Isolation and identification of plant growth-promoting endophytic bacteria from Eclipta prostrata (L.) L. and its metagenomic evidence through functional annotation

The roots of two legume species (Tephrosia purpurea and Tephrosia villosa) that grew wild on dry sandy soils of Binh Thuan province were sources for isolating plant growth-promoting endophytic bacteria. Semi-solid LGI medium was used for the isolation of nitrogen-fixing bacteria from root extracts. All bacterial isolates isolates were evaluated for their ability to solubilize calcium orthophosphate on solid NBRIP medium and their ability to produce IAA in Burk's liquid medium supplemented with 100 mg/L tryptophan. The possibilities of nitrogen fixation, phosphate solubilization and IAA synthesis were all quantitative examined by colorimetric method. Twenty-two bacterial isolates of T. purpurea and 18 isolates of T. villosa were capable of nitrogen fixation in the range of 1.94 to 2.81 mg/L NH4+, whereas only 18 isolates of T. purpurea and 16 isolates of T. villosa showed phosphate solubilization in the range of 12.30 – 48.90 mg/L P2O5, and IAA production in the range of 0.38 – 12.72 mg/L. Sixteen outstanding bacterial isolates of the two legume species were identified by MALDI-TOF technique. The results showed that 13 isolates had high similarity with five bacterial genera including Klebsiella, Cronobacter, Enterobacter, Burkholderia, and Bacillus with score values in the range of 2.070 – 2.411.

In addition to rhizobia, other non-symbiotic endophytic bacteria also have been simultaneously isolated from the same root nodules. The existence of non-symbiotic endophytic bacteria in leguminous root nodules is a universal phenomenon. The vast majority of studies have detected endophytic bacteria in other plant tissues. In contrast, little systemic observation has been made on the non-symbiotic endophytic bacteria within leguminous root nodules. The present investigation was carried out to isolate plant growth-promoting endophytic non-symbiotic bacteria from indigenous leguminous Sphaerophysa salsula and their influence on plant growth. A total of 65 endophytic root nodule-associated bacteria were isolated from indigenous legume S. salsula growing in the northwestern arid regions of China. When combining our previous work with the current study, sequence analysis of the nifH gene revealed that the strain belonging to non-nodulating Bacillus pumilus Qtx-10 had genes similar to those of Rhizobium leguminosarum Qtx-10-1. The results indicated that horizontal gene transfer could have occurred between rhizobia and non-symbiotic endophyties. Under pot culture conditions, out of the 20 representative endophytic isolates, 15 with plant growth-promoting traits, such as IAA production, ACC deaminase, phosphate solubilization, chitinase, siderophore, and fungal inhibition activity showed plant growth-promoting activity with respect to various plant parameters such as chlorophyll content, fresh weight of plant, shoot length, nodule number per plant and average nodule weight per plant when co-inoculated with rhizobial bioinoculant Mesorhizobium sp. Zw-19 under N-free culture conditions. Among them, Bacillus pumilus Qtx-10 and Streptomyces bottropensis Gt-10 were excellent plant growth-promoting bacteria, which enhanced the seeding fresh weight by 87.5% and the shoot length by 89.4%, respectively. The number of nodules grew more than 31.89% under field conditions. Our findings indicate the frequent presence of these non-symbiotic endophytic bacteria within root nodules, and that they help to improve nodulation and nitrogen fixation in legume plants through synergistic interactions with rhizobia.

The isolation and identification of plant growth-promoting endophytic bacteria (PGPEB) from Achyranthes bidentata roots have profound theoretical and practical implications in ecological agriculture, particularly as bio-inoculants to address challenges associated with continuous monoculture. Our research revealed a significant increase in the abundance of these beneficial bacteria in A. bidentata rhizosphere soil under prolonged monoculture conditions, as shown by bioinformatics analysis. Subsequently, we isolated 563 strains of endophytic bacteria from A. bidentata roots. Functional characterization highlighted diverse plant growth-promoting traits among these bacteria, including the secretion of indole-3-acetic acid (IAA) ranging from 68.01 to 73.25 mg/L, phosphorus and potassium solubilization capacities, and antagonistic activity against pathogenic fungi (21.54%-50.81%). Through 16S rDNA sequencing, we identified nine strains exhibiting biocontrol and growth-promoting potential. Introduction of a synthetic microbial consortium (SMC) in pot experiments significantly increased root biomass by 48.19% in A. bidentata and 27.01% in replanted Rehmannia glutinosa. These findings provide innovative insights and strategies for addressing continuous cropping challenges, highlighting the practical promise of PGPEB from A. bidentata in ecological agriculture to overcome replanting obstacles for non-host plants like R. glutinosa, thereby promoting robust growth in medicinal plants.

