Chief EditorT. Mohapatra
Print ISSN 0367-8245
Online ISSN 0976-058X
NAAS Rating 5.20
Full Research Article
Ignatzschineria cameli Strain KAUPDF7-A First Report on Plant Growth Promoting Rhizobacteria (PGPR) from the Post-flood Affected Soils of Kerala
- Email firstname.lastname@example.org
First Online 26-06-2023|
Background: Kerala flood in 2018 and 2019 had reduced the yield in many agricultural plots of Attapadi, Kerala, India. The scope of the study was to identify the potential native plant growth promoting rhizobacteria from the post flood-affected sites to rejuvenate the nutrient depleted soils.
Methods: The present study was carried out in the department of agricultural microbiology, College of Agriculture, Kerala Agricultural University, Vellanikara, Kerala during 2020-2022. Bacteria were isolated from post flood-affected agricultural soils of Attapadi in Palakkad district of Kerala, India. Three morphologically distinct isolates were screened for cellulase, laccase and dehydrogenase to select the best bacterial isolate that could produce the multifunctional enzymes for rejuvenation of flood-affected soils. The isolate were also screened for plant growth promotion traits such as; indole acetic acid (IAA), phosphate solubilizing ability, nitrogen fixing ability and potassium solubilizing ability.
Result: The most promising isolate was identified as Ignatzschineria cameli, which was found to be a high indole acetic acid producer along with phosphate and potassium solubilizing ability and revealed as the first report of PGPR from post flood-affected soils of Kerala.
Plant growth regulators (PGRs) mediate interactions between plant growth promoting microorganisms and the rhizosphere region of soil, which benefits the plants and might result in the geochemical cycling of soil nutrients (Rana et al., 2011). The decline of production in the impacted agricultural fields may be related to the loss of these potential microbial biomass which resulted in the depletion of soil nutrients. In order to improve the growth and yield of agricultural crops and maintain the sustainability of agro-ecosystems, it is crucial to screen and select effective PGPRs and use them in the integrated nutrient management strategies. However, more research are required because the long-term implications of are still unknown (Mace et al., 2016).
To identify potential PGPRs with soil rejuvenating ability, screening for their enzyme producing ability is relevant. The soil benefits from cellulose, which is the most prevalent polysaccharide in plant cell walls (Richards, 1988). Exo, endo and b-glucosidase, which make up the important enzyme cellulase, cooperate to break down the substrates of cellulose polymers. After cellulose, lignin is the most prevalent source of soil-based carbon (Kubicek, 2012). According to Datta et al., (2017), laccases and peroxidase enzymes can depolymerize phenolic and non-phenolic polymer lignin as well as the insoluble lignin. They can also cause lignin degradation by low molecular weight free radicals like OH. Dehydrogenases are only found within cells and they play a key role in the early stages of soil oxidation (Januszek et al., 2014) by transferring hydrogen from organic substrates to an inorganic acceptor (Zhang et al., 2010).
In the present studies, the test location was a two-acre tract in Thazhesambarcode, Attapadi (11.071808, 76.571502), interplanted with banana, arecanut, coconut and pepper. According to the farmer, the region’s overall crop yield was 50% lower than in previous years. The property experienced flooding in 2018 and 2019 due to its proximity to the river. The isolates TSFAB1, TSFAB2 and TSFAB3 (three morphologically different bacterial isolates obtained from the flood-affected locations of Thazhesambarcode, Attapadi) were screened for the enzymes, cellulase, laccase and dehydrogenase.
The primary objective of the study was to identify a potentially beneficial multifunctional isolate that can promote plant growth in the flood-affected areas. The found isolate may also be used to revitalize nutrient-depleted soils, perhaps increasing the reduced crop output in the afflicted areas.
