The endophytic colonization of sugarcane by G. diazotrophicus represents the first established case in which a monocot plant hosts a diazotrophic bacterial species, supplies significant amounts of fixed nitrogen for plant growth. Several bacterial endophytes have been isolated from a single plant (Zinniel et al., 2002). The tissue type and location of endophytes within plants, affect the bacterial community and their frequency of colonization. Bhoonobtong et al. (2012) isolated endophytic bacteria Bacillus amyloliquefaciens from T. cordifolia and characterize its antimicrobial activity. Kaur and Vyas (2017) isolated thirty five bacterial endophytes from six stem samples of T. cordifolia collected from three different districts of Punjab, India. They further carried research on Pseudomonas sp. and explored this bacterial species for phosphate solubilization and plant growth promoting activity.
The present investigation pertains to isolation of G. diazotrophicus from different parts of elite sugarcane cultivars, collected from Gene Park of sugarcane research institute, RPCAU, Pusa in Samastipur district of Bihar, which is situated in middle gangetic plains of India region and characterization of the isolates for their efficiency in vitro. Out of 115 isolates screened only 15 efficient isolates were screened out based on nitrogen fixing capacity, phytoharmone production and solubilization of phosphorous and zinc as well as production of low molecular weight organic acid in broth. An isolate from sugarcane GdS08S fixed highest amount of nitrogen i.e., 153µg of N/mg of Carbon used and also produced maximum concentration of Indole acetic acid (7.58µg/ml) while isolate GdS15S produced maximum concentration of Gibberellic acid (8.19µg/25ml). The isolate GdS07R showed highest phosphorus solubilization zone of diameter 4.22 cm whereas zone of zinc solubilisation of 4.52 cm. The G. diazotrophicus which is present in intercellular space of plant tissue and in addition to N fixation, performed the function of biosynthesis of phytoharmone (IAA), promote rooting and improve sugarcane growth under field condition. The GA syntheses help in cell elongation which ultimately increases the cane length. The best 15 screened isolate have been tested for production of different organic acid (Tartaric, Fumaric, Malonic, Gluconic, Citric, and Lactic) and titratable acidity which play pivotal role in solubilization of insoluble phosphorous and zinc.
The exploration of G. diazotrophs from different crops paves the way to reduce the cost incurred on nitrogenous fertilizers as well as minimizes the risk of pollution created by continuous application and their leaching. Though G. diazotrophicus was first isolated from sugarcane (Cavalcante and Dobereiner, 1988), several literatures showed its wide occurrence in different crops (Jimenez - Salgado et al., 1997; Loganathan et al., 1999; Hernandez et al., 2000). Many isolates of G. diazotrophicus from different crops (sugarcane, maize, pineapple and carrot) and these plants contain in their cell sap considerable amount of sucrose so they can survive in the plant system and proliferate. The selective biochemical tests clearly points out the occurrence of G. diazotrophicus. These putative endophytic strains of G. diazotrophicus also exhibited appreciable amount of nitrogen fixation phytoharmone production, solubilization of zinc and phosphorous.
The endophytes play an important role in sustainable crop production; probable mechanism of growth promotion may be the production of growth hormone. The IAA and gibberellins have been found in the cultures of G. diazotrophicus and also reported by Bastian et al., 1998. Till date Azospirillum is the promising diazotroph recommended to graminaceous crops as biofertilizer. All the isolates exhibited significant amount of phosphate and zinc solubilizing capacity. Alloway, (2001) also reported that the inclusion of solubilizing bacteria for P and Zn, as a microbial inoculants in crop production techniques performs beneficial role in India, where high incidence (more than 70%) of zinc deficiency occurred. Thus, understanding basic mechanisms behind the growth of crop may help to minimize the cost of sugarcane production e.g. phosphorus and zinc are the nutrients that are essentially required by the sugarcane crops but they are relatively transformed into insoluble/ sparingly soluble form by complex soil reactions and become major portion of the applied nutrient, unavailable to plants. In order to improve the status of nutrients, G. diazotrophicus may be used in combination with less expensive materials such rock phosphate and zinc insoluble compounds/ores. Along with its capacity to fix nitrogen and produce growth hormones, G. diazotrophicus will also play a new role in the stimulation of plant development in the upcoming years. Thus the studies on G. diazotrophicus may be diversified, several basic growth promoting properties of these bacteria may be elucidated. According to Fisher and Newton (2005), the molybdenum-dependent mechanism (Mo-nitrogenase) that G. diazotrophicus uses to fix nitrogen may deliver a significant amount of fixed nitrogen to its host. Several scientific studies revealed that certain cultivars of sugarcane are capable of having up to 200 kg N per hectare fixed by G. diazotrophicus, meeting approximately more than half of the crop’s nitrogen requirement without the application of additional fertilizers (Boddey et al., 2001; Lima et al., 1987). Earlier reports revealed the unique feature of G. diazotrophicus that it does not contain a nitrate reductase protein. Therefore, the bacterium is capable of nitrogen fixation in crops that are supplemented with either nitrate-based fertilizers or with low amounts of ammonium-based fertilizers (Eskin et al., 2014).
