Eder Marques

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.

Sensitivity to copper in Xanthomonas campestris pv. viticola

O cancro bacteriano, causado por Xanthomonas campestris pv. viticola, afeta o cultivo irrigado de videira no Vale do Submedio Sao Francisco, nos estados de Pernambuco e Bahia. O manejo da doenca tem sido realizado atraves de um conjunto de praticas que inclui a aplicacao de cupricos. Este tem sido o unico metodo de controle quimico disponivel e o mais utilizado nas areas de ocorrencia da doenca. O objetivo deste estudo foi avaliar a sensibilidade ao cobre de estirpes de X. campestris pv. viticola coletadas em diferentes localidades, entre os anos de 1998 a 2006. Detectou-se variabilidade na sensibilidade ao oxicloreto de cobre e ao sulfato de cobre entre as 21 estirpes testadas. A concentracao minima inibitoria variou entre 10 e 60 µg/mL Cu2+, para os dois produtos. De forma geral, observouse uma evolucao no crescimento da tolerância ao cobre ao longo dos anos, com as estirpes brasileiras apresentando maior tolerância que a estirpe-tipo, coletada em 1972 na India. As diferencas observadas em sensibilidade ao cobre entre as estirpes de X. campestris pv. viticola podem levar, com o uso continuo de compostos cupricos na regiao, a selecao e dominância das estirpes mais tolerantes na populacao bacteriana.

New isolates of Trichoderma antagonistic to Sclerotinia sclerotiorum

Forty-nine isolates of Trichoderma from the Brazilian Midwest were evaluated for their antagonistic activity in vitro against Sclerotinia sclerotiorum (causal agent of white mold), which were then identified based on their nuclear ribosomal ITS sequences. Paired culture tests showed that all isolates exhibited some antagonism, with a maximum of 77% mycelial inhibition and complete inhibition of sclerotia production. Two isolates were found to be the most promising biocontrol agents, considering both antagonistic parameters (CEN1253 - T. koningiopsis and CEN1265 - T. brevicompactum). Five different species were identified: T. harzianum (23), T. spirale (9), T. koningiopsis (8), T. brevicompactum (7) and T. asperellum (2). These isolates are stored in the Embrapa Fungi Collection for Biological Control and the information obtained in the experiments will be incorporated into the database of biological assets within the genetic resources information system (Allele) and be made available for further studies.

Virulência de estirpes (biovar 1 e 2T) de Ralstonia solanaceraum a Eucalyptus spp.

The bacterial wilt of Eucalyptus (Ralstonia solanacearum Race 3 biovar 2T) was reported in field of Eucalyptus hybrid urograndis. The objective of this research was to evaluate the virulence of two R. solanacearum strains to 17 Eucalyptus species, using the seed microbiolization method for pathogen inoculation. The results of the statistical analyses showed that virulence varied between the two R. solanacearum strains. The R3bv2T virulence was higher than the one in R1bv1 in the following Eucalyptus species: urograndis, E. urophylla, E. grandis x E. camaldulensis, E. grandis, E. microcorys, E. deanei, and E. phaeotricha. The virulence of the R1bv1 strain was higher only in E. pellita. Among species of Eucalyptus the following ones were more susceptibles: to R3bv2T strain - urograndis, E. grandis and E. paniculata, and to R1bv1 strain - E. cloeziana, E. paniculata, E. botryoides, E. pellita, E. propinqua and E. resinifera.

Bactérias extremófilas facultativas melhorando a germinabilidade de sementes de Eucalyptus urophylla S.T. blake

In this study the effect of seed microbiolization with facultative extremophile bacteria (Bacillus sp. and Enterobacter sp.), isolated in previous work, from extreme conditions of pH and NaCl and able to increase eucalyptus phytomass, on seed germinability of Eucalyptus urophylla S.T. Blake was evaluated. Mean germination time, speed of germination and coefficient of speed germination were measured to evaluate germinability. The experiment was conducted in a completely randomized design including 11 treatments (five strains of Bacillus sp., five of Enterobacter sp. and one control without bacteria), formed by 8 replicates with 25 seeds each. The results showed that Bacillus sp. strains UnB 1366 and UnB 1374 significantly reduced the lower mean germination time and higher coefficients and speed of germination in relation to the other strains.

BIOCONTROL OF TEAK CANKER CAUSED BY Lasiodiplodia theobromae

Teak is a forest species that has assumed great importance in Brazil, where it has found excellent conditions for development since its introduction into the country in the 1960s. However, phytosanitary problems are beginning to threaten the production of this timber species. An example is teak canker, caused by the fungus Lasiodiplodia theobromae (Lt), which has only recently been reported in Brazil, and for which, therefore, there are no recommended control methods. Thus, this study evaluated the control of this pathogen, investigating the potential of the biocontrol agents (BCAs) Trichoderma spp., Bacillus sp. and Enterobacter sp., initially through in vitro assays and, subsequently, with in vivo tests. According to the in vitro assay results, the Trichoderma isolates CEN162 and CEN1153 and the strain of Bacillus sp. (UnB1366) were the treatments that stood out, as they were able to completely inhibit mycelial growth of some isolates of Lt. When these isolates were tested in a preventive way, the control levels varied depending on the Lt isolate and the antagonist-clone interaction, where CEN162 (T. asperellum) and UnB166 (Bacillus sp.) showed 100% control. Thus, there is a positive correlation between the in vitro and in vivo tests, since the same BCAs stood out. Although good levels of control have been obtained with the BCAs used, it can be concluded that there is a variation in the antagonism to different Lt isolates or even in the antagonist-clone interaction, corroborating the information available in the scientific literature on this plant-pathogenic fungus.

Identification and pathogenicity of Pseudomonas cichorii associated with a bacterial blight of gerbera in the Federal District

In 2013, leaf samples of gerbera plants showing symptoms of bacterial blight were collected in cut-flower high tunnels, in the settlement of nucleo rural alexandre gusmao, located in brazlândia, distrito federal, brazil. seven isolates obtained were subjected to phenotypic and molecular characterization, including pathogenicity tests, lopat, and partial sequencing of the 16s rdna gene. all isolates were gram-negative, aerobic, oxidase-positive, produced fluorescent pigment, induced hypersensitivity in tobacco leaves, used sorbitol and glutamate and were pathogenic to 24 different plant species. results of these tests and analysis of the sequences of rdna showed 100% identity with pseudomonas cichorii. to our knowledge, this is the first report of p. cichorii in gerbera in the federal district.