Multispecies Bacterial Bio-Input: Tracking and Plant-Growth-Promoting Effect on Lettuce var. sagess.

The use of multispecies bacterial bio-inputs is a promising strategy for sustainable crop production over the use of single-species inoculants. Studies of the use of multispecies bio-inputs in horticultural crops are scarce, not only on the growth-promoting effects of each bacterium within the formulation, but also on their compatibility and persistence in the root environment. In this work, we described that a multispecies bacterial bio-input made up of Azospirillum argentinense Az39, Gluconacetobacter diazotrophicus PAL-5, Pseudomonas protegens Pf-5 and Bacillus sp. Dm-B10 improved lettuce plant growth more effectively than when these strains were inoculated as single-species bio-inputs. Bacteria persisted together (were compatible) and also colonized seedling roots of lettuce plants grown in controlled conditions. Interestingly, colonization was highly related to an early and enhanced growth of seedlings grown in the nursery. A similar effect on plant growth was found in lettuce plants in a commercial greenhouse production in the peri-urban area of La Plata City, Buenos Aires, Argentina. To our knowledge, this is the first study demonstrating that a synthetic mixture of bacteria can colonize and persist on lettuce plants, and also showing their synergistic beneficial effect both in the nursery greenhouse as well as the commercial production farm.

Volatile organic compounds profile synthesized and released by endophytes of tomato (Solanum lycopersici L.) and their antagonistic role.

The endophytic microbiome uses mechanisms such as the secretion of diffusible antibiotic molecules, synthesis and release of volatile organic compounds, and/or toxins to protect plants. The aim of this research was to study the volatile organic compounds (VOCs) profile as well as the diffusible secondary metabolites produced and released by endophytic bacteria isolated from tomato plants that in in-vitro assays prevented growth of pathogenic fungi. Bacteria belonging to seven genera (Acinetobacter, Arthrobacter, Bacillus, Microbacterium, Pantoea, Pseudomonas, and Stenotrophomonas) were isolated from different tissues of tomato plants with and without symptoms of Gray leaf spot, a disease provoked by Stemphylium lycopersici. In vitro, antagonistic assays were performed and the effect of volatile and soluble compounds released by endophytic bacteria on the growth of pathogenic fungi was determined. The VOCs synthesized by the endophytes were extracted, identified and quantified. These isolates representatives of seven bacterial genera inhibited the growth of three fungal pathogens of tomato S. lycopersici, Alternaria alternata and Corynespora cassiicola, which was related to the synthesis of soluble compounds as well as VOCs. Endophytes synthesize and release different VOCs, probably due to the different type of interaction that each bacterium establishes with the fungus, presenting a range of fungal growth inhibition.

Bradyrhizobium japonicum, B. elkanii and B. diazoefficiens Interact with Rice (Oryza sativa), Promote Growth and Increase Yield.

Bradyrhizobium is a genus of plant growth-promoting rhizobacteria (PGPR) that have been studied for several decades mainly for the ability to fix diazotrophic nitrogen after having been established endosymbiotically inside root nodules of the legumes of Fabaceae. The aim of this work was to evaluate the capability of Bradyrhizobium to promote the growth of crops belonging to other families, in this case, rice (Oryza sativa), both in laboratory and in field trials. For laboratory test, surface-sterilized rice seeds were soaked with cultures of each strain and planted in pots. Plant length and dry weight were measured after 35 days. For the field test, rice seeds of varieties Yeruá La Plata and Gurí INTA were inoculated with the three best strains observed in the laboratory test and planted in plots. After 60 days of growth, plant length and dry weight were measured. At harvest time, we measured the dry weight of the aerial part, yield and thousand-grain weight. Inoculation with any of the three species described provoked significant increments compared to the uninoculated control at least in one of the parameters measured, both in the laboratory and in the field tests. Bradyrhizobium japonicum E109 was the strain that promoted rice growth the most in the lab while Bradyrhizobium elkanii SEMIA 587 was the strain that promoted rice growth the most in the field, with increments in yield of approximately 1000 kg/ha. Data obtained suggest that the Bradyrhizobium species promoted all rice growth and yield.

Endomycobiome associated with females of the planthopper Delphacodes kuscheli (Hemiptera: Delphacidae): A metabarcoding approach.

