Marisa A.S.V. Ferreira

Caracterização morfocultural e molecular de isolados de Colletotrichum gloeosporioides patogênicos ao mamoeiro

Twenty-nine monoconidial cultures of Colletotrichum isolated from papaya (Carica papaya) petioles and fruits were characterized by conidial and appressoria morphology, colony color, growth rate, sensitivity to benomyl, presence of setae, presence of the teleomorph, PCR with taxon-specific primers and analysis of PCR-RFLP of the ITS region. The 29 isolates from papaya were identified as C. gloeosporioides, based mainly on conidial and appressoria morphology, with most isolates producing cylindrical and/or obclavate conidia and entirely or weakly lobed appressoria, in contrast with the strawberry (Fragaria x ananassa) isolate of C. acutatum, which produced fusiform conidia and circular appressoria with entire edges. Presence of setae, teleomorphic stage, colony color, sensitivity to benomyl and growth rate were variable among isolates and influenced by the culture medium. All papaya isolates and four isolates (C. gloeosporioides and C. acutatum) from other hosts, mango (Mangifera indica), strawberry and apple (Malus domestica), were pathogenic to papaya fruits cv. Sunrise Solo, producing similar symptoms, but with variability in aggressiveness. PCR with C. gloeosporioides-specific primer, CgInt, confirmed the identity of four papaya isolates. Two other isolates reacted with C. acutatum-specific primer, CaInt2. The majority of papaya isolates (23), however, did not react with any of the primers tested. In contrast, RFLP analysis of the amplified ITS region with RsaI, generated distinct patterns that could differentiate between the two species, C. gloeosporioides and C. acutatum, and showed uniformity among papaya isolates.

Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil

Leafroll is an economically important disease affecting grapevines (Vitis spp.). Nine serologically distinct viruses, Grapevine leafroll-associated virus-1 through 9, are associated with this disease. The present study describes the coat protein gene sequence of four GLRaV-3 isolates occurring in the Sao Francisco River basin, Northeastern Brazil. The viral RNA was extracted from GLRaV-3 ELISA-positive plants and the complete coat protein gene was amplified by RT-PCR. Sequences were generated automatically and compared to the complete coat protein sequence from North American (NY1) and Chinese (Dawanhong No2 and SL10) GLRaV-3 isolates. The four studied isolates, named Pet-1 through 4, showed deduced amino acid identities of 98-100% (Pet-1 through 3) and 95% (Pet-4) with North American and Chinese isolates. A total of seventeen amino acid substitutions was detected among the four characterized isolates in comparison to the NY1, Dawanhong No.2 and SL10 sequences. The results indicated the existence of natural variation among GLRaV-3 isolates from grapevines, also demonstrating a lack of correlation between sequence data and geographic origin. This variability should be considered when selecting regions of the viral genome targeted for reliable and consistent virus molecular detection.

Simultaneous detection and identification of the Xanthomonas species complex associated with tomato bacterial spot using species‐specific primers and multiplex PCR

To establish protocols for the simultaneous detection and identification of Xanthomonas species causing tomato bacterial spot.

Development of a molecular method for detection and identification of Xanthomonas campestris pv. viticola

In order to develop a molecular method for detection and identification of Xanthomonas campestris pv. viticola (Xcv) the causal agent of grapevine bacterial canker, primers were designed based on the partial sequence of the hrpB gene. Primer pairs Xcv1F/Xcv3R and RST2/Xcv3R, which amplified 243- and 340-bp fragments, respectively, were tested for specificity and sensitivity in detecting DNA from Xcv. Amplification was positive with DNA from 44 Xcv strains and with DNA from four strains of X. campestris pv. mangiferaeindicae and five strains of X. axonopodis pv. passiflorae, with both primer pairs. However, the enzymatic digestion of PCR products could differentiate Xcv strains from the others. None of the primer pairs amplified DNA from grapevine, from 20 strains of nonpathogenic bacteria from grape leaves and 10 strains from six representative genera of plant pathogenic bacteria. Sensitivity of primers Xcv1F/Xcv3R and RST2/Xcv3R was 10 pg and 1 pg of purified Xcv DNA, respectively. Detection limit of primers RST2/Xcv3R was 104 CFU/ml, but this limit could be lowered to 102 CFU/ml with a second round of amplification using the internal primer Xcv1F. Presence of Xcv in tissues of grapevine petioles previously inoculated with Xcv could not be detected by PCR using macerated extract added directly in the reaction. However, amplification was positive with the introduction of an agar plating step prior to PCR. Xcv could be detected in 1 µl of the plate wash and from a cell suspension obtained from a single colony. Bacterium identity was confirmed by RFLP analysis of the RST2/Xcv3R amplification products digested with Hae III.

Molecular characterization of Brazilian strains of Xanthomonas campestris pv. viticola by rep-PCR fingerprinting

Bacterial canker of grapevine (Vitis vinifera), caused by Xanthomonas campestris pv. viticola was first detected in Brazil in 1998, affecting grapevines in the Sao Francisco river basin, state of Pernambuco. The disease was also reported in Juazeiro, Bahia and later in Piaui and Ceara. Due to its limited geographical distribution and relatively recent detection in Brazil, very little is known about the pathogens biology and diversity. Repetitive DNA based-PCR (rep-PCR) profiles were generated from purified bacterial DNA of 40 field strains of X. campestris pv. viticola, collected between 1998 and 2001 in the states of Pernambuco, Bahia and Piaui. Combined analysis of the PCR patterns obtained with primers REP, ERIC and BOX, showed a high degree of similarity among Brazilian strains and the Indian type strain NCPPB 2475. Similar genomic patterns with several diagnostic bands, present in all strains, could be detected. Fingerprints were distinct from those of strains representing other pathovars and from a yellow non-pathogenic isolate from grape leaves. The polymorphism observed among the Brazilian strains allowed their separation into five subgroups, although with no correlation with cultivar of origin, geographic location or year collected.

