Unravelling Promising Diazotrophic Microbiota of Rauvolfia tetraphylla L.  with Existing Conventional Nitrogen-Fixing Microbial Partners

Introduction

The rampant use of agricultural fertilizers to meet the demand of food for burgeoning population has remained a contentious issue, especially when the world is faced with serious concerns like  climate change, Greenhouse effect, depleting land resources and fossil fuels. Early 1950s witnessed pioneering efforts by Norman Boralug, who introduced the Green Revolution model to address food security concerns. This model among other tenets, emphasized on the adoption of nitrogenous fertilizers in the farmers’ fields.  Although the model became a pervasive aspect of agriculture, its excessive use proved unsustainable, because the application was undertaken without comprehensive and long-term assessment of  their ecological, economic, and agronomic consequences.^1,2 The impact of green revolution in agriculture was overwhelming as the production enhanced and mortality rate decreased, but the sustainability of Green revolution itself  was at stake because  enhancing agricultural production remained the main focus, while other concerns like environmental sustainability and nutritional value of the produce were overlooked.^2,3 Among other environmental hazards,  the nitrogen fertilizer industry and distribution chain of fertilizers contributed to 5 percent of the total nitrous oxide (a potent  Greenhouse gas)  emissions.⁴ In 2023 alone, the emissions from pre and post agricultural production of fertilizer  manufacturing is estimated to be 94.26kt,  32.37 kt,  2.01 kt and  0.423 kt  for China,  USA, Brazil and India respectively.⁵ Nitrogenous fertilizers  add nearly 150 million tons reactive nitrogen including HONO,  NH₃  and NO (nitrous acid, ammonia and nitric oxide) globally, and this is expected to increase further to 600 million tons in the coming years.⁶ Adding to the nitrogen footprint is also the nitrate runoff, that pollutes soil and water bodies and increases chances of  blue baby syndrome or methemoglobinemia and carcinomas in children and adults respectively. The changes in the chemistry of the atmospheric air on the health of man and ecosystems costs approximately USD 340 billion to USD 3.4 trillion yearly (https://www.unep.org/news-and-stories/story/four-reasons-why-world-needs-limit-nitrogen-pollution, accessed on 29.11.2025). So, it is essential to bring down nitrogen foot print and for this, nitrogenous fertilizers must be substituted by other ecofriendly and sustainable options like biofertilizers or microbial inoculants.

Nitrogen is the most abundant gas, comprising nearly 78 percent of the total atmospheric gases, contributing to vital component of biomolecules viz. amino acids, proteins, chlorophyll, nucleic acids, nitrogenous bases, alkaloids and numerous secondary metabolites in plants. However, plants are unable to access it directly from the environment. Diazotrophs are group of nitrogen-fixing microorganisms including bacteria, archaea and blue green algae, that play crucial role in reducing atmospheric nitrogen to plant usable forms like ammonium, referred as biological nitrogen fixation (BNF) through nitrogenase enzyme, a metalloprotein complex encoded by the nifH gene.⁷ This process harnesses the physiological potential of nitrogen-fixers that find application in biofertilizer formulations. Diazotrophs have been isolated from the root nodules of leguminous plants, but recent years have seen their exploration from the non-legumes also. Most pioneering work on nitrogen fixing bacteria was undertaken by Johanna Döbereiner, who originally hailed from Czechoslovakia, but later in 1953 moved to Brazil. Due to the contributions of Döbereiner and group, Brazil stands at the apex, surpassing many other countries for research on diazotrophic bacteria.⁸  Although, conventional nitrogen fixers like Rhizobia, Azotobacter, Azospirillum, Azolla–Anabaena symbiosis etc. routinely find application as commercial inoculants, there are a vast number of other less explored diazotrophs with multiple PGP (plant growth promoting) traits, that can be developed into bioinoculants.⁹  PGPR (plant growth promoting rhizobacteria) inoculants possess PGP traits like fixation of atmospheric nitrogen, solubilization of iron by siderophore production, solubilization of phosphate, synthesis of IAA etc.¹⁰  The incorporation of diazotrophic bacteria into agricultural practices is ecofriendly and sustainable, and  can reduce reliance on chemical fertilizers and therefore,  nitrous oxide emissions during the production and distribution chain of nitrogenous fertilizers.¹¹

Rauvolfia  tetraphylla (barachandrika  or devil pepper)  belongs to Dogbane family/ Apocyanaceae  and serves as a validated substitute to  Rauvolfia serpentina or  Sarpagandha due to their comparable alkaloid profile.^{12, 13}  The plant originated in some parts of Northern and Southern America, and it was later introduced to India, China, Nepal, Taiwan, Vietnam and some parts of Australia.¹⁴ Rauvolfia. serpentina was popularized  as an antihypertensive, antimicrobial and also for the treatment of insomnia,  anxiety,  etc. due to the presence of reserpine, a monoterpene indole alkaloid.¹⁵ Because of its medicinal value, Rauvolfia serpentina was ruthlessly exploited by the pharmaceutical industry, which drastically brought down its numbers, resulting in its inclusion in the endangered category by the IUCN.¹⁶  Recent in silico studies have shown that leaves  of R.  tetraphylla L. possess antiviral properties.¹⁷ In the Bhagalpur district (25.2372° N, 86.9746° E) of Bihar, India   R. tetraphylla L. is found growing abundantly in wild patches.

