Biodiversity and antimicrobial potential of bacterial endophytes from halophyte Salicornia brachiata

Extreme natural habitats like halophytes, marsh land, and marine environment are suitable arena for chemical ecology between plants and microbes having environmental impact. Endophytes are an ecofriendly option for the promotion of plant growth and to serve as sustainable resource of novel bioactive natural products. The present study, focusing on biodiversity of bacterial endophytes from Salicornia brachiata, led to isolation of around 336 bacterial endophytes. Phylogenetic analysis of 63 endophytes revealed 13 genera with 27 different species, belonging to 3 major groups: Firmicutes, Proteobacteria, and Actinobacteria. 30% endophytic isolates belonging to various genera demonstrated broad-spectrum antibacterial and antifungal activities against a panel of human, plant, and aquatic infectious agents. An endophytic isolate Bacillus amyloliquefaciens 5NPA-1, exhibited strong in-vitro antibacterial activity against human pathogen Staphylococcus aureus and phytopathogen Xanthomonas campestris. Investigation through LC–MS/MS-based molecular networking and bioactivity-guided purification led to the identification of three bioactive compounds belonging to lipopeptide class based on ¹H-, ¹³C-NMR and MS analysis. To our knowledge, this is the first report studying bacterial endophytic biodiversity of Salicornia brachiata and the isolation of bioactive compounds from its endophyte. Overall, the present study provides insights into the diversity of endophytes associated with the plants from the extreme environment as a rich source of metabolites with remarkable agricultural applications and therapeutic properties.

     

Biotechnological potential of plant growth-promoting bacteria from the roots and rhizospheres of endemic plants in ironstone vegetation in southeastern Brazil

Microorganisms associated with plants have a great biotechnological potential, but investigations of these microorganisms associated with native plants in peculiar environments has been incipient. The objective of this study was to analyze the plant growth-promoting bacteria potential of cultivable bacteria associated with rare plants from the ferruginous rocky fields of the Brazilian Iron Quadrangle. The roots and rhizospheres of nine endemic plants species and samples of a root found in a lateritiric duricrust (canga) cave were collected, the culturable bacteria isolated and prospected for distinct biotechnological and ecological potentials. Out of the 148 isolates obtained, 8 (5.4%) showed potential to promote plant growth, whereas 4 (2.7%) isolates acted as biocontrol agents against Xanthomonas citri pathotype A (Xac306), reducing the cancrotic lesions by more than 60% when co-inoculated with this phytopathogen in Citrus sinensis plants. Moreover, other 4 (2.7%) isolates were classified as potential bioremediation agents, being able to withstand high concentrations of arsenite (5 mM As³⁺) and arsenate (800 mM As⁵⁺), by removing up to 35% and 15% of this metalloid in solution, respectively. These same four isolates had a positive influence on the growth of both the roots and the aerial parts when inoculated with tomato seeds in the soil contaminated with arsenic. This is the first time that an investigation highlights the potentialities of bacteria associated with rare plants of ferruginous rocky fields as a reservoir of microbiota of biotechnological and ecological interest, highlighting the importance of conservation of this area that is undergoing intense anthropic activity.

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Molecular characterization of diazotrophic and denitrifying bacteria associated with mangrove roots

An analysis of the molecular diversity of N(2) fixers and denitrifiers associated with mangrove roots was performed using terminal restriction length polymorphism (T-RFLP) of nifH (N(2) fixation) and nirS and nirK (denitrification), and the compositions and structures of these communities among three sites were compared. The number of operational taxonomic units (OTU) for nifH was higher than that for nirK or nirS at all three sites. Site 3, which had the highest organic matter and sand content in the rhizosphere sediment, as well as the lowest pore water oxygen concentration, had the highest nifH diversity. Principal component analysis of biogeochemical parameters identified soil texture, organic matter content, pore water oxygen concentration, and salinity as the main variables that differentiated the sites. Nonmetric multidimensional scaling (MDS) analyses of the T-RFLP data using the Bray-Curtis coefficient, group analyses, and pairwise comparisons between the sites clearly separated the OTU of site 3 from those of sites 1 and 2. For nirS, there were statistically significant differences in the composition of OTU among the sites, but the variability was less than for nifH. OTU defined on the basis of nirK were highly similar, and the three sites were not clearly separated on the basis of these sequences. The phylogenetic trees of nifH, nirK, and nirS showed that most of the cloned sequences were more similar to sequences from the rhizosphere isolates than to those from known strains or from other environments.     

