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Reverse transcription-quantitative PCR (RT-qPCR) is an analytical tool for gene expression quantification. Reference genes are not yet available for gene expression analysis during interactions of Ralstonia solanacearum with ‘Hawaii 7996’ (the most stable source of resistance in tomato). Here, we carried out a multi-algorithm stability analysis of eight candidate reference genes during interactions of ‘Hawaii 7996’ with one incompatible/avirulent and two compatible/virulent (= resistance-breaking) bacterial isolates. Samples were taken at 24- and 96-h post-inoculation (HPI). Analyses were performed using the ∆∆Ct method and expression stability was estimated using BestKeeper, NormFinder, and geNorm algorithms. TIP41 and EF1α (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and TIP41 (with BestKeeper), were the best combinations for mRNA normalization in incompatible interactions at 24 HPI and 96 HPI. The most stable genes in global compatible and incompatible interactions at 24 HPI and 96 HPI were PDS and TIP41 (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and PDS / EXP (with BestKeeper). Global analyses on the basis of the three algorithms across 20 R. solanacearum -tomato experimental conditions identified UBI3, TIP41 and ACT as the best choices as reference tomato genes in this important pathosystem.

The pathovar of causes bacterial canker of grapevine. This disease was first recorded in India in 1972, and later in Brazil in 1998, where its distribution is currently restricted to the northeastern region. A multilocus sequence analysis (MLSA) based on seven housekeeping genes and a multilocus variable number of tandem repeat analysis (MLVA) with eight loci were performed in order to assess the genetic relatedness among strains from India and Brazil. Strains isolated in India from three related pathovars affecting Vitaceae species and pathogenic strains isolated from sp. found in bacterial canker-infected vineyards in Brazil were also included. MLSA revealed lack of diversity in all seven genes and grouped grapevine and Amaranthus strains in a monophyletic group in . The VNTR (variable number of tandem repeat) typing scheme conducted on 107 strains detected 101 haplotypes. The total number of alleles per locus ranged from 5 to 12. A minimum spanning tree (MST) showed that Brazilian strains were clearly separated from Indian strains, which showed unique alleles at three loci. The two strains isolated from symptomatic sp. presented unique alleles at two loci. STRUCTURE analyses revealed three groups congruent with MST and a fourth group with strains from India and Brazil. Admixture among populations were observed in all groups. MST, STRUCTURE and e-BURST analyses showed that the strains collected in 1998 belong to two distinct groups, with predicted founder genotypes from two different vineyards in the same region. This suggest that one introduction of grape planting materials contaminated with genetically distinct strains took place, which was followed by pathogen adaptation. Genome sequencing of one Brazilian strain confirmed typical attributes of pathogenic xanthomonads and allowed the design of a complementary VNTR typing scheme dedicated to pv. that will allow further epidemiological survey of this genetically monomorphic pathovar.

Bacterial fruit blotch, caused by Acidovorax citrulli , is a serious threat to the watermelon crop in Brazil. To date, there are no disease-resistant varieties, thus requiring research seeking sources of resistance. To select genotypes with potential use in the management of fruit blotch, the resistance level of watermelon genotypes belonging to the Cucurbits Germplasm Active Bank for the Brazilian Northeast (Banco Ativo de Germoplasma de Cucurbitáceas para o Nordeste Brasileiro—BAG) of Embrapa Semiárido was evaluated at different plant developmental stages: seeds (74 genotypes), seedlings and plants before flowering (29 genotypes) as well as plants during flowering and fruiting (seven genotypes). The genotypes were evaluated for the incidence or severity of the disease, which was estimated with the aid of descriptive scales. Additionally, A. citrulli transmission was determined in seeds derived from symptomatic and asymptomatic fruits. No watermelon genotype was immune to fruit blotch, and the majority showed variations in resistance responses. However, the genotypes BGCIA 979, BGCIA 34 and Sugar Baby showed high levels of resistance at most stages of plant development, thereby suggesting that these genotypes possess fruit blotch resistance genes that could be used in breeding programs. Seeds from symptomatic and asymptomatic fruits of the seven tested genotypes showed transmission rates of A. citrulli up to 35.3 % and 8.7 %, respectively. These results confirm that asymptomatic fruits can harbor contaminated seeds that are responsible for the transmission of the bacteria.

The phylogenetic relationships and distribution of Ralstonia pseudosolanacearum and Ralstonia solanacearum sequevars were analyzed in 108 strains of Solanaceae in three mesoregions of the Pernambuco State, Brazil. These mesoregions are characterized by a semiarid climate and account for 88.20% of the area of the state. Ralstonia pseudosolanacearum (phylotype I, biovar 3) was predominant in Agreste, and R. solanacearum (phylotype II, biovar 1) was predominant in São Francisco and Sertão. Two haplotypes of R. pseudosolanacearum were identified by analysis of the haplotype network of the partial egl gene sequences and were phylogenetically related to sequevars I-17 and I-18. Five haplotypes of R. solanacearum were identified; two haplotypes were related to sequevar IIA-50, and three haplotypes were related to new sequevars IIA-61, IIA-62, and IIA-63. In Brazil, sequevars I-17, IIA-61, IIA-62, and IIA-63 were detected exclusively in the semiarid climate of Pernambuco, unlike sequevars I-18 and IIA-50, which were detected in other regions of Brazil. Furthermore, R. pseudosolanacearum (phylotype I) and sequevar IIA-50 were detected in the Sertão mesoregion. Sequevar I-18, IIA-61, and IIA-62 strains were prevalent in the Agreste mesoregion, and sequevars I-17 and IIA-50 were prevalent in the São Francisco mesoregion. The virulence variability of the strains toward tomato was related to species and sequevars. Introduced R. pseudosolanacearum strains of sequevar I-18 had higher estimated virulence compared to that of the native R. solanacearum strains of sequevar IIA-50. This is the first study that analyzes the variability of Ralstonia spp. in the semiarid region of Brazil.