The seeds of Rauvolfia  tetraphylla L. have poor germination inspite of  83.1% viability of seeds in nature. Hence, multiplication through seeds and regeneration  by stem cuttings is difficult due to occurrence of phenolic compounds in the seed and also limited rooting.¹⁸ Besides, reserpine extraction from roots of  Rauvolfia tetraphylla requires sacrificing the whole plant, making its sustainable utilization difficult. Therefore, it is necessary to bring this plant into cultivation and application of microbial inoculants based on diazotrophs with PGP traits like IAA synthesis, iron solubilization and phosphate solubilization can lead to plant growth promotion and also increase concentration of bioactives. Mishra and Kumar have critically reviewed the interactions of  rhizospheric microbes and endophytes in Rauvolfia sp. and they could be of immense potential in sustainable agriculture.¹⁹

Among the major nitrogen fixing bacteria is Azotobacter, which is frequently used in formulations of biofertilizers, but it suffers from several pitfalls like inconsistent performance due to variations in soil physicochemical parameters and hence may not be effective in enhancing alkaloid level and ameliorating growth. Hence, exploring rhizomicrobiota of R. tetraphylla L. other than the conventional ones can provide useful insights for the development of microbial inoculant/s specially designed for this medicinal plant.

Materials and Methods

 Collection of rhizospheric soil

Healthy plants of Rauvolfia tetraphylla L.  growing in the Botanical Garden of University Department of Botany, Tilka Manjhi Bhagalpur University were carefully uprooted. The plants were kept in sterile polybags, with their rhizospheric soil intact and taken to the Biolab at  Bihar Agricultural University, Sabour within a few hours of collection. By vigourous shaking, bulk soil was removed, and the remaining soil adhering to the root (the rhizospheric soil) was used for the isolation of diazotrophs from the rhizosphere.

 Isolation of  diazotrophic bacterial groups

The rhizospheric soil was obtained by vigorous shaking in 1 L 0.9 percent NaCl for 10 minutes. The contents were put on orbital shaker at 300 rpm, for 90 minutes and then centrifuged to homogenize bacterial content in the supernatant, while the soil particles pelleted at the bottom.²⁰ Using 1 mL of the supernatant, serial dilution was carried out until reaching 10^-4 dilution. Usually 10^-3 or 10^-4  dilutions are taken  since at higher dilutions colonies are crowded and might overlap, whereas at  dilutions lesser than this, the colonies may be very few to count or almost absent from a plate. Ideally, the number of colonies on the plate should be from 30-300 for statistical accuracy. Hence 10^-3 or 10^-4  dilutions are preferred for enumeration and isolation of bacteria.^{21, 22}

 Isolation was done on Jensen’s agar media (procured from HiMedia) supplemented with bromothymol blue (BTB).²³ 100µL of each dilution was spread on the petriplates containing the sterile media in the laminar flow hood. After spreading the plates were incubated in inverted position in an incubator at 28°C till the appearance of visible colonies. The incorporation of BTB as a pH indicator is similar to that in NFb (nitrogen-free bromothymol blue) media for visual screening of diazotrophs.²⁴ Change in colour of the media around the colonies to  blue colour is regarded as nitrogen-fixation.^25,26 Isolates were purified and preserved in 20% glycerol stocks until further study.

Screening for PGP traits

Screening for siderophore production 

For qualitative screening for siderophore production, the selected diazotrophs were spot inoculated on Chrome Azurol Sulfonate (CAS) agar media (procured from HiMedia) and incubated at 28°C for 3-5 days. Development of orange halos around bacterial colonies indicated positive siderophore production.²⁷

 Screening for IAA production

50 μL of glycerol stock was inoculated in 5ml of nutrient broth medium containing 5 mM L-tryptophan for 48 h  at 28°C. Thereafter, 500 μL of freshly prepared Salkowski reagent (1 ml of 0.5 M FeCl₃ in 50 ml 35% HClO₄) was added to each tube and incubated in dark for 30 minutes. Development of pink colour indicated positive IAA production.²⁸

 Screening for phosphate solubilization

This was done on Pikovskaya’s agar medium (procured from HiMedia) containing tricalcium phosphate. Bacterial isolates were spot inoculated and incubated for 48 hours at a temperature of 28°C. Development of clear halo zones around isolates indicated positive phosphate solubilization.²⁹

 Morphological  characterization of selected isolates

Pure cultures (48-72 h old) were studied for their colony characteristics viz.  colour, shape, size, margin etc. on Jensens agar media amended with  BTB.