Chapter 23 - Plant growth promoting bacteria (PGPB): applications and challenges in bioremediation of metal and metalloid contaminated soils

Effects of inoculation of plant-growth promoting bacteria on Ni uptake by Indian mustard

The application of plant growth-promoting bacteria (PGPB) is vital for sustainable agriculture with continuous world population growth and an increase in soil salinity. Salinity is one of the severe abiotic stresses which lessens the productivity of agricultural lands. Plant growth-promoting bacteria are key players in solving this problem and can mitigate salinity stress. The highest of reported halotolerant Plant growth-promoting bacteria belonged to Firmicutes (approximately 50%), Proteobacteria (40%), and Actinobacteria (10%), respectively. The most dominant genera of halotolerant plant growth-promoting bacteria are Bacillus and Pseudomonas. Currently, the identification of new plant growth-promoting bacteria with special beneficial properties is increasingly needed. Moreover, for the effective use of plant growth-promoting bacteria in agriculture, the unknown molecular aspects of their function and interaction with plants must be defined. Omics and meta-omics studies can unreveal these unknown genes and pathways. However, more accurate omics studies need a detailed understanding of so far known molecular mechanisms of plant stress protection by plant growth-promoting bacteria. In this review, the molecular basis of salinity stress mitigation by plant growth-promoting bacteria is presented, the identified genes in the genomes of 20 halotolerant plant growth-promoting bacteria are assessed, and the prevalence of their involved genes is highlighted. The genes related to the synthesis of indole acetic acid (IAA) (70%), siderophores (60%), osmoprotectants (80%), chaperons (40%), 1-aminocyclopropane-1-carboxylate (ACC) deaminase (50%), and antioxidants (50%), phosphate solubilization (60%), and ion homeostasis (80%) were the most common detected genes in the genomes of evaluated halotolerant plant growth-promoting and salinity stress-alleviating bacteria. The most prevalent genes can be applied as candidates for designing molecular markers for screening of new halotolerant plant growth-promoting bacteria.

Identification and isolation of plant growth-promoting bacteria (PGPB) are critical steps toward understanding the role of these bacteria in stress tolerance in plants. This procedure also provides essential knowledge about the microbes needed to formulate effective biofertilizers. This chapter describes culture-dependent and culture-independent strategies to identify and isolate PGPB. The culture-dependent strategy commonly involves growing PGPB on general and selective media. However, the culture-independent strategy involves next-generation sequencing technologies. A combination of both strategies would identify the structure of the bacterial communities and isolate bacteria from their environments. Therefore, this chapter describes a comprehensive strategy where the methods are sequentially applied to identify and isolate epiphytic and endophytic PGPB from a particular environmental sample. However, a single procedure can also be employed to identify and isolate a specific type of PGPB.

PGPB enhance plant growth through nitrogen fixation, IAA production, phosphorus and pottasium solubilization. PGPB as biofertilizer provide an alternative for chemical fertilizer to reduce environmental damage. PGPB in consortium could be more effective instead of single strain inoculant. The aim of this study is to investigate compatibility, design soil bacteria consortium isolated from rubber-Canna intercropping plantation and evaluate the consortium’s potential as biofertilizer. Functional roles of PGPB was tested such as nitrogen fixation, potassium and phosphate solubilization, and IAA production. Detection of pathogenic bacteria was tested by Blood Agar method. Compatibility test was performed by cross streak method. Bacteria were identified using sequencing of 16srRNA gene and verified using BLAST to database of NCBI. The results showed that among 19 bacterial isolates, all showed the nitrogen-fixing activity, G5 had the highest phosphorus and potassium solubilization index, whereas Citrobacter braakii strain 167 produced the highest IAA concentration. Compatibility analysis showed that Citrobacter freundii strain LMG 3246, Citrobacter braakii strain DSM 17596 and G5 are compatible as a bacteria consortium and may developed as biofertilizer. This study found a possible new and beneficial biofertilizer formulation to enhance plant growth and to reduce the application of chemical fertilizer.

