Biological nitrogen fixation associated with sugar cane and rice: Contributions and prospects for improvement

¹⁵N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (<150 kg N ha^-1 year^-1) from biological nitrogen fixation (BNF). This may be due to the fact that this crop in Brazil has been systematically bred for high yields with low fertilizer N inputs. In the case of wetland rice, N balance experiments performed both in the field and in pots suggest that 30 to 60 N ha^-1 crop^-1 may be obtained from plant-associated BNF and that different varieties have different capacities to obtain N from this source. ¹⁵N₂ incorporation studies have proved that wetland rice can obtain at least some N from BNF and acetylene reduction (AR) assays also indicate differences in N₂-fixing ability between different rice varieties. However in situ AR field estimates suggest plant-associated BNF inputs to be less than 8 kg N ha^-1 crop^-1. The problems associated with the use of the ¹⁵N dilution technique for BNF quantification are discussed and illustrated with data from a recent study performed at EMBRAPA-CNPAB. Although many species of diazotrophs have been isolated from the rhizosphere of both sugarcane and wetland rice, the recent discovery of endophytic N₂-fixing bacteria within roots, shoots and leaves of both crops suggests, at least in the case of sugarcane, that these bacteria may be the most important contributors to the observed BNF contributions. In sugarcane both Acetobacter diazotrophicus and Herbaspirillum spp. have been found within roots and aerial tissues and these microorganisms, unlike Azospirillum spp. and other rhizospheric diazotrophs, have been shown to survive poorly in soil. Herbaspirillum spp. are found in many graminaceous crops, including rice (in roots and aerial tissue), and are able to survive and pass from crop to crop in the seeds. The physiology, ecology and infection of plants by these endophytes are fully discussed in this paper. The sugarcane/endophytic diazotroph association is the first efficient N₂-fixing system to be discovered associated with any member of the gramineae. As yet the individual roles of the different diazotrophs in this system have not been elucidated and far more work on the physiology and anatomy of this system is required. However, the understanding gained in these studies should serve as a foundation for the improvement/development of similar N₂-fixing systems in wetland rice and other cereal crops.     

Diversity of Culturable Plant Growth-Promoting Bacterial Endophytes Associated with Sugarcane Roots and Their Effect of Growth by Co-Inoculation of Diazotrophs and Actinomycetes

Application of environmentally friendly agents to reduce the use of chemicals and to enhance growth of plants is an ultimate goal of sustainable agriculture. The use of plant growth-promoting endophytes has become of great interest as a way to enhance plant growth and additionally protect plants from phytopathogens. In this study, 135 isolates of endophytic bacteria including actinomycetes were isolated from roots of commercial sugarcane plants cultivated in Thailand and were characterized for plant growth-promoting (PGP) traits. Based on morphological and 16S rRNA sequence analysis, the endophytes were distributed into 14 genera of which the most dominant species belong to Bacillus, Enterobacter, Microbispora, and Streptomyces. Two strains of endophytic diazotrophs, Bacillus sp. EN-24 and Enterobacter sp. EN-21; and two strains of actinomycetes, Microbispora sp. GKU 823 and Streptomyces sp. GKU 895, were selected based on their PGP traits including 1-aminocyclopropane-1-decarboxylate deaminase, indole-3-acetic acid, nitrogen fixation, phosphate solubilization, and siderophore production for evaluation of sugarcane growth enhancement by individual and co-inoculation. Sixty days after co-inoculation by endophytic diazotrophs and actinomycetes, the growth parameters of sugarcane plants were significantly greater than that of individual and un-inoculated plants. The results indicated that these endophytes have high potential as PGP agents that could be applied to promote sugarcane growth and could be developed as active added value biofertilizers in the future.     

Native bacterial endophytes promote host growth in a species-specific manner; phytohormone manipulations do not result in common growth responses

Background

All plants in nature harbor a diverse community of endophytic bacteria which can positively affect host plant growth. Changes in plant growth frequently reflect alterations in phytohormone homoeostasis by plant-growth-promoting (PGP) rhizobacteria which can decrease ethylene (ET) levels enzymatically by 1-aminocyclopropane-1-carboxylate (ACC) deaminase or produce indole acetic acid (IAA). Whether these common PGP mechanisms work similarly for different plant species has not been rigorously tested.