A metabarcoding approach was performed aimed at identifying fungi associated with Delphacodes kuscheli (Hemiptera: Delphacidae), the main vector of "Mal de Río Cuarto" disease in Argentina. A total of 91 fungal genera were found, and among them, 24 were previously identified for Delphacidae. The detection of fungi that are frequently associated with the phylloplane or are endophytes, as well as their presence in digestive tracts of other insects, suggest that feeding might be an important mechanism of their horizontal transfer in planthoppers. This study draws the baseline for future research regarding mutualistic associations present in D. kuscheli as well as their physiological role in the life cycle of this important pest that might lead to developing new management strategies to keep insects populations under control.

The endosphere bacteriome of diseased and healthy tomato plants.

Here we analyze the microbial community of healthy and diseased tomato plants to evaluate its impact on plant health. The organisms found in all samples mainly belonged to 4 phyla: Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The Proteobacteria were the highest relative abundant within the endophytic communities of different plant organs of diseased tomato. Among endophytic bacteria of tomato, only a few taxa could be cultured. Here we showed that only a few taxa of bacteria inhabiting tomato plants could be cultured and that all plant organs have a highly diverse endophytic bacterial, whose activity might affect plant growth and development as well as health. The roots seem to be an important barrier for microbes and leaves appear to be the organs with the higher diversity which is incidentally related to plant health. Fruits also contain a complex bacterial community that appeared to be unaffected by foliar diseases such as gray leaf spot at least under the conditions studied.

Fungal Mitogenomes: Relevant Features to Planning Plant Disease Management.

Mitochondrial genomes (mt-genomes) are characterized by a distinct codon usage and their autonomous replication. Mt-genomes encode highly conserved genes (mt-genes), like proteins involved in electron transport and oxidative phosphorylation but they also carry highly variable regions that are in part responsible for their high plasticity. The degree of conservation of their genes is such that they allow the establishment of phylogenetic relationships even across distantly related species. Here, we describe the mechanisms that generate changes along mt-genomes, which play key roles at enlarging the ability of fungi to adapt to changing environments. Within mt-genomes of fungal pathogens, there are dispensable as well as indispensable genes for survival, virulence and/or pathogenicity. We also describe the different complexes or mechanisms targeted by fungicides, thus addressing a relevant issue regarding disease management. Despite the controversial origin and evolution of fungal mt-genomes, the intrinsic mechanisms and molecular biology involved in their evolution will help to understand, at the molecular level, the strategies for fungal disease management.

Biotransformation of grape pomace from Vitis labrusca by Peniophora albobadia LPSC # 285 (Basidiomycota).

Grape pomace from Vitis labrusca is an important sub-product of the "American table wine" industry. It is recalcitrant to degradation, and its accumulation is a serious problem with negative environmental impacts. We analyzed the ability of five white-rot fungi to transform this residue in-vitro. Mass loss and phenol removal in grape pomace treated with each fungus were compared after 30-day solid-state fermentation. Since Peniophora albobadia isolate LPSC 285 was the fungus that showed the highest degradative ability and the lowest free phenol levels in the residue transformed, we selected this fungus to monitor its effect on this residue after 30, 60, and 90 days of incubation. We analyzed mass loss of the residue caused by the fungus activity and its chemical changes using Fourier transform infrared spectroscopy. After 90 days of incubation, Peniophora albobadia isolate LPSC 285 reduced grape pomace mass by 20.48%, which was associated with degradation of polysaccharides and aromatic structures. We concluded that Peniophora albobadia LPSC # 285 isolate is a promising fungus to transform grape pomace from Vitis labrusca under solid-state fermentation conditions.

Fungal transformation and reduction of phytotoxicity of grape pomace waste.

Grape pomace (GP) from Vitis labrusca, the main byproduct from "American table wine" production, is recalcitrant to degradation, and its accumulation is a serious problem with negative environmental impacts. In this work, transformation of grape pomace using a steam pretreatment followed by incubation of GP during a 90-day period with six different fungi were evaluated. Several fungi tested reduced the phytotoxicity of water-soluble fraction (WSFd) from steam-pretreated GP after 90 days' incubation to lettuce and tomato seeds. U. botrytis caused the largest effective phytotoxicity reduction of WSFd (used in the concentration range of 10-1.25% p/v) and was the only fungus causing the removal of monoaromatic compounds. Therefore, this procedure with U. botrytis effectively reduces the availability of phytotoxic monoaromatic compounds in GP, which opens a way for the development of guidelines for the management of these wastes and their potential use as organic amendments in agricultural soil.

