Federico Battistoni

Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp.

The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifHi::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1-carboxylate oxidase synthase, peroxidase precursor, PR10-a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important.

New betaproteobacterial Rhizobium strains able to efficiently nodulate Parapiptadenia rigida (Benth.) Brenan.

ABSTRACT Among the leguminous trees native to Uruguay, Parapiptadenia rigida (Angico), a Mimosoideae legume, is one of the most promising species for agroforestry. Like many other legumes, it is able to establish symbiotic associations with rhizobia and belongs to the group known as nitrogen-fixing trees, which are major components of agroforestry systems. Information about rhizobial symbionts for this genus is scarce, and thus, the aim of this work was to identify and characterize rhizobia associated with P. rigida. A collection of Angico-nodulating isolates was obtained, and 47 isolates were selected for genetic studies. According to enterobacterial repetitive intergenic consensus PCR patterns and restriction fragment length polymorphism analysis of their nifH and 16S rRNA genes, the isolates could be grouped into seven genotypes, including the genera Burkholderia, Cupriavidus, and Rhizobium, among which the Burkholderia genotypes were the predominant group. Phylogenetic studies of nifH, nodA, and nodC sequences from the Burkholderia and the Cupriavidus isolates indicated a close relationship of these genes with those from betaproteobacterial rhizobia (beta-rhizobia) rather than from alphaproteobacterial rhizobia (alpha-rhizobia). In addition, nodulation assays with representative isolates showed that while the Cupriavidus isolates were able to effectively nodulate Mimosa pudica, the Burkholderia isolates produced white and ineffective nodules on this host.

Exploring the Function of Alcohol Dehydrogenases During the Endophytic Life of Azoarcus Sp. Strain BH72

The endophytic bacterium Azoarcus sp. strain BH72 is capable of colonizing the interior of rice roots, where it finds suitable physicochemical properties for multiplying and fixing nitrogen. Because these properties are poorly understood, a microtiter-plate-based screening of a transcriptional gfp (green fluorescent protein) fusion library of Azoarcus sp. grown under different conditions was performed. Monitoring of the GFP activity allowed the identification of a gene highly expressed in medium supplemented with ethanol. Sequence analysis revealed that this gene encodes a pyrrolo-quinoline quinone-dependent alcohol dehydrogenase (ADH). Inspection of the complete genome sequence of the Azoarcus sp. strain BH72 identified seven additional genes encoding putative ADH, indicating that BH72 is well equipped to survive in different environmental conditions offering various alcohols as carbon source. Analyses of these eight putative ADH showed that expression of three was induced by ethanol, of which two were also expressed inside rice roots. The fact that waterlogged plants such as rice accumulate ethanol suggests that ethanol occurs in sufficiently high concentration within the root to induce expression of bacterial ADH. Disruption of these two ADH evoked a reduced competitiveness to the wild type in colonizing rice roots internally. Thus, it is likely that ethanol is an important carbon source for the endophytic life of Azoarcus sp.

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.

The oil-contaminated soil diazotroph Azoarcus olearius DQS-4T is genetically and phenotypically similar to the model grass endophyte Azoarcus sp. BH72

The genome of Azoarcus olearius DQS-4T , a N2 -fixing Betaproteobacterium isolated from oil-contaminated soil in Taiwan, was sequenced and compared with other Azoarcus strains. The genome sequence showed high synteny with Azoarcus sp. BH72, a model endophytic diazotroph, but low synteny with five non-plant-associated strains (Azoarcus CIB, Azoarcus EBN1, Azoarcus KH32C, A. toluclasticus MF63T and Azoarcus PA01). Average Nucleotide Identity (ANI) revealed that DQS-4T shares 98.98% identity with Azoarcus BH72, which should now be included in the species A. olearius. The genome of DQS-4T contained several genes related to plant colonization and plant growth promotion, such as nitrogen fixation, plant adhesion and root surface colonization. In accordance with the presence of these genes, DQS-4T colonized rice (Oryza sativa) and Setaria viridis, where it was observed within the intercellular spaces and aerenchyma mainly of the roots. Although they promote the growth of grasses, the mechanism(s) of plant growth promotion by A. olearius strains is unknown, as the genomes of DQS-4T and BH72 do not contain genes for indole acetic acid (IAA) synthesis nor phosphate solubilization. In spite of its original source, both the genome and behaviour of DQS-4T suggest that it has the capacity to be an endophytic, nitrogen-fixing plant growth-promoting bacterium.

Endophytic colonization of sugarcane (Saccharum officinarum) by the novel diazotrophs Shinella sp. UYSO24 and Enterobacter sp. UYSO10

AimsSugarcane is a multipurpose crop mostly used in Uruguay for bioethanol production. It requires high amounts of N fertilization for optimal growth, which causes environmental degradation and high production costs. Previously, a bacterial collection associated with surface-sterilized stems of sugarcane was characterized for in vitro plant growth-promoting (PGP) traits. The aims of this study were (1) to determine if selected isolates from the collection are sugarcane growth promoters and (2) to determine if they are true endophytes of sugarcane.MethodsPlant growth promotion assays were used to study the effects of selected isolates on sugarcane plantlets. Light microscopy, transmission electron, and scanning electron microscopy (TEM, SEM) were employed to describe the structure of the interaction between the plant growth-promoting bacteria and the plants. qPCR was used to quantify the bacteria residing in the inner plant tissues.ResultsEnterobacter sp. UYSO10 and Shinella sp. UYSO24 were confirmed to have a PGP effect on the commercial sugarcane cv. LCP 85384. Both strains were defined as true endophytes of sugarcane plants with this being the first case for a strain in the genus Shinella in grasses.ConclusionsThese data will contribute to the final development of a sugarcane PGP inoculant based on endophytic plant growth-promoting bacteria.

Identification and characterization of the part of the bacterial community associated with field-grown tall fescue ( Festuca arundinacea ) cv. SFRO Don Tomás in Uruguay

The aims of this study were to isolate, characterize and identify the native culturable putative endophytic bacterial community associated with tall fescue (Festuca arundinacea) cv. SFRO Don Tomás, cultivated in Uruguay, and to study the effects of inoculation on cv. SFRO Don Tomás and the commercial cv. Tacuabé. A total of 342 isolates were collected from surface-sterilized roots, stems and seeds of healthy cv. SFRO Don Tomás. The functional ability of the isolates to produce indole, to solubilize minerals (P, Fe, K) and to biologically fix molecular nitrogen (N2) was determined. Several infection traits, such as the ability to produce proteases, peroxidases, cellulases and hemicellulases, were identified in the isolates. Selected bacterial isolates were identified by 16S rRNA sequencing and shown to belong to a broad spectrum of genera, including Bacillus, Microbacterium, Curtobacterium, Streptomyces, Acidovorax, Variovorax, Acinetobacter, Pseudomonas, Pantoea, Rhanella and Xanthomonas. Plant growth promotion assays shown that ten isolates were able to promote the growth of cv. SFRO Don Tomás under gnotobiotic conditions, thereby highlighting the potential of these isolates in biotechnological applications as inoculant for this cultivar which is highly adapted to dry and cold seasons.