Methodology/ Principal Findings

We isolated bacterial endophytes from field-grown Solanum nigrum; characterized PGP traits (ACC deaminase activity, IAA production, phosphate solubilization and seedling colonization); and determined their effects on their host, S. nigrum, as well as on another Solanaceous native plant, Nicotiana attenuata. In S. nigrum, a majority of isolates that promoted root growth were associated with ACC deaminase activity and IAA production. However, in N. attenuata, IAA but not ACC deaminase activity was associated with root growth. Inoculating N. attenuata and S. nigrum with known PGP bacteria from a culture collection (DSMZ) reinforced the conclusion that the PGP effects are not highly conserved.

Conclusions/ Significance

We conclude that natural endophytic bacteria with PGP traits do not have general and predictable effects on the growth and fitness of all host plants, although the underlying mechanisms are conserved.     

Carbon mineralization in soil of roots from twenty crop species,as affected by their chemical composition and botanical family

Background and aims

Plant growth-promoting rhizobacteria (PGPR) have been widely studied for agricultural applications. One aim of this study was to isolate cadmium (Cd)-tolerant bacteria from nodules of Glycine max (L.) Merr. grown in heavy metal-contaminated soil in southwest of China. The plant growth-promoting (PGP) traits and the effects of the isolate on plant growth and Cd uptake by legume and non-legume plants in Cd-polluted soil were investigated.

Methods

Cd-tolerant bacteria were isolated by selective media. The isolates were identified by 16S rRNA gene and phylogenetic analysis. The PGR traits of the isolates were evaluated in vitro. Cd in soil and plant samples was determined by ICP-MS.

Results

One of the most Cd-tolerant bacteria simultaneously exhibited several PGP traits. Inoculation with the PGPR strain had positive impacts on contents of photosynthesis pigments and mineral nutrients (Fe or Mg) in plant leaves. The shoot dry weights of Lolium multiflorum Lam. increased significantly compared to uninoculated control. Furthermore, inoculation with the PGPR strain increased the Cd concentrations in root of L. multiflorum Lam. and extractable Cd concentrations in the rhizosphere, while the Cd concentrations in root and shoot of G. max (L.) Merr. significantly decreased.

Conclusions

This study indicates that inoculation with Cd-tolerant PGPR can alleviate Cd toxicity to the plants, increase Cd accumulation in L. multiflorum Lam. by enhancing Cd availability in soils and plant biomass, but decrease Cd accumulation in G. max (L.) Merr. by increasing Fe availability, thus highlighting new insight into the exploration of PGPR on Cd-contaminated soil.     

Evidence from field nitrogen balance and 15N natural abundance data for the contribution of biological N2 fixation to Brazilian sugarcane varieties

Aims

In Brazil N fertilization of sugarcane (Saccharum spp.) is low compared to most other countries. ¹⁵N-aided studies and the occurrence of many N₂-fixing bacteria associated with cane plants suggest significant contributions from biological N₂ fixation (BNF). The objective of this study was to evaluate BNF contributions to nine cane varieties under field conditions using N balance and ¹⁵N natural abundance techniques.

Methods

The field experiment was planted near Rio de Janeiro in 1989, replanted in 1999 and harvested 13 times until 2004. Soil total N was evaluated at planting and again in 2004. Samples of cane leaves and weeds for the evaluation of ¹⁵N natural abundance were taken in 2000, 2003 and 2004.

Results

N accumulation of the commercial cane varieties and a variety of Saccharum spontaneum were persistently high and N balances (60 to 107?kg?N ha^?1?yr^?1) significantly (p?<?0.05) positive. The δ¹⁵N of leaf samples were lower than any of the weed reference plants and data obtained from a greenhouse study indicated that this was not due to the cane plants tapping into soil of lower ¹⁵N abundance at greater depth.

Conclusion

The results indicate that the Brazilian varieties of sugarcane were able to obtain at least 40?kg?N ha^?1?yr^?1 from BNF.     

Molecular diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing PGPR from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) rhizosphere

Aims

The present study was planned to investigate the diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing bacteria from the rhizosphere of wheat plants and subsequent evaluation of selected PGPR on growth enhancement of wheat seedlings under drought and saline conditions.

Methods

ACC deaminase producing plant growth promoting rhizobacteria (PGPR) were isolated from the rhizosphere of wheat and identified using 16S rRNA gene sequence analysis. Isolates were evaluated for various direct and indirect plant growth promoting (PGP) traits. Plant inoculation experiment was conducted using isolates IG 19 and IG 22 in wheat to assess their plant growth promotion potential under salinity and drought stress.

