Multifarious Indigenous Diazotrophic Rhizobacteria of Rice (Oryza sativa L.) Rhizosphere and Their Effect on Plant Growth Promotion

A diverse group of rhizobacteria persists in the rhizospheric soil, on the surface of roots, or in association with rice plants. These bacteria colonize plant root systems, enhance plant growth and crop yield. Indigenous rhizobacteria are known to promote soil health, grain production quality and serve as sustainable bioinoculant. The present study was aimed to isolate, identify and characterize indigenous plant growth promoting (PGP) diazotrophic bacteria associated with the rhizosphere of rice fields from different areas of Jammu and Kashmir, India. A total of 15 bacteria were isolated and evaluated for various PGP traits, antagonistic activity against phytopathogens, production of hydrolytic enzymes and biofilm formation under in-vitro conditions. The majority of the isolated bacteria were Gram-negative. Out of 15 bacterial isolates, nine isolates produced IAA (12.24 ± 2.86 to 250.3 ± 1.15 μg/ml), 6 isolates exhibited phosphate solubilization activity (36.69 ± 1.63 to 312.4 ± 1.15 μg/ml), 7 isolates exhibited rock phosphate solubilization while 5 isolates solubilized zinc (10–18 mm), 7 isolates showed siderophore production, 8 isolates exhibited HCN production, 6 isolates exhibited aminocyclopropane-1-carboxylate (ACC) deaminase activity, 13 isolates exhibited cellulase activity, nine isolates exhibited amylase and lipase activity and six isolates exhibited chitinase activity. In addition, 5 isolates showed amplification with the nifH gene and showed a significant amount of nitrogenase activity in a range of 0.127–4.39 μmol C2H4/mg protein/h. Five isolates viz., IHK-1, IHK-3, IHK-13, IHK-15 and IHK-25 exhibited most PGP attributes and successfully limited the mycelial growth of Rhizoctonia solani and Fusarium oxysporum in-vitro. All the five bacterial isolates were identified based on morphological, biochemical and 16S rDNA gene sequencing study, as Stenotrophomonas maltophilia, Enterobacter sp., Bacillus sp., Ochrobactrum haematophilum and Pseudomonas aeruginosa. Rice plants developed from seeds inoculated with these PGP strains individually had considerably higher germination percentage, seed vigor index and total dry biomass when compared to control. These findings strongly imply that the PGP diazotrophic bacteria identified in this work could be employed as plant growth stimulators in rice.

Multiple Metabolic Phenotypes as Screening Criteria Are Correlated With the Plant Growth-Promoting Ability of Rhizobacterial Isolates

Efficient screening method is the prerequisite for getting plant growth-promoting (PGP) rhizobacteria (PGPR) which may play an important role in sustainable agriculture from the natural environment. Many current traditional preliminary screening criteria based on knowledge of PGP mechanisms do not always work well due to complex plant–microbe interactions and may lead to the low screening efficiency. More new screening criteria should be evaluated to establish a more effective screening system. However, the studies focused on this issue were not enough, and few new screening criteria had been proposed. The aim of this study was to analyze the correlation between the metabolic phenotypes of rhizobacterial isolates and their PGP ability. The feasibility of using these phenotypes as preliminary screening criteria for PGPR was also evaluated. Twenty-one rhizobacterial isolates were screened for their PGP ability, traditional PGP traits, and multiple metabolic phenotypes that are not directly related to PGP mechanisms, but are possibly related to rhizosphere colonization. Correlations between the PGP traits or metabolic phenotypes and increases in plant agronomic parameters were analyzed to find the indicators that are most closely related to PGP ability. The utilization of 11 nutrient substrates commonly found in root exudates, such as D-salicin, β-methyl-D-glucoside, and D-cellobiose, was significantly positively correlated with the PGP ability of the rhizobacterial isolates. The utilization of one amino acid and two organic acids, namely L-aspartic acid, α-keto-glutaric acid, and formic acid, was negatively correlated with PGP ability. There were no significant correlations between four PGP traits tested in this study and the PGP ability. The ability of rhizobacterial isolates to metabolize nutrient substrates that are identical or similar to root exudate components may act as better criteria than PGP traits for the primary screening of PGPR, because rhizosphere colonization is a prerequisite for PGPR to affect plants.

