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Applied and Environmental Microbiology, July 2003, p. 4249-4255, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.4249-4255.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection and Diversity Assessment of Xylella fastidiosa in Field-Collected Plant and Insect Samples by Using 16S rRNA and gyrB Sequences

Jorge L. M. Rodrigues,1* M. E. Silva-Stenico,1 J. E. Gomes,1 J. R. S. Lopes,2 and S. M. Tsai1

Centro de Energia Nuclear na Agricultura,1 Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, ESALQ, Universidade de São Paulo, Piracicaba, São Paulo 13400-970, Brazil2

Received 13 November 2002/ Accepted 16 April 2003

The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.

* Corresponding author. Mailing address: Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 300, Piracicaba, SP 13400-970, Brazil. Phone: 55-19-3429-4600. Fax: 55-19-3429-4822. E-mail: jorgelmr{at}cena.usp.br.

Applied and Environmental Microbiology, July 2003, p. 4249-4255, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.4249-4255.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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