The hyperaccumulator Sedum plumbizincicola harbors metal-resistant endophytic bacteria that improve its phytoextraction capacity in multi-metal contaminated soil

Endophyte-assisted phytoremediation has recently been suggested as a successful approach for ecological restoration of metal contaminated soils, however little information is available on the influence of endophytic bacteria on the phytoextraction capacity of metal hyperaccumulating plants in multi-metal polluted soils. The aims of our study were to isolate and characterize metal-resistant and 1-aminocyclopropane-1-carboxylate (ACC) utilizing endophytic bacteria from tissues of the newly discovered Zn/Cd hyperaccumulator Sedum plumbizincicola and to examine if these endophytic bacterial strains could improve the efficiency of phytoextraction of multi-metal contaminated soils. Among a collection of 42 metal resistant bacterial strains isolated from the tissues of S. plumbizincicola grown on Pb/Zn mine tailings, five plant growth promoting endophytic bacterial strains (PGPE) were selected due to their ability to promote plant growth and to utilize ACC as the sole nitrogen source. The five isolates were identified as Bacillus pumilus E2S2, Bacillus sp. E1S2, Bacillus sp. E4S1, Achromobacter sp. E4L5 and Stenotrophomonas sp. E1L and subsequent testing revealed that they all exhibited traits associated with plant growth promotion, such as production of indole-3-acetic acid and siderophores and solubilization of phosphorus. These five strains showed high resistance to heavy metals (Cd, Zn and Pb) and various antibiotics. Further, inoculation of these ACC utilizing strains significantly increased the concentrations of water extractable Cd and Zn in soil. Moreover, a pot experiment was conducted to elucidate the effects of inoculating metal-resistant ACC utilizing strains on the growth of S. plumbizincicola and its uptake of Cd, Zn and Pb in multi-metal contaminated soils. Out of the five strains, B. pumilus E2S2 significantly increased root (146%) and shoot (17%) length, fresh (37%) and dry biomass (32%) of S. plumbizincicola as well as plant Cd uptake (43%), whereas Bacillus sp. E1S2 significantly enhanced the accumulation of Zn (18%) in plants compared with non-inoculated controls. The inoculated strains also showed high levels of colonization in rhizosphere and plant tissues. Results demonstrate the potential to improve phytoextraction of soils contaminated with multiple heavy metals by inoculating metal hyperaccumulating plants with their own selected functional endophytic bacterial strains.

Influence of organic fertilization on the number of culturable diazotrophic endophytic bacteria isolated from sugarcane

The numbers of culturable diazotrophic endophytic bacteria (CDEB) from roots stems and leaves of sugarcane submitted to organic inorganic or no fertilization were compared In order to determine the size of the N(2) fixing populations the Most Probable Number technique (MPN) was used The quantification of diazotrophic bacteria by using the acetylene reduction assay (ARA) was more accurate than observing the bacterial growth in the vials to confirm N(2) fixing capability the detection of gene nifH was performed on a sample of 105 Isolated bacteria The production of extracellular enzymes involved in the penetration of the plants by the bacteria was also studied The results showed that organic fertilization enhances the number of CDEB when compared with conventional fertilization used throughout the growing season The maximum number of bacteria was detected in the roots Roots and stems presented the greatest number of CDEB in the middle of the cropping season and in leaves numbers varied according to the treatment Using two pairs of primers and two different methods the nifH gene was found in 104 of the 105 tested isolates Larger amounts of pectinase were released by isolates from sugarcane treated with conventional fertilizers (66%) whereas larger amounts of cellulase were released by strains isolated from sugarcane treated with organic fertilizers (80%) (C) 2010 Elsevier Masson SAS All rights reserved

Volatiles produced by soil-borne endophytic bacteria increase plant pathogen resistance and affect tritrophic interactions

