Transcriptomic Response of the Diazotrophic Bacteria Gluconacetobacter diazotrophicus Strain PAL5 to Iron Limitation and Characterization of the fur Regulatory Network.

Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicusfur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicusfur protein was able to complement an E. colifur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus.

Selection and validation of reference genes for RT-qPCR indicates that juice of sugarcane varieties modulate the expression of C metabolism genes in the endophytic diazotrophic Herbaspirillum rubrisubalbicans strain HCC103.

Quantitative reverse transcription PCR (RT-qPCR) is an important tool for evaluating gene expression. However, this technique requires that specific internal normalizing genes be identified for different experimental conditions. To date, no internal normalizing genes are available for validation of data analyses for Herbaspirillum rubrisubalbicans strain HCC103, an endophyte that is part of the sugarcane consortium inoculant. This work seeks to identify and evaluate suitable reference genes for gene expression studies in HCC103 grown until middle log phase in sugarcane juice obtained from four sugarcane varieties or media with three different carbon sources. The mRNA levels of five candidate genes (rpoA, gyrA, dnaG, recA and gmK) and seven target genes involved in carbon metabolism (acnA, fbp, galE, suhB, wcaA, ORF_0127.0101 and _0127.0123) were quantified by RT-qPCR. Analysis of expression stability of these genes was carried out using geNorm and Normfinder software. The results indicated that the HCC103 dnaG and gyrA genes are the most stable and showed adequate relative expression level changes among the different sugarcane juices. The highest expression level was seen for ORF_0127.0101, which encodes a sugar transporter, in juice from sugarcane variety RB867515 and glucose as the carbon source. The suhB gene, encoding SuhB inositol monophosphatase, had a higher relative expression level on 0.5% glucose, 100% sugarcane juice from variety RB867515 and 0.5% aconitate. Together the results suggest that dnaG and gyrA genes are suitable as reference genes for RT-qPCR analysis of strain HCC103 and that juice from different sugarcane varieties modulates the expression of key genes involved in carbon metabolism.

Selection and evaluation of reference genes for RT-qPCR expression studies on Burkholderia tropica strain Ppe8, a sugarcane-associated diazotrophic bacterium grown with different carbon sources or sugarcane juice.

Among the members of the genus Burkholderia, Burkholderia tropica has the ability to fix nitrogen and promote sugarcane plant growth as well as act as a biological control agent. There is little information about how this bacterium metabolizes carbohydrates as well as those carbon sources found in the sugarcane juice that accumulates in stems during plant growth. Reverse transcription quantitative PCR (RT-qPCR) can be used to evaluate changes in gene expression during bacterial growth on different carbon sources. Here we tested the expression of six reference genes, lpxC, gyrB, recA, rpoA, rpoB, and rpoD, when cells were grown with glucose, fructose, sucrose, mannitol, aconitic acid, and sugarcane juice as carbon sources. The lpxC, gyrB, and recA were selected as the most stable reference genes based on geNorm and NormFinder software analyses. Validation of these three reference genes during strain Ppe8 growth on the same carbon sources showed that genes involved in glycogen biosynthesis (glgA, glgB, glgC) and trehalose biosynthesis (treY and treZ) were highly expressed when Ppe8 was grown in aconitic acid relative to other carbon sources, while otsA expression (trehalose biosynthesis) was reduced with all carbon sources. In addition, the expression level of the ORF_6066 (gluconolactonase) gene was reduced on sugarcane juice. The results confirmed the stability of the three selected reference genes (lpxC, gyrB, and recA) during the RT-qPCR and also their robustness by evaluating the relative expression of genes involved in glycogen and trehalose biosynthesis when strain Ppe8 was grown on different carbon sources and sugarcane juice.

Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus.

TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

Bacillus endophytic strains control Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici in tomato cv. Perinha.

