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.

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.

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.

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."

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.

Coriolopsis rigida, a potential model of white-rot fungi that produce extracellular laccases.

In the last two decades, a significant amount of work aimed at studying the ability of the white-rot fungus Coriolopsis rigida strain LPSC no. 232 to degrade lignin, sterols, as well as several hazardous pollutants like dyes and aliphatic and aromatic fractions of crude oil, including polycyclic aromatic hydrocarbons, has been performed. Additionally, C. rigida in association with arbuscular mycorrhizal fungi appears to enhance plant growth, albeit the physiological and molecular bases of this effect remain to be elucidated. C. rigida's ability to degrade lignin and lignin-related compounds and the capacity to transform the aromatic fraction of crude oil in the soil might be partially ascribed to its ligninolytic enzyme system. Two extracellular laccases are the only enzymatic components of its lignin-degrading system. We reviewed the most relevant findings regarding the activity and role of C. rigida LPSC no. 232 and its laccases and discussed the work that remains to be done in order to assess, more precisely, the potential use of this fungus and its extracellular enzymes as a model in several applied processes.

Morphological and molecular characterization of a fungus, Hirsutella sp., isolated from planthoppers and psocids in Argentina.

A mycosed planthopper, Oliarus dimidiatus Berg (Hemiptera: Cixiidae), and two psocids, Heterocaecilius sp. (Psocodea: Pseudocaeciliidae) and Ectopsocus sp. (Ectopsocidae), were collected from Los Hornos and La Plata, Buenos Aires, Argentina between February and September 2007. Observations of mycelia growing on the host revealed that the putative fungal parasite had synnemata supporting monophialidic conidiogenous cells. Likewise, in vitro fungal cultures presented characteristics typical of the fungus Hirsutella citriformis Speare (Ascomycota: Hypocreales: Clavicipitaceae). The identity of the isolated fungi characterized based on morphological aspects was complemented by means of the internal transcribed spacer sequences. The sequences of both isolates were highly homologous to those of Cordyceps sp. (Fries) Link and Ophiocordyceps sinensis (Berkely) G.H. Sung, J.M. Sung, Hywel-Jones, and Spatafora (Ophiocordycipitaceae). We additionally confirmed that both isolates had the ability to infect and kill adults of Delphacodes kuscheli Fennah (Hemiptera: Delphacidae) after 10 days. Therefore, based on the morphology of the isolated fungi, their ribosomal internal transcribed spacer sequence, and their ability to parasite insects, we conclude that the fungi isolated belong to the genus Hirsutella and might have biotechnological potential.

Cladosporium cladosporioides LPSC 1088 produces the 1,8-dihydroxynaphthalene-melanin-like compound and carries a putative pks gene.

Cladosporium cladosporioides is a dematiaceous fungus with coloured mycelia and conidia due to the presence of dark pigments. The purpose of this study was to characterize the dark pigments synthetized by Cladosporium sp. LPSC no. 1088 and also to identify the putative polyketide synthase (pks) gene that might be involved in the pigment biosynthesis. Morphological as well as molecular features like the ITS sequence confirmed that LPSC 1088 is Cladosporium cladosporioides. UV-visible, Fourier Transform Infrared (FTIR) and Electron Spin Resonance (ESR) spectroscopy analysis as well as melanin inhibitors suggest that the main dark pigment of the isolate was 1,8 dihydroxynaphthalene (DHN)-melanin-type compound. Two commercial fungicides, Difenoconazole and Chlorothalonil, inhibited fungal growth as well as increased pigmentation of the colonies suggesting that melanin might protect the fungus against chemical stress. The pigment is most probably synthetized by means of a pentaketide pathway since the sequence of a 651 bp fragment, coding for a putative polyketide synthase, is highly homologous to pks sequences from other fungi.

Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua / Methods for the detection of Agrobacterium from plant, soil and water samples

El género Agrobacterium incluye especies ftopatógenas que inducen la formación de agallas en el cuello o la proliferación de raíces en cabellera en más de 600 especies de dicotiledóneas, y especies no patógenas cuyo hábitat natural es el suelo. Como no es posible erradicar a las especies patógenas y habida cuenta de que más del 80 % de las infecciones puede provenir de viveros, es importante evitar la diseminación de la enfermedad. Por ello, el objetivo de este trabajo ha sido desarrollar técnicas sensibles y precisas que, aisladamente o combinadas, permitan detectar la presencia de especies y biovares de Agrobacterium a partir de muestras de material vegetal, suelo y agua. Se comprobó que con la estrategia combinada de realizar aislamientos en los medios semiselectivos D1, D1-M y YEM-RCT; PCR multiplex sobre el gen 23S ADNr; PCR específca sobre los genes virC1 y virC2 y bioensayos en plántulas de pimiento cv. California Wonder y en hojas cortadas de kalanchoe, se reduce la posibilidad de obtener falsos negativos y/o falsos positivos. Por lo expuesto, esta combinación de técnicas constituye una herramienta adecuada para el diagnóstico de cepas patógenas de Agrobacterium a partir de distintos tipos de muestras.

The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly diffcult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identifcation techniques by using a set of semi-selective and differential culture media in combination with a specifc PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specifc PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes.

[Methods for the detection of Agrobacterium from plant, soil and water samples]. / Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua.

The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly difficult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identification techniques by using a set of semi-selective and differential culture media in combination with a specific PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specific PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes.

Differential regulation of laccase gene expression in Coriolopsis rigida LPSC No. 232.

Two laccase isoenzyme genes (lcc2 and lcc3) from the white-rot fungus Coriolopsis rigida were cloned, and together with the previously described lcc1, their transcript levels were analysed by Quantitative RT-PCR in order to study their expression patterns under a range of putative inducers (Cu(2+), Mn(2+), Fe(3+), 2,6-dimethoxy-1,4-benzoquinone, H(2)O(2,) caffeine, amphotericin B and syringic acid). The highest induction was observed for lcc1 in presence of copper, and thus, a kinetic study was performed to analyze its effect on temporary lcc1 gene expression. Our results showed that upregulation due to copper was linked to growth stage, being highest during the trophophase and decreasing during the idiophase. Amphotericin B increased levels of transcripts of lcc1 and lcc2, syringic acid upregulated lcc1 and lcc3 and 2,6-dimethoxy-1,4-benzoquinone induced lcc2 and lcc3. Possible reasons for why laccase genes from C. rigida are differentially regulated at the transcriptional level are discussed.

Pseudocercospora griseola causing angular leaf spot on Phaseolus vulgaris produces 1,8-dihydroxynaphthalene-melanin.

Pseudocercospora griseola is the causal agent of angular leaf spot of common bean (ALS). It has undergone parallel coevolution with its host and two major groups have been defined, "Andean" (P. griseola f. griseola) and "Mesoamerican" (P. griseola f. mesoamericana). The aim of this study was to analyze the nature and the level of the dark pigment synthesized by the representatives of each group. After 21 days of incubation on potato dextrose agar medium, P. griseola f. griseola isolate S3b developed colonies with diameters of 17.5 +/- 1.3 mm and concentric rings of pigmentation. Isolate T4 of P. griseola f. mesoamericana presented smaller colonies (9.9 +/- 0.3 mm) with a uniform dark-gray color. Both isolates, S3b and T4, produced the same pigment, a 1,8-dihydroxynaphthalene-melanin, although different in quantity and structural features as suggested by the IR spectrum. The P. griseola f. griseola isolate S3b had a higher growth rate and melanin content as well as smaller sensitivity to melanin synthesis inhibitors compared to the isolate T4 of P. griseola f. mesoamericana. These results suggest a possible link between melanin and growth in P. griseola.