Molecular Detection of Ralstonia solanacearum to Facilitate Breeding for Resistance to Bacterial Wilt in Potato.

Potato bacterial wilt is caused by the devastating bacterial pathogen Ralstonia solanacearum. Quantitative resistance to this disease has been and is currently introgressed from a number of wild relatives into cultivated varieties through laborious breeding programs. Here, we present two methods that we have developed to facilitate the screening for resistance to bacterial wilt in potato. The first one uses R. solanacearum reporter strains constitutively expressing the luxCDABE operon or the green fluorescent protein (gfp) to follow pathogen colonization in potato germplasm. Luminescent strains are used for nondestructive live imaging, while fluorescent ones enable precise pathogen visualization inside the plant tissues through confocal microscopy. The second method is a BIO-multiplex-PCR assay that is useful for sensitive and specific detection of viable R. solanacearum (IIB-1) cells in latently infected potato plants. This BIO-multiplex-PCR assay can specifically detect IIB-1 sequevar strains as well as strains belonging to all four R. solanacearum phylotypes and is sensitive enough to detect without DNA extraction ten bacterial cells per mL in complex samples.The described methods allow the detection of latent infections in roots and stems of asymptomatic plants and were shown to be efficient tools to assist potato breeding programs.

An Integrative Approach for the Characterization of Plant-Pathogenic Streptomyces spp. Strains Based on Metabolomic, Bioactivity, and Phylogenetic Analysis.

Actinomycetes are generally recognized as a diverse group of gram-positive, mycelium-forming, soil bacteria that play an important role in mineralization processes and plant health, being Streptomyces the most well-known genus from this group. Although plant pathogenicity is a rare attribute in this genus, some species have significant impact worldwide due to their ability to cause important crop diseases such as potato common scab (CS). In this work, an integrative approach was applied to investigate the pathogenic potential of Streptomyces spp. isolates obtained from a local collection of actinomycetes isolated from potato fields. Secretion of phytotoxic compounds was verified in most pathogenic strains from our collection (27 out of 29), and we followed metabolomic analysis to investigate those phytotoxins. We first evaluated the production of the known phytotoxins thaxtomin A (TXT) and desmethylmensacarcin (DMSN) in phytotoxic Streptomyces spp. by HPLC analysis, resulting in 17 TXT and 6 DMSN producers. In addition, NMR-based metabolomic models were able to classify strains according to their phytotoxicity, and metabolomic data was also used to infer chemotaxonomy within pathogenic species. A correlation between phylogeny and the production of distinct phytotoxins was found, supporting the idea that there are "species specific" metabolites produced by this genus. The recently discovered polyketide DMSN was associated unequivocally with S. niveiscabiei strains and was not produced by other species in the growth conditions employed. Two S. niveiscabiei and two S. puniciscabiei phytotoxic strains that did not produce TXT nor DMSN suggest the production of other kind of metabolites involved in phytotoxicity, and allowed the prioritization of these strains for further chemical studies. Indeed, we found two S. niveiscabiei strains whose supernatants were not phytotoxic in the radish assay, suggesting other pathogenic mechanisms involved. We believe our work will be useful to help understand relations between metabolites and phylogenetic clades within actinomycetes.

Ectopic Spacer Acquisition in Streptococcus thermophilus CRISPR3 Array.

Streptococcus thermophilus relies heavily on two type II-A CRISPR-Cas systems, CRISPR1 and CRISPR3, to resist siphophage infections. One hallmark of these systems is the integration of a new spacer at the 5' end of the CRISPR arrays following phage infection. However, we have previously shown that ectopic acquisition of spacers can occur within the CRISPR1 array. Here, we present evidence of the acquisition of new spacers within the array of CRISPR3 of S. thermophilus. The analysis of randomly selected bacteriophage-insensitive mutants of the strain Uy01 obtained after phage infection, as well as the comparison with other S. thermophilus strains with similar CRISPR3 content, showed that a specific spacer within the array could be responsible for misguiding the adaptation complex. These results also indicate that while the vast majority of new spacers are added at the 5' end of the CRISPR array, ectopic spacer acquisition is a common feature of both CRISPR1 and CRISPR3 systems in S. thermophilus, and it can still provide phage resistance. Ectopic spacer acquisition also appears to have occurred naturally in some strains of Streptococcus pyogenes, suggesting that it is a general phenomenon, at least in type II-A systems.

Minthostachys verticillata essential oil activates macrophage phagocytosis and modulates the innate immune response in a murine model of Enterococcus faecium mastitis.

Minthostachys verticillata essential oil (EO) is a natural product that reports immunomodulatory effects on human T cells as well as anti-inflammatory activity. Bovine mastitis is a worldwide disease, mainly caused by bacteria, affecting milk quality and yield, leading to high economic losses. Environmental pathogens, as Enterococcus faecium, are implicated in the disease. Antibiotic therapy is adequate, although it can leave residues in milk, causing problems in human health. The search of immunomodulatory substances for bovine mastitis treatment is a promising alternative strategy. The aim of this study was to characterize the effect of M. verticillata EO on macrophage phagocytosis and evaluate its immunomodulatory and protective effects in mice challenged with E. faecium. The results showed that EO activated macrophage phagocytosis mechanisms inducing reactive oxygen species production. Moreover, EO modulated the innate immune response in mammary glands of female Balb/c mice challenged with E. faecium decreasing the infiltration of polymorphonuclear neutrophils and IL-1ß and TNF-α mRNA expression. In addition, EO increased the expression of IL-10 in the last hours of infection. Treatment with EO did not increase the number of activated CD4+ or CD8+ T cells or the production of specific antibodies. These results suggest that EO play an important role in helping to resolve the infection in the first hours without activating adaptive immunity. In addition, a marked decrease of the bacterial count in the glands of mice treated with EO was observed. A natural product such as M. verticillata EO could have a potential use to control bovine mastitis.