Arsenic-resistant bacteria associated with roots of the wild Cirsium arvense (L.) plant from an arsenic polluted soil,and screening of potential plant growth-promoting characteristics

A rhizobacterial community, associated with the roots of wild thistle Cirsium arvense (L.) growing in an arsenic polluted soil, was studied by fluorescence in situ hybridization (FISH) analysis in conjunction with cultivation-based methods. In the bulk, rhizosphere, and rhizoplane fractions of the soil, the qualitative picture obtained by FISH analysis of the main phylogenetic bacterial groups was similar and was predominantly comprised of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The arsenic-resistant isolates belonged to 13 genera, the most abundant being those of Bacillus, Achromobacter, Brevundimonas, Microbacterium, and Ochrobactrum. Most bacteria grew in the presence of high arsenic concentrations (over 100 mM arsenate and 10 mM arsenite). Most strains possessed the ArsC, ArsB and ACR3 genes homologous to arsenate reductase and to the two classes of arsenite efflux pumps, respectively, peculiar to the ars operon of the arsenic detoxification system. ArsB and ACR3 were present simultaneously in highly resistant strains. An inconsistency between 16S rRNA phylogenetic affiliations and the arsenate reductase sequences of the strains was observed, indicating possible horizontal transfer of arsenic resistance genes in the soil bacterial community. Several isolates were able to reduce arsenate and to oxidise arsenite. In particular, Ancylobacter dichloromethanicum strain As3-1b possessed both characteristics, and arsenite oxidation occurred in the strain also under chemoautotrophic conditions. Some rhizobacteria produced siderophores, indole acetic acid and 1-amino-cyclopropane-1-carboxylic acid deaminase, thus possessing potential plant growth-promoting traits.     

Plant growth-promoting potential of endophytic bacteria isolated from roots of coastal sand dune plants

Endophytic bacteria associated with the roots of coastal sand dune plants were isolated, taxonomically characterized, and tested for their plant growth-promoting activities. Ninety-one endophytic bacterial isolates were collected and assigned to 17 different genera of 6 major bacterial phyla based on partial 16S rDNA sequence analyses. Gammaproteobacteria represented the majority of the isolates (65.9%), and members of Pseudomonas constituted 49.5% of the total isolates. When testing for antagonism towards plant pathogenic fungi, 25 strains were antagonistic towards Rhizoctonia solani, 57 strains were antagonistic towards Pythium ultimum, 53 strains were antagonistic towards Fusarium oxysporum, and 41 strains were antagonistic towards Botrytis cinerea. Seven strains were shown to produce indole acetic acid (IAA), 33 to produce siderophores, 23 to produce protease, 37 to produce pectinase, and 38 to produce chitinase. The broadest spectra of activities were observed among the Pseudomonas strains, indicating outstanding plant growth-promoting potential. The isolates from C. kobomugi and M. sibirica also exhibited good plant growth-promoting potential. The correlations among individual plant growth-promoting activities were examined using phi coefficients, and the resulting data indicated that the production of protease, pectinase, chitinase, and siderophores was highly related.     

Genetics of phosphate solubilization and its potential applications for improving plant growth-promoting bacteria

Plant growth-promoting bacteria (PGPB) are soil and rhizosphere bacteria that can benefit plant growth by different mechanisms. The ability of some microorganisms to convert insoluble phosphorus (P) to an accessible form, like orthophosphate, is an important trait in a PGPB for increasing plant yields. In this mini-review, the isolation and characterization of genes involved in mineralization of organic P sources (by the action of enzymes acid phosphatases and phytases), as well as mineral phosphate solubilization, is reviewed. Preliminary results achieved in the engineering of bacterial strains for improving capacity for phosphate solubilization are presented, and application of this knowledge to improving agricultural inoculants is discussed.     

The biosynthetic potential of plant roots

The contribution of roots to the biology of the whole plant is being reevaluated in the light of classical and recent findings. In addition to their role in water and nutrient uptake and in symbiotic associations, plant roots also synthesize a remarkable variety of secondary metabolites. These chemicals, many of which are used as pharmaceuticals, agrichemicals, flavors, dyes, or fragrances, may help the plant cope with biotic and abiotic stress. Root cultures are being used as experimental systems to explore both root-specific secondary metabolites and their biological significance. They may also provide future systems for commercial production of plant specialty chemicals.     

Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr.

