Paenibacillus apii sp. nov., a novel nifH gene-harbouring species isolated from the rhizospheres of vegetable plants grown in different regions of northern China.

Two nifH gene-harbouring bacterial strains were isolated from rhizospheres of different vegetable plants grown in different regions of northern PR China. The two strains possessed almost identical 16S rRNA gene sequences. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 99.21 and 93.6% respectively, suggesting they belong to one species. Based on 16S rRNA gene phylogeny, the two strains were clustered together with Paenibacillus rhizophilus 7197T, Paenibacillus sabinae T27T and Paenibacillus forsythiae T98T, but on a separate branch. Novelty of the species was confirmed by ANI and dDDH comparisons between the type strain 7124T and its closest relatives, since the obtained values were considerably below the proposed thresholds for the species delineation. The genome size of strain 7124T was 5.40 Mb, comprising 5050 predicted genes with a DNA G+C content of 52.3 mol%. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids. The major cellular fatty acids were anteiso-C15  :  0 (52.9%) and C16  :  0 (23.4 %). Menaquinone-7 was reported as the major respiratory quinone. The diamino acid in the cell-wall peptidoglycan was found to be meso-diaminopimelic acid. Based on phylogenetic, genomic, chemotaxonomic and phenotypic data, the two isolates are considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus apii sp. nov. is proposed, with 7124T (=DSM 103172T=CGMCC 1.15689T) as type strain.

Pseudomonas aeruginosa Ld-08 isolated from Lilium davidii exhibits antifungal and growth-promoting properties.

A plant growth-promoting and antifungal endophytic bacteria designated as Ld-08 isolated from the bulbs of Lilium davidii was identified as Pseudomonas aeruginosa based on phenotypic, microscopic, and 16S rRNA gene sequence analysis. Ld-08 exhibited antifungal effects against Fusarium oxysporum, Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi. Ld-08 showed the highest growth inhibition, i.e., 83.82±4.76% against B. dothidea followed by 74.12±3.87%, 67.56±3.35%, and 63.67±3.39% against F. fujikuroi, B. cinerea, and F. oxysporum, respectively. The ethyl acetate fraction of Ld-08 revealed the presence of several bioactive secondary metabolites. Prominent compounds were quinolones; 3,9-dimethoxypterocarpan; cascaroside B; dehydroabietylamine; epiandrosterone; nocodazole; oxolinic acid; pyochelin; rhodotulic acid; 9,12-octadecadienoic acid; di-peptides; tri-peptides; ursodiol, and venlafaxine. The strain Ld-08 showed organic acids, ACC deaminase, phosphate solubilization, IAA, and siderophore. The sterilized bulbs of a Lilium variety, inoculated with Ld-08, were further studied for plant growth-promoting traits. The inoculated plants showed improved growth than the control plants. Importantly, some growth parameters such as plant height, leaf length, bulb weight, and root length were significantly (P ≤0.05) increased in the inoculated plants than in the control un-inoculated plants. Further investigations are required to explore the potential of this strain to be used as a plant growth-promoting and biocontrol agent in sustainable agriculture.

Characterization of an Endophytic Antagonistic Bacterial Strain Bacillus halotolerans LBG-1-13 with Multiple Plant Growth-Promoting Traits, Stress Tolerance, and Its Effects on Lily Growth.

Microbial inoculants are an important tool for increasing arable land productivity and decreasing mineral fertilizer application. This study was aimed at isolating and identifying endophytic antagonistic bacteria from lily (Lilium davidii var. unicolor) roots grown in Northwestern China and at evaluating their antifungal activity and plant growth-promoting characteristics. For this purpose, endophytic bacteria were isolated from plant roots, and plant growth-promoting strains were identified. One bacterial strain, isolated from the root part, was identified as Bacillus halotolerans based on 16S rRNA gene sequence analysis and was designated as LBG-1-13. The strain showed antagonistic activities against important plant pathogens of lily including Botrytis cinerea, Botryosphaeria dothidea, and Fusarium oxysporum. The highest percentage of growth inhibition, i.e., 71.65 ± 2.39%, was observed for LBG-1-13 against Botryosphaeria dothidea followed by 68.33 ± 4.70% and 48.22 ± 4.11% against Botrytis cinerea and Fusarium oxysporum, respectively. Meanwhile, the isolated strain also showed plant growth-promoting traits such as the production of indole-3-acetic acid (IAA), siderophore, ACC deaminase, and phosphate solubilization activity. The strain showed ACC deaminase activity and was able to cleave 58.41 ± 2.62 nmol α-ketobutyrate (mg protein)-1 min-1. The strain exhibited tolerance to salt and drought stress in an in vitro experiment. The strain LBG-1-13 was able to grow in the presence of 10% NaCl and 20% polyethylene glycol (PEG) in the growth medium. Inoculation of Lilium varieties, Tresor and Bright Diamond, with LBG-1-13 enhanced plant growth under greenhouse and field conditions, respectively. All these results demonstrated that Bacillus halotolerans LBG-1-13 could be utilized as a good candidate in the biocontrol of lily disease and plant growth promotion in sustainable agriculture.