Biochemical and molecular characterization of selected diazotrophs

Isolates showing in vitro nitrogen-fixation by their visible blue colour on Jensens agar media amended with BTB were screened for citrate utilization, lysine and ornithine utilization, urease detection, phenylalanine deamination, nitrate reduction, H₂S production, and carbohydrate metabolism tests (glucose, adonitol, lactose, arabinose, and sorbitol) using  HiAssorted™ Biochemical Test Kit KB002 procured from HiMedia.³⁰

Extraction of genomic DNA from Bacterial isolates

From glycerol stocks, the  bacterial isolates  were revived in nutrient broth medium till the appearance of turbidity/ visible growth. The cultures were centrifuged  at 10,000 rpm for 10 minutes. Following centrifugation, the supernatant was discarded, and bacterial cells were pelleted down. From the pellets, extraction of genomic DNA  was performed using HiPurA^® Genomic DNA Purification Kit. The protocol provided by the manufacturer was followed, and upon isolation, the quality of separated DNA sequences  was checked using agarose gel electrophoresis. The genomic DNA  of the isolates in the gel were visualized by UV transilluminator.

Amplification of  16S r RNA gene with 16S universal Primers

Using purified DNA as template, 16S rRNA of the isolates were amplified  using  primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT3′). The resulting amplicons were resolved on a 1.5% TAE-agarose gel. Thereafter, the amplicons were purified and  sequencing was performed using ABI 3500 XL Genetic Analyzer.³¹ The generated sequences were allowed to overlap to get a concatenated sequence using CodonCode Aligner (Version 12.0.4). Thereafter,  the assembled sequences were converted into FASTA format files and subjected to alignment in public database using the EzBioCloud server (last updated on 21.04.2025) for identification of closely related species.³² Later, comparisons were carried on NCBI BLAST server  to determine the closest phylogenetic neighbour and the sequences were deposited in NCBI GenBank using the submission portal to obtain accession numbers. Alignment was done using Clustal W and phylogenetic tree was made using MEGA 12 software (version 12.0.14).³³

 Results and Discussion

 Isolation and screening for Nitrogen Fixation

Qualitative screening for nitrogen fixation was done on BTB containing Jensens media. The production of distinct blue colour within 2-5 days of incubation by the isolates, was taken as confirmatory test for nitrogen fixation. The study yielded 79 isolates and they were designated with codes RT1 to RT79. Among them, 10 isolates produced distinct blue colour on BTB containing Jensens media, but the fixation was higher among the isolates RT2, RT34, RT40A, RT71, RT77, RT78 and RT79.  Fig 1. Showing high nitrogen fixation by isolates RT71, RT77 and RT78 on BTB containing Jensens media. The results for in vitro screening for nitrogen fixation and PGP traits are highlighted in Table 1.

The isolate RT2 showed strong nitrogen fixation changing the colour of the Jensens medium containing BTB from forest green to blue and then to yellow and this aligns well with the earlier findings in Stenotrophomonas maltophilia.³⁴ Stenotrophomonas maltophilia  belongs to Gammaproteobacteria  and earlier research has implicated their role in cystic fibrosis and also nosocomial infections, frequently leading to morbidity.^{35, 36, 37}  In sharp contrast to these studies, several strains of  S. maltophilia harbour the rhizosphere and possess PGP traits like nitrogen fixation, phosphate solubilization, production of plant growth regulators, siderophore production etc. and are also involved in biofortification and degradation of xenobiotic compounds.³⁸ S. maltophilia have also been isolated from the roots of  Arachis hypogea where they have shown to regulate physiological and biochemical responses, and improve tolerance to stress,  under limiting nitrogen conditions.³⁹ Earlier the biofertilizer and biocontrol properties of  S. maltophilia  in sustainable cultivation of wheat has also been reported .⁴⁰

The first  report of isolation of Bacillus rugosus was done from a marine sponge.⁴¹ In another study in rice, although not reported as a diazotroph, B.  rugosus  occurred as an endophyte and it was found to provide tolerance to  multiple stress.⁴² B.  rugosus  RT34 has shown for nitrogen fixation on BTB containing Jensens media, however further validation for nitrogen fixation would require amplification of nif gene. Among the Bacilli members isolated in this study, Bacillus stercorsis RT40A was earlier isolated from Magnaporthe oryzae in rice and tomato wilts respectively, and they were found effective in controlling blast fungus in rice  and Fusarial wilts in tomato.^43,44 Although not shown to be a diazotroph in previous studies, B.  stercorsis RT 40A showed in vitro nitrogen fixation on BTB containing Jensens agar medium. Another PGPR, Bacillus cabrialesii was isolated from Mexican wheat varieties but its diazotrophic potential has not been reported yet.⁴⁵ In sharp contrast, the diazotrophic capacity of Bacillus cabrialesii RT51 on Jensens media containing BTB has been evidenced in our study. Another member, Lysobacter prati RT71 has shown high nitrogen fixation activity and this aligns with the earlier observation in E4 Lysobacter strain.⁴⁶ The earliest report of isolation of Lysobacter prati  was from plateau meadow.⁴⁷ The observation of nitrogen fixation by R. oryzihabitans aligns with the previous reports for the same being isolated from rice endosphere.⁴⁸ Previously,  Metapseudomonas lalkuanensis, was isolated  from contaminated soil in  Lalkuan in India .⁴⁹ Although literature does not mention it as a nitrogen fixer, but our studies have shown isolate Metapseudomonas lalkuanensis RT76 to be moderately fixing nitrogen.  Nitrogen fixing ability of Pseudomonas hunanensis RT77 aligns with the nitrogen fixing ability of the same isolate from soybean.⁵⁰ Pseudomonas hunanensis SPT26 was earlier reported to secrete antifungal substances that could control wilt diseases in tomato and was proposed as promising rhizobacteria for the formulation of microbial inoculant.⁵¹ In the present study the release of siderophores by Pseudomonas hunanensis  RT77 may be effective in controlling phytopathogens. Pseudomonas  mosselii isolated from rice rhizosphere is a promising biocontrol agent against rice blast fungus Magnaporthe oryzae and it was also reported to enhance yield and nutrient acquisition in rice.^52,53 P. mosselii isolated from durian rhizosphere was also reported as a nitrogen fixer and produced IAA from rhizosphere of durian rhizosphere.⁵⁴ In our study, P.  mosselii  RT78 did not solubilize phosphate but showed nitrogen fixation and siderophore production in the in vitro assay. Bacillus tequilensis RT79 although showing strong nitrogen fixation in our study, has not been reported as a diatroph from previous studies.