Endophytic fungi are plant symbionts that produce a variety of beneficial metabolites for plant growth and protection against herbivory and pathogens. Fourteen fungal samples were isolated from the roots of soybean cultivar Daemangkong and screened on waito-c rice for their plant growth-promoting capacity. Twelve of the fungal isolates promoted plant growth, while two inhibited it. The fungal isolate DK-1-1 induced maximum plant growth in both waito-c rice and soybean. The plant growth promotion capacity of DK-1-1 was higher than the wild type Gibberella fujikuroi. Gibberellin (GA) analysis of culture filtrate of DK-1-1 showed the presence of higher amounts of bioactive GA₃, GA₄, and GA₇ (6.62, 2.1 and 1.26 ng/mL, respectively) along with physiologically inactive GA₅, GA₁₅, GA₁₉, and GA₂₄. Phylogenetic analysis of 18S rDNA sequence identified the fungal isolate as a new strain of Cladosporium sphaerospermum. Gibberellin production and plant growth-promoting ability of genus Cladosporium are reported for the first time in the present study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gut bacteria of cockroaches are a potential source of antibacterial compound(s)

Here, we hypothesized that the microbial gut flora of animals/pests living in polluted environments, produce substances to thwart bacterial infections. The overall aim of this study was to source microbes inhabiting unusual environmental niches for potential antimicrobial activity. Two cockroach species, Gromphadorhina portentosa (Madagascar) and Blaptica dubia (Dubia) were selected. The gut bacteria from these species were isolated and grown in RPMI 1640 and conditioned media were prepared. Conditioned media were tested against a panel of Gram‐positive (Methicillin‐resistant Staphylococcus aureus, Streptococcus pyogenes, Bacillus cereus) and Gram‐negative (Escherichia coli K1, Salmonella enterica, Serratia marcescens, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, as well as the protist pathogen, Acanthamoeba castellanii. The results revealed that the gut bacteria of cockroaches produce active molecule(s) with potent antibacterial properties, as well as exhibit antiamoebic effects. However, heat‐inactivation at 95°C for 10 min had no effect on conditioned media‐mediated antibacterial and antiamoebic properties. These results suggest that bacteria from novel sources i.e. from the cockroach's gut produce molecules with bactericidal as well as amoebicidal properties that can ultimately lead to the development of therapeutic drugs.

Significance and Impact of the Study

The bacteria isolated from unusual dwellings such as the cockroaches' gut are a useful source of antibacterial and antiamoebal molecules. These are remarkable findings that will open several avenues in our search for novel antimicrobials from unique sources. Furthermore studies will lead to the identification of molecules to develop future antibacterials from insects.     

Isolation of ACC deaminase-producing habitat-adapted symbiotic bacteria associated with halophyte Limonium sinense (Girard) Kuntze and evaluating their plant growth-promoting activity under salt stress

Background and aims

Many plant growth-promoting endophytes (PGPE) possessing 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity can reduce the level of stress ethylene and assist their host plants cope with various biotic and abiotic stresses. However, information about the endophytic bacteria colonizing in the coastal halophytes is still very scarce. This study aims at isolating efficient ACC deaminase-producing plant growth-promoting (PGP) bacterial strains from the inner tissues of a traditional Chinese folk medicine Limonium sinense (Girard) Kuntze, a halophyte which has high economic and medicinal values grown in the coastal saline soils. Their PGP activity and effects on host seed germination and seedling growth under salinity stress were also evaluated.

Methods

A total of 126 isolates were obtained from the surface sterilized roots, stems and leaves of L. sinense (Girard) Kuntze. They were initially selected for their ability to produce ACC deaminase as well as other PGP properties such as production of indole-3-acetic acid (IAA), N₂-fixation, and phosphate-solubilizing activities and subsequently identified by the 16S rRNA gene sequencing. For selected strains, seed germination, seedling growth, and flavonoids production in axenically growth L. sinense (Girard) Kuntze seedlings at different NaCl concentrations (0–500 mM) were quantified.

Results

Thirteen isolates possessing ACC deaminase activity were obtained. The 16S rRNA gene sequencing analysis showed them to belong to eight genera: Bacillus, Pseudomonas, Klebsiella, Serratia, Arthrobacter, Streptomyces, Isoptericola, and Microbacterium. Inoculation with four of the selected ACC deaminase-producing strains not only stimulated the growth of the host plant but also influenced the flavonoids accumulation. All four strains could colonize and can be re-isolated from the host plant interior tissues.

Conclusions

These results demonstrate that ACC deaminase-producing habitat-adapted symbiotic bacteria isolated from halophyte could enhance plant growth under saline stress conditions and the PGPE strains could be appropriate as bioinoculants to enhance soil fertility and protect the plants against salt stress.     