Plant pathogenic Xanthomonas species attack a wide range of agriculture crops and is capable of surviving on weeds. In this study, Xanthomonas strains obtained from weeds grown withing vegetable crop fields in the state of Pernambuco, Northeast Brazil, were identified. Bacterial colonies resembling Xanthomonas were obtained from Aeollanthus suaveolens (strain CCRMXe03), Amaranthus lividus (CCRMXe01 and CCRMXe02), Sida glomerata (CCRMXe04), and Emilia fosbergii (CCRMXe04). All weeds but E. fosbergii exhibited lesions on the leaves. Phylogenetic analysis of the gyrB and rpoD genes and PCR-specific assays allowed to identify X. euvesicatoria pv. euvesicatoria (CCRMXe01, CCRMXe02, CCRMXe03, and CCRMXe04) and X. campestris pv. campestris (CCRMXcc371). All strains but CCRMXe03 were pathogenic to its host of origin. All X. euvesicatoria pv. euvesicatoria strains were pathogenic to leaves and fruits of tomato and bell pepper. Two strains, CCRMXe03 and CCRMXe04, were capable of inducing soft rot in fruits. Amylolytic activity was found in all strains and two strains (CCRMXe03 and CCRMXe04) degraded pectate. Strain CCRMXcc371 was pathogenic to cabbage, kale, cauliflower, and broccoli. This work provides new knowledge of hosts for two important plant pathogenic bacteria for vegetable crops.

The etiology of onion scale rot caused by bacteria in the semi-arid region of northeastern Brazil is unclear, which complicates disease control. Forty-five bacterial strains collected from the main onion producing regions in the states of Pernambuco and Bahia were identified by sequencing and phylogenetic analysis of the 16S rRNA gene and were characterized by rep-PCR and pathological behaviour on onion. The strains were grouped into three clades: 29 strains in clade I formed by the Burkholderia cepacia complex, 10 strains in clade II formed by Burkholderia gladioli, and six strains in clade III formed by Pseudomonas aeruginosa. Rep-PCR analysis grouped the strains in 31 clusters at 70% similarity. However, it was not possible to identify the three clades by rep-PCR analysis. Inoculation of wounded onion scales showed that the strains from the B. cepacia complex and B. gladioli were more aggressive on onion scales than those from P. aeruginosa. Based on the phylogenetic identification performed in this study, we conclude that scale rot of onion bulb in the semi-arid region of northeastern Brazil is caused by bacteria from the B. cepacia complex, B. gladioli, and P. aeruginosa. In addition, more than one species of B. cepacia complex may be associated with the disease in this region.

Biofilms are dense surface-associated communities formed by microorganisms. Formation of these structures by the plant pathogenic bacterium Xanthomonas campestris pv. viticola (bacterial canker of grapevine) had not previously been studied. The ability of seven strains of this bacterium to adhere to abiotic surfaces and to form biofilms in vitro was evaluated under different conditions. The surfaces tested were polystyrene and glass using microtiter plates and tubes, respectively. Four liquid culture media were used, nutrient-dextrose-yeast extract (NYD), yeast extract-dextrose-calcium carbonate (YDC), Kado 523 (KADO) and Luria-Bertani (LB). The biofilm architecture was examined by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Seven strains adhered to polystyrene in the microtiter plates and formed biofilms in all culture media at weak, moderate, and strong levels. In glass tubes, only strains Xcv229 and Xcv158 formed biofilms. SEM of Xcv229 and Xcv158 revealed typical biofilm architectures. CLSM showed that only Xcv229 formed an initial matrix structure characteristic of biofilms. The X. campestris pv. viticola strains exhibited different levels of biofilm formation in different culture media, of which LB and KADO were the best. Therefore, bacterial growth in polystyrene microtiter plates using LB and KADO media is a good qualitative method for the detection of biofilms of this pathogen.

ABSTRACT We report here the complete genome sequences of two Ralstonia pseudosolanacearum strains, isolated from the warm northeast region of Brazil. They display divergent (compatible versus incompatible) interactions with the resistant tomato line Hawaii 7996. Polymorphisms were detected in a subset of effector genes that might be associated with these contrasting phenotypes.

Background Finding the best extraction method of proteins from lysed cells is the key step for detection and identification in all proteomics applications. These are important to complement the knowledge about the mechanisms of interaction between plants and phytopathogens causing major economic losses. To develop an optimized extraction protocol, strains of Acidovorax citrulli , Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum were used as representative cells in the study of phytopathogenic bacteria. This study aims to compare four different protein extraction methods, including: Trizol, Phenol, Centrifugation and Lysis in order to determine which are more suitable for proteomic studies using as parameters the quantity and quality of extracted proteins observed in two-dimensional gels. Results The bacteria studied showed different results among the tested methods. The Lysis method was more efficient for P. carotovorum subsp. carotovorum and R. solanacearum phytobacteria, as well as simple and fast, while for A. citrulli , the Centrifugation method was the best. This evaluation is based on results obtained in polyacrylamide gels that presented a greater abundance of spots and clearer and more consistent strips as detected by two-dimensional gels. Conclusions These results attest to the adequacy of these proteins extraction methods for proteomic studies.

ABSTRACTHere, we report the complete 5.3-Mb genome sequence of Xanthomonas campestris pv. viticola (CCRMXCV 80), which causes grapevine (Vitis vinifera L.) bacterial canker. Genome data will improve our understanding of the strain’s comparative genomics and epidemiology, and help to further define plant protection and quarantine procedures.