Detection of Phytoplasma by Polymerase Chain Reaction of Insect Feeding Medium and Its Use in Determining Vectoring Ability

Abstract

A polymerase chain reaction (PCR)-based method was developed for the detection of phytoplasma in insect feeding medium (sucrose). A correlation was established between the transmissibility of Flavescence dorée phytoplasma in the experimental leafhopper vector Euscelidius variegatus and its detection by PCR in the insect feeding medium. However, phytoplasma were detected in the insects' bodies 3 weeks before they began to transmit. Hence, PCR assays of the sucrose medium reflected phytoplasma vectoring ability probably by detecting it in the insect saliva, whereas detection of phytoplasma in the insect's body did not identify it as a vector. The assay was applied to two field-collected leafhoppers suspected of being phytoplasma vectors in Israel (Orosius albicinctus and Anaceratagallia laevis). The presence of phytoplasma in the body of specimens of the latter species was assayed by PCR in 1999. Phytoplasmas were detected in insects' bodies throughout the year, with no specific seasonal pattern. In the saliva, however, no phytoplasma could be detected in the autumn. This seasonal pattern supported the validity of the feeding-medium tests and their correlation to the insect's ability to transmit phytoplasma. Transmission assays indicated, to our knowledge for the first time, that O. albicinctus and A. laevis are vectors of phytoplasma in Israel. A simple PCR-based assay is thus provided, circumventing the need for tedious biological assays and enabling epidemiological studies of phytoplasma transmissibility on a large scale.

Leafhoppers and cixiids in phytoplasma-infected carrot fields: Species composition and potential phytoplasma vectors

“…Field collected A. laevis could transmit the yellows phytoplasma to Catharantus roseus and potato plants (Tanne et al, 2001) and recently laboratory-reared A. ribauti adults transmit stolbur phytoplasma to Vicia faba seedlings (Riedle-Bauer et al, 2008).…”

Identification and Characterization of Phytoplasmal Genes, Employing a Novel Method of Isolating Phytoplasmal Genomic DNA

“…We adapted a method of phytoplasma isolation from artificial feeding medium (J. Zhang, S. Miller, C. Hoy, X. Zhou, and L. Nault, Phytopathology 88, abstr. S84, 1998), described by Tanne et al (53), whereby phytoplasmas are isolated from the insect vector's saliva and their genomic DNA is extracted. White microcentrifuge tubes (1.5 ml) were used as insect chambers.…”

“…Recently, for the purposes of demonstrating the vector's ability to transmit phytoplasma, we devised a method of collecting insect saliva and showed, by PCR analysis of rRNA genes, that phytoplasmal DNA can be easily detected in that saliva (53). Based on those findings, we describe a novel approach to isolating phytoplasmas and their genomic DNAs and validate it by isolating and characterizing 78 new putative phytoplasmal open reading frames (ORFs).…”

Role of Cacopsylla pyri in the epidemiology of pear decline in Spain

“…The caps were filled with 200 ll of 5% sucrose, 0.5% sorbitol and 9.4 mg l )1 of NCTC 135 medium (Sigma) then sealed with Parafilm (M. Maixner, Institut fu¨r Pflanzenschutz im Weinbau, Germany, personal communication). Sucrose medium has been reported as suitable for studying the transmission of phytoplasmas by leafhoppers (Tanne et al, 2001). Each tube, containing an individual psyllid, was kept at 23-25°C until the insect died or for a maximum of 72 h. Time of death was recorded.…”