorial for recycling of biosolid waste, using various processes and containers. Recently, more importance is being given to bio- composting, considering the efficiency of microorganisms in enhancing degradation of organic substrates by their multiple mode of action. Hence, this study was taken up to explore microorganisms which can enhance the process of aerobic composting of biosolid waste. Isolation of potential decomposing microorganisms was attempted from different compost samples including coir pith compost, kitchen waste compost and Oushadhi ayurvedic compost. A total of 14 isolates were obtained from different compost samples. All the isolates were assigned names depending upon the type of microorganism and the source from which they were isolated. Based on the ability to degrade the chemical components in selective medium, four isolates (BaBc-1, BaCp-1, BaOu-1 and AcOu-1) and four reference cultures (Bacillus subtilis, Bacillus niabensis, Gongronella butleri and Trichoderma asperellum) were selected for quantitative assay. Enzyme assay was carried out for selected isolates and the isolate G. butleri exhibited highest cellulase activity. BaBc-1, B. subtilis and BaOu-1 recorded significantly higher β- 1, 3 glucanase activity. Glucosidase activity was found to be significantly high in G. butleri, T. asperellum, BaBc-1 and B. subtilis. Significantly higher laccase, amylase and pectinase activity was recorded in BaOu-1, BaBc-1 and AcOu-1 respectively. Maximum protease activity was recorded in fungal isolates G. butleri and T. asperellum. Potential isolates were further subjected to cultural, morphological, biochemical and molecular characterization. The isolate BaBc-1 showed maximum homology to Bacillus subtilis, BaCp-1 to Bacillus cereus, BaOu-1 to Bacillus sp. and the actinomycete isolate AcOu-1 to Streptomyces roseofulvus. The compatible combinations of selected isolates with high enzyme activity were selected for formulation of microbial consortia and the consortia were evaluated for degrading vegetable waste under in vitro condition. All the inoculated treatments showed faster degradation compared to uninoculated control. Based on visual observations, per cent weight reduction, enzyme activity and microbial population on 21 DAI in flask culture, consortium II (B. subtilis BaBc-1+ T.asperellum+ Bacillus sp. BaOu-1) and consortium IV (B. subtilis+ G. butleri +B. subtilis BaBc-1) were selected for pilot scale experiment. The efficiency of selected consortia was evaluated in KAU smart biobin along with cow dung slurry and uninoculated treatment. In T1 (B. subtilis BaBc-1+ T.asperellum+ Bacillus sp. BaOu-1) compost formation was initiated within 17 days after inoculation. Based on the volume reduction, duration of composting process, yield of compost, microbial population and phytotoxicity of compost, consortium I (B. subtilis BaBc-1+ T.asperellum+ Bacillus sp. BaOu-1) was selected as best performing consortium in KAU smart biobin. Hence, this consortial formulation was selected for large scale experiment in Thumburmuzhi composting units. Cow dung was used as inoculum in positive control and uninoculated treatment served as negative control. The treatment T1 (B. subtilis BaBc-1+ T.asperellum+ Bacillus sp. BaOu-1) recorded maximum temperature (640C) during composting period, faster volume reduction and maximum microbial population in compost. Based on these results, T1 was found to be the best treatment in Thumburmuzhi composting unit. The study revealed that, consortial formulation of B. subtilis BaBc-1, T. asperellum and Bacillus sp. BaOu-1 could be exploited for enhancing degradation of biosolid waste in aerobic composting. This can be used in future for the management of agricultural and municipal solid waste. The plant growth promoting (PGP) activities of these isolates could be an added advantage in improving the growth and yield of plants.