Fusarium wilt is one of main phytopathology attacking tomato (Solanum lycopersicum L.) plantations in Brazil. Plant rhizosphere and endophytic beneficial microorganism are well known as plant growth promoters and biocontrol agents. The present study aims to evaluate the potential of different Bacillus strains as biocontrol agent to Fusarium oxysporum f. sp. lycopersici Race 3 strains; and also as plant growth promoting bacteria on Solanum lycopersicum cv Perinha. Different in vitro and greenhouse experiments were carried out to evaluate the direct and indirect bacterial-fungus antagonism, and they inoculation effects on plant traits. In vitro direct, metabolites, and volatile antagonism analysis demonstrated that B. toyonensis BR 10491(FORT 02) presented a broad antagonism to all tested race 3 FOL strains while B. megaterium BR 10466 (FORT 12), B. aryabhattai BR 10494 (FORT 25), B. stratosphericus BR 10438 (FORT 29) and B. cereus BR 10493 (FORT 113.1) strains showed significant antagonistic activity for at least two applied methods. Greenhouse pot experiments demonstrated a significant BCA effect of FORT 113.1 and FORT 02 against FOL Race 3 Fus 1302 strain during different tomato development stages (seedling, vegetative, and reproductive). Bacillus cereus (FORT 113.1) showed significantly higher shoot and height fresh weight, Chlorophyll a and Chlorophyll b content, stomata conductance, water use efficiency, and also a lower xylem infection percentage during vegetative and reproductive stages. Antioxidant enzymatic components analysis demonstrated a synergic effect of Fusarium and Bacillus inoculation, leading to a higher superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activity. In conclusion, the results suggest that strain FORT113.1 could be considered as a good candidate for production of new biofungicide with high potential to augment the existing biocontrol strategies.

The Effects of Gluconacin on Bacterial Tomato Pathogens and Protection against Xanthomonas perforans, the Causal Agent of Bacterial Spot Disease.

As agricultural practices become more sustainable, adopting more sustainable practices will become even more relevant. Searching for alternatives to chemical compounds has been the focus of numerous studies, and bacteriocins are tools with intrinsic biotechnological potential for controlling plant diseases. We continued to explore the biotechnological activity of the bacteriocin Gluconacin from Gluconacetobacter diazotrophicus, PAL5 strain, by investigating this protein's antagonism against important tomato phytopathogens and demonstrating its effectiveness in reducing bacterial spots caused by Xanthomonas perforans. In addition to this pathogen, the bacteriocin Gluconacin demonstrated bactericidal activity in vitro against Ralstonia solanacearum and Pseudomonas syringae pv. tomato, agents that cause bacterial wilt and bacterial spots, respectively. Bacterial spot control tests showed that Gluconacin reduced disease severity by more than 66%, highlighting the biotechnological value of this peptide in ecologically correct formulations.

Stimulatory effects of defective and effective 3-indoleacetic acid-producing bacterial strains on rice in an advanced stage of its vegetative cycle.

The production of 3-indoleacetic acid (IAA) by plant growth-promoting bacteria (PGPR) stimulates root development and plant growth. In addition, morphological changes such as an increased root ramification and root hair production improves nutrient absorption and biomass accumulation. The objective of this work was to evaluate the effect of IAA-producing strains on rice in an advanced stage of its vegetative cycle. Rice was inoculated with Gluconacetobacter diazotrophicus PAL 5 and its lao- mutant, deficient in auxin production, Azospirillum baldaniorum Sp 245, and Escherichia coli DH10b. Both the mutant and wild-type G. diazotrophicus stimulated root elongation, area, volume, and diameter. However, the lao- mutant strain was the only one capable of increasing the number of roots. In turn, inoculation with A. baldaniorum had no significant effect on plant development. The inoculation with E. coli led to changes in root volume, area, and diameter, and a response that may be related to the stress caused by its presence. We conclude that the inoculation with G. diazotrophicus stimulates the root system's growth independently of their IAA production ability, suggesting that a metabolite other than IAA is responsible for this effect at advanced stages of the rice's vegetative cycle.

Herbaspirillum seropedicae strain HRC54 expression profile in response to sugarcane apoplastic fluid.

