Elineide Barbosa de Souza

Moko Disease-Causing Strains of Ralstonia solanacearum from Brazil Extend Known Diversity in Paraphyletic Phylotype II

The epidemic situation of Moko disease-causing strains in Latin America and Brazil is unclear. Thirty-seven Ralstonia solanacearum strains from Brazil that cause the Moko disease on banana and heliconia plants were sampled and phylogenetically typed using the endoglucanase (egl) and DNA repair (mutS) genes according to the phylotype and sequevar classification. All of the strains belonged to phylotype II and a portion of the strains was typed as the Moko disease-related sequevars IIA-6 and IIA-24. Nevertheless, two unsuspected sequevars also harbored the Moko disease-causing strains IIA-41 and IIB-25, and a new sequevar was described and named IIA-53. All of the strains were pathogenic to banana and some of the strains of sequevars IIA-6, IIA-24, and IIA-41 were also pathogenic to tomato. The Moko disease-causing strains from sequevar IIB-25 were pathogenic to potato but not to tomato. These results highlight the high diversity of strains of Moko in Brazil, reinforce the efficiency of the egl gene to reveal relationships among these strains, and contribute to a better understanding of the diversity of paraphyletic Moko disease-causing strains of the R. solanacearum species complex, where the following seven distinct genetic clusters have been described: IIA-6, IIA-24, IIA-41, IIA-53, IIB-3, IIB-4, and IIB-25.

CHARACTERIZATION OF RALSTONIA SOLANACEARUM CAUSING BACTERIAL WILT IN BELL PEPPER IN THE STATE OF PERNAMBUCO, BRAZIL

Seventy-seven bacterial strains obtained from wilted bell pepper plants from the Agreste and Mata mesoregions of Pernambuco State (northeastern Brazil), were identified as Ralstonia solanacearum using multiplex PCR. The strains were further analyzed to characterize their biochemical, physiological, and molecular diversities. The biovars and biotypes were determined biochemically, and a molecular characterization of the strains was made using multiplex PCR, rep-PCR, and ISSR. The genotypic diversity was assessed, considering the number of genotypes observed and how they were distributed throughout the populations, for their differences in terms of richness, evenness and diversity. Strains of biovar 3, biotype 8 and phylotype I were predominant (97.40%), but biovar 1, biotypes 3 and 6 and phylotype II were also present. Rep-PCR analysis using REP and BOX primers, and ISSR showed similarities among the majority of the strains; however, these primers did not allow strain separation by biovars, biotypes, phylotypes or areas. Analysis of the genotypic diversity revealed a moderate diversity in the overall population, with a high variability in the strains from the same municipality.

REDUCTION OF THE SEVERITY OF ANGULAR LEAF SPOT OF COTTON MEDIATED BY SILICON

Angular leaf spot, caused by Xanthomonas citri subsp. malvacearum, is the major bacterial disease of cotton in Brazil and other countries. This study evaluated the effectiveness of silicon (Si) for the control of the disease and the possible resistance mechanisms triggered by this element. Calcium silicate (CaSiO3) was incorporated into the soil at concentrations of 0.00, 0.25, 0.50, 1.50 and 3.00 g of SiO2 kg-1 soil 25 days before planting. Leaves of 33-day-old plants were inoculated by infiltration with 0.5 ml of a bacterial suspension (108 CFU ml-1). Components of resistance were evaluated every two days up to 10 days post inoculation, when plant development and the accumulation of Si and calcium were determined. H2O2 production and the activity of enzymes related to plant defenses were analyzed 6, 12 and 24 h post inoculation in +/-Si plants. In vitro inhibition of pathogen growth was also assessed. No significant difference was found among treatments regarding incubation period, disease incidence or bacterial growth inhibition. However, with the application of 1.50 g of SiO2 kg-1 of soil, a reduction in disease severity (54.9%) was observed along an increase in plant height (7%). There was no detectable accumulation of Si in the cotton leaves. In the presence of Si (1.80 g SiO2 kg-1), the level of soluble proteins and the activity of the enzymes SOD, APX, guaiacol- peroxidase, PAL and b-Glu increased, whereas there was a decrease of H2O2. The angular leaf spot reduction observed may be associated with these modification, suggesting resistance induction.