Volatile organic compounds (VOCs) released by soil microorganisms influence plant growth and pathogen resistance. Yet, very little is known about their influence on herbivores and higher trophic levels. We studied the origin and role of a major bacterial VOC, 2,3-butanediol (2,3-BD), on plant growth, pathogen and herbivore resistance, and the attraction of natural enemies in maize. One of the major contributors to 2,3-BD in the headspace of soil-grown maize seedlings was identified as Enterobacter aerogenes, an endophytic bacterium that colonizes the plants. The production of 2,3-BD by E. aerogenes rendered maize plants more resistant against the Northern corn leaf blight fungus Setosphaeria turcica. On the contrary, E. aerogenes-inoculated plants were less resistant against the caterpillar Spodoptera littoralis. The effect of 2,3-BD on the attraction of the parasitoid Cotesia marginiventris was more variable: 2,3-BD application to the headspace of the plants had no effect on the parasitoids, but application to the soil increased parasitoid attraction. Furthermore, inoculation of seeds with E. aerogenes decreased plant attractiveness, whereas inoculation of soil with a total extract of soil microbes increased parasitoid attraction, suggesting that the effect of 2,3-BD on the parasitoid is indirect and depends on the composition of the microbial community.

Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion.

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry

Endophytic bacteria for biocontrol of coffee leaf rust (Hemileia vastatrix).

2007

Endophytic microorganisms from coffee tissues as plant growth promoters and biocontrol agents of coffee leaf rust

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endophytic microorganisms isolated from coffee leaves, roots and branches as plant growth promoters and biocontrol agents of coffee leaf rust

Suppression of plant diseases and growth promotion due to the action of endophytic microorganisms has been demonstrated in several pathosystems. Experiments under controlled conditions involving 234 endophytic bacteria and fungi isolated from coffee leaves, roots and branches were conducted with the objective of evaluating the germination inhibition of Hemileia vastatrix urediniospores, the control of coffee leaf rust development in tests with leaf discs and on plastic bags seedling, and to promote growth of coffee seedlings. None of the fungal isolates induced plant growth or reduced disease severity. The bacterial isolates (identified by the fatty acids profile analysis) 85G (Escherichia fergusonii), 161G, 163G, 160G, 150G (Acinetobacter calcoaceticus) and 109G (Salmonella enterica) increased plant growth, the maximum being induced by 85G. This isolate produced in vitro phosphatase and indol acetic acid. In assay to control rust on coffee leaf disc, nine bacterial isolates, 64R, 137G, 3F (Brevibacillus choshinensis), 14F (Salmonella enterica), 36F (Pectobacterium carotovorum), 109G (Bacillus megaterium), 115G (Microbacterium testaceum), 116G and 119G (Cedecea davisae) significantly reduced disease severity, when applied 72 or 24h before challenging with the pathogen. In seedling tests most disease severity reduction was achieved by the isolates 109G and 119G. There was no correspondence between the organisms that promoted seedling growth and those that reduced rust severity on seedlings or leaf discs.

Eucalyptus growth promotion by endophytic Bacillus spp

Clonal eucalyptus plantings have increased in recent years; however, some clones with high production characteristics have vegetative propagation problems because of weak root and aerial development. Endophytic microorganisms live inside healthy plants without causing any damage to their hosts and can be beneficial, acting as plant growth promoters. We isolated endophytic bacteria from eucalyptus plants and evaluated their potential in plant growth promotion of clonal plantlets of Eucalyptus urophylla x E. grandis, known as the hybrid, E. urograndis. Eighteen isolates of E. urograndis, clone 4622, were tested for plant growth promotion using the same clone. These isolates were also evaluated for indole acetic acid production and their potential for nitrogen fixation and phosphate solubilization. The isolates were identified by partial sequencing of 16S rRNA. Bacillus subtilis was the most prevalent species. Several Bacillus species, including B. licheniformis and B. subtilis, were found for the first time as endophytes of eucalyptus. Bacillus sp strain EUCB 10 significantly increased the growth of the root and aerial parts of eucalyptus plantlets under greenhouse conditions, during the summer and winter seasons.

