Range of Phytoplasma Concentrations in Various Plant Hosts as Determined by Competitive Polymerase Chain Reaction

Abstract

For competitive polymerase chain reaction (PCR), an internal standard DNA template was developed that consisted of a highly conserved, internally deleted 16S rDNA fragment of an aster yellows phytoplasma. The internal standard was calibrated using a quantified culture of Acholeplasma laidlawii. Serial dilutions of the internal standard and fixed amounts of target templates from infected plants were coamplified with the same primers, and the products obtained were quantified using an enzyme-linked immunosorbent assay procedure. Analysis of the data revealed that the phytoplasma concentration in the plants examined differed by a factor of about 4 x 10(6). Phytoplasma concentrations of 2.2 x 10(8) to 1.5 x 10(9) cells per g of tissue were identified in periwinkles infected with various phytoplasmas. High to moderate concentrations were detected in Malus domestica (apple) genotypes infected with the apple proliferation phytoplasma, Alnus glutinosa (alder) genotypes infected with the alder yellows phytoplasma, and most aster yellows-infected Populus (poplar) genotypes examined. Very low phytoplasma concentrations, ranging from 370 to 34,000 cells per g of tissue, were identified in proliferation-diseased apple trees on resistant rootstocks 4551 and 4608, yellows-diseased Quercus robur (oak) trees, and Carpinus betulus (hornbeam) trees. Such low concentrations, which corresponded to about 4 to 340 cells in the reaction mixture, could only be detected and quantified by nested PCR.

‘Candidatus Phytoplasma pini’, a novel taxon from Pinus silvestris and Pinus halepensis

“…This may be explained by very low phytoplasma titres and/or poor preservation of the organisms in the tissue examined. It is well established that phytoplasma numbers in infected plants may be so low that microscopic detection is difficult or impossible (Berges et al, 2000;Caudwell & Kuszala, 1992;Kartte & Seemüller, 1991).…”

First identification of the pathogen causing tumor malformations in evergreen oaks in Spain

“…Different species of Quercus are known to be hosts of phytoplasmas and to develop the disease after infection (Seemüller and Schneider, 2004;Marcone et al, 1999;Berges et al, 2000). Nevertheless, the described symptomatology does not match the one observed in Q. ilex.…”

Polymerase Chain Reaction for Phytoplasmas Detection

“…Palmano (2001) (134) demonstrated importance of proper identification of plant parts sampling; in this case the leaves have to show obvious symptoms but without being necrotic or completely yellow. In addition, variance in phytoplasma titters between infected plants of the same species has been observed by Berges et al (2000) (15) and may be caused by different stages of development and age of plants.…”

“…Many of the economically important diseases are those of woody plants, including coconut lethal yellowing, peach X-disease, grapevine yellows, and apple proliferation. Following their discovery, phytoplasmas have been difficult to detect due to their low concentration especially in woody hosts and their erratic distribution in the sieve tubes of the infected plants (15). First detection technique which indicated presence of some intercellular disorder was based on graft transmission of the pathogen to healthy indicator plants.…”