High-throughput allelic discrimination of B and Q biotypes of the whitefly, Bemisia tabaci, using TaqMan allele-selective PCR.

BACKGROUND B and Q biotypes of the whitefly, Bemisia tabaci (Gennadius), are generally regarded as the most significant given their global distribution and strong resistance to insecticides. Since these biotypes can coexist and differ markedly in their insecticide resistance profiles, a rapid but reliable means of discriminating between them would be a valuable complement to resistance monitoring and management programmes. Recently, PCR-based methods have been developed to determine the biotype status of B. tabaci populations. However, these require post-amplification procedures, which increase time and labour. RESULTS The authors have developed an allelic discrimination real-time PCR assay using fluorescent dye-labelled probes to distinguish the B and Q biotypes. The assay targets a single nucleotide polymorphism (SNP) in the mitochondrial cytochrome oxidase I (mtCOI) gene. To evaluate the assay, DNA was extracted from individual whiteflies of six known biotype strains, and all scored correctly as either a B or Q biotype. As further validation, 72 individuals from field samples collected in different parts of the world were also tested by the assay. No failed reactions were observed, with all 72 samples scoring clearly as either the B or Q biotype. CONCLUSION The development of this rapid and high-throughput assay has important potential for routine monitoring of B and Q biotypes on ornamental plants and for the screening of B. tabaci populations in countries where these biotypes are not yet established.

[1]  J. Brown,et al.  First report of the Q biotype ofBemisia tabaci in Japan by mitochondrial cytochrome oxidase I sequence analysis , 2006, Phytoparasitica.

[2]  D. Bosco,et al.  PCR-RFLP identification ofBemisia tabaci biotypes in the Mediterranean Basin , 2006, Phytoparasitica.

[3]  S. Kontsedalov,et al.  Biotype Q ofBemisia tabaci identified in Israel , 2003, Phytoparasitica.

[4]  K. Žanić,et al.  Note: Current status ofBemisia tabaci in Coastal Croatia , 2008, Phytoparasitica.

[5]  B. Cong,et al.  THE INTRODUCTION OF THE EXOTIC Q BIOTYPE OF BEMISIA TABACI FROM THE MEDITERRANEAN REGION INTO CHINA ON ORNAMENTAL CROPS , 2006 .

[6]  D. Bosco,et al.  PCR-RFLP identification of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in the Mediterranean Basin. , 2006 .

[7]  S. Morin,et al.  DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics , 2005, Bulletin of Entomological Research.

[8]  R. Nichols,et al.  New Challenges to Management of Whitefly Resistance to Insecticides in Arizona , 2005 .

[9]  D. Frohlich,et al.  Bemisia argentifolii is a race of B. tabaci (Hemiptera: Aleyrodidae): the molecular genetic differentiation of B. tabaci populations around the world , 2005, Bulletin of Entomological Research.

[10]  R. Nauen,et al.  Resistance of insect pests to neonicotinoid insecticides: current status and future prospects. , 2005, Archives of insect biochemistry and physiology.

[11]  S. Kontsedalov,et al.  Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance. , 2005, Archives of insect biochemistry and physiology.

[12]  M. Williamson,et al.  High-throughput detection of knockdown resistance in Myzus persicae using allelic discriminating quantitative PCR. , 2004, Insect biochemistry and molecular biology.

[13]  R. ffrench-Constant,et al.  Detection of insecticide resistance-associated mutations in cat flea Rdl by TaqMan-allele specific amplification , 2004 .

[14]  David R. Jones.  Plant Viruses Transmitted by Whiteflies , 2003, European Journal of Plant Pathology.

[15]  J. Brown,et al.  Characterization and distribution of esterase electromorphs in the whitefly,Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) , 1995, Biochemical Genetics.

[16]  R. Nauen,et al.  Toxicological and mechanistic studies on neonicotinoid cross resistance in Q-type Bemisia tabaci (Hemiptera: Aleyrodidae). , 2002, Pest management science.

[17]  D. Navia,et al.  Genetic diversity of Bemisia tabaci (Genn.) Populations in Brazil revealed by RAPD markers , 2002 .

[18]  M. Cervera,et al.  Genetic relationships among biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae) based on AFLP analysis , 2000, Bulletin of Entomological Research.

[19]  K. Livak,et al.  Allelic discrimination using fluorogenic probes and the 5' nuclease assay. , 1999, Genetic analysis : biomolecular engineering.

[20]  F. Beitia,et al.  Biotype determination of Spanish populations of Bemisia tabaci (Hemiptera: Aleyrodidae) , 1997 .

[21]  Irina Afonina,et al.  Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder , 1997, Nucleic Acids Res..

[22]  R. Rosell,et al.  Geminivirus transmission and biological characterisation of Bemisia tabaci (Gennadius) biotypes from different geographic regions , 1994 .

[23]  David H. Headrick,et al.  Description of a Species of Bemisia (Homoptera: Aleyrodidae) , 1994 .

[24]  J. Brown,et al.  Regional Distribution, Insecticide Resistance, and Reciprocal Crosses Between the A and B Biotypes of Bemisia Tabaci , 1993 .

[25]  A. Devonshire,et al.  Insensitive Acetylcholinesterase and Esterase Polymorphism in Susceptible and Resistant Populations of the Tobacco Whitefly Bemisia tabaci (Genn.) , 1993 .

[26]  J. Brown,et al.  Variation in biological characteristics and esterase patterns among populations of Bemisia tabaci, and the association of one population with silverleaf symptom induction , 1991 .