GENETIC VARIABILITY OF SUGARCANE-ASSOCIATED DIAZOTROPHIC BACTERIA CAPABLE OF INORGANIC PHOSPHATE SOLUBILIZING

The sugarcane is a culture of great importance for the Brazilian agriculture. Every year this culture consumes great amounts of nitrogen and phosphate fertilizers. However, the use of plant growth-promoting bacteria can reduce the use of the chemical fertilizers, contributing to the economy and the environment conservation. So, the goal of this study was to select sugarcane-associated diazotrophic bacteria able to solubilize inorganic phosphate and to evaluate the genetic diversity of these bacteria. A total of 68 diazotrophic bacteria, leaf and root endophytic and rizoplane, of three sugarcane varieties. The selection of inorganic phosphate solubilizing diazotrophic bacteria was assayed by the solubilization index (SI) in solid medium containing insoluble phosphate. The genetic variability was analyzed by the BOX-PCR technique. The results showed that 74% of the diazotrophic strains were able to solubilize inorganic phosphate, presenting classes of different SI. The results showed that the vegetal tissue and the genotype plant influenced in the interaction between phosphate solubilizing diazotrophic bacteria and sugarcane plants. BOX-PCR revealed high genetic variability among the strains analyzed. So, sugarcane-associated diazotrophic bacteria express the capacity to solubilize inorganic phosphate and they present high genetic diversity.

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Background and aims Endophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants. Methods A total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique. Results The putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA. Conclusion Endophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

Isolation and selection of plant growth-promoting bacteria associated with sugarcane

v. 46, n. 2, p. 149-158, apr./jun. 2016.

Indole acetic acid and ACC deaminase fromendophytic bacteria improves the growth of Solanum lycopersicum

Background: Endophytic bacteria are ubiquitous in all plant species contributing in host plant\'s nutrient uptake and helping the host to improve its growth. Moringa peregrina which is a medicinal plant, growing in arid region of Arabia, was assessed for the presence of endophytic bacterial strains. Results: PCR amplification and sequencing of 16S rRNA of bacterial endophytes revealed the 5 endophytic bacteria, in which 2 strains were from Sphingomonas sp.; 2 strains from Bacillus sp. and 1 from Methylobacterium genus. Among the endophytic bacterial strains, a strain of Bacillus subtilis LK14 has shown significant prospects in phosphate solubilization (clearing zone of 56.71 mm after 5 d), ACC deaminase (448.3 ± 2.91 nM α-ketobutyrate mg-1 h-1) and acid phosphatase activity (8.4 ± 1.2 nM mg-1 min-1). The endophytic bacteria were also assessed for their potential to produce indole-3-acetic acid (IAA). Among isolated strains, the initial spectrophotometry analysis showed significantly higher IAA production by Bacillus subtilis LK14. The diurnal production of IAA was quantified using multiple reactions monitoring method in UPLC/MS–MS. The analysis showed that LK14 produced the highest (8.7 μM) IAA on 14th d of growth. Looking at LK14 potentials, it was applied to Solanum lycopersicum , where it significantly increased the shoot and root biomass and chlorophyll (a and b) contents as compared to control plants. Conclusion: The study concludes that using endophytic bacterial strains can be bio-prospective for plant growth promotion, which might be an ideal strategy for improving growth of crops in marginal lands.