Cultivable Endophytic Bacteria in Seeds of Dongxiang Wild Rice and Their Role in Plant-Growth Promotion

Dongxiang wild rice (Oryza rufipogon Griff.) germplasm is a precious resource for the improvement of agronomic traits in rice. Rice seeds also harbor a diverse endophytic bacterial community, and their interactions with their hosts and each other can influence plant growth and adaptability. Here, we investigated the community composition of cultivable endophytic bacteria obtained from the surface-sterilized seeds of Dongxiang wild rice and screened them for plant growth-promoting traits. Phylogenetic analysis of 16S rRNA gene sequences indicated that the 47 isolates were affiliated with five classes and 13 discrete genera, and Bacillus and Microbacterium predominated. Evaluations of plant growth promoting (PGP) traits showed that 45 endophytic bacteria isolates produced between 3.37 and 90.11 μg mL−1 of Indole-3-acetic acid (IAA), with the highest yield of 90.11 μg mL−1 (Fse28). Further, 37 of the isolates were able to solubilize mineral phosphate, while 28 other isolates had the ability of N2-fixation, 17 isolates possessed 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity with the highest yield of 20.72 μmol mg−1 protein h−1 (Fse35), and 17 isolates were also able to produce siderophores. The two strains Fse28 and Fse35 had multiple PGP traits that significantly improved the agronomic traits (root length, shoot length, dry matter, and chlorophyll content) of cultivated rice seedlings. Our results illustrate the rich diversity of seed endophytic bacteria in Dongxiang wild rice and their potential for developing novel efficient bioinoculants to enhance soil fertility and favor seedling growth.

Isolation, Identification and Evaluation of Indigenous Plant Growth Promoting Bacterium Klebsiella pneumoniae PNE1

Application of chemical fertilizer is an integral practice to optimize crop productivity, but the dominant use of chemical fertilizers contributes largely to the deterioration of the environment, leads to loss of soil fertility, increases pollution, and causes hazardous diseases. Hence, the chemical fertilizers, pesticides and other supplements are being replaced by the plant growth promoting bacteria (PGPB) due to their improved potency and environment friendly nature. Plant growth-promoting bacteria (PGPB) can enhance plant growth by a wide variety of mechanisms like Phosphate (P) solubilization, Potassium solubilisation, siderophore production, biological nitrogen fixation and Indole acetic acid (IAA) production. The Klebsiella species is also known to exhibit important PGP traits like solubilization of phosphate, phytohormone production and good germination potential. In present study the Klebsiella pneumoniae PNE1 was selected from the isolates obtained from vegetable waste collected from Kadi market. The isolate was selected on the basis of its ability for Nitogen fixation, Phosphate solubilization, Potassium solubilization, IAA production, EPS production and biopolymer degradation. The molecular identification through 16S rRNA gene sequence, confirmed the isolate as Klebsiella pneumonia PNE1. Quantitative analysis of ammonia production revealed that isolate Klebsiella pneumonia PNE1 produced 0.5 µg/ml of ammonia (NH3) on 6th day of incubation and produced 0.09 µg/ml Nitrite after 8th day of incubation. The Phosphate solubilisation Index (SI) of the isolate was 4.16 and the isolate released 177.50 μg/ml Phosphate. The qualitative estimation of Potassium solubilisation by the isolate Klebsiella pneumoniae PNE1 in terms of Potassium solubilisation zone was found to increase gradually from day 1 to 7 days and was maximum at 2nd day with a KSI of 3.6. The isolate Klebsiella pneumoniae PNE1 released 29.94 mg/l Potassium on 21th day of incubation respectively. The IAA production was found to be 94.96 µg/ml. The maximum the EPS yield was 11.3 mg/ml. The Klebsiella pneumonia PNE1 had capacity to degrade Cellulose, Pectin and Xylan i.e. all biopolymers tested. The antibiotic susceptibility test indicated that isolate was sensitive to all 22 antibiotics tested. The Klebsiella pneumonia PNE1 thus, shows important plant growth promoting traits and can be used in a bio-fertilizer formulation for sustainable agriculture.

Unraveling the Association between Metabolic Changes in Inter-Genus and Intra-Genus Bacteria to Mitigate Clubroot Disease of Chinese Cabbage