Results

Thirty-eight ACC deaminase producing PGPR were isolated which belonged to 12 distinct genera and falling into four phyla γ-proteobacteria, β-proteobacteria, Flavobacteria and Firmicutes. Klebsiella sp. was the most abundant genera and followed by Enterobacter sp. The isolates exhibited ACC deaminase activities ranging from 0.106–0.980 μM α- ketobutyrate μg protein^?1 h^?1. The isolates showed multiple PGP traits such as IAA production, phosphate, zinc, potassium solubilization and siderophore production. Enterobacter cloacae (IG 19) and Citrobacter sp. (IG 22) inoculated wheat seedlings showed notable increases in fresh and dry biomass under non-stress as well as under stressed condition.

Conclusion

To the best of our knowledge this is the first report of presence of ACC deaminase activity and other PGP traits from the genus Citrobacter and Empedobacter. Our finding revealed that the γ-proteobacteria group dominated the wheat rhizosphere. Plant inoculation with PGPR could be a sustainable approach to alleviate abiotic stresses in wheat plants. These native PGPR isolates could be used as potential biofertilizers for sustainable agriculture.

     

Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates

Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37°C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria.     

Isolation,characterization and plant growth promotion effects of putative bacterial endophytes associated with sweet sorghum (Sorghum bicolor (L) Moench)

Sweet sorghum (Sorghum bicolor) is cultivated in Uruguay in complementation with sugarcane (Saccharum officinarum) as a feedstock for bioethanol production. It requires the application of high levels of chemical fertilizer for optimal growth, which causes environmental degradation. Plant growth-promoting (PGP) bacteria are of biotechnological interest since they can improve the growth of several important agronomical crops. Of particular interest are endophytes, which are those bacteria that can be detected at a particular moment within the internal tissues of healthy plants from where they can promote their growth. The aims of this work were to isolate and characterize, as well as identify putatively endophytic bacteria associated with sweet sorghum (cv-M81E), and also to study the inoculation effects of selected isolates on sorghum growth. A collection of 188 putative endophytes from surface-sterilized stems and roots was constructed and characterized. Bacterial isolates were shown to belong to different genera including Pantoea, Enterobacter, Pseudomonas, Acinetobacter, Stenotrophomonas, Ralstonia, Herbaspirillum, Achromobacter, Rhizobium, Chryseobacterium, Kocuria, Brevibacillus, Paenibacillus, Bacillus and Staphylococcus. PGP and infection features were investigated in vitro, and revealed some promising biotechnological candidates. In addition, isolates UYSB13 and UYSB45 showed PGP effects in greenhouse assays. This work provides the basis for further studies under field conditions, with the final aim of developing an effective inoculant for sorghum.     

Diversity of rhizospheric and endophytic bacteria isolated from dried fruit of Ficus carica

There is currently an increasing demand for the characterization of endophytic bacteria isolated from different parts of plants (rhizosphere, roots, fruit, leaf) in order to improve the organic agriculture practices. The current research was performed to identify both rhizospheric bacteria isolated from the rhizosphere of Ficus carica in three different sites in the north of Tunisia and endophytic bacteria isolated from dried figs. We then characterized them for a diversity of plant growth-promoting (PGP) activities. A collection of 120 isolates from rhizospheric soil and 9 isolates from dried figs was obtained and purified. 16SrDNA gene amplification of rhizospheric bacteria revealed significant diversity and allowed for the assigning of the isolates to 6 phyla: Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Representative strains of the collection (90 strains) were tested for numerous PGP activities and resistance to abiotic stresses. The most common PGP trait for all bacteria from the three regions was siderophore production (62%), followed by cellulase (38%), then protease activity (37%), then by lipases activity (17%) and lastly by solubilization of phosphates (9%). Twenty -three strains that showed most PGP traits were selected, 8 strains presented 12 or more, and 15 strains displayed between 7 and 11 of 17 PGP activities. The majority of the isolates manifested a possible adaptation to abiotic stress and unfavorable environments. PCR-DGGE analysis of soil rhizosphere of the three sites allowed also for the acquisition of a Cluster analysis of rhizospheric bacterial communities. Our current study identified and characterized for the first time in Tunisia rhizospheric and endophytic bacteria from dried fruit of Ficus carica.     