联合固氮菌Klebsiella oxytoca SA2和水稻幼苗的相互作用

从内蒙流沙地先锋植物沙竹（Psammochloa mongokca）内分离到一株内生细菌，经鉴定定名为Klebsiella oxytoca SA-2 K.oxytoca SA-2兼性厌氧固氮，NH4+抑制其固氮酶合成。部分抑制固氮酶活性;N03 -抑制其固氮酶的合成和活性。 60℃灭活K.oxytoca SA-2整体菌免疫兔子得到抗血清。免疫印迹表明此抗血清具K.oxytoca SA-2种特异性。石蜡切片免疫金银染色结合显微观察发现K.oxytoca SA-2定殖于沙竹叶鞘薄壁细胞和叶片的薄壁细胞内。 K.oxytoca SA-2在半固体培养基中接种水稻幼苗，限菌培养21天，根内重新分离的数量达l06 cfu/g．FWroot，但K.oxytoca SA-2在富养的土壤中生长良好，表现为兼性内生菌。 限菌培养水稻（Oryza sativa）幼苗，石蜡切片免疫金银染色结合显微观察研究了的K.oxytoca SA-2的侵染特性．K.oxytoca SA-2可以通过侧根发生处和表皮细胞胞间层进入根内，在皮层薄壁细胞间隙大量定殖，在解体和看似完整的薄壁细胞内也有定殖，在根和茎中柱内K.oxytoca SA-2进入了木质部导管。在根基，K.oxytoca SA-2大量侵入了已解体的内皮层和中柱鞘细胞，植物细胞在K.oxytoca SA-2侵入后解体，可能表现为严格的局部超敏反应。 接种K.oxytoca SA-2 21天，水稻地上苗部分没有发现肉眼和显微可见的病症。与对照相比，接种K.oxytoca SA-2显著促进限氮培养水稻幼苗的生长。由于K.oxytoca SA-2在限碳限氮培养基和水稻幼苗共培养时能分泌NH4+和植物激素，它可能通过向水稻幼苗提供氮素和分泌植物激素促进植物生长。而且用固氮酶铁蛋白抗血清进行免疫金银染色发现定殖在根基皮层薄壁细胞胞间层和细胞间隙，木质部导管和茎基木质部导管的K.oxytoca SA-2可以表达固氮酶，固氮参与了K.oxytoca SA-2在水稻幼苗中的内生。 培养基内碳源（苹果酸）和培养温度对K.oxytoca SA-2和水稻幼苗相互作用的影响也进行了研究。 研究表明，K.oxytoca SA-2作为兼性内生固氮菌，能够和植物紧密联合，并在植物体内开拓一个有利的生态位固氮，而且K.oxytoca SA-2可以分泌NH4+和植物激素，在和植物相互作用中使植物受益。