Figure 1: Petriplates showing pure cultures of   RT 71, 77 and 78 showing nitrogen fixing ability on Jensens media containing BTBClick here to view Figure

Screening of rhizospheric diazotrophic bacteria for Plant Growth Promoting Trait-IAA synthesis

The principal auxin, Indole-3-acetic acid (IAA) is  an important phytohormone regulating plant growth and development  by  cell enlargement and division, tissue differentiation, and responses to light, and also controls many aspects of  plant development including stimulation of root growth and nutrient acquisition from soil.  The capacity to produce the phytohormone indole-3-acetic acid (IAA) is widespread among bacteria that inhabit diverse environments such as soils, fresh and marine waters, and plant and animal hosts. Among the ten diazotrophic isolates, all the isolates produced IAA, but it was significantly higher for the RT2, RT34, RT76 and RT79. This was revealed by the intensity of colour produced using Salkowski’s  reagent, followed by incubation in dark. Therefore, it is expected that incorporating any of these isolates in a biofertilizer formulation will provide IAA to the host plants and improve  root architecture and branching and also help in facilitating  accumulation of bioactive. Enhanced IAA production by RT34 is consistent with the Bacillus rugosus isolated from Arachis hypogea.⁵⁵ Similarly RT2 showed higher production of IAA and this observation is in line with IAA released by S. maltophilia isolated from rhizosphere of chilli.⁵⁶ Stenotrophomonas, isolated from poplar  also showed auxin production in the in vitro condition similar to RT2, but there it existed as an endophyte , and in this study it was isolated from the rhizosphere.⁵⁷ Higher production of  IAA by Metapseudomonas lalkuanensis RT76 in this study has not been reported in any previous study. IAA production by P. mosselii RT 78 aligns with the production of IAA  the same isolate from Agave americana.⁵⁸ Similarly, Bacillus tequilensis from soybean rhizosphere was shown to produce IAA , which is similar to B. tequilensis  RT79 from R. tetraphylla .⁵⁹  Growth responses of IAA producing rhizobacteria on dicots and monocots by pot experiments would be further required to validate their potential in agriculture.

Screening of diazotrophs for siderophore production

In soils, iron occurs mostly as Fe³⁺ oxy-hydroxides and must be reduced to Fe²⁺ to be easily assimilated by plants and microorganisms. In agriculture, this is usually accomplished by synthetic chelates, which are costly and sometimes may even lead to toxicity when used in excess.⁶⁰ Besides this, alkalinity of soils in arid regions limit iron availability to plants leading to reduced crop yields, chlorosis etc. by disrupting the photosynthetic machinery. Certain rhizospheric bacteria release siderophores which help in enhancing iron availability to plants. The siderophores complexes with iron, diminishing its availability to pathogens like Fusarium oxysporum and  Rhizoctonia solani, thereby exerting their plant growth promoting effect. Qualitative screening for siderophore production relied on visual examination of  the halo zone around orange-yellow colonies produced by the isolates on CAS media  followed by incubation for 3-5 days at 28°C.  All the nitrogen fixing isolates, except RT51 and RT71 produced siderophores. S. maltophilia RT2 showed efficient iron solubilization and probably it was useful in scavenging iron from the soil.⁶¹ Rhizobium oryzihabitans RT74 also showed orange halo on CAS plate confirming siderophore production, however efficiency of solubilization is lesser as compared to other diazotrophs, and this observation is similar to earlier reports regarding siderophore production, in addition to IAA and ACC deaminase.⁴⁸ Bacillus rugosus RT34 is also positive for siderophore release, and this observation is in line with the observation in rice, where the same isolate also  provides tolerance to salinity and drought stress.⁶² Another Bacilli isolate, Bacillus tequilensis RT79 from R. tetraphylla was evidenced to solubilize iron and this observation was in line with earlier research focussing on biocontrol properties of  this isolate against Fusarium and it also showed production of IAA and siderophore.⁶³ Members of  Pseudomonas generally produce siderophores under iron limiting conditions.⁶⁴ M. lalkuanensis RT76 has been shown to produce siderophore P. mosselii RT78 produced siderophore  and this was also observed in  P. mosselii from durian rhizosphere.⁵⁴  Rhizobacteria producing  siderophores control phytopathogens by secreting antimicrobial compounds.⁶⁵