Bacterial transcriptome profiling in the presence of plant fluids or extracts during microbial growth may provide relevant information on plant-bacteria interactions. Here, RNA sequencing (RNA-Seq) was used to determine the transcriptomic profile of Herbaspirillum seropedicae strain HRC54 at the early stages of response to sugarcane apoplastic fluid. Differentially expressed gene (DEG) analysis was performed using the DESeq2 and edgeR packages, followed by functional annotation using Blast2GO and gene ontology enrichment analysis using the COG and KEGG databases. After 2 h of sugarcane apoplastic fluid addition to the H. seropedicae HRC54 culture, respectively, 44 and 45 genes were upregulated and downregulated. These genes were enriched in bacterial metabolism (e.g., oxidoreductase and transferase), ABC transporters, motility, secretion systems, and signal transduction. RNA-Seq expression profiles of 12 genes identified in data analyses were verified by RT-qPCR. The results suggested that H. seropedicae HRC54 recognized sugarcane apoplastic fluid as the host signal, and some DEGs were closely involved at the early stages of the establishment of plant-bacteria interactions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02848-y.

Endophytic Bacillus Bacteria Living in Sugarcane Plant Tissues and Telchin licus licus Larvae (Drury) (Lepidoptera: Castniidae): The Symbiosis That May Open New Paths in the Biological Control.

Bacteria of the genus Bacillus can colonize endophytically and benefit several crops including the control of some pest orders. In view of the benefits provided by these microorganisms and in order to find out an efficient biotechnological control for the giant borer, our interest in studying the microorganisms in symbiosis with sugarcane and the giant borer has arisen, since there is no efficient chemical or biological control method for this pest. Therefore, endophytic Bacillus strains were isolated from three sugarcane niches (apoplast fluid, central internode cylinder and roots) and also from the giant borer larvae living inside sugarcane varieties grown in the Northeast region of Brazil. The taxonomical characterization (16S rRNA) of 157 Gram-positive isolates showed that 138 strains belonged to the Bacillus genus. The most representative species were phylogenetically closely related to B. megaterium (11.5%) followed by B. safensis (10.8%), B. cereus (8.9%), B. oleronius (8.9%), B. amyloliquefaciens (7.0%), and B. pacificus (6.4%). BOX-PCR analyses showed very distinct band pattern profiles suggesting a great diversity of Bacillus species within the sugarcane niches and the digestive tract, while the B. cereus group remained very closely clustered in the dendrogram. According to XRE biomarker analysis, eleven strains (FORCN005, 007, 008, 011, 012, 014, 067, 076, 092, 093, and 135) correspond to B. thuringiensis species. Additional studies using conserved genes (glp, gmk, pta, and tpi) indicated that most of these strains were phylogenetically closely related to B. thuringiensis and may be considered different subspecies. In conclusion, this study suggests that the culturable Bacillus species are greatly diversified within the plant niches and showed Bacillus species in the digestive tract of the giant borer for the first time. These results open new perspectives to understand the role and functions played by these microorganisms in symbiosis with this pest and also the possibility of developing an efficient biological control method for the giant borer using strains identified as the B. thuringiensis species.

The nematicide Serratia plymuthica M24T3 colonizes Arabidopsis thaliana, stimulates plant growth, and presents plant beneficial potential.

Nine bacterial strains were previously isolated in association with pinewood nematode (PWN) from wilted pine trees. They proved to be nematicidal in vitro, and one of the highest activities, with potential to control PWN, was showed by Serratia sp. M24T3. Its ecology in association with plants remains unclear. This study aimed to evaluate the ability of strain M24T3 to colonize the internal tissues of the model plant Arabidopsis thaliana using confocal microscopy. Plant growth-promoting bacteria (PGPB) functional traits were tested and retrieved in the genome of strain M24T3. In greenhouse conditions, the bacterial effects of all nematicidal strains were also evaluated, co-inoculated or not with Bradyrhizobium sp. 3267, on Vigna unguiculata fitness. Inoculation of strain M24T3 increased the number of A. thaliana lateral roots and the confocal analysis confirmed effective bacterial colonization in the plant. Strain M24T3 showed cellulolytic activity, siderophores production, phosphate and zinc solubilization ability, and indole acetic acid production independent of supplementation with L-tryptophan. In the genome of strain M24T3, genes involved in the interaction with the plants such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinolytic activity, and quorum sensing were also detected. The genomic organization showed ACC deaminase and its leucine-responsive transcriptional regulator, and the activity of ACC deaminase was 594.6 nmol α-ketobutyrate µg protein-1 µl-1. Strain M24T3 in co-inoculation with Bradyrhizobium sp. 3267 promoted the growth of V. unguiculata. In conclusion, this study demonstrated the ability of strain M24T3 to colonize other plants besides pine trees as an endophyte and displays PGPB traits that probably increased plant tolerance to stresses.