Bacterial Biosynthesis of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase and Indole-3-Acetic Acid (IAA) as Endophytic Preferential Selection Traits by Rice Plant Seedlings

In this study, bacteria were isolated from the rhizosphere and inside the roots and nodules of berseem clover plants grown in the field in Iran. Two hundred isolates were obtained from the rhizosphere (120 isolates), interior roots (57 isolates), and nodules (23 isolates) of clover plants grown in rotation with rice plants. Production of chitinase, pectinase, cellulase, siderophore, salicylic acid, hydrogen cyanide, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, solubilization of phosphate, antifungal activity against various rice plant pathogen fungi, N₂ fixation, and colonization assay on rice seedlings by these strains was evaluated and compared (endophytic isolates vs. rhizosphere bacteria). The results showed both the number and the ability of plant growth-promoting (PGP) traits were different between endophytic and rhizosphere isolates. A higher percentage of endophytic isolates were positive for production of IAA, ACC deaminase, and siderophore than rhizosphere isolates. Therefore, it is suggested that clover plant may shape its own associated microbial community and act as filters for endophyte communities, and rhizosphere isolates with different (PGP) traits. We also studied the PGP effect of the most promising endophytic and rhizosphere isolates on rice seedlings. A significant relationship among IAA and ACC deaminase production, the size of root colonization, and plant growth (root elongation) in comparison with siderophore production and phosphate solubilization for the isolates was observed. The best bacterial isolates (one endophytic isolate and one rhizosphere isolate), based on their ability to promote rice growth and colonize rice roots, were identified. Based on 16S rDNA sequence analysis, the endophytic isolate CEN7 and the rhizosphere isolate CEN8 were closely related to Pseudomonas putida and Pseudomonas fluorescens, respectively. It seems that PGP trait production (such as IAA, ACC deaminase) may be required for endophytic and rhizosphere competence as compared to other PGP traits in rice seedlings under constant flooded conditions. The study also shows that the presence of diverse rhizobacteria with effective growth-promoting traits associated with clover plants may be used for sustainable crop management under field conditions.     

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.     

Screening of plant growth promoting Rhizobacteria isolated from sunflower (Helianthus annuus L.)

Background and Aims

This study was aimed at assessing the diversity of putatively diazotrophic rhizobacteria associated with sunflower (Helianthus annuus L.) cropped in the south of Brazil, and to examine key plant growth promotion (PGP) characteristics of the isolates for the purposes of increasing plant productivity.

Methods

299 strains were isolated from the roots and rhizosphere of sunflower cultivated in five different areas using N-free media. 16S rDNA PCR-RFLP and 16S rRNA partial sequencing were used for identification and the Shannon index was used to evaluate bacterial diversity. Production of siderophores and indolic compounds (ICs), as well phosphate solubilization activities of each isolate were also evaluated in vitro. On the basis of multiple PGP activities, eight isolates were selected and tested for their N-fixation ability, and their capacity as potential PGPR on sunflower plants was also assessed.

Results

All except three Gram-positive strains (phylum Actinobacteria) belonged to the Gram-negative Proteobacteria subgroups [Gamma (167), Beta (78), and Alpha (50)] and the family Flavobacteriaceae (1)]. Shannon indexes ranged from 0.96 to 2.13 between the five sampling sites. Enterobacter and Burkholderia were the predominant genera isolated from roots and rhizosphere, respectively. Producers of siderophores and ICs were widely found amongst the isolates, but only 19.8% of them solubilized phosphate. About 8% of the isolates exhibited all three PGP traits, and these mostly belonged to the genus Burkholderia. Four isolates were able to stimulate the growth of sunflower plants under gnotobiotic conditions.

Conclusions

Enterobacter and Burkholderia were the dominant rhizospheric bacterial genera associated with sunflower plants. Inoculation with isolates belonging to the genera Achromobacter, Chryseobacterium, Azospirillum, and Burkholderia had a stimulatory effect on plant growth.     

A combination of humic substances and Herbaspirillum seropedicae inoculation enhances the growth of maize (Zea mays L.)