Isolation of the potential endophytic bacteria from plants that are grown in sodic soils, their diversity and screening their plant growth-promoting activities for testing their efficiency as bio inoculants for the problem soils were studied. Twelve different isolates were selected out of 35 endophytic bacterial isolates on the basis of their richness in the endophytic population of different crop plants and the nucleotide sequences of 16S rRNA genes were observed. The endophytic bacteria for their plant growth-promoting traits such as nitrogen fixation, IAA production, ACC deaminase production, phosphate solubilization, siderophore secretion and antifungal activity were assessed. The best performing strains were evaluated using principal component analysis (PCA). The phylogenetic tree from the endophytic bacterial isolates of different crops grown in sodic soil was categorized into three clusters i.e. Firmicutes, β-Proteobacteria and γ- Proteobacteria. The PCA results identified two potential endophytic strains viz., Ma11 (Bacillus mojavensis) and Ri 2 (Pseudomonas fluorescens) from sodic soil-grown crops. The present findings revealed that the isolates, Ma 11 and Ri 2 were capable of improving crop growth under sodicity. These multifaceted endophytic bacterial isolates would be developed as new inoculants which will be highly suitable for sodic soils. Bangladesh J. Bot. 51(2): 271-280, 2022 (June)

The present study was carried out for the isolation and characterization of endophytic bacteria from chickpea nodules. A total of 107 endophytes were isolated from five districts of Haryana using three media, viz. YEMA for rhizobia, Pikovskaya and TSA for non-rhizobial isolates. The endophytes were then screened for various growth promoting traits like IAA production, Phosphate solubilization, Siderophore production, ACC utilization, Potassium solubilization and tolerance to NaCl concentrations. Total six endophytes, one rhizobium (HM2) and five non-rhizobial endophytes (RE6, BE13, ME3, HE5, HE7) were selected based on of plant growth promoting traits. Furthermore, the most promising non-rhizobial endophytes RE6 and BE13, compatible with Rhizobial isolate HM2 were Pseudomonas protegens and Bacillus boroniphilus using 16s rDNA sequencing. Both the isolates are non-pathogenic to humans and thus, are potential plant probiotics that can be used as biofertilizers.

Plant growth-promoting bacteria (PGPB) are found in plant tissues and promote plant growth by secretion of hormones and enzymes, or by facilitating the nutrient uptake. This study assessed forty PGPB isolates to determine their effects on maize and sorghum growth. These isolates were also compared with uninoculated plants, as negative (-N; without N fertilization) and positive (+N; with N fertilization) controls. Plant height, stem diameter, shoot and root dry mass, leaf N accumulation and chlorophyll content were evaluated. For both the maize and sorghum, the height, stem diameter and shoot dry mass in plants inoculated with PGPB were similar to those of uninoculated plants supplied with N, and the responses for root mass were higher than in plants supplied with N. However, the PGPB isolates did not promote N accumulation and chlorophyll content similar to those of uninoculated plants supplied with N. The IPACC26 and IPACC30 isolates, both identified as Bacillus subtilis, resulted in better responses for plant growth and N accumulation than the other isolates.

Mangroves are a potential source of plant growth-promoting bacteria (PGPB) due to their unique habitat. Exploring potential endophytes as biofertilizers to reduce dependence on chemical fertilizers. This study aims to determine the effect of IAA-producing endophytic bacteria and phosphate solubilizers from mangrove roots on the germination of mung beans (Vigna radiata L.). The bacteria were isolated from mangrove roots in the Bagek Kembar Mangrove Ecosystem Area, Sekotong, West Lombok. The study began with the isolation of endophytic bacteria, followed by characterization, including morphological identification, cell morphology (via Gram staining), and physiological properties (biochemical testing). The potential of endophytic bacteria to produce IAA was tested qualitatively (using colorimetry) and quantitatively (using a spectrophotometer at a wavelength of 530 nm). Additionally, the ability of endophytic bacteria to solubilize phosphate was tested both qualitatively (using a screening method) and quantitatively (using a spectrophotometer at a wavelength of 880 nm). To test the effect of endophytic bacteria on soybean germination, green soybean seeds were grown on modified Murphy medium for 5 days, with the observed parameters being plant height, root length, fresh weight, and dry weight. The results of the green soybean germination observations were analyzed using One-Way ANOVA. Based on the research results, 11 endophytic bacterial isolates were found to be capable of producing IAA in the range of 16.88 to 30.28 ppm. Meanwhile, in terms of phosphate solubility, based on the screening results, 6 out of 11 isolates were able to solubilize phosphate with an average dissolved phosphate concentration of 17.63 ppm on the 4th day of incubation. The results of the analysis of the effect of IAA-producing bacteria and phosphate solubilizers on the germination of green beans showed a significant effect on plant height, root length, fresh weight, and dry weight of green bean plants.