The strain has increased nitrogen fixation activity in the testing, results resembled with the work of Nong et al. (2022) which, where characterized under various physiological and nutritional sources. The nitrogenase activity of the strain was substantially significant between the temperature ranges of 28oC to 37oC with the maximum at 32oC. In addition, glucose, sucrose, ethanol and mannitol were used as carbon sources. With 5% sucrose, the strain showed enhanced nitrogenase activity, however glucose had similar tendencies at different doses (1–10%). The nitrogenase activity reached at 4 and 6 g/L of (NH4)2SO4 and KNO3, respectively, in terms of nitrogen sources. Plant hormones play important role in the resistance response to endophytic bacterial colonization (Ibort et al., 2018). Different plant hormones (IAA and GA) can exert their biological functions synergistically or antagonistically through complex and cross connected network like regulatory pathways to regulate the process of plant development (Berens et al., 2017). A plant growth promoting substance, such as indole-3-acetic acid, known to be produced by G. diazotrophicus, could be a nitrogen fixation independent factor (Lee et al., 2004).
Studies related to the production of low molecular organic acids have shown that tartaric, gluconic and fumaric acids were major organic acids produced by G. diazotrophicus. The result is comparable to that reported by Illmer and Schinner (1995), who found that Aspergillus niger produces little quantities of gluconic acid. However, it contradicts the reports by Whitelaw et al. (1999) that gluconic acid was not produced by phosphate solubilizing microbes (PSM) strains used in their experiments. Venkateswarlu et al. (1994) detected lactic acid from Aspergillus niger while none of the two isolates produced lactic acid in the present study. Aspergillus flavus produced malonic, tartaric and gluconic acid with low, moderate and low intensity respectively.
On the basis that the kind and quantity of acid generated relies on the phosphate solubilizing strain, medium composition, growth conditions, and a number of other parameters. Phosphate solubilizing microorganisms used glucose as carbon source to produce organic acids. These results are similar to Nautial et al. (1999). They stated that more organic acids are produced by microorganisms when glucose was used as carbon source. Based on the total amount of organic acids and the quality of the organic acids produced by the phosphate-solubilizing bacterial isolates, GdS15S was found to be the most effective isolate, followed by GdS02R and GdS18L. This is because calcium was most strongly chelated by a tribasic acid (citric acid), but less strongly by aliphatic monobasic acids (gluconic acid), malic acid, and tartaric acid. Thus citric acid produced by GdS15S followed GdS02R and GdS18L were most powerful chelator of calcium than other organic acids produced by different bacterial isolates, Wada et al. (2021).
As conclusions, our data show that 15 efficient isolates were prooved best on the basis of plant growth promoting activities like nitrogen fixing ability, production of IAA and GA, P and Zn solubilization. The endophytic bacteria isolated from different parts of sugarcane, in that we found root isolated stains were performed best in organic acid production as well as P and Zn solubilization, but in case of phytoharmone production and nitrogen fixing ability were performed best in stem isolated strains. So, finally G. diazotrophicus has prooved a potential biofertilizer for enhancing nutrient (N, P and Zn) use efficiency in sugarcane crop which is considered as a high input crop.