Clubroot disease caused by the obligate parasite Plasmodiophora brassicae is a serious threat to cabbage production worldwide. Current clubroot control primarily relies on a fungicide, but this has a negative impact on the environment and the use of a single biocontrol agent cannot efficiently control the disease. Thus, the combined application of different biocontrol agents has been proposed as a promising alternative. In this study, we used bacterial biocontrol agents as a co-culture (inter-genus and intra-genus) and mono-culture to mitigate the clubroot disease of Chinese cabbage. We evaluated their biocontrol effect and plant growth promoter (PGP) traits in in vitro and in vivo experiments. This study revealed that the inter-genus bacterial co-culture significantly suppresses the incidence of clubroot disease and enhances plant growth compared with intra-genus and mono-culture. In pairwise interaction, we observed that Bacillus cereus BT-23 promotes the growth of Lysobacter antibioticus 13-6 (inter-genus bacterial co-culture), whereas L. capsici ZST1-2 and L. antibioticus 13-6 (intra-genus microbial co-culture) are antagonists to each other. Furthermore, a total of 5575 metabolites, 732 differentially expressed metabolites (DEMs), and 510 unique metabolites were detected through the LC-MS/MS technique in the bacterial co-culture. The number of unique metabolites in inter-genus bacterial co-culture (393 metabolites) was significantly higher than in the intra-genus bacterial co-culture (117 metabolites). Further analysis of DEMs showed that the DEMs were mainly involved in four kinds of metabolism pathways, i.e., carbohydrate metabolism, amino metabolism, nucleotide metabolism, and metabolism of cofactors and vitamins. The contents of some secondary metabolites with biocontrol activity and plant growth-promoting functions were increased in inter-genus bacterial co-culture, indicating that inter-genus bacterial co-culture has a solid potential to suppress clubroot disease. We conclude that the inter-genus bacterial interaction changes the community metabolism and improves several secondary metabolites functions with respect to disease control and PGP ability.

Differential Protein Expression Analysis of Two Sugarcane Varieties in Response to Diazotrophic Plant Growth-Promoting Endophyte Enterobacter roggenkampii ED5

Plant endophytic bacteria have many vital roles in plant growth promotion (PGP), such as nitrogen (N) fixation and resistance to biotic and abiotic stresses. In this study, the seedlings of sugarcane varieties B8 (requires a low concentration of nitrogen for growth) and GT11 (requires a high concentration of nitrogen for growth) were inoculated with endophytic diazotroph Enterobacter roggenkampii ED5, which exhibits multiple PGP traits, isolated from sugarcane roots. The results showed that the inoculation with E. roggenkampii ED5 promoted the growth of plant significantly in both sugarcane varieties. 15N detection at 60 days post-inoculation proved that the inoculation with strain ED5 increased the total nitrogen concentration in the leaf and root than control in both sugarcane varieties, which was higher in B8. Biochemical parameters and phytohormones in leaf were analyzed at 30 and 60 days after the inoculation. The results showed that the inoculation with E. roggenkampii ED5 improved the activities of superoxide dismutase (SOD), catalase (CAT), NADH-glutamate dehydrogenase (NADH-GDH), glutamine synthetase (GS), and endo-β-1,4-glucanase, and the contents of proline and indole acetic acid (IAA) in leaf, and it was generally more significant in B8 than in GT11. Tandem Mass Tags (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to perform comparative proteomic analysis in the sugarcane leaves at 30 days after inoculation with strain ED5. A total of 27,508 proteins were detected, and 378 differentially expressed proteins (DEPs) were found in the treated sugarcane variety B8 (BE) as compared to control (BC), of which 244 were upregulated and 134 were downregulated. In contrast, a total of 177 DEPs were identified in the treated sugarcane variety GT11 (GE) as compared to control (GC), of which 103 were upregulated and 74 were downregulated. The DEPs were associated with nitrogen metabolism, photosynthesis, starch, sucrose metabolism, response to oxidative stress, hydrolase activity, oxidative phosphorylation, glutathione metabolism, phenylpropanoid metabolic process, and response to stresses in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. To the best of our knowledge, this is the first proteomic approach to investigate the molecular basis of the interaction between N-fixing endophytic strain E. roggenkampii ED5 and sugarcane.

Bacterial Seed Endophytes of the Holoparasitic Cistanche Armena and C. Phelypaea (Orobanchaceae) from Saline Habitats: Semi-Deserts vs Flooded Coasts

The current study compares the bacterial seed endomicrobiomes of the endemic holoparasitic plant species Cistanche armena and C. phelypaea. Both species thrive in habitats that are saline but with a very different soil water status. The study aims to uncover how environmental conditions influence the diversity of the bacterial communities of seeds of these holoparasites and also highlights the physiological activities, several enzymatic and PGP traits of culturable endophytes that may support the tolerance of their hosts to abiotic stresses. A combination of culture-dependent and molecular techniques was employed for the identification of the seed endomicrobiome (culturable and unculturable). From the seeds of both parasitic plant species closely related strains were isolated. Sixteen phyla, 323 genera and 710 bacterial species were identified, mainly Gram negative, halotolerant bacteria with an environmental origin. Most of the isolates were endospore forming, halotolerant and alkaliphile Bacillus spp. which suggests that the endophytic bacteria of C. armena and C. phelypaea seeds possess traits that are correlated with the natural habitat of their hosts. The Paenibacillus strains from both plant species demonstrate similar biochemical traits. Although the seed endophytic microbiomes of C. armena and C. phelypaea contain a high number of common bacterial taxa, also remarkable differences exist. We demonstrated that the diversity of the bacterial communities is related to the environmental conditions, water status or abiotic stresses.