Identification of an intermediate for 1,8-dihydroxynaphthalene-melanin synthesis in a race-2 isolate of Fulvia fulva (syn. Cladosporium fulvum).

Fulvia fulva (syn. Cladosporium fulvum, Mycosphaerellaceae) is a dematiaceous fungus that causes tomato leaf mould. It is characterized by its biotrophic lifestyle and the synthesis of the bianthraquinone secondary metabolite, cladofulvin. The aim of the study was to characterize the dark pigment photochemically synthesized by F. fulva and to elucidate its biochemical pathway. We isolated a black pigment from in vitro cultures of the fungus. We determined the pigment to be 1,8-dihydroxynaphthalene (DHN)-melanin based on its chemical and photochemical characteristics, as well as the presence of flaviolin, when fungal reductases were inhibited by tricyclazole. Furthermore, the pks1 gene involved in pigment synthesis has a KS domain already associated with DHN-melanin. Our findings support the relevance of studying melanization in F. fulva.

Draft Genome Sequence of Bacillus thuringiensis Strain m401, Isolated from Honey in Argentina.

We report here the 6,092,003-bp draft genome sequence of Bacillus thuringiensis strain m401, a tetracycline-resistant isolate recovered from honey. The isolate contained three plasmids of 8,307 bp, 9,934 bp, and 69,561 bp and a tetracycline resistance gene with high homology to tet45 in a contig of 236,180 bp.

Draft Genome Sequences of Sporulating (CIDEFI-213) and Nonsporulating (CIDEFI-212) Strains of Stemphylium lycopersici.

Stemphylium lycopersici (Pleosporales) is a pathogenic fungus found on a broad range of plant hosts. It is one of the causal agents of gray leaf spot disease in tomato that causes severe yield reductions and economic losses worldwide. Here, we present the draft genome sequences and the gene annotations of two strains of S. lycopersici, nonsporulating strain CIDEFI-212 and sporulating strain CIDEFI-213.

Melanins from two selected isolates of Pseudocercospora griseola grown in-vitro: Chemical features and redox activity.

Pseudocercospora griseola is the causal agent of Angular Leaf Spot (ALS), a disease of common bean. Due to its coevolution with beans, two major groups have been defined, "Andean" (P. griseola f. griseola) and "Mesoamerican" (P. griseola f. mesoamericana). The aim of this study was to characterize the dark pigment, melanin, synthetized by a selected isolate of each genic group of P. griseola when grown on Potato-dextrose broth. P. griseola f. griseola isolate S3b and P. griseola f. mesoamericana isolate T4 produced 1.7 ±â€¯0.6 and 4.1 ±â€¯0.9 mg of melanin per g of dry biomass, respectively. Although both melanins possessed similar UV-visible absorption spectroscopic pattern, P. griseola f. mesoamericana isolate T4 melanin had a lower UV-visible absorption, higher reducing activity and metal chelating ability than melanin from P. griseola f. griseola isolate S3b. However, when the size of the sample was 10 mg S3b melanin had a higher content of free phenolic groups. Furthermore, cell wall polysaccharides modified in melanin the availability of active phenolic groups, which was dependent on the fungal isolate and the size of the sample. Therefore, the amount and chemical features of melanin as well as its deposition in mycelium walls within isolates is different, which might explain the different pigmentation and physiological behaviours of these representatives of the two groups of P. griseola.

Screening of bacterial endophytes as potential biocontrol agents against soybean diseases.

AIMS: This research was aimed at identifying and characterizing endophytic micro-organisms associated with soybean that have antimicrobial activity towards soybean pathogens. METHODS AND RESULTS: Soybean plants were collected from field trials in four locations of southern Brazil that were cultivated with conventional (C) and transgenic glyphosate-resistant (GR) soybeans. Endophytic bacteria isolated from roots, stems and leaves of soybeans were evaluated for their capacity to inhibit fungal and bacterial plant pathogens and 13 micro-organisms were identified with antagonistic activity. Approximately 230 bacteria were isolated and identified based on the 16S rRNA and rpoN gene sequences. Bacteria isolated from conventional and transgenic soybeans were significantly different not only in population diversity but also in their antagonistic capacity. Thirteen isolates showed in vitro antagonism against Sclerotinia sclerotiorum, Phomopsis sojae and Rhizoctonia solani. Bacillus sp. and Burkholderia sp. were the most effective isolates in controlling bacterial and fungal pathogens in vitro. Extracts and precipitates from culture supernatants of isolates showed different patterns of inhibitory activity on growth of fungal and bacterial pathogens. CONCLUSIONS: Bacillus sp. and Burkholderia sp. were the most effective isolates in controlling fungal pathogens in vitro, and the activity is mainly due to peptides. However, most of the studied bacteria showed the presence of antimicrobial compounds in the culture supernatant, either peptides, bacteriocins or secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be significant to develop tools for the biological control of soybean diseases. The work brought to the identification of micro-organisms such as Bacillus sp. and Burkholderia sp. that have the potential to protect crops in order to enhance a sustainable management system of crops. Furthermore, the study provides the first evidences of the influence of management as well as the genetics of glyphosate-resistant soybean on the diversity of bacterial endophytes of soybean phytobiome.