Molecular and Biochemical Characterization, Antimicrobial Activity, Stress Tolerance, and Plant Growth-Promoting Effect of Endophytic Bacteria Isolated from Wheat Varieties.

Endophytic bacteria have been utilized as an alternative source to chemical fertilizers and pesticides to enhance plant productivity and defense mechanisms against biotic and abiotic stress. Five endophytic bacterial strains were isolated from the seeds of three different Pakistani wheat varieties (Ghaneemat-e-IBGE, Atta-Habib, and Siren). The isolated strains AH-1, S-5, S-7, GI-1, and GI-6 showed phylogenetic similarity with Bacillus altitudinis, B. aryabhattai, B. wiedmannii, Pseudomonas aeruginosa, and Burkholderia gladioli, respectively. All strains showed catalase activity (except AH-1) and Indole-3-acetic acid production, with the highest concentration (16.77 µg·mL-1) found for GI-6, followed by S-5 (11.5 µg·mL-1), nitrogen assimilation (except S-7), phosphorus solubilization (except S-7 and AH-1), and ability to produce siderophores, with maximum productions for GI-6 (31 ± 3.5 psu) and GI-1 (30 ± 2.9 psu). All five analyzed strains possessed antimicrobial activity, which was particularly strong in GI-6 and S-5 against Klebsiella pneumonia, Escherichia coli, and Bacillus subtilis. Increasing salinity stress with NaCl negatively affected the bacterial growth of all isolates. However, strains GI-6 and S-5 showed salt tolerance after three days of incubation. A drought tolerance test resulted in a negative impact of poly ethylene glycol on bacterial growth, which was, however, less pronounced in GI-6 strain. The GI-6 strain revealed growth-promoting effects on inoculated wheat plants.

Identification and characterization of thermophilic amylase producing bacterial isolates from the brick kiln soil.

The present experiment was designed to isolate bacterial strains from the brick kiln soil and to check the activity and enzyme kinetics of amylase from these isolates. The bacterial colonies were isolated from soil samples through the serial dilution method. The bacterial isolates were identified through morphological, electron microscopic and molecular analysis. The 16S ribosomal RNA sequences of the isolates IR-1, IR-2, IR-3, IR-8, and IR-9 showed high similarities with Bacillus tequilensis, Bacillus paramycoides, Proteus alimentorum, Bacillus wiedmannii, and Pseudomonas aeruginosa, respectively. All of the bacterial isolates showed a positive catalase activity except IR-9. Furthermore, the isolates showed variable antagonistic effects against different bacterial pathogens. All of the strains produced indole acetic acid (IAA), and the concentrations increased in the presence of tryptophan application. The isolates showed the amylase enzyme activity and maximum activity of isolates was achieved in 4% starch concentration. The IR-9 isolate showed the highest amylase activity of 5.9 U/ml. The V max values of the extracellular amylase from different bacterial isolates ranged between 12.90 and 50.00 IU ml-1. The lowest K m value of 6.33 mg starch was recorded for IR-8 and the maximum K cat value of 2.50 min-1 was observed for IR-3. The amylase activity of the isolates was significantly affected by a range of different incubation time, temperature, and pH values. Further tests are required before the potential utilization of these isolates for amylase production, and in the biopesticide and biofertilizer applications.

Characterization of Endophytic Fungi, Acremonium sp., from Lilium davidii and Analysis of Its Antifungal and Plant Growth-Promoting Effects.