Figure 2: Plates highlighting iron solubilization on CAS media by RT2, RT34. RT40A and RT77.Click here to view Figure

Screening of diazotrophs for phosphate solubilization

After nitrogen, phosphorus is the second most important macronutrient that are needed by lants for growth and development. But sometimes, phosphorus availability to plants is also limited because it occurs in combined form with metal complexes. Phosphate-solubilizing bacteria (PSB) are applied as inoculants in fields to improve bioavailability of phosphate from the soil by converting organic and insoluble forms of phosphorus into soluble forms. Some diazotrophs, in addition to fixing nitrogen, also contribute to phosphorus solubilization through the release of organic acids and enzymes, further contributing to plant growth.  It has been  suggested that several nitrogen-fixing strains of Azotobacter  vinelandii have demonstrated promising phosphate-solubilizing capabilities and they could be used as superior candidates for biofertilizer development.⁶⁶ In rice, phosphate-solubilizing diazotrophic bacteria like Herbaspirillum and Burkholderia  were isolated and they were found to improve nitrogen use efficiency too.⁶⁷  In our study, diazotrophic isolates RT2, RT40A, and RT51   showed solubilization zone around colonies on Pikovskaya’s agar and the efficiency of phosphate solubilization was almost comparable for isolates  RT2, RT40A and RT51.

The ability of Stenotrophomonas maltophilia RT2 to solubilize phosphate aligns with previous research, where this isolate was also used for reclamation of wastes.⁶⁸  S. maltophilia was earlier isolated from Porang,  and it was able to fix nitrogen and also solubilize phosphate.⁶⁹Among the Bacilli isolates, Bacillus stercoris RT40 A and B. cabrialessi RT51 have shown phosphate solubilization but this PGP trait is not shown by the other two Bacilli isolates B. rugosus and B. tequilensis. The production of  IAA and phosphate solubilization by RT40A,  have previously  been reported in Bacillus stercoris.⁴⁴Pseudomonas hunanensis RT77 has shown no phosphate solubilization in our study, however the same isolate has shown other PGP traits in addition to phosphate solubilization in earlier studies.⁷⁰ Although none of the Pseudomonas isolates have shown  phosphate solubilization in our study, nevertheless phosphate solubilizing bacteria including Bacillus, Pseudomonas, Serratia and Enterobacter find application in the development of microbial inoculants and can be used to boost plant growth and development.⁷¹

Figure 3: Plates showing screening for phosphate solubilization on Pikovskaya’s agar medium. Note : Although RT34 forms a big colony but the halo is missing, so it is considered negative for phosphate solubilization.Click here to view Figure

Table 1: The table highlights PGP traits of the diazotrophs. The efficiency of each bacterial partner is indicated by + (low), ++ (moderate), or +++ (high) or ++++(very high) and the absence of PGP trait is indicated by ‘– ‘ sign.

Bacterial Isolate	Nitrogen Fixation	Siderophore Production	Phosphate Solubilization	IAA Production
RT2	++++	+++	++	+++
RT34	+++	+++	–	+++
RT40A	+++	+++	++	+
RT51	++	–	++	++
RT71	++++	–	–	+
RT74	++	+	–	+
RT76	++	+	–	+++
RT77	+++	+++	–	+
RT78	+++	++++	–	+
RT79	+++	++++	–	+++

 Carbon utilization pattern of  selected nitrogen fixing bacterial isolates

Different isolates showed varying responses for the utilization of different carbohydrates. Except RT34, RT40A and RT74, none of the isolates were capable of utilizing glucose. Adonitol utilization was shown by isolate RT 74 only. None of the isolates utilized lactose except RT74. Isolates RT34, RT40A, RT74, RT77 and RT78 showed utilization of arabinose. Only one isolate RT74 was capable of utilizing sorbitol. The results for carbon utilization pattern is highlighted in table 2.

Stenotrophomonas maltophilia RT2 was found to be  non-fermentative and this is consistent with the results of Swapna and Audipudi.⁵⁶ Glucose and arabinose utilization by B. rugosus is consistent with the findings in Arachis hypogea Rabari et al.⁵⁵ B. rugosus RT34 was not able to utilize lactose, but  B. rugosus from Arachis hypogea was found to utilize lactose.⁵⁵  One of the plausible reasons for this could be that certain bacteria prefer using simpler sugars, and may require ATP to breakdown disaccharide and therefore sticks to the former. Studies on adonitol utilization revealed that RT34 was not able to utilize adonitol, however previous studies have not revealed anything   on the utilization of adonitol by this isolate. Figueroa-Brambila et al., have reported glucose fermentation by B. cabrialessi but  this is not the case with B. cabrialesii  RT51.⁷² Previous studies have not explicitly mentioned studies on sugar utilization by B. cabrialessi.  Lysobacter prati RT71 did not utilize sorbitol, arabinose and lactose and these results are consistent with the previous findings.⁴⁷  The ability of Bacillus stercorsis  RT40A for citrate utilization and glucose fermentation is consistent  with earlier findings while negative nitrate reduction is contrary to the earlier findings reported so far.⁷³ R. oryzihabitans RT74 was found to ferment arabinose, sorbitol, lactose, adonitol and glucose is in sync with the earlier findings.⁴⁸ Metapseudomonas lalkuanensis RT76 did not  utilize sorbitol, any sugar in our study , but it showed utilization of glucose, arabinose, lactose, adonitol, and sorbitol as earlier reports.⁴⁹ Pseudomonas hunanensis  RT77 and P. mosselli RT78 utilized only arabinose and not any other sugars and this is a characteristic for several Pseudomonads . Bacillus tequilensis RT79  did not utilize any of the carbon sources tested including glucose, sorbitol, adonitol, lactose and arabinose and this observation is in sharp contrast to earlier studies indicating that phenotypic variability could be common.⁷⁴

Table 2: Shows the ability of diazotrophs to utilize various carbon sources.