Nodulation and Delayed Nodule Senescence: Strategies of Two Bradyrhizobium Japonicum Isolates with High Capacity to Fix Nitrogen.

The purpose of this work was to study further two Bradyrhizobium japonicum strains with high nitrogen-fixing capacity that were identified within a collection of approximately 200 isolates from the soils of Argentina. Nodulation and nitrogen-fixing capacity and the level of expression of regulatory as well as structural genes of nitrogen fixation and the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene of the isolates were compared with that of E109-inoculated plants. Both isolates of B. japonicum, 163 and 366, were highly efficient to fix nitrogen compared to commercial strain E109. Isolate 366 developed a higher number and larger biomass of nodules and because of this fixed more nitrogen. Isolate 163 developed the same number and nodule biomass than E109. However, nodules developed by isolate 163 had red interiors for a longer period, had a higher leghemoglobin content, and presented high levels of expression of acdS gene, that codes for an ACC deaminase. In conclusion, naturalized rhizobia of the soils of Argentina hold a diverse population that might be the source of highly active nitrogen-fixing rhizobia, a process that appears to be based on different strategies.

Fusarium equiseti LPSC 1166 and its in vitro role in the decay of Heterostachys ritteriana leaf litter.

The role of microorganisms in litter degradation in arid and semi-arid zones, where soil and water salinization is one of the main factors limiting carbon turnover and decay, remains obscure. Heterostachys ritteriana (Amaranthaceae), a halophyte shrub growing in arid environments such as "Salinas Grandes" (Córdoba, Argentina), appears to be the main source of organic matter in the area. Little is known regarding the microorganisms associated with H. ritteriana, although they are a potential source of enzymes such as cellulolytic ones, which might be important in biotechnological fields such as bioethanol production using ionic liquids. In the present study, by studying the microbiota growing on H. ritteriana leaf litter in "Salinas Grandes," we isolated the cellulolytic fungus Fusarium equiseti LPSC 1166, which grew and degraded leaf litter under salt stress. The growth of this fungus was a function of the C substrate and the presence of NaCl. Although in vitro the fungus used both soluble and polymeric compounds from H. ritteriana litter and synthesized extracellular ß-1,4 endoglucanases, its activity was reduced by 10% NaCl. Based on these results, F. equiseti LPSC 1166 can be described as a halotolerant cellulolytic fungus most probably playing a key role in the decay of H. ritteriana leaf litter in "Salinas Grandes."

The mitochondrial genome of the plant-pathogenic fungus Stemphylium lycopersici uncovers a dynamic structure due to repetitive and mobile elements.

Stemphylium lycopersici (Pleosporales) is a plant-pathogenic fungus that has been associated with a broad range of plant-hosts worldwide. It is one of the causative agents of gray leaf spot disease in tomato and pepper. The aim of this work was to characterize the mitochondrial genome of S. lycopersici CIDEFI-216, to use it to trace taxonomic relationships with other fungal taxa and to get insights into the evolutionary history of this phytopathogen. The complete mitochondrial genome was assembled into a circular double-stranded DNA molecule of 75,911 bp that harbors a set of 37 protein-coding genes, 2 rRNA genes (rns and rnl) and 28 tRNA genes, which are transcribed from both sense and antisense strands. Remarkably, its gene repertoire lacks both atp8 and atp9, contains a free-standing gene for the ribosomal protein S3 (rps3) and includes 13 genes with homing endonuclease domains that are mostly located within its 15 group I introns. Strikingly, subunits 1 and 2 of cytochrome oxidase are encoded by a single continuous open reading frame (ORF). A comparative mitogenomic analysis revealed the large extent of structural rearrangements among representatives of Pleosporales, showing the plasticity of their mitochondrial genomes. Finally, an exhaustive phylogenetic analysis of the subphylum Pezizomycotina based on mitochondrial data reconstructed their relationships in concordance with several studies based on nuclear data. This is the first report of a mitochondrial genome belonging to a representative of the family Pleosporaceae.