The present study was aimed at isolating endophytic fungi from the Asian culinary and medicinal plant Lilium davidii and analyzing its antifungal and plant growth-promoting effects. In this study, the fungal endophyte Acremonium sp. Ld-03 was isolated from the bulbs of L. davidii and identified through morphological and molecular analysis. The molecular and morphological analysis confirmed the endophytic fungal strain as Acremonium sp. Ld-03. Antifungal effects of Ld-03 were observed against Fusarium oxysporum, Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi. The highest growth inhibition, i.e., 78.39 ± 4.21%, was observed for B. dothidea followed by 56.68 ± 4.38%, 43.62 ± 3.81%, and 20.12 ± 2.45% for B. cinerea, F. fujikuroi, and F. oxysporum, respectively. Analysis of the ethyl acetate fraction through UHPLC-LTQ-IT-MS/MS revealed putative secondary metabolites which included xanthurenic acid, valyl aspartic acid, gancidin W, peptides, and cyclic dipeptides such as valylarginine, cyclo-[L-(4-hydroxy-Pro)-L-leu], cyclo(Pro-Phe), and (3S,6S)-3-benzyl-6-(4-hydroxybenzyl)piperazine-2,5-dione. Other metabolites included (S)-3-(4-hydroxyphenyl)-2-((S)-pyrrolidine-2-carboxamido)propanoic acid, dibutyl phthalate (DBP), 9-octadecenamide, D-erythro-C18-Sphingosine, N-palmitoyl sphinganine, and hydroxypalmitoyl sphinganine. The strain Ld-03 showed indole acetic acid (IAA) production with or without the application of exogenous tryptophan. The IAA ranged from 53.12 ± 3.20 µg ml-1 to 167.71 ± 7.12 µg ml-1 under different tryptophan concentrations. The strain was able to produce siderophore, and its production was significantly decreased with increasing Fe(III) citrate concentrations in the medium. The endophytic fungal strain also showed production of organic acids and phosphate solubilization activity. Plant growth-promoting effects of the strain were evaluated on in vitro seedling growth of Allium tuberosum. Application of 40% culture dilution resulted in a significant increase in root and shoot length, i.e., 24.03 ± 2.71 mm and 37.27 ± 1.86 mm, respectively, compared to nontreated control plants. The fungal endophyte Ld-03 demonstrated the potential of conferring disease resistance and plant growth promotion. Therefore, we conclude that the isolated Acremonium sp. Ld-03 should be further investigated before utilization as a biocontrol agent and plant growth stimulator.

Isolation and characterization of plant growth-promoting endophytic bacteria Bacillus stratosphericus LW-03 from Lilium wardii.

In the present study, a new strain of Bacillus stratosphericus LW-03 was isolated from the bulbs of Lilium wardii. The isolated endophytic strain LW-03 exhibited excellent antifungal activity against common plant pathogens, such as Fusarium oxysporum, Botryosphaeria dothidea, Botrytis cinerea, and Fusarium fujikuroi. The growth inhibition percentage of Botryosphaeria dothidea was 74.56 ± 2.35%, which was the highest, followed by Botrytis cinerea, Fusarium fujikuroi, and Fusarium oxysporum were 71.91 ± 2.87%, 69.54 ± 2.73%, and 65.13 ± 1.91%, respectively. The ethyl acetate fraction revealed a number of bioactive compounds and several of which were putatively identified as antimicrobial agents, such as 4-hydroxy-2-nonenylquinoline N-oxide, sphingosine ceramides like cer(d18:0/16:0(2OH)), cer(d18:0/16:0), and cer(d18:1/0:0), di-peptides, tri-peptide, cyclopeptides [cyclo(D-Trp-L-Pro)], [cyclo (Pro-Phe)], dehydroabietylamine, oxazepam, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine like compound (PC(0:0/20:4), phosphatidylethanolamine (PE(18:1/0:0)), 3-Hydroxyoctadecanoic acid, 7.alpha.,27-Dihydroxycholesterol, N-Acetyl-d-mannosamine, p-Hydroxyphenyllactic acid, Phytomonic acid, and 2-undecenyl-quinoloin-4 (1H). The LW-03 strain exhibits multiple plant growth-promoting traits, including the production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, and nitrogen fixation activity. The beneficial effects of the endophytic strain LW-03 on the growth of two lily varieties were further evaluated under greenhouse conditions. Our results revealed plant growth-promoting activity in inoculated plants relative to non-inoculated control plants. The broad-spectrum antifungal activity and multiple plant growth-promoting properties of Bacillus stratosphericus LW-03 make it an important player in the development of biological fertilizers and sustainable agricultural biological control strategies.

Variovorax beijingensis sp. nov., a novel plant-associated bacterial species with plant growth-promoting potential isolated from different geographic regions of Beijing, China.