Óleos essenciais e extratos vegetais no controle da podridão mole em alface crespa

The effect of oils and plant extracts was evaluated for controlling soft rot in lettuce and assessed the influence of these products in physico-chemical characteristics of this vegetable. In the in vitro tests, filter paper discs were soaked in eleven essential oils (0.0, 0.25, 0.50, 0.75 and 1%) and twenty plant extracts (10, 40, 70 and 100%) being deposited on a culture medium containing Pectobacterium carotovorum subsp. carotovorum (strain Pcc A1). The zones of inhibition were measured after 24 and 48 h of incubation. In the greenhouse, plants of cv. Veneranda were treated with eleven oils (0.5 and 1%), twenty extracts (10%) and Mycoshield® (3 g L-1) and after 72 h were inoculated with Pcc A1. We evaluated the disease severity with an interval of six hours until 48 h, and the area under the disease progress curve (AUDPC) was calculated. In a similar experiment plants treated with Corymbia citriodora oil, seven plant extracts and Mycoshield® were analyzed for soluble solids, vitamin C, acidity and pH. Strain Pcc A1 was not inhibited in vitro. Two oils (C. citriodora and C. sinensis) and seven extracts (Parkinsonia aculeata, Chamaecrista cytisoides, Sida galherensis, Polygala violaceae, C. desvauxii and Pityrocarpa moniliformis) significantly reduced disease severity in comparison to control, without differing from Mycoshield®. On the other hand AACPD was reduced by eight oils and ten extracts. It is worth to notice that severity and AUDPC were reduced by eucalyptus oil at 0.5% (3.6, 83.35) and by the extract of S. galherensis (3.5, 88.25) with the same efficiency as Mycoshield® when compared to control (7.5, 132.3). The levels of vitamin C, acidity and pH were not altered in leaves of lettuce treated with eucalyptus oil and extracts of P. aculeata and C. cytisoides.

Polyphasic Characterization of Pigmented Strains of Xanthomonas Pathogenic to Cashew Trees

The export of cashew (Anacardium occidentale) nuts generates millions of dollars for the Brazilian economy annually. However, production may be limited by the occurrence of diseases that affect cashew trees, such as Xanthomonas spot and angular leaf spot, which are caused by pigmented strains of Xanthomonas and Xanthomonas citri pv. anacardii, respectively. Thirty-one pigmented strains of Xanthomonas were characterized for phenotypic, pathogenic, and molecular attributes. These strains were similar to X. citri pv. anacardii in phenotypical characteristics, sensitivity to antibiotics and copper compounds used in agriculture, epidemiology, and repetitive sequence-based polymerase chain reaction (rep-PCR) profiles. When inoculated into Brazilian pepper, cashew, mango, and hog plum seedlings, the pigmented strains of Xanthomonas and X. citri pv. anacardii produced similar symptoms. However, the pigmented strains of Xanthomonas were more aggressive toward cashew plants than toward the other hosts tested, which confirms their specificity. We conclude that pigmented strains of Xanthomonas are very aggressive on cashew trees and should not be considered casual pathogens of these hosts. Moreover, based on our results from rep-PCR and IS1595-PCR amplification, we suggest that these strains constitute a variant of X. citri pv. anacardii.

Characterization and variability of soft rot-causing bacteria in Chinese cabbage in North Eastern Brazil.

SUMMARY Yield of Chinese cabbage (Brassica pekinensis) may be limited by the occurrence of the soft rot caused by pectinolytic bacteria. Thirty-nine bacterial isolates associated with soft rot in Chinese cabbage, obtained from northeastern Brazil, were identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) based on biochemical tests and URP-PCR. The variability of all isolates was assessed with reference to disease components, i.e. incubation period (IP), initial severity (ISEV), final severity (FSEV) and area under the disease progress curve (AUDPC), sensitivity to 12 antibiotics and the banding pattern of REP, ERIC and BOX in Rep-PCR. Based on IP, ISEV, FSEV and AUDPC, the isolates were distributed in six similarity groups after cluster analysis. There was significant correlation (P≤0.05) between IP and ISEV. Based on the sensitivity to antibiotics, Pcc isolates were distributed in 14 groups. Significant correlations between sensitivity to gentamicin and IP (r = -0.41), as well as between sensitivity to clindamycin and FSEV (r = -0.45) were detected. There was high genetic variability among the 39 isolates based on the molecular markers. A total of 32 similarity groups were formed. No significant correlations were found between the linkage distances of molecular markers and either the disease components or antibiotic sensitivity. Overall, there was high variability in populations of Pcc affecting Chinese cabbage in north-eastern Brazil.

Selection of a protein solubilization method suitable for phytopathogenic bacteria: a proteomics approach.

BackgroundFinding 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.ResultsThe 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.ConclusionsThese results attest to the adequacy of these proteins extraction methods for proteomic studies.

Biofumigation with essential oils for managing bacterial wilt of sweet peppers.