Bacteriomes of the corn leafhopper, Dalbulus maidis (DeLong & Wolcott, 1923) (Insecta, Hemiptera, Cicadellidae: Deltocephalinae) harbor Sulcia symbiont: molecular characterization, ultrastructure, and transovarial transmission.

In this study, we surveyed the bacteriome-associated microbiota of the corn leafhopper Dalbulus maidis by means of histological, ultrastructural, and molecular analyses. Amplification and sequencing of 16S rDNA genes revealed that the endosymbiont "Candidatus Sulcia muelleri" (Phylum Bacteroidetes) resides in bacteriomes of D. maidis. Phylogenetic analysis showed that the sequence was closely allied to others found in representatives of the subfamily Deltocephalinae. We failed to amplify other sequences as "Candidatus Nasuia deltocephalinicola," a co-primary symbiont frequently associated to deltocephaline leafhoppers. In addition, a metagenetic analysis carried out in order to investigate the presence of other bacteriome-associated bacteria of D. maidis showed that the sequence of Sulcia accounted for 98.56 % of all the sequences. Histological and ultrastructural observations showed that microorganisms harbored in bacteriomes (central syncytium and cytoplasm of uninucleate bacteriocytes) look like others Sulcia described in hemipteran species and they were transovarially transmitted from mother to offspring which is typical of obligate endosymbionts. The only presence of Sulcia in the bacteriomes of D. maidis was discussed.

Antagonism of entomopathogenic fungi by Bacillus spp. associated with the integument of cicadellids and delphacids.

Entomopathogenic fungi are potential tools to biocontrol cicadellids and delphacids, two groups of insects that cause extensive damage to agricultural crops. However, bacteria living on the host cuticle may inhibit fungal growth. In the present work, following the molecular characterization of 10 strains of Bacillus isolated from the integument of cicadellids and delphacids, we selected isolates of the fungi Beauveria bassiana and Metarhizium anisopliae that are resistant to the antimicrobials secreted by these bacterial strains. The antagonistic activity of the 10 bacterial isolates belonging to the genus Bacillus (i.e., B. amyloliquefaciens, B. pumilus, and B. subtilis) against 41 isolates of Bea. bassiana and 20 isolates of M. anisopliae was investigated in vitro on tryptic soy agar using the central disk test. With this approach, isolates of Bea. bassiana and M. anisopliae resistant to antagonistic bacteria were identified that can be further developed as biological control agents.

Draft Genome Sequence and Gene Annotation of Stemphylium lycopersici Strain CIDEFI-216.

Stemphylium lycopersici is a plant-pathogenic fungus that is widely distributed throughout the world. In tomatoes, it is one of the etiological agents of gray leaf spot disease. Here, we report the first draft genome sequence of S. lycopersici, including its gene structure and functional annotation.

A Survey on Occurrence of Cladosporium fulvum Identifies Race 0 and Race 2 in Tomato-Growing Areas of Argentina.

The presence of Cladosporium fulvum (syn. Passalora fulva), causal agent of tomato leaf mold, was confirmed in the two main greenhouse-production areas for tomato in Argentina. Using both morphological characters and internal transcribed spacer sequencing, we confirmed the presence of physiological races of this pathogen. A diagnostic multiplex polymerase chain reaction (PCR) was also developed, using primers derived from C. fulvum avirulence (Avr) genes. In all, 20 isolates of Cladosporium spp. were obtained as monospore cultures and 12 were identified as C. fulvum. By this method, we showed that, of these 12 isolates, 5 were race 0 (carrying functional Avr2, Avr4, Avr4E, and Avr9 genes) and 7 were race 2 (lacking the Avr2 gene). Race identity was confirmed by testing their virulence on a set of tomato differentials carrying different Cf resistance genes. All Avr genes could be amplified in single or multiplex PCR using DNA isolated from in vitro grown monospore cultures but only three Avr could be amplified when genomic DNA was isolated from C. fulvum-infected necrotic leaf tissue.