	Glucose	Adonitol	Lactose	Arabinose	Sorbitol
RT 2	–	–	–	–	–
RT 34	+	–	–	+	–
RT 40A	+	–	–	+	–
RT 51	–	–	–	–	–
RT 71	–	–	–	–	–
RT 74	+	+	+	+	+
RT 76	–	–	–	–	–
RT 77	–	–	–	+	–
RT 78	–	–	–	+	–
RT 79	–	–	–	–	–

Biochemical Characterization of the selected diazotrophs

All the isolates were capable of utilizing lysine as the sole nitrogen source except RT71. Citrate utilization was shown by the isolates RT34, RT40A, RT71, RT74, RT76, RT77, RT78 and RT79. All the isolates utilized ornithine except RT71 and RT79. All the isolates showed urease activity except RT71. None of the isolates were capable of phenylalanine deamination. Nitrate reduction was shown only by the isolates RT74  and RT78. Only RT77  produced H₂S. Overall, the biochemical profiling of the isolates highlight their functional potential in the rhizosphere and are in agreement with the earlier findings, and minor variations have also been observed. In Table 3 biochemical characterization of the selected nitrogen fixers are mentioned.

S. maltophilia was found positive for nitrate reduction, but in our study it did not show nitrate reduction.⁷⁵ Further, urease positive result by RT2 is found to be consistent with earlier studies. Similarly, positive responses to lysine, ornithine, urease positive and negative for phenylalanine deaminase utilization is consistent with the results.⁵⁶ While negative nitrate reduction in RT2 is in sharp contrast to positive tests for nitrate reduction by Stenotrophomonas,  probably indicating that nitrate reduction may not be a useful feature of all Stenotrophomonas isolates.⁵⁶ Citrate utilization by Bacillus stercorsis  RT40A is consistent  with earlier findings while negative nitrate reduction is contrary to the earlier findings reported.⁷³

B. cabrialesii RT51 in sharp contrast to previous studies was neither able to reduce nitrite to nitrate, nor was able to use citrate, which possibly may be due to strain variations.⁴⁵ Negative lysine utilization, ornithine utilization and urease is consistent, while negative nitrate reduction of L. prati RT71 is not in sync with the observation of Fang et al.⁴⁷ R. oryzihabitans RT74 showing ornithine utilization, urease, nitrate reduction is similar to the observation.⁴⁸ Metapseudomonas lalkuanensis RT76 utilized citrate as earlier but  did not show nitrate reduction in sharp contrast to earlier studies by Thorat et al.  where it was positive for nitrate reduction.⁴⁹ P. hunanensis RT77 showed positive citrate utilization, negative nitrate reduction and this was  consistent with the reports of Gao et al.⁷⁶ P.  mosselii RT78 showed similar traits to P. hunanensis except negative nitrate reduction and positive hydrogen sulfide production for the former, and vice versa for P. mosselii RT78. Bacillus tequilensis RT79 utilized citrate, ornithine, lysine, showed urease activity and  nitrate reduction, and this is similar to the observation of Gatson et al.⁷⁴

Table 3: Shows biochemical characterization of the selected nitrogen fixers

	Citrate utilization	Lysine Utilization	Ornithine utilization	Urease Activity	Phenylalanine Deamination	Nitrate Reduction	H2S Production
RT2	–	+	+	+	–	–	–
RT34	+	+	+	+	–	–	–
RT40A	+	+	+	+	–	–	–
RT51	–	+	+	+	–	–	–
RT71	+	–	–	–	–	–	–
RT74	+	+	+	+	–	+	–
RT76	+	+	+	–	–	–	–
RT77	+	+	+	+	–	–	+
RT78	+	+	+	+	–	+	–
RT79	+	+	–	+	–	–	–

 Morphological traits of selected isolates

For all the isolates, colonies were round with smooth consistency, shape and their margins for certain isolates exceeded 1mm in diameter, and for some they were less than 1mm. Colour of colonies were generally white except RT34 and RT74, which were yellow in colour. The isolates were all rod shaped, and Gram negative, except Bacilli isolates RT34, RT40A, RT51 and RT79, which were Gram positive. The colonies of the diazotrophs were mostly slimy or mucoid, probably a mechanism to prevent nitrogenase enzyme from inactivation due to oxygen. Colony morphology and Grams reaction were similar to previous findings for all the isolates. The morphological characters of selected diazotrophs are mentioned in Table 4.

Table 4: Shows morphological characteristics of the selected diazotrophs.