Microbe-mediated recovery of salt marshes contaminated with oil hydrocarbons

Salt marshes are highly productive intertidal habitats that serve as nursery grounds for many commercially and economically important species. Because of their location and physical and biological characteristics, salt marshes are considered to be particularly vulnerable to anthropogenic inputs of oil hydrocarbons. Sediment contamination with oil is especially dangerous for salt marsh vegetation, since low molecular weight aromatic hydrocarbons can affect plants at all stages of development. However, the use of vegetation for bioremediation (phytoremediation), by removal or sequestration of contaminants, has been intensively studied. Phytoremediation is an efficient, inexpensive and environmental friendly approach for the removal of aromatic hydrocarbons, through direct incorporation by the plant and by the intervention of degrading microbial populations in the rhizosphere (microbe-assisted phytoremediation). Rhizosphere microbial communities are enriched in important catabolic genotypes for degradation of oil hydrocarbons (OH) which may have a potential for detoxification of the sediment surrounding the roots. In addition, since rhizosphere bacterial populations may also internalize into plant tissues (endophytes), rhizocompetent AH degrading populations may be important for in planta AH degradation and detoxification. The present study involved field work and microcosms experiments aiming the characterization of relevant plant-microbe interactions in oilimpacted salt marshes and the understanding of the effect of rhizosphere and endosphere bacteria in the role of salt marsh plants as potential phytoremediation agents. In the field approach, molecular tools were used to assess how plant species- and OH pollution affect sediment bacterial composition [bulk sediment and sediment surrounding the roots (rhizosphere) of Halimione portulacoides and Sarcocornia perennis subsp. perennis] in a temperate estuary (Ria de Aveiro, Portugal) chronically exposed to OH pollution. In addition, the 16S rRNA gene sequences retrieved in this study were used to generate in silico metagenomes and to evaluate the distribution of potential bacterial traits in different microhabitats. Moreover, a combination of culture-dependent and -independent approaches was used to investigate the effect of oil hydrocarbons contamination on the structure and function of endophytic bacterial communities of salt marsh plants.Root systems of H. portulacoides and S. perennis subsp. perennis appear to be able to exert a strong influence on bacterial composition and in silico metagenome analysis showed enrichment of genes involved in the process of polycyclic aromatic hydrocarbon (PAH) degradation in the rhizosphere of halophyte plants. The culturable fraction of endophytic degraders was essentially closely related to known OH-degrading Pseudomonas species and endophytic communities revealed sitespecific effects related to the level of OH contamination in the sediment. In order to determine the effects of oil contamination on plant condition and on the responses in terms of structure and function of the bacterial community associated with plant roots (rhizosphere, endosphere), a microcosms approach was set up. The salt marsh plant Halimione portulacoides was inoculated with a previous isolated Pseudomonas sp. endophytic degrader and the 2-methylnaphthalene was used as model PAH contaminant. The results showed that H. portulacoides health and growth were not affected by the contamination with the tested concentration. Moreover, the decrease of 2-methylnaphthalene at the end of experiment, can suggest that H. portulacoides can be considered as a potential plant for future uses in phytoremedition approaches of contaminated salt marsh. The acceleration of hydrocarbon degradation by inoculation of the plants with the hydrocarbon-degrading Pseudomonas sp. could not, however, be demonstrated, although the effects of inoculation on the structure of the endophytic community observed at the end of the experiment indicate that the strain may be an efficient colonizer of H. portulacoides roots. The results obtained in this work suggest that H. portulacoides tolerates moderate concentrations of 2-methylnaphthalene and can be regarded as a promising agent for phytoremedition approaches in salt marshes contaminated with oil hydrocarbons. Plant/microbe interactions may have an important role in the degradation process, as plants support a diverse endophytic bacterial community, enriched in genetic factors (genes and plasmids) for hydrocarbon degradation.

Signal transduction-related responses to phytohormones and environmental challenges in sugarcane

Background: Sugarcane is an increasingly economically and environmentally important C4 grass, used for the production of sugar and bioethanol, a low-carbon emission fuel. Sugarcane originated from crosses of Saccharum species and is noted for its unique capacity to accumulate high amounts of sucrose in its stems. Environmental stresses limit enormously sugarcane productivity worldwide. To investigate transcriptome changes in response to environmental inputs that alter yield we used cDNA microarrays to profile expression of 1,545 genes in plants submitted to drought, phosphate starvation, herbivory and N-2-fixing endophytic bacteria. We also investigated the response to phytohormones (abscisic acid and methyl jasmonate). The arrayed elements correspond mostly to genes involved in signal transduction, hormone biosynthesis, transcription factors, novel genes and genes corresponding to unknown proteins.Results: Adopting an outliers searching method 179 genes with strikingly different expression levels were identified as differentially expressed in at least one of the treatments analysed. Self Organizing Maps were used to cluster the expression profiles of 695 genes that showed a highly correlated expression pattern among replicates. The expression data for 22 genes was evaluated for 36 experimental data points by quantitative RT-PCR indicating a validation rate of 80.5% using three biological experimental replicates. The SUCAST Database was created that provides public access to the data described in this work, linked to tissue expression profiling and the SUCAST gene category and sequence analysis. The SUCAST database also includes a categorization of the sugarcane kinome based on a phylogenetic grouping that included 182 undefined kinases.Conclusion: An extensive study on the sugarcane transcriptome was performed. Sugarcane genes responsive to phytohormones and to challenges sugarcane commonly deals with in the field were identified. Additionally, the protein kinases were annotated based on a phylogenetic approach. The experimental design and statistical analysis applied proved robust to unravel genes associated with a diverse array of conditions attributing novel functions to previously unknown or undefined genes. The data consolidated in the SUCAST database resource can guide further studies and be useful for the development of improved sugarcane varieties.