MATERIALS AND METHODS
The bacterial isolates were isolated on Soil Extract Agar by serial dilution and plate count method (Aneja, 2003). The population was recorded and the isolates with varying colony morphology were purified and preserved for further work. The three selected isolates were screened for 3 enzymes, i.e. dehydrogenase (TTC method) (Thalmann, 1968), Laccase (Bavendamm, 1928) and Cellulase (Sazci and Erenler, 1986) by agar plate based screening method. Based on the maximum multi-functional enzyme activities, one isolate (TSFAB1) was further screened for plant growth promoting traits. Indole Acetic Acid (IAA) was quantified for the selected microbial isolate, TSFAB1 using Salkowski’s reagent method (Gordon and Weber, 1951). Nitrogen was quantified by Kjeldhal method (Kjeldahl, 1883), phosphorous with ANSA reagent (Olsen, 1982) and potassium quantified using flame photometer (Knudsen et al., 1982).
Colony morphology of the selected isolate was analyzed in nutrient agar. Microscopic analysis by Gram’s staining and spore staining was performed. The motility of the isolate was monitored by hanging drop method and in SIM medium with triphenyl-tetrazolium chloride (TTC). Biochemical characteristics were analyzed for indole utilization (I), methyl red (MR), Voges-Proskauer (VP), fermentation of sugars (glucose, fructose, lactose, sucrose, maltose and D-mannitol), catalase, oxidase and citrate utilization (Basak and Shetty, 2021).
DNA of the selected bacteria was isolated using NucleoSpin Tissue Kit following manufacturer’s instructions. The isolated DNA of bacteria was amplified using 16S rRNA primers (16S-RS-F, Forward primer CAGGCCTA ACA CATGCAAGTC and 16S-RS-R Reverse primer GGGC GGWGTGTACAAGGC). The PCR amplification was carried out in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems). ExoSAP-IT treated PCR products were sequenced in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) using the BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, USA) following manufactures protocol. The sequence quality was checked using Sequence Scanner Software v1 (Applied Biosystems). Sequence alignment and required editing of the obtained sequences were carried out using Geneious Pro v5.1 (Drummond et al., 2010).
RESULTS AND DISCUSSION
Colony characteristics of the isolate showed that the colonies were small, off-white in color with a raised elevation with an excavation in the centre, irregular form and with undulate margin (Fig 2). The cells were gram negative short rods (Fig 3). They were non-sporulating, highly motile and oxidase positive but could not utilize citrate. Voges-Proskauer (VP) tests showed positive reaction, whereas indole (I) production and methyl red (MR) test were negative. The isolate could utilize glucose, fructose and sucrose, partial fermentation of mannitol and maltose recorded. Lactose was not utilized by the isolate (Fig 4).
The potential multifunctional enzymatic ability revealed that TSFAB1 was a maximum dehydrogenase and cellulase producer with efficient laccase production. Based on the maximum multifunctional enzyme producing ability, the isolate selected (TSFAB1) were further screened for the four plant growth promoting parameters; IAA, nitrogen, phosphorus and potassium. Plant growth promoting rhizobacteria (PGPR) fixes atmospheric nitrogen, dissolves soil-insoluble phosphates and stimulates the release of various phytohormones that promote plant development (Mahanty et al., 2017). The plant growth promoting traits (phosphate solubilization, indole acetic acid production (IAA), potassium solubilization and nitrogen utilization) analyzed showed that the TSFAB1 produce 92.75 µg/ml/hour indole acetic acid in Luria Bertani broth containing 0.05% L-Tryptophan at room temperature. TSFAB1 also produced phosphate (8.8 µg/ml), potassium (4.42 µg/ml) and nitrogen (0.91µg/ml) (Table 1). IAA enhances plant development and yield. The generation of the trait in the medium also depends on tryptophan concentration, bacterial stage, incubation and media conditions (Bessai et al., 2022). Rhizobium species (142 μg IAA/ml) (Kumar et al., 2012) Pseudomonas aeuruginoasa (116±0.13 and 108±0.26 ìg IAA/ml) (Uzma et al., 2022) and Bacillus safesnsis (85.70±3.55 µg IAA/ml) (Lakshmanaan et al., 2022) are the common PGPR species which are reported with higher quantities of indole acetic acid (IAA).