	Colony shape	Colony size	Margin of colony	Colony elevation	Chromogenesis	Surface of Colony	Texture	Cell Shape	Grams Staining
RT2	Round	>1mm	Entire	Raised	White to pale yellow	Smooth	Mucoid	Rod	Negative
RT34	Round	>1mm	Entire	Convex	yellow	Rugose	Butyrous	Rod	Positive
RT40A	Round	>1mm	Undulate	Convex	Creamy white	Wrinkled	Rough and dry	Rod	Positive
RT51	Round	>1mm	Entire	Convex	White	Rough	Slimy	Rod	Positive
RT71	Round	<1mm	Entire	Raised	Off white	Smooth and shiny	Mucoid	Rod	Negative
RT74	Round	>1mm	Entire	Raised	Yellow	Smooth and shiny	Slimy	Rod	Negative
RT76	Round	>1mm	Entire	Flat	Round	Smooth and moist	Mucoid	Rod	Negative
RT77	Round	>1mm	Entire	Flat	White	Smooth and moist	Slimy	Rod	Negative
RT78	Round	>1mm	Entire	Flat	White	Smooth and moist	Slimy	Rod	Negative
RT79	Round	>1mm	Entire	Convex	White	Rough	Mucoid	Rod	Positive

Molecular Phylogenetic Identification of Diazotrophs by 16S rRNA gene sequencing

Molecular phylogenetic diversity studies of  diazotrophs by 16S r RNA gene sequencing, confirmed four species of  Bacilli, three Pseudomonads, one each of  Lysobacter, Stenotrophomonas and Rhizobium,  showing 100%  sequence similarity with the corresponding strains on NCBI. Fig 4: showed amplification of conserved 16S rRNA gene of the rhizospheric isolates, with M indicating 1 kb marker. Table 5 below highlights 16S rRNA based identification of diazotrophs using EzBiocloud and NCBI. NCBI Accession numbers of the isolates are also mentioned. Fig 5 – In fig 5 phylogenetic tree showing isolated diazotrophic strains from the rhizosphere of R. tetraphylla L. have been depicted. Bootstrap values are indicated on the branches and scale bar showing the number of substitutions per site is indicated below the tree.

Figure 4: Amplification of 16S rRNA gene of the diazotrophs, with M indicating marker of 1 kb DNA plus ladder are shown.Click here to view Figure

Table 5: Molecular identification of diazotrophs based on EzBiocloud type strains and NCBI reference sequences.

Isolate code	Homologous microorganism(% identity)(Accession no. of NCBI reference seq.)	Identified organism	GenBank Accession Number
RT2	Stenotrophomonas maltophilia M 15 (100%) ( LT222224.1)	Stenotrophomonas maltophilia RT2	PX363496.1
RT34	Bacillus rugosus strain SPB7(100%)(NR_181236.1)	Bacillus rugosus RT34	PX363497.1
RT40A	Bacillus stercoris strain JCM 30051 (100%)  (MN536904.1)	Bacillus stercoris RT40A	PX363498.1
RT51	Bacillus cabrialesii strain TE3 (100%) ( MK462260.1 )	Bacillus cabrialessi RT51	PX363499.1
RT71	Lysobacter prati  SYSU H10001 (Type) (100%)(MN181427)	Lysobacter prati RT71	PX363500.1
RT74	Rhizobium oryzihabitans strain M15 (100%) MT023790.1	Rhizobium oryzihabitans RT74	PX363501.1
RT76	Metapseudomonas lalkuanensis strain PE08(100%) (MF943158.1 )	Metapseudomonas lalkuanensis RT76	PX363502.1
RT77	Pseudomonas hunanensis strain LV(100%)( JX545210.1)	Pseudomonas hunanensis RT77	PX363503.1
RT78	Pseudomonas mosselii strain CIP 105259  (100%) (AF072688.2)	Pseudomonas mosselii RT78	PX363504.1
RT79	Bacillus tequilensis  KCTC13622 (100%)( AYTO01000043.1) (100%)	Bacillus tequilensis RT79	PX363505.1

Figure 5: Phylogram/ phylogenetic tree showing relationship of isolated diazotrophic strains from the rhizosphere of R. tetraphylla L.Click here to view Figure

Tree was made by the Neighbouring joining approach using MEGA 12. The numbers indicated on the branch indicate the bootstrap confidence values which means how many times this grouping was repeated when the tests were replicated 1,000 times. GenBank accession numbers are also indicated before the type strains. The phylogenetic distances were calculated by the Kimura 2-parameter method and are shown as units of substitution of bases at each site, modelled using gamma distribution (shape parameter = 1.00). Construction of phylogenetic tree revealed that bootstrap values ranged from 75-100%, with 75% considered reliable and 100% representing very strong statistical support for the grouping of clades.