Two plant-associated bacterial strains were isolated from Beijing, China. The two strains possessed almost identical 16S rRNA gene sequences. However, REP-PCR fingerprint patterns discriminated that they were not from one clonal origin. The average nucleotide identity (ANI) value and the digital DNA-DNA hybridization (dDDH) value between the two strains were 99.4% and 94.7%, respectively, suggesting that they belonged to the same species. The 16S rRNA gene phylogeny analysis indicated that the two strains belonged to the genus Variovorax and were closely related to V. paradoxus NBRC 15149T and V. boronicumulans BAM-48T. Their phylogenetic relationship were confirmed in both phylogenetic trees constructed with house-keeping gene sequences and concatenated core genes of the genome. The ANI and dDDH comparisons among 502T and the most related type strains showed values below the accepted threshold for species discrimination. The genome sizes of strains 502T and T529 were 6.76 and 6.69 Mbp, respectively. The strain 502T had 6,227 predicted genes with DNA G+C content of 67.4 %. The respiratory quinone was ubiquinone-8 and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphospatidylglycerol. The major fatty acids of strain 502T were C10: 03-OH (26.2%), C16:0 (12.9%), C17:0 cyclo (14.5%) and summed feature 3 (21.4%). Furthermore, both strains showed the potential of plant growth promotion. Based on these results, the two isolates could be considered to represent a novel species of the genus Variovorax, for which the name Variovorax beijingensis sp. nov., is proposed, with 502T (= DSM 106862T = CGMCC 1.16560T) as the type strain.

The Endophytic Bacteria Bacillus velezensis Lle-9, Isolated from Lilium leucanthum, Harbors Antifungal Activity and Plant Growth-Promoting Effects.

Bacillus velezensis is an important plant growth-promoting rhizobacterium with immense potential in agriculture development. In the present study, Bacillus velezensis Lle-9 was isolated from the bulbs of Lilium leucanthum. The isolated strain showed antifungal activities against plant pathogens like Botryosphaeria dothidea, Fusarium oxysporum, Botrytis cinerea and Fusarium fujikuroi. The highest percentage of growth inhibition i.e., 68.56±2.35% was observed against Fusarium oxysporum followed by 63.12 ± 2.83%, 61.67 ± 3.39% and 55.82 ± 2.76% against Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi, respectively. The ethyl acetate fraction revealed a number of bioactive compounds and several were identified as antimicrobial agents such as diketopiperazines, cyclo-peptides, linear peptides, latrunculin A, 5α-hydroxy-6-ketocholesterol, (R)-S-lactoylglutathione, triamterene, rubiadin, moxifloxacin, 9-hydroxy-5Z,7E,11Z,14Zeicosatetraenoic acid, D-erythro-C18-Sphingosine, citrinin, and 2- arachidonoyllysophosphatidylcholine. The presence of these antimicrobial compounds in the bacterial culture might have contributed to the antifungal activities of the isolated B. velezensis Lle- 9. The strain showed plant growth-promoting traits such as production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, and nitrogen fixation and phosphate solubilization. IAA production was accelerated with application of exogenous tryptophan concentrations in the medium. Further, the lily plants upon inoculation with Lle-9 exhibited improved vegetative growth, more flowering shoots and longer roots than control plants under greenhouse condition. The isolated B. velezensis strain Lle-9 possessed broad-spectrum antifungal activities and multiple plant growth-promoting traits and thus may play an important role in promoting sustainable agriculture. This strain could be developed and applied in field experiments in order to promote plant growth and control disease pathogens.

Identification and characterization of a novel multi-stress responsive gene in Arabidopsis.

Abiotic stresses especially salinity, drought and high temperature result in considerable reduction of crop productivity. In this study, we identified AT4G18280 annotated as a glycine-rich cell wall protein-like (hereafter refer to as GRPL1) protein as a potential multistress-responsive gene. Analysis of public transcriptome data and GUS assay of pGRPL1::GUS showed a strong induction of GRPL1 under drought, salinity and heat stresses. Transgenic plants overexpressing GRPL1-3HA showed significantly higher germination, root elongation and survival rate under salt stress. Moreover, the 35S::GRPL1-3HA transgenic lines also showed higher survival rates under drought and heat stresses. GRPL1 showed similar expression patterns with Abscisic acid (ABA)-pathway genes under different growth and stress conditions, suggesting a possibility that GRPL1 might act in the ABA pathway that is further supported by the inability of ABA-deficient mutant (aba2-1) to induce GRPL1 under drought stress. Taken together, our data presents GRPL1 as a potential multi-stress responsive gene working downstream of ABA.