The isolate (TSFAB1) were identified using morphological and biochemical characters and confirmed by 16SrRNA sequencing as Ignatzschineria cameli. The species was frequently described as being linked to infectious illnesses in humans and animals (Barker et al., 2014; Le Brun et al., 2015; Heddema et al., 2016). Ignatzschineria cameli was confirmed as the isolate by molecular (16S rRNA) sequencing. The 16SrRNA gene partial sequence obtained was searched using National Centre for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) and found 100% similarity with Ignatzschineria sp. and Ignatzschineria cameli. The sequence was 763 bases in length. The obtained partial sequence was submitted to National Centre for Biotechnology Information (NCBI) Gen Bank (Accession number: OP435588). According to Park et al., (2021), an Ignatzschineria sp. identified was a highly auxin (IAA) producing bacterial strain found in Korea and had an impact on plant growth. However, the species was not specified. According to our research, Ignatzschineria cameli, a potentially multifunctional isolate with PGPR activities, was found in flood affected soil samples from Thazhesambarcode, Attapadi Kerala. The isolate has reportedly been linked to necrotic foot tissue in camels and related maggots in the United Arab Emirates (Tsang et al., 2018). The isolate is a first report as PGPR from flood affected areas of Kerala.
CONFLICT OF INTEREST
- Aneja, K.R. (2003). Staining and Biochemical Techniques. In: Experiments in Microbiology Plant Pathology and Biotechnology (4th ed.), New Age International Ltd, New Delhi. 157-162.
- Barker, H.S., Snyder, J.W., Hicks, A.B., Yanoviak, S.P., Southern, P., Dhakal, B.K., Ghimire, G.R. and Couturier, M.R. (2014). First case reports of Ignatzschineria (Schineria) indica associated with myiasis. Journal of Clinical Microbiology. 52: 4432-4434. DOI:10.1128/JCM.02183-14.
- Basak, S. and Shetty, P.H. (2021). Conventional Microbial Counting and Identification Techniques. In: Techniques to Measure Food Safety and Quality [Khan, M.S., Shafiur Rahman, M. (eds.)]. Springer, Cham. DOI: 10.1007/978-3-030- 68636-9_4.
- Bavendamm, W. (1928). Uber dad vorkommen und den nachweisvon oxydasen bei holzstorenden pilzen. Z Ptlkrankh Schulz. 38: 257-276.
- Bessai, S.A., Bensidhoum, L. and Nabti, E. (2022). Optimization of IAA production by telluric bacteria isolated from northern Algeria. Biocatalysis and Agricultural Biotechnology. 41: 102379. DOI:10.1016/j.bcab.2022.102319.
- Datta, R., Kelkar, A., Baraniya, D., Molaei, A., Moulick, A., Meena, R.S. and Formanek, P. (2017). Enzymatic degradation of lignin in soil: A review. Sustainability. 9(7): DOI: 10.3390 /su9071163, 2017.
- Drummond, A.J., Ashton, B., Buxton, S., Cheung, M., Cooper, A. et al. (2010). Geneious v5.5, Available: http://www. geneious.com.
- Gordon, S.A. and Weber, R.P. (1951). Colorimetric estimation of indole acetic acid. Plant Physiol. 26: 192-195.
- Heddema, E., Janssen, F., van Westreenen, H. (2016). A case of Ignatzschineria bacteraemia in an unconscious man from the Netherlands. JMM Case Reports. 3:e005043.
- Januszek, K., Blonska, E., Dluga, J., Socha, J. (2014). Dehydrogenase activity of forest soils depends on the assay used. International Agrophyics. 29: 47-59.
- Kjeldahl, J. (1883) A new method for the determination of nitrogen in organic matter. Zeitschrift für Analytische Chemie, 22: 366-382. DOI: 10.1007/BF01338151.
- Knudsen, D., Peterson, G.A. and Pratt, P.F. (1982). Lithium, sodium and potassium. In: Methods of Soil Analysis, Part 2: Chemical and Microbiological Properties. [Page, A.L., Miller, R.H. and Keeney, D.R. (eds.)], American Society of Agronomy, Inc and Soil Science Society of America, Inc, Madison, Wisconsin, U.S.A, 225-246.
- Kubicek, C.P.B. (2012). The Plant Biomass In: Fungi and Lignocellulosic Biomass. [Kubicek, C.P.B. (eds.)], Wiley-Blackwell, Oxford, UK. 1-28.