Sugarcane apoplast fluid modulates the global transcriptional profile of the diazotrophic bacteria Paraburkholderia tropica strain Ppe8.

The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium.

Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

Validation of reference genes for RT-qPCR analysis in Herbaspirillum seropedicae.

The RT-qPCR technique needs a validated set of reference genes for ensuring the consistency of the results from the gene expression. Expression stabilities for 9 genes from Herbaspirillum seropedicae, strain HRC54, grown with different carbon sources were calculated using geNorm and NormFinder, and the gene rpoA showed the best stability values.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium

Abstract Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75 Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

Determinação da diversidade genética entre acessos de algodão por meio de marcadores microssatélites / Genetic diversity determination among accessions of cotton by microsatellites markers

A utilização de marcadores moleculares microssatélites ou SSR (Simples Sequência Repetitiva) tem se mostrado uma excelente técnica para identificação de cultivares, análise genealógica e de distância genética entre organismos. O conhecimento da diversidade genética entre grupos de genitores por meio da utilização de marcadores moleculares vem permitindo estabelecer diferenças entre acessos de algodão, facilitando a seleção de genitores a serem empregados em programas de melhoramento. O trabalho teve por objetivo avaliar a diversidade genética entre acessos provenientes do Banco de germoplasma de algodão da Empresa de Pesquisa de Minas Gerais (EPAMIG) por meio de marcadores moleculares SSR. Os 37 oligonucleotídeos da série BNL amplificaram um total de 106 alelos. Uma média de 2,86 alelos por locus foi detectada, com uma média de conteúdo informativo de polimorfismo (PIC) de 0,068. A maior taxa de heterozigosidade (0,15) foi obtida para o acesso Dendera e (0,12) para o acesso Giza 80. As menores taxas de heterozigosidade (0,05) foram observadas para os acessos T-7044 e Coker-310. A partir das estimativas de dissimilaridade de Nei a maior distância genética foi obtida entre os acessos Giza 80 e T-7044 (0,88), e a menor distância (0,042) entre os acessos SL 24-82885 e o T-295. O dendrograma gerado apresentou a formação de nove grupos distintos, sendo os acessos Giza 80 e Dendera os mais divergentes entre os todos os estudados.

The use of microsatellite molecular markers or SRS (Simple Repetitive Sequence) has proved to be an excellent technique for cultivar identification, genealogical analysis and genetic distance between organisms. The knowledge of genetic diversity among groups of parents through the use of molecular markers has allowed establishing differences among accessions of cotton, facilitating the selection of parents to be used in breeding programs. The study aimed to assess the genetic diversity among accessions from the germplasm bank Cotton Research Company of Minas Gerais (EPAMIG) using SRS markers. The 37 primers from BNL series amplified a total of 106 alleles. An average of 2.86 alleles per locus was detected with average polymorphism information content (PIC) of 0.068. The highest rate of heterozygosity (0.15) was obtained for access Dendera and (0.12) for Giza 80. The lowest rates of heterozygosity (0.05) were observed for the accessions Coker T-7044-310. From the Nei ´s dissimilarity estimation the largest genetic distance was observed between Giza 80 and T-7044 (0.88), and the shortest distance (0.042) between accessions 24-82885 SL and T-295. The dendrogram generated showed the formation of nine distinct groups, accesses Giza 80 and Dendera were the most divergent among all studied.