Seven plant growth-promoting bacteria (PGPB) were isolated from extracts of surface-sterilized Sedum alfredii Hance. Among the seven isolates, the strain SaRB5 identified as Stenotrophomonas maltophilia through 16S rDNA sequence analysis, exhibited highest levels of heavy metal resistance and plant growth-promoting traits. SaRB5 tolerated high concentrations of cadmium (Cd) (1.5 mg·kg-1) and demonstrated production of indole-3-acetic acid and siderophores when grown with or without Cd. Whole genome sequence alignment and comparative genomic analysis revealed thirty genes associated with plant growth and Cd tolerance metabolism in strain SaRB5, including trpA, trpB, czcB, and others. Pot experiments indicated that SaRB5-treated willow biomass increased by 71.7 % and extracted 129.2 % more Cd. Meanwhile,the inoculation of SaRB5 enhanced the release of organic acids, amino acids, and salicylic acid from plants into the soil, consequently facilitating the recruitment of specific beneficial microbiota at the rhizoplane (including Bradyrhizobium, Mesorhizobium, Romboutsia, and Turicibacter). The recruitment of Bradyrhizobium and Mesorhizobium and the strong upregulation of the nifH gene suggest that SaRB5 inoculation favored efficient nitrogen-fixing microbes at the rhizoplane. Romboutsia and Turicibacter had an impact on the mobilization of iron-manganese oxide-bound Cd and its uptake. This study suggests that SaRB5 enhances phytoremediation not only through the direct effects of the inoculum itself but also by impacting the indigenous rhizoplane bacterial community.

Plant growth–promoting bacteria (PGPB) can play an essential role as biofertilizers to increase pasture efficiency and reduce the application of agrochemicals. Plant growth can be potentialized when these bacteria are combined with silica nanoparticles (SiNPs). The present study aimed to evaluate the effect of PGPB associated with SiNPs on the growth of bahiagrass (Paspalum notatum) seedlings. The PGPB were isolated from rhizospheric soils and leaves of Paspalum spp. grown in the tropical high‐altitude region of Brazil and selected by their ability to fix nitrogen, solubilize phosphate, and synthesize indoleacetic acid (IAA). They were identified as Alcaligenes faecalis, Enterobacter asburiae, and Serratia marcescens by 16S rDNA sequencing. Spherical SiNPs (85 nm in diameter) were synthesized by the hydrolysis of the silicon precursor tetraethyl orthosilicate (TEOS), characterized by infrared spectroscopy and scanning electron microscopy (SEM) and applied at 5% (0.05 mg·mL−1) and 10% (0.1 mg·mL−1) concentrations. Disinfected P. notatum seeds were treated with PGPB, SiNPs, and PGPB + SiNPs and cultivated in magenta boxes containing peat, sand, and perlite. The seedlings were evaluated for their germination percentage, root length, shoot length, root dry weight, and shoot dry weight. Disinfected seeds subjected to the same treatments were also grown in Petri dishes containing 0.7% agarose. The roots of the seedlings in Petri dishes were stained with diaminobenzidine tetrahydrochloride (DAB) and visualized using a light microscope to confirm bacterial colonization. The three strains without SiNPs promoted the growth of P. notatum seedlings. S. marcescens treatment presented the greatest shoot length, and both concentrations of nanosilica with PGPB improved or maintained root lengths. Treatments of S. marcescens and E. asburiae with 10% SiNPs showed 100% seed germination. Seedlings inoculated with 10% SiNPs with S. marcescens and E. asburiae alone showed the highest shoot dry weight, and all treatments increased root dry weight compared to the control. The 10% SiNPs’ concentration inoculated with S. marcescens and A. faecalis positively affected P. notatum seedlings’ growth. This study suggests that nanosilica can be applied with PGPB to improve the development of bahiagrass and reduce the need for applications of agrochemicals.

Plant growth-promoting bacteria (PGPB) have become an important subject of research to increase maize production. The PGPB consortium should provide more benefits than single or dual inoculation. This study aimed to investigate the effect of a PGPB consortium on improving maize growth and yield. The field experiment used a split-plot design. The main plot consisted of three maize varieties (Talenta, Pertiwi-3, and Bisma), and the subplot consisted of three formulations of PGPB consortia [endophytic bacteria isolates, <em>Acetobacter</em> sp., cellulolytic, and ligninolytic (F1); endophytic bacteria isolates<em>, </em><em>Azospirillum </em>sp., cellulolytic, and ligninolytic (F2); and endophytic bacteria isolates, cellulolytic, ligninolytic, <em>Acetobacter </em>sp., and <em>Azospirillum </em>sp. (F3)] and one control. PGPB consortia formulation did not influence maize growth significantly, but maize varieties did. Pertiwi-3 showed the highest value in all growth variables, followed by Bisma and Talenta, respectively. The effect of PGPB consortia formulation upon ear fresh and dry weight depends upon the maize variety, and Pertiwi-3 showed the highest value in yield variables. PGPB consortia formulation 2 was the most effective to apply for Pertiwi-3 cultivation, while PGPB consortia formulation 3 produced higher yields for Talenta and Bisma. These findings indicated that specific PGPB formula could improve the yield for specific maize varieties.