Encapsulation of plant growth-promoting bacteria in alginate beads enriched with humic acid

The key to achieving successful, reproducible results following the introduction of beneficial microbes into soil relies on the survival rate of the inoculated bacteria in a heterogeneous soil environment and hence an improved encapsulation method was developed. Owing to the constraints associated with the inoculum formulation, in this study, encapsulation of a plant growth promoting bacteria (PGPB) isolate Bacillus subtilis CC-pg104 was attempted with alginate by enriching the bead microenvironment with humic acid. High viability of the encapsulated bacteria was observed with minimum cell loss upon storage for 5 months. Steady and constant cell release from the bead was observed for 1 week at different pH. Encapsulated cells remained active as evidenced by their ability to solubilize calcium phosphate in vitro. Successful plant growth promotion of lettuce by the encapsulated bacteria under gnotobiotic and sterile environment was also achieved. Feasibility of this improved encapsulation technique is mainly due to the dual benefits of humic acid to microbe and plant and its chemical properties allowing an easy mixing with alginate without interfering in the formation of the alginate gel beads by cross-linking with Ca2+ ions. Thus, the encapsulation method described in this study can be effectively used to protect the PGPB inoculum from adverse conditions of the soil for their successful establishment in the rhizosphere.     

Characterization of plant growth-promoting bacteria associated with rice cropped in iron-stressed soils

Plant growth-promoting rhizobacteria (PGPR) are able to promote plant growth using a wide variety of mechanisms as well as provide bioprotection against biotic and abiotic stresses. The objectives of this study were to isolate and characterize putative PGPR associated with rice cultivars with a distinct tolerance to iron toxicity grown in two areas: one area with a well-established history of iron toxicity and another without iron toxicity. Bacterial strains were selectively isolated based on their growth in selective media and were identified by partial sequencing of their 16S rRNA genes. Bacterial isolates were evaluated for their ability to produce indolic compounds, siderophores, and ACC deaminase and to solubilize tricalcium phosphates. In vitro biological nitrogen fixation was evaluated for the bacterial isolates used in the inoculation experiments. A total of 329 bacterial strains were isolated. The composition of the bacterial genera and the occurrence of different plant growth-promoting (PGP) traits were significantly affected by the iron conditions and by the cultivar. Strains belonging to the Burkholderia and Enterobacter genera were the most abundant of all the Gram-negative isolates, and those belonging to the Paenibacillus and Bacillus genera were the most abundant of the Gram-positive isolates. A large number of putative PGPR belonging to different bacterial genera presented several PGP traits. Strains belonging to the Burkholderia, Chryseobacterium, and Ochrobactrum genera contributed to plant growth as well as to enhanced nutrient uptake of the rice plants in in vivo experiments. Growth and nutrient uptake of plants inoculated with isolate FeS53 (Paenibacillus sp.) in the presence of an iron excess were similar to those of plants submitted to the control iron condition, indicating that this bacterium can mitigate the effects caused by iron stress.     

Characterization of a novel plant growth-promoting bacteria strain Delftia tsuruhatensis HR4 both as a diazotroph and a potential biocontrol agent against various plant pathogens

A novel, plant growth-promoting bacterium Delftia tsuruhatensis, strain HR4, was isolated from the rhizoplane of rice (Oryza sativa L., cv. Yueguang) in North China. In vitro antagonistic assay showed this strain could suppress the growth of various plant pathogens effectively, especially the three main rice pathogens (Xanthomonas oryzae pv. oryzae, Rhizoctonia solani and Pyricularia oryzae Cavara). Treated with strain HR4 culture, rice blast, rice bacterial blight and rice sheath blight for cv. Yuefu and cv. Nonghu 6 were evidently controlled in the greenhouse. Strain HR4 also showed a high nitrogen-fixing activity in N-free Döbereiner culture medium. The acetylene reduction activity and ¹⁵N₂-fixing activity (N₂FA) were 13.06 C₂H₄ nmol ml⁻¹ h⁻¹ and 2.052 ¹⁵Na.e.%, respectively. The nif gene was located in the chromosome of this strain. Based on phenotypic, physiological, biochemical and phylogenetic studies, strain HR4 could be classified as a member of D. tsuruhatensis. However, comparisons of characteristics with other known species of the genus Delftia suggested that strain HR4 was a novel dizotrophic PGPB strain.     