The earliest derived lineage is represented by the Bacilli clade, while the other taxa were derived later.  Basal taxon is Bacillus tequilensis RT79 meaning it diverged earliest as compared to other sister taxa.  The diazotrophs based on Bacilli (Phylum – Firmicutes)  clustered together in the same clade and while the Pseudomonads (Phylum – Gammaproteobacteria) clustered in a separate  clade. Two other diazotrophs, Stenotrophomonas maltophilia RT2 and Lysobacter prati  RT71, belonging to Gammaproteobacteria have also been isolated and they form a separate clade showing 100% bootstrap value. Two Pseudomonas isolates RT77 and RT78 clustered together in the same clade showing 100% bootstrap values. Metapseudomonas lalkuanensis RT76 and the two isolates Pseudomonas hunanensis RT77 and P.  mosselli RT78 were derived from a common ancestor, again showing consistent 100% bootstrap value. Rhizobium oryzihabitans RT74 (Alphaproteobacteria) and all the members of Gammaproteobacteria/ Pseudomonads evolved from a common ancestor and this is supported by a strong bootstrap value of 100%.

Conclusion

The rhizosphere of R. tetraphylla L. harboured immense diversity of diazotrophs showing multiple PGP traits.  A total of 79 rhizospheric isolates were obtained on Jensen’s agar media and among these 10 isolates showed positive nitrogen-fixing capacity indicated by their ability to change forest green colour to blue on BTB containing  Jensens media. The rhizospheric isolates from R. tetraphylla L. belonged to Bacilli, Pseudomonads, Lysobacter, Stenotrophomonas and Rhizobium. A careful analysis of Table 1 indicates that RT2 and  RT40A are showing all the PGP traits including nitrogen fixation, siderophore production, phosphate solubilization and IAA synthesis. Although RT2 and RT40A showed comparable PGP traits,  but IAA production by RT2 is comparatively higher than RT40A. Hence, based on PGP traits highlighted in table 1, we recommend  that Stenotrophomonas maltophilia RT2 is the best isolate and it can be further studied for its colonization and  plant growth promoting effects in in vivo conditions.

Stenotrophomonas maltophilia RT2 strain is expected to provide increased levels of  fixed nitrogen to Rauvolfia plants that can be assimilated into biomolecules including monoterpene indole alkaloids(reserpine),  nitrogenous bases, amino acids, proteins and nucleic acids, enhancing root and shoot biomass and also accommodating elevated concentration of alkaloids. Iron is  an important micronutrient in plants, and also a component of  electron transport system (cytochromes), chlorophyll and ferredoxin etc., and plants need to maintain iron homeostasis. Enhanced IAA release by RT2 in the rhizosphere of R. tetraphylla is further expected to increase root branching and its architecture, to provide home for the  biosynthesis and accumulation of alkaloids. Hence, developing an microbial inoculant based on RT2 is expected to manifest overall health benefits to Rauvolfia. Screening for phosphate solubilization on Pikovskaya’s agar  medium has explicitly revealed the potential of RT2 for phosphate solubilization, and therefore developing inoculants based on RT2 can help in improving phosphate flux into molecules like ATP, NADPH, phospholipids, nucleic acid backbone  etc. Overall such studies can be useful in addressing sustainable cultivation of not only medicinal plants but also other crops. Whole genome sequencing studies of RT 2 should also be performed to know about the presence of gene/s and gene products responsible for PGP activities. Besides, since the isolate has previously been reported to cause nosocomial infections, it would be necessary to carry out functional genomic studies to ascertain its pathogenicity in humans. Besides, annotation of genes in whole genome of the organism would be useful in identifying other potential uses of this bacteria like production of secondary metabolites. Further studies are required to test the efficiency of  this isolate  by the acetylene reduction assay. Various combinations of isolates can be made into microbial consortia to study microbial synergies in plant growth promotion. Besides this, assessment of increase in alkaloid level following inoculation into tissue culture raised R. tetraphylla L.  plants to ascertain increment in the alkaloid level could be beneficial to the pharmaceutical industry, which at present faces scarcity of raw materials due to their over exploitation and switching to substitutes. Thus,  isolation and identification of diazotrophs particularly unconventional ones, with excellent PGP traits, from the rhizosphere of R. tetraphylla  points out that they could be complementing the conventional nitrogen-fixing microbial partners by adapting to diverse ecological niches. These endeavours would be valuable in addressing SDG 2030 by reducing excessive reliance on chemical fertilizers, and promote ecological sustainability in the production of medicinal plants and bioactives.

Acknowledgement

The present work was conducted at the Biolab, Bihar Agricultural University (BAU), Sabour, Bhagalpur. We sincerely thank Prof. Anshuman Kohli, Director, Department of Soil Science and Agricultural Chemistry, for granting permission to carry out this research. Our gratitude extends to Dr. Rajiv Rakshit, Associate Professor and Senior Scientist at Bihar Agricultural University, Sabour for providing necessary laboratory facilities that were instrumental in the successful completion of this work.

The authors also sincerely wish to express their heartfelt thanks to Biologia Research Pvt. Ltd., Karnal, Haryana for their invaluable co-operation  in the smooth completion of all sequencing-related work.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

16S rRNA gene sequences of the various diazotrophs have been deposited in the GenBank database and can be retrieved from their accession numbers.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval. 

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required. 

Permission to reproduce material from other sources

Not Applicable

Authors’ contribution

• Manisha Mishra: Study design, conceptualization, methodology, data analysis, investigation, writing-original draft, revision and editing
• Sandeep Kumar: Study design, methodology, data analysis, revision and editing
• Arvind Kumar: Study design, methodology and supervision 

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