Inoculation and detection of Xanthomonas axonopodis pv. malvacearum in cotton seeds (Gossypium hirsutum L.) / âInoculaÃÃo e detecÃÃo de Xanthomonas axonopodis pv. malvacearum em sementes de algodÃo (Gossypium hirsutum L.)â

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

ABSTRACT BARBOSA, J. F. Inoculation and detection of Xanthomonas axonopodis pv. malvacearum in cotton seeds (Gossypium hirsutum L.). 2007. 135p. Thesis (Doctorate in Agronomy/Plant Pathology) â Universidade Federal de Lavras, Minas Gerais, Brazil. The objectives in the present study were to develop a technique for artificial inoculation of cotton seeds with Xanthomonas axonopodis pv. malvacearum (Xam), through the use of the osmotic conditioning technique, aiming to obtain uniformly contaminated/infected seeds, with preservation of the germination potential of the seeds, as well as to establish a safe, efficient and fast methodology for the detection of the bacterium in cotton seeds. The variability of isolates of Xam was assessed through the artificial inoculation of cotton plants, hypersensibility in tobacco, tomato and sweet pepper plants and amplification of DNA with the primers 16S and 23S, ERIC and REP. A semi-selective culture medium was developed for the detection of Xam in cotton seeds and the efficiency of the indirect immunofluorescence technique was tested in the detection of this bacterium. In the artificial inoculation with the osmotic conditioning technique it was verified that the solutes mannitol and sucrose had no statistical difference in relation to percentage of seed germination and bacterial growth. The osmotically modified 523 medium with potential of -0.85 and -1.0 MPa presented the strongest effect on the inhibition of seed germination. The inoculation through osmotic conditioning technique was efficient and the 24 hours seed exposition to the bacterial inoculum allowed the recovering of seeds with 84.5% incidence of that pathogen. Electromicrographs (taken in a Scanning Electron Microscope) revealed the presence of Xam associated with the seed peel in all the inoculation periods. The incidence of the bacterium in the seeds, detected by direct plating in semi-selective medium, was higher in the inoculation method that used osmotic conditioning, with level of 88% occurrence, with no statistical difference for the percentage of cotyledons with symptoms of bacterial blight amongst the inoculation methods used. Differences in virulence amongst the isolates in cotton were observed, with four distinct groups. In relation to HR reaction, the tomato plants were the most adequate host for the identification of the Xam pathogenic strains. The primers 16S and 23S amplified fragments of approximately 680 bp. The PCR amplification using the REP, repetitive unit, was effective to all Xam strains, however, the number of band patterns in each strain was significantly lower when compared with the ERIC oligonucleotides. Using the 523 medium of Kado and Heskett (1970) as the basal medium, the following selective medium was developed: 30 g of mannitol, 4 g of yeast extract, 8 g of hydrolyzed casein, 2 g of K2HPO4, 0.3 g of MgSO4.7H2O, 15 g of agar, 1000 mL of distilled water, with addition of 50 mg of cefalexin, 50 mg of cefadroxil, 10 mg of chlorathalonil and 300 ÂL of triadimenol after autoclaving. This medium and the semi-selective culture medium of Metha et al., (2005) and Dezordi (2006), were all effective in recovering of Xam from the seeds. The efficiency of the indirect immunofluorescence technique also was tested by using three types of seed extracts (crude, centrifuged and filtered in Millipore membrane), with the observation of the bacterium in all extracts when using this technique.

ASSUNTO(S)

algodÃo sementes cotton mancha angular bacterial blight seeds bactÃrias fitopatologia

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