Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake

Background and Aims

Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (P_i) in vitro and in association with rice plants cultivated in pots.

Methods

Forty-nine isolates were tested for the ability to solubilize P_i on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing ¹⁵N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the ¹⁵N content accumulated in plant tissues were measured at 135 days after planting.

Results

Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized P_i on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L^?1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the ¹⁵N-labeled fertilizer.

Conclusion

These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.     

Serologic survey of West Nile virus in horses from Central-West,Northeast and Southeast Brazil

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.     

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes

Aims: To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. Methods and Results: Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but β‐glucuronidase (GUS)‐stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5–plant interaction. PAL5 could be isolated from the root surface (10⁸ CFU g^?1) and from surface‐disinfected root and stem tissues (10⁴ CFU g^?1) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. Conclusion: The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. Significance and Impact of the Study: These tools are of use to: (i) study PAL5 mutants affected in bacteria–plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.     

CAG repeat polymorphism in androgen receptor gene is not directly associated with polycystic ovary syndrome but influences serum testosterone levels

Hyperandrogenemia has been the most consistent feature of polycystic ovary syndrome (PCOS). Androgens exert their effects through androgen receptors (ARs). The expansion of the codon CAG trinucleotide repeat polymorphism in exon 1 of the AR gene represents a type of genetic alteration associated with changes in the AR gene function. The purpose of this study was to establish a possible association of the AR gene CAG repeat length polymorphism with PCOS, and its influence on clinical and biochemical androgen traits. Two hundred and fourteen Croatian women with PCOS and 209 healthy control women of reproductive age were enrolled. Phenotypic hyperandrogenism, BMI and waist to hip ratio were recorded. Hormonal profiles, fasting insulin and glucose levels were measured on cycle days 3-5. Genotyping of the CAG repeat polymorphism in the AR gene was performed. We found no significant difference in the mean CAG repeat number between the PCOS patients and controls (22.1±3.4 vs. 21.9±3.2, P=0.286). There was a positive correlation between the CAG repeat length and total testosterone (TT) in the PCOS group (R=0.225, P=0.015). A multiple linear regression model using mean CAG repeat length, BMI, age and HOMA-IR as predictors explained 8.5% (adjusted R2) of the variability in serum TT levels. In this model the CAG repeat polymorphism was found to be a significant predictor of serum TT levels in PCOS patients (P=0.015). The logistic regression analysis revealed that the CAG repeat length is not a significant predictor of hirsutism and acne status (P=0.921 and P=0.437, respectively). The model was adjusted for serum TT, free testosterone, androstendione and DHEAS levels as independent variables, which were also not found to be significant predictors of hirsutism (P=0.687, P=0.194, P=0.675 and P=0.938, respectively) or acne status (P=0.594, P=0.095, P=0.290 and P=0.151, respectively). In conclusion, the AR CAG repeat polymorphism is not a major determinant of PCOS in the Croatian population, but it is a predictor of serum TT level variability in women with PCOS.     

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.     

The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants

We investigated the effects of an autumn sowing of contrasting cover crops (oats, rye and a combination of oats and rye) on soil aggregate stability, mycorrhizal colonization, phosphorus uptake and yield of sweet corn planted the following summer. Rye is a common cover crop in the middle Atlantic region of the United States of America. It grows slowly in the autumn, survives the winter, grows rapidly in the spring and flowers in the summer. Thus, herbicide is commonly used to kill rye prior to planting spring crops. Oats, in contrast, grows rapidly in the autumn but is killed by frost during the winter. Thus, with oats, potentially less herbicide is needed to prepare the field for spring planting. When compared to fallow, oats was as effective as rye in increasing mycorrhizal colonization of sweet corn, density of mycorrhizal hyphae, and soil aggregate stability. An oats cover crop may thus be a viable alternative to rye. The combination of cover crops (rye and oats), however, was significantly better than single species of cover crops in terms of sweet corn mycorrhizal colonization, P uptake and yield of sweet corn.     

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.     

Technical approaches to inoculate micropropagated sugar cane plants were Acetobacter diazotrophicus

Micropropagated plantlets of sugar cane were inoculated with the N2-fixing bacterium Acetobacter diazotrophicus. Various modifications on the basic plant culture medium MS were made for the plant/bacteria association. The protocol required the inoculation of the bacteria at the end of the rooting period in a medium without hormones or vitamins, and with the concentration of sugar and mineral nutrients reduced by a factor of 10. Individual plants were inoculated with A. diazotrophicus and maintained under the appropriate light and temperature condition used for micropropagation up to 7 days. The system favored the infection and the establishment of the bacteria within the plant tissue. Bacteria colonized the plant tissue and accumulated in inter-cellular cavities and the region of lateral root emergence and also colonizes the xylem vessels. The inoculated plantlets were subsequently transferred to the acclimatization phase and after 30 days it was possible to isolate the bacteria from plant tissue. This protocol permitted studies of infection and comparison among strains.     

Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria

It is well described that the beneficial interactions between plants and bacteria are genotype and site specific. Brazilian sugarcane varieties can obtain up to 70% of their nitrogen requirement from biological nitrogen fixation (BNF), and this contribution is related to the Brazilian breeding and selection processes, by example of the variety SP70-1143. In this study the effect of two inoculation mixtures containing diazotrophic bacteria in our earlier pot experiment was evaluated with two sugarcane varieties, a known responder, SP70-1143, and a newly selected variety, SP81-3250, to investigate the sugarcane genotype effect and the role of the mixtures. The sugarcane varieties SP70-1143 and SP81-3250 were grown under commercial field conditions at three sites with contrasting soil types: an Alfisol, an Oxisol and an Ultisol that means a low, medium and high natural fertility respectively. The stem yield and BNF contribution in response to bacterial inoculation were influenced by the strain combinations in the inoculum, the plant genotype, and the soil type and nitrogen fertilization, confirming the genetic and environmental influence in PGP-bacteria interactions. Inoculation effects on the BNF contribution and stem yield increased in the variety SP70-1143 grown in the Alfisol without nitrogen fertilization for three consecutive crops, and it was equivalent to the annual nitrogen fertilization. The plants grown in the Oxisol showed small increases in the productivity of the variety SP70-1143, and in the Ultisol the sugarcane plants presented even decreases in the stem productivity due to inoculation with diazotrophic bacteria mixtures. The results demonstrate the feasibility of the inoculation technology using diazotrophic bacteria in micropropagated sugarcane varieties grown in soils with low to medium levels of fertility. In addition, the results also indicated that specific plant – bacteria – environment combinations are needed to harness the full benefits of BNF. Section Editor: C. P. Vance