Background

Endophytic diazotrophic bacteria colonize several non-leguminous plants and promote plant growth. Different mechanisms are involved in bacteria-induced plant growth promotion, including biological nitrogen fixation (BNF), mineral solubilization, production of phytohormones, and pathogen biocontrol. Herbaspirillum seropedicae is a broad-host-range endophyte that colonizes sugarcane, rice, wheat, sorghum, and maize, and has been used as a biofertilizer. Contrasting results between greenhouse and field experiments have prompted efforts to improve the consistency of the plant response to microbial stimulation.

Aims

The aim of this study was to evaluate the effect of the presence of humic substances on inoculation of maize (Zea mays L.) with H. seropedicae.

Methods

Two experiments were conducted: one in the greenhouse using sand and nutrient solution and the other a field trial in soil with low natural fertility and to which was applied N in the form of urea (50 kg ha^?1). In the greenhouse, pre-emerging seeds were inoculated with a solution of H. seropedicae (10⁹ cells mL^?1) in the presence of humic substances isolated from vermicompost (10, 20, or 30 mg C?L^?1); in the field trial, bacteria combined with humate were added as a foliar spray (450 L?ha^?1).

Results

At early stages (7 and 45 days old) in the greenhouse, the treatment activated plant metabolism including enhancement of plasma membrane H⁺-ATPase activity, alteration of sugar and N metabolism, and greater net photosynthesis. The number of viable bacterial cells was higher in root tissues when inoculation was in the presence of soluble humic substances. Foliar application of endophytic diazotrophic bacteria and humic substances increased maize grain production 65 % under field conditions. These results show a promising use of humic substances to improve the benefit of endophytic diazotrophic inoculation.     

Transfer RNA-dependent asparagine biosynthesis in Gluconacetobacter diazotrophicus and its influence on biological nitrogen fixation

Background and aims

Gluconacetobacter diazotrophicus is a nitrogen-fixing endophytic bacterium isolated from sugarcane, rice, elephant grass, sweet potato, coffee, and pineapple. These plants have high level of asparagine, which promotes microbial growth and inhibits nitrogenase activity. The regulation of intracellular concentrations of this amino acid is essential for growth and biological nitrogen fixation (BNF) in this diazotroph; however its asparagine metabolic pathway has not yet been clearly established.

Methods

The work reported here is the first to demonstrate the use of an alternative route for asparaginyl-tRNA (Asn-tRNA) and asparagine formation in an endophytic nitrogen-fixing bacterium by using in silico and in vitro analysis.

Results

The indirect route involves transamidation of incorrectly charged tRNA via GatCAB transamidase. Nitrogenase activity was completely inhibited by 20?mM Asn in LGI-P medium, which in contrast promotes protein synthesis and microbial growth.

Conclusions

The analysis carried out in this work shows that intracellular levels of asparagine regulate the expression of nitrogenase nifD gene (GDI0437), suggesting that the presence of an alternative route to produce asparagine might give the G. diazotrophicus a tighter control over cell growth and BNF, and may be of importance in the regulation of the endophytic plant-microbe interaction.     

Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N₂-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N₂-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and ¹⁵N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N₂-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N₂ fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N₂-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.     

Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings

Study of endophytic bacteria within plant seeds is very essential and meaningful on account of their heritability and versatility. This study investigated Bacillus bacterial communities within the seeds of four commercial tomato varieties, by 16S rRNA gene PCR-RFLP (restriction fragment length polymorphism). Phylogenetic analysis of 16S rRNA gene sequences indicated that the 22 representative isolates belonged to five species of genus Bacillus and the bacterial compositions showed remarkable differences among tomato varieties. Isolates exhibited multiple plant growth promoting (PGP) traits: 37 % of indole-3-acetic acid production; 37 % of phosphate solubilization; 24 % of siderophores production; 85 % of potential nitrogen fixation and 6 % of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Isolate HYT-12-1 was shown to have highest ACC deaminase activity (112.02 nmol α-ketobutyrate mg^?1 protein h^?1) among the five ACC deamiase producing strains. 16S rRNA gene sequencing indicated that the isolate HYT-12-1 shared the highest sequence similarity (100 %) with B. subtilis. PGP experiments under gnotobiotic and greenhouse conditions revealed the ability of strain HYT-12-1 to enhance the growth of tomato seedlings. This is the first study to describe endophytic Bacillus communities within tomato seeds, and the results suggest that B. subtilis strain HYT-12-1 would have a great potential for industrial application as biofertilizer in the future.     