Bacterial wilt of sweet pepper, caused by Ralstonia solanacearum (Rs), is an important disease in Brazil. The effectiveness of bergamot, blue gum, cinnamon, clary sage, copal, fennel, lemon, lemongrass, lemon-scented eucalyptus, mint, palmarosa and sweet orange essential oils was evaluated for disease control via biofumigation. Soil infested by Rs CGM-8 was biofumigated with all oils (0.14%, v:v) in the greenhouse, and with bergamot, sweet orange and palmarosa oils in the field for four days. Variables evaluated were: latency period (LP50), incidence, bacterial wilt index (BWI), area under the disease progress curve (AUDPC), plant development, Rs population in the soil, soil characteristics and the in vitro growth of Rs. Palmarosa, bergamot and sweet orange oils increased LP50 (15%), reduced BWI (60%) and AUDPC (64.4%), in the greenhouse. In the field, only the palmarosa oil increased LP50 (38%), reduced BWI (36%) and AUDPC (38%), and increased the number of fruits per plant. Only greenhouse soils biofumigated with bergamot and sweet orange oils presented significant high sodium levels. The in vitro growth of Rs was reduced in 66.9% by palmarosa oil. These results indicate that palmarosa essential oil is a potential alternative for the management of bacterial wilt of sweet peppers.

Polyphasic analysis of Acidovorax citrulli strains from northeastern Brazil

Bacterial fruit blotch (BFB) of cucurbit plants is caused by Acidovorax citrulli and represents a serious concern to melon (Cucumis melo L.) growers worldwide, including those in Brazil. Thirty-four A. citrulli strains from different melon production areas of northeastern Brazil were characterized for their virulence on melon fruits and their substrate utilization and molecular profiles. Based on the analysis of BFB severity on melon fruits, the A. citrulli strains were divided into three groups, classified as mildly, moderately or highly virulent. Although host-related groups were not observed, the watermelon and ‘melao-pepino’ strains exhibited only low or moderate virulence on melon fruit. Substrate utilization profiles revealed that 94 % of the 95 tested compounds were used by A. citrulli strains as a carbon source. Overall, based on substrate utilization, low variability was observed with no relationship to host of origin. The formation of one group of A. citrulli strains based on Repetitive Sequence-based PCR (rep-PCR) analysis confirmed the low variability observed in the substrate utilization analyses. Bayesian inference based on the analysis of 23S rDNA partial sequence data resulted in one well-supported clade and clustered the strains with the A. citrulli-type species with high posterior probability support. Based on the markers used, the Brazilian A. citrulli strains belong to a single group, which corresponds to the previously described Group I for this bacterium in the United States.

USE OF SILICON FOR REDUCING THE SEVERITY OF BACTERIAL WILT OF SWEET PEPPER

Bacterial wilt of sweet pepper, caused by Ralstonia solanacearum (Rs) race 1, is a limiting disease in northern and northeastern Brazil. The present study evaluated the effect of silicon (Si) on the disease in the sweet pepper cv. Enterprise, which was grown in a substrate containing 0.00, 0.25, 0.50, 1.50, or 3.00 g SiO2 kg-1and was subsequently transplanted into soil infested with Rs CGM-8. The following variables were evaluated: latency period (LP50); incidence; bacterial wilt index (BWI); area under the disease progress curve (AUDPC); biomass; accumulation of Ca+2, Mg+2, and Si; total protein and enzymatic activity; chemical characteristics of the substrate; and in vitro bacterial growth. A dose of 2.95 g Si kg-1substrate increased the LP50 (33.6%) and reduced the BWI (98%) and AUDPC (93.7%); this treatment also increased Ca+2 levels in the shoots and reduced Mg+2levels in the shoots and the roots. Supplementation with various doses of Si yielded maximum increases in the shoot fresh biomass (121.8%), the root fresh biomass (83.6%), and the shoot dry biomass (84.9%); increased the concentrations of total protein, catalase, ascorbate peroxidase, and chitinase; caused the accumulation of Si in the shoots and substrate; increased the pH, Na+, and K+; and decreased P in the substrate. The putative mechanisms of action of Si included a direct effect on colonization of the pathogen, an indirect effect on the plant’s development, increased Ca+2 absorption, and signaled for the production of plant defence enzymes. Therefore, the production of sweet pepper seedlings in a substrate containing calcium silicate (2.95 g SiO2 kg-1) could be utilized as an alternative cultivation practice for the management of bacterial wilt disease. More research is needed to elucidate the exact mechanisms of silicon and its effects on latent infections and on bacterial cells and population.