Isolation and Characterization of Plant Growth-Promoting Endophytic Bacteria Paenibacillus polymyxa SK1 from Lilium lancifolium.

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium that has immense potential to be used as an environmentally friendly replacement of chemical fertilizers and pesticides. In the present study, Paenibacillus polymyxa SK1 was isolated from bulbs of Lilium lancifolium. The isolated endophytic strain showed antifungal activities against important plant pathogens like Botryosphaeria dothidea, Fusarium oxysporum, Botrytis cinerea, and Fusarium fujikuroi. The highest percentage of growth inhibition, i.e., 66.67 ± 2.23%, was observed for SK1 against Botryosphaeria dothidea followed by 61.19 ± 3.12%, 60.71 ± 3.53%, and 55.54 ± 2.89% against Botrytis cinerea, Fusarium fujikuroi, and Fusarium oxysporum, respectively. The metabolite profiling of ethyl acetate fraction was assessed through the UHPLC-LTQ-IT-MS/MS analysis, and putative identification was done with the aid of the GNPS molecular networking workflow. A total of 29 compounds were putatively identified which included dipeptides, tripeptides, cyclopeptides (cyclo-(Leu-Leu), cyclo(Pro-Phe)), 2-heptyl-3-hydroxy 4-quinolone, 6-oxocativic acid, anhydrobrazilic acid, 1-(5-methoxy-1H-indol-3-yl)-2-piperidin-1-ylethane-1,2-dione, octadecenoic acid, pyochelin, 15-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid, (Z)-7-[(2R,3S)-3-[(2Z,5E)-Undeca-2,5-dienyl]oxiran-2-yl]hept-5-enoic acid, arginylasparagine, cholic acid, sphinganine, elaidic acid, gossypin, L-carnosine, tetrodotoxin, and ursodiol. The high antifungal activity of SK1 might be attributed to the presence of these bioactive compounds. The isolated strain SK1 showed plant growth-promoting traits such as the production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, nitrogen fixation, and phosphate solubilization. IAA production was strongly correlated with the application of exogenous tryptophan concentrations in the medium. Furthermore, inoculation of SK1 enhanced plant growth of two Lilium varieties, Tresor and White Heaven, under greenhouse condition. In the light of these findings, the P. polymyxa SK1 may be utilized as a source of plant growth promotion and disease control in sustainable agriculture.

The complete chloroplast genome sequence of Lilium speciosum Var. gloriosoides, an important breeding parent.

Lilium speciosum var. gloriosoides is an important breeding parent with high ornamental and edible value in worldwide. In this study, we reported a complete chloroplast genome of L. speciosum var. gloriosoides, which was de novo assembled using the next-generation sequencing data. The whole genome is 152,912 bp in length and includes one large single copy (LSC) region of 70,693 bp, one small single copy (SSC) region of 17,517 bp, and a pair of inverted repeat (IR) region of 26,539 bp. A total of 131 functional genes were encoded, consisting of 76 protein-coding genes, 36 transfer RNA genes, and eight ribosomal RNA genes. The overall AT content of the chloroplast genome is 63.00%. In the maximum likelihood, a strong phylogenetic signal showed that L. speciosum var. gloriosoides is a species of Lilium, which is the first major genome in section Archelirion.

Utilization of genes encoding osmoprotectants in transgenic plants for enhanced abiotic stress tolerance

Global agriculture in the context of growing and expanding populations is under huge pressure to provide increased food, feed, and fiber. The recent phenomenon of climate change has further added fuel to the fire. It has been practically established now that the global temperature has been on the increase with associated fluctuations in annual rainfall regimes, and the resultant drought and flood events and increasing soil and water salinization. These challenges would be met with the introduction and utilization of new technologies coupled with conventional approaches. In recent years, transgenic technology has been proved very effective in terms of production of improved varieties of crop plants, resistant to biotic stresses. The abiotic stresses such as salt and drought are more complex traits, controlled by many genes. Transgenic plant development for these stresses has utilized many single genes. However, much emphasis has been placed on genes catalyzing the biosynthetic pathways of osmoprotectants. This review focuses on the current status of research on osmoprotectant genes and their role in abiotic stress tolerance in transgenic plants.