茅苍术叶片可培养内生细菌多样性及其促生潜力

对江苏省道地药材茅苍术叶片可培养内生细菌的多样性及其固氮、解磷、解钾、产生长素的能力进行研究。依据菌落形态的不同,共分离得到52株内生细菌。能正常传代培养的45株内生细菌经ARDRA分析后归入14个聚类簇,簇内菌株的BOX-PCR指纹图谱相似度不高,在属水平上显示出茅苍术内生细菌丰富的多样性。各聚类簇代表菌株16S rDNA的序列分析表明分离得到的内生细菌与泛菌属、微杆菌属、短杆菌属、农杆菌属、假单胞菌属、芽孢杆菌属细菌亲缘关系相近,优势内生细菌与假单胞菌属细菌亲缘关系相近。45株能正常传代培养的内生细菌中,有10株能够在无氮培养基上正常生长,具固氮潜力。使用nifH基因通用引物对其基因组进行扩增后,除ALEB 33外,其它9株内生细菌均可获得与nifH基因片段大小相近的条带。分别使用NBRIP培养基和蒙金娜有机磷培养基筛选后获得19株和15株能够溶解磷酸钙和卵磷脂的内生细菌,其中ALEB 43溶解无机磷的能力最强,达(251.43±6.55)mg/L;ALEB 4A溶解有机磷的能力最强,达(23.63±1.46)mg/L。部分内生细菌溶解无机磷的能力与其产酸能力呈正相关,而菌株溶解有机磷的能力却无此相关性。通过硅酸盐培养基的筛选,获得具有解钾潜力的菌株24株。43株内生细菌能够将色氨酸转化为生长素,其中ALEB 44产生长素的能力最强,达(268.44±10.12)μg/mL。本研究首次揭示了江苏省道地药材茅苍术体内丰富的内生细菌资源及其促生长潜力,对进一步阐述茅苍术与内生菌之间的相互关系具有重要意义。     

Whole genome sequence of the multi-resistant plant growth-promoting bacteria Streptomyces sp. Z38 with potential application in agroindustry and bio-nanotechnology

The genus Streptomyces is widely recognized for its biotechnological potential. Due to a need to improve crops, clean up the environment and produce novel antimicrobial molecules exploiting Streptomyces has become a priority. To further explore the biotechnological potential of these organisms we analyzed the genome of the strain Streptomyces sp. Z38 isolated from contaminated roots tissues. Our analysis not only confirmed the ability of the strain to produce plant growth promoting traits but also a range of mechanisms to cope with the toxic effect of heavy metals through genes involved in metal homeostasis and oxidative stress response. The production of silver nanoparticles indicated that Streptomyces sp. Z38 may find utility in Green, Grey and Red biotechnology.     

Inoculation with plant growth-promoting bacteria (PGPB) improves salt tolerance of maize seedling

Our objective was to evaluate the role of plant growth-promoting bacteria to protect maize (Zea mays L.) plants against salt damage. Bacillus aquimaris DY-3 based on their 16S rDNA sequences, the most tolerant to salinity and the synthesis of indole acetic acid was selected for further studies. Strain was inoculated on maize roots growing in sterilized sand under salt stress conditions (1% NaCl). After one week, plant growth was promoted by bacterial inoculation regardless of salt stress and non-salt stress. Chlorophyll content, leaf relative water content, accumulation of proline, soluble sugar and total phenolic compound, and activities of superoxide dismutase, catalase, peroxidase and ascorbate peroxidase were enhanced, while lipid peroxidation levels and Na⁺ content were decreased. The results showed that B. aquimaris DY-3 alleviated the salt stress in maize, likely through the integration of the antioxidant enzymes and the non-antioxidant systems that improve the plant response. Hence, the application of indole acetic acid synthesizing plant growth-promoting bacteria may represent an important alternative approach to decrease the impact of salt stress on crops.     

Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen-supply

Summary The population size of diazotrophic bacteria naturally associated with the maize rhizosphere can be affected by biotic and environmental factors. In this work we have evaluated the effect of two genotypes of maize, with and without nitrogen fertilization, on the population dynamics and distribution of diazotrophic bacteria associated with maize plants over different plant ontogenic stages. The study was carried out in a field experiment with and without nitrogen fertilization, using two maize cultivars (Santa Helena 8447 and Santa Rosa 3063) previously selected from 32 maize cultivars for the lowest and highest response to nitrogen fertilization, respectively. Microbiological and molecular approaches were used to characterize the diazotrophic bacterial population structure. Bacterial population was assessed by the most probable number using semi-solid N-free media, and by DNA isolation from soil and plant tissue followed by amplification of nifH gene fragments using nested PCR, and by RFLP analysis using the restriction endonucleases TaqI and HaeIII. The dynamics of the diazotrophic bacterial population were affected by the ontogenic stage of the maize plants, but not by the cultivar type. Roots were the preferred site of colonization, independent of cultivar type and growth stage. During the first stage of maize growth, addition of nitrogen fertilizer negatively affected the diazotrophic bacterial population.