Isolation and structural characterization of a non-diketopiperazine phytotoxin from a potato pathogenic Streptomyces strain.

Two Streptomyces spp. strains responsible for potato common scab infections in Uruguay which do not produce diketopiperazines were identified through whole-genome sequencing, and the virulence factor produced by one of them was isolated and characterized. Phylogenetic analysis showed that both pathogenic strains can be identified as S. niveiscabiei, and the structure of the phytotoxin was elucidated as that of the polyketide desmethylmensacarcin using MS and NMR methods. The metabolite is produced in yields of ∼200 mg/L of culture media, induces deep necrotic lesions on potato tubers, stuns root and shoot growth in radish seedlings, and is comparatively more aggressive than thaxtomin A. This is the first time that desmethylmensacarcin, a member of a class of compounds known for their antitumor and antibiotic activity, is associated with phytotoxicity. More importantly, it represents the discovery of a new virulence factor related to potato common scab, an economically-important disease affecting potato production worldwide.

Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031.

Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain's virulence.

Efficacy of corn silage inoculants on the fermentation quality under farm conditions and their influence on Aspergillus parasitucus, A. flavus and A. fumigatus determined by q-PCR.

Laboratory-scale silos were prepared to evaluate the efficacy of two different lactic acid bacteria (LAB) on the fermentation quality and mycobiota of corn silage. Their influence on Aspergillus species' variability by using the q-PCR technique was studied. Silage inoculated with Lactobacillus rhamnosus RC007 or L. plantarum RC009 were compared with uninoculated silage. Silos were opened after 1, 7, 45, 90 and 120 days after ensiling. At the end of the ensiling period, silos were left open for 7 days to evaluate aerobic stability. Rapid lactic acid production and decline in pH values were seen in the early stages of fermentation in silage inoculated with L. rhamnosus RC007. After aerobic exposure, a significant decline in lactic acid content was observed in untreated and L. plantarum RC009-inoculated silages. Counts for yeasted and toxigenic fungus remained lower, after aerobic exposure, in L. rhamnosus RC007-inoculated silage, in comparison with L. plantarum RC009 and uninoculated silages. Comparing the influence exerted by both BAL, it was observed that L. rhamnosus RC007 was more efficient at inhibiting the three fungal species tested whose DNA concentrations, determined by q-PCR, oscillated near the initial value (pre-ensiling maize). The ability of L. rhamnosus RC007 to produce lactic acid rapidly and the decline in pH values in the early stages of the fermentation along with the reduction of yeast and mycotoxicogenic fungus after aerobic exposure shows its potential as a bio-control inoculant agent in animal feed.

New type IV pili-related genes involved in early stages of Ralstonia solanacearum potato infection.

This study provides insights into the pathogenesis of Ralstonia solanacearum, in particular with regards to strains belonging to phylotype IIB, sequevar 1 (IIB-1) and their interaction with potato, its natural host. We performed a comparative genomic analysis among IIB-1 R. solanacearum strains with different levels of virulence in order to identify candidate virulence genes. With this approach, we identified a 33.7-kb deletion in a strain showing reduced virulence on potato. This region contains a cluster of six genes putatively involved in type IV pili (Tfp) biogenesis. Functional analysis suggests that these proteins contribute to several Tfp-related functions such as twitching motility and biofilm formation. In addition, this genetic cluster was found to contribute to early bacterial wilt pathogenesis and colonization fitness of potato roots.

A novel, sensitive method to evaluate potato germplasm for bacterial wilt resistance using a luminescent Ralstonia solanacearum reporter strain.

Several breeding programs are under way to introduce resistance to bacterial wilt caused by Ralstonia solanacearum in solanaceous crops. The lack of screening methods allowing easy measurement of pathogen colonization and the inability to detect latent (i.e., symptomless) infections are major limitations when evaluating resistance to this disease in plant germplasm. We describe a new method to study the interaction between R. solanacearum and potato germplasm that overcomes these restrictions. The R. solanacearum UY031 was genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. Colonization of this reporter strain on different potato accessions was followed using life imaging. Bacterial detection in planta by this nondisruptive system correlated with the development of wilting symptoms. In addition, we demonstrated that quantitative detection of the recombinant strain using a luminometer can identify latent infections on symptomless potato plants. We have developed a novel, unsophisticated, and accurate method for high-throughput evaluation of pathogen colonization in plant populations. We applied this method to compare the behavior of potato accessions with contrasting resistance to R. solanacearum. This new system will be especially useful to detect latency in symptomless parental lines before their inclusion in long-term breeding programs for disease resistance.

Biodegradation of agroindustrial wastes by Pleurotus spp for its use as ruminant feed

The increasing expansion of agro-industrial activity has led to the accumulation of a large quantity of lignocellulosic residues all over the world. In particular, large quantities of rice straw (300.000 t) and citric bagasse (50.000 t) are annually produced in Uruguay. In this work we present the study of the bioconversion of these substrates with the edible mushroom Pleurotus spp so as to increase nutritional values and digestibility for its use as animal feed. The SSF process was optimized and the products after different periods of mushroom growth were evaluated. The microbial counts (cfu/g) for the inoculated substrates 44 days after incubation were 15 x 10(4), < 10 and < 10 for aerobic microorganisms, coliforms and E. coli, respectively. After 14 days of SSF the percentage of dry matter, ADF and NDF decreased, and the content of protein increased. These results show that vegetal cell-wall components were degraded during the period of mushroom incubation. PCR - RFLP analysis of the ITS region was used to characterize the Pleurotus species produced in Uruguay and discriminate between DNAs of Pleurotus ostreatus 814 and other fungi from the different substrates.