- Kumar, P. and Ram, M. (2012). Production of indole acetic acid by rhizobium isolates from Vigna trilobata (L) Verdc. African J. Microbiol Res. 6(27): 5536-5541.
- Lakshmanan, R., Poyil, M.M., Kalaimurugan, D., Sivasankar, P., Ponmurugan, K. and Venkatesan, S. (2022). Optimization, characterization and quantification of indole acetic acid produced by a potential plant growth promoting rhizobacterium Bacillus safensis YKS2 from Yercaud Hills, Eastern Ghats. Journal of Pure Applied Microbiology. 16(3): 1998-2009. DOI: 10.22207/JPAM.16.3.50.
- Le Brun, C., Gombert, M., Robert, S., Mercier, E., Lanotte, P. (2015). Association of necrotizing wounds colonized by maggots with Ignatzschineria associated septicemia. Emerging Infectious Diseases. 21: 1881-1883.
- Mace, O.G., Steinauer, K., Jousset, A., Eisenhauer, N. and Scheu, S. (2013). Flood-induced changes in soil microbial functions as modified by plant diversity. PLoS ONE. 11: e0166349. DOI: 10.1371/Journal.pone.0166349, 2016.
- Mahanty, T., Bhattacharjee, S., Goswami, M., Bhattacharyya, P., Das, B., Ghosh, A. and Tribedi, P. (2017). Biofertilizers: A potential approach for sustainable agriculture development. Environmental Science and Pollution Research. 24: 3315- 3335. DOI: 10.1007/s11356-016-8104-0.
- Olsen, S.R. and Sommers. L.E. (1982). “Phosphorus,” In: Methods of Soil Analysis, Part 2: Chemical and Microbiological Properties. [Page, A.L., Miller, R.H. and Keeney, D.R. (eds.)], American Society of Agronomy, Inc and Soil Science Society of America, Inc, Madison, Wisconsin, U.S.A. 403-430.
- Park, S., Kim, A.L., Hong, Y.K., Shin, J. and Joo, S. (2021). A highly efficient auxin-producing bacterial strain and its effect on plant growth. Journal of Genetic Engineering and Biotechnology. 19: 179. DOI: 10.1186/s43141-021-00252-w.
- Rana, A., Saharan, B., Joshi, M., Prasanna, R., Kumar, K. and Nain, L. (2011). Identification of multi-trait PGPR isolates and evaluating their potential as inoculants for wheat. Annals of Microbiology. 61: 893-900. DOI: 10.1007/ s13213-011-0211-z.
- Richards, B. (1988). The microbiology of terrestrial ecosystems. Journal of Ecology. 5: DOI: 10.2307/2260337.
- Sazci, A. and Erenler, K. (1986). Detection of cellulolytic fungi by using Congo red as an indicator: A comparative study with the dinitrosalicyclic acid reagent method. Journal of Applied Microbiology. 61: 559 -562.
- Thalmann, A. (1968). Zur Methodik der Bestimmung der Dehydroge naseaktivität im Boden mittels Triphenyltetrazolium chlorid (TTC). Landwirtschaft Forschung. 21: 249-258.
- Tsang, C.C., Tang, J.Y.M., Fong, J.Y.H., Kinne, J., Lee, H.H., Joseph, M., Jose, S., Schuster, R.K., et al. (2018). Ignatzschineria cameli sp. nov., isolated from necrotic foot tissue of dromedaries (Camelus dromedarius) and associated maggots (Wohlfahrtia species) in Dubai. International Journal of Systemic and Evolutionary Microbiology. 68: 3627-3634. DOI: 10.1099/ijsem.0.003046.
- Uzma, M., Iqbal, A. and Hasnain, S. (2022). Drought tolerance induction and growth promotion by indole acetic acid producing Pseudomonas aeruginosa in Vigna radiata. PLoS One. 17(2): e0262932. DOI: 10.1371/Journal.pone. 0262932.
- Zhang, N., He, X., Gao, Y., Wang, H., Ma, D., Zhang, R., Yang, S. (2010). Pedogenic carbonate and soil dehydrogenase activity in response to soil organic matter in Artemisia ordosica community. Pedosphere. 20: 229-235.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.