Ocorrência e caracterização do complexo de espécies causadoras da mancha bacteriana do tomateiro no Alto Vale do Rio do Peixe, SC

Occurrence and characterization of the species complex causing tomato bacterial spot in “Alto Vale do Rio do Peixe”, SC, Brazil The aim of this study was to identify at the species level Xanthomonas strains causing tomato bacterial spot in the region of “Alto Vale do Rio do Peixe”, state of Santa Catarina, Brazil, as well as to determine their in vitro sensitivity to copper. Species were determined by similarity analysis of genomic profiles generated by BOX-PCR and sensitivity to copper was established using the CYE medium supplemented with copper sulfate at concentrations of 50, 100 and 200 μg/mL. Of the 44 isolates, 80% were identified as X. gardneri, 11% as X. perforans and 9% as X. vesicatoria. According to the response to copper, the isolates were divided into four classes (S, sensitive; MS, moderately sensitive; MI, moderately insensitive; I, insensitive). Regarding the sensitivity to copper, 98% of all isolates were sensitive at 200 μg/mL, suggesting that the recommended dosage of copper-based products registered for the crop may still provide effective control of the different bacterial species.

Diversidade patogênica e molecular de Ralstonia solanacearum da região amazônica brasileira

Pathogenic and molecular diversity of Ralstonia solanacearum isolates from the Brazilian Amazon The diversity among 70 isolates of Ralstonia solanacearum collected from tomato and other hosts in the Brazilian Amazon was evaluated. Firstly, the isolates were identified at the biovar level and their virulence assessed by inoculating seedlings of tomato, sweet pepper and Amazon chicory (Eryngium foetidum). Fifty-three isolates were identified as biovar 1, four as biovar N2 and 13 as biovar 3, therefore confirming the prevalence of biovar 1 associated with tomato in the North Region of Brazil. Cluster analysis of the isolates allowed their separation into different virulence classes. On tomato, 44.3% of them were highly virulent, 37.1% were moderately virulent and 18.6% were weakly virulent. On peppers, 20% of the isolates were highly virulent, 27.1% moderately virulent and 52.9% were weakly virulent. When inoculated on Amazon chicory, only the chicory isolate caused wilt, thus revealing an uncommon specificity for R. solanacearum. Fortysix isolates from tomato and 18 from ten other hosts, collected in flooded and non-flooded areas, were then compared by BOX-PCR. Genomic fingerprints were highly polymorphic. Five groups were identified, without any clear-cut correlations among them with host of origin, biovar, ecosystem or geographic origin. The isolate obtained from Eryngium foetidum was the most divergent, with only 6.4% similarity to the other isolates. The tomato isolates were separated into three groups. All four biovar N2 isolates were grouped together and separated from the isolates representing the other biovars present in the Amazon region. Additional keywords: bacterial wilt, Lycopersicon esculentum, repetitive DNA, rep-PCR.

Low genetic diversity among pathogenic strains of Erwinia psidii from Brazil

Erwinia psidii causes bacterial disease of guava in Brazil. Phenotypic and molecular characterization through rep-PCR fingerprinting of 42 strains from different geographical regions showed that E. psidii populations in Brazil have a low level of genetic diversity and suggest that contaminated plant material is the main source for pathogen dissemination in the country.

Control of Post-harvest Anthracnose Infection in Guava (Psidium guajava) Fruits with Phosphites, Calcium Chloride, Acetyl Salicylic Acid, Hot Water, and 1-MCP

The control of anthracnose (Colletotrichum simmondsii) during the post-harvest stage in guava fruits (Psidium guajava L.) was performed by the application of phosphites [phosphite-K (40% P2O5 and 30% K2O) and phosphite-Ca (10.7% P2O5, 3.89% Ca, and 0.5% B)] including the Carbendazim as reference, calcium chloride (CaCl2), acetyl salicylic acid (ASA), hot water (HW), and 1-methylcyclopropene (1-MCP). These treatments were applied individually or in combination each other with two or three compounds. The evaluated parameters were diameter of anthracnose lesion (DL), number of lesions (NL), and fruit quality (fresh weight loss, pH, total soluble solids, and titrable acidity]. The fruits were disinfested, inoculated, and maintained in an incubator containing fluorescent lights at 75 µmol·m-2·s-1 (25°C, 12h photoperiod) for 5 days and were then analyzed. The results showed that the DL and the NL were reduced following treatments, and that the HW (47°C for 20 min) was the strongest and the 1-MCP treatment was the least effective. The physico-chemical characteristics of fruits were affected by some treatments without compromising fruit quality. The combination of treatments was also able to alleviate the anthracnose effect on fruits compared to individual treatments and the control without affect the fruit quality. The combinations which included the HW treatment showed the best performance to control this disease, particularly when combined with the 1-MCP and phosphite.

Specific primers for Xanthomonas vesicatoria, a tomato bacterial spot causal agent

Xanthomonas vesicatoria is a member of the species complex associated with tomato bacterial spot. New and specific primers for X. vesicatoria were developed and validated. The primers were highly specific and detection was positive using purified bacterial DNA, bacterial suspensions and foliar lesions. These primers represent an additional tool for detection and identification of one of the species involved in this important disease complex.