Diversity of Endophytic Bacteria Associated with Different Cultivars of Medicinal Plant Arctium Lappa L. and Their Potential for Host Plant Growth Promoting

Purpose Arctium lappa L. is one of the medicinal and food homologous plants in China, which is rich in nutrients and medicinal ingredients. The use of plant growth promoting (PGP) endophytic bacteria is a useful alternative in agricultural production to reduce the use of chemical fertilizers. The aim of this study was to analysis the diversity of endophytic bacteria in different cultivars of A. lappa L. collected from two different geographical locations in China and evaluate PGP traits of the isolates and their potential PGP ability in greenhouse condition. Methods Endophytic bacterial community was investigated by culture-dependent and culture-independent methods. Isolates were screened and investigated for multiple PGP traits, and representative strains were inoculated host seedlings to evaluate the growth promoting effect. Results A total of 348 endophytic bacteria were obtained and they distributed into four phyla, 30 genera and 73 different species. In addition, high throughput sequencing revealed more abundant bacterial community, including 17 bacterial phyla, and 207 different known genera. A high proportion of PGP traits were detected, including production of indole acetic acid, siderophore, ammonia and phosphate solubilization. Four representative strains with multiple PGP traits of the most prevalent genera were further selected for host inoculation and growth promoting evaluation, and they significantly increase seedlings length, root length and fresh weight. Conclusion This study demonstrated that A. lappa L. harbors abundant endophytic bacteria, and plant genotype and geographical origin affect their composition. Moreover, some endophytic bacteria showed good potential for the development of microbial fertilizer in the future.

Plant Growth Promotion Diversity in Switchgrass-Colonizing, Diazotrophic Endophytes

Endophytic nitrogen-fixing (diazotrophic) bacteria are essential members of the microbiome of switchgrass (Panicum virgatum), considered to be an important commodity crop in bioenergy production. While endophytic diazotrophs are known to provide fixed atmospheric nitrogen to their host plant, there are many other plant growth-promoting (PGP) capabilities of these organisms to be demonstrated. The diversity of PGP traits across different taxa of switchgrass-colonizing endophytes is understudied, yet critical for understanding endophytic function and improving cultivation methods of important commodity crops. Here, we present the isolation and characterization of three diazotrophic endophytes: Azospirillum agricola R1C, Klebsiella variicola F10Cl, and Raoultella terrigena R1Gly. Strains R1C and F10Cl were isolated from switchgrass and strain R1Gly, while isolated from tobacco, is demonstrated herein to colonize switchgrass. Each strain exhibited highly diverse genomic and phenotypic PGP capabilities. Strain F10Cl and R1Gly demonstrated the highest functional similarity, suggesting that, while endophyte community structure may vary widely based on host species, differences in functional diversity are not a clearly delineated. The results of this study advance our understanding of diazotrophic endophyte diversity, which will allow us to design robust strategies to improve cultivation methods of many economically important commodity crops.

Potassium mobilization and plant growth promotion by soil bacteria isolated from different agroclimatic zones of Odisha, India

<p class="042abstractstekst"><span lang="EN-US">Potassium is essential for plant metabolism; improves immunity to stress and increase crop productivity. Soil contains insoluble form of potassium, which is unavailable for plant absorption. Potash mobilizing bacteria (KMB) solubilise complex potassium and make it available to plant. KMB with plant growth promoting (PGP) traits could enhance growth and crop productivity. Here we attempt to screen KMBs with PGP traits from different agroclimatic zones of Odisha and study dynamics of potassium in soil. Isolation of KMB and determination of PGP traits was performed with standard protocols. Pot culture experiment was aimed to study their effect on sunflower crop. Available soil potassium was quantified using inductively coupled plasma-optical emission spectrometry (ICP-OES). Thirty KMBs were isolated from different agro-climatic zones of Odisha, out of which 6 isolates exhibited maximum PGP traits. Moreover, after adding inoculums the available soil potassium decreased over 0 to 30 days as compared to control, with increase in shoot length. T7 (consortium) reported maximum (144 %) increase in shoot length. Available soil potassium content decreased with increase in time. A maximum decrease was reported in T7 (26.31 %), suggesting potassium accumulation by plant.</span></p>