The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists

Background and aim

Nitrogen-fixing bacteria or diazotrophs have been isolated for many years using different formulations of N-free semi-solid media. However, the strategies used to isolate them, and the recipes of these media, are scattered through the published literature and in other sources that are more difficult to access and which are not always retrievable. Therefore, the aim of this work was to collate the various methods and recipes, and to provide a comprehensive methodological guide and their use by the scientific community working in the field of biological nitrogen fixation (BNF), particularly with non-leguminous plants.

Methods

Procedures used for bacterial counting and identification either from rhizosphere soil or on the surface of, or within, plant tissues (to access “endophytic” bacteria) are presented in detail, including colony and cell morphologies. More importantly, appropriate recipes available for each N-free semi-solid culture medium that are used to count and isolate various diazotrophs are presented.

Results

It is recognized by those working in the field of BNF with non-legumes that the development of the N-free semi-solid medium has allowed a tremendous accumulation of knowledge on the ecology and physiology of their associated diazotrophs. At least 20 nitrogen-fixing species have been isolated and identified based on the enrichment method originally developed by Döbereiner, Day and collaborators in the 70’s. In spite of all the advances in molecular techniques used to detect bacteria, in most cases the initial isolation and identification of these diazotrophs still requires semi-solid media.

Conclusions

The introduction of the N-free semi-solid medium opened new opportunities for those working in the area of BNF with non-legumes not only for elucidating the important role played by their associated microorganisms, but also because some of these bacteria that were isolated using semi-solid media are now being recommended as plant growth-promoting inoculants for sugarcane (Saccharum sp.), maize (Zea mays) and wheat (Triticum aestivum) in Brazil and other countries. Further progress in the field could be made by using a combination of culture-independent molecular community analyses, in situ activity assessments with probe-directed enrichment, and isolation of target strains using modified or standard semi-solid media.     

Isolation and identification of endophytic bacteria from root tissues of Salvia miltiorrhiza Bge. and determination of their bioactivities

Endophytic bacteria are microorganisms that live in host plants, but do not cause diseases to the hosts. This study examined the occurrence, distribution, growth-promoting and antifungal activities of endophytes in the root of Salvia miltiorrhiza Bge. Six endophytic bacterial strains, which belong to genera of Pseudomonas, Rhizobium, Bacillus and Novosphingobium, were isolated from the root of healthy S. miltiorrhiza. Cell suspension (approx. 10⁹ cell?·?ml^?1) of two isolates and cell-free fermentation filtrate of four isolates substantially promoted the growth of hypocotyl and radicle of muskmelon seeds. The cell-free fermentation filtrate of six isolates had no inhibiting effect on tested pathogenic fungi, namely Fusarium solani, F. oxysporum f. sp. vasinfectum and F. oxysporum. Six compounds were isolated from one of the six endophytic bacteria, namely, Bacillus aryabhattai, and two of these compounds displayed certain antifungal activity against three tested S. miltiorrhiza pathogens. Our work indicates that endophytic bacteria occur in the root of S. miltiorrhiza, and that associated bacterial isolates have growth-promoting effect on muskmelon seeds and are expected to be a potential source for bioactive metabolites.     

High diversity of culturable Burkholderia species associated with sugarcane

Sugarcane is an important crop around the world. Burkholderia genus has emerged as an important plant associated bacteria in the last years. In this study, the occurrence of Burkholderia species associated with two sugarcane varieties cultivated in Mexico was assessed. Burkholderia species were isolated with and without diazotrophs enrichment from sugarcane. Burkholderia strains were identified using a semi-selective set of primers and clustered by restriction analysis of 16S rRNA. The isolates were characterized by 16S rRNA, recA and nifH sequence analysis, whole-cell protein patterns, and plant-growth promotion (PGP) characteristics. Diazotrophic B. unamae and B. tropica were predominant using diazotroph enrichment method. Non-diazotrophic B. cepacia complex (Bcc) species were predominant without enrichment. Among non-diazotrophs, B. tropica was identified. The diazotrophic Burkholderia species exhibit in vitro PGP activities: biosynthesis of indolic compounds, phosphate solubilization, siderophores production and acdS gene presence, which encodes the enzyme ACC (1-aminocyclopropane-1-carboxylate) deaminase. The present study confirms the broad environmental and geographic distribution of diazotrophic B. unamae and B. tropica, and reveals the riches of Bcc and other Burkholderia species associated with sugarcane field-grown in Mexico. This work also shows the potential activities in PGP.