Amplification of plasmid DNA to detect plant pathogenic mycoplasmalike organisms

Abstract

The polymerase chain reaction (PCR) was employed to develop a specific assay for plant pathogenic mycoplasmalike organisms (MLOs). A cloned fragment of a plasmid from a severe strain of western aster yellows (AY)-MLO was sequenced to identify oligonucleotide primers for PCR. Amplified DNA fragments of the predicted size were obtained from DNA extracted from plants and insects infected with pear decline MLO, beet leafhopper-transmitted virescence agent, elm yellows MLO and several AY-MLO strains. No amplification occurred from healthy leafhopper or plant DNA. The PCR-based assay was over 500 times more sensitive than a hybridisation-based assay which utilised a cloned AY plasmid fragment as a probe. With the PCR-based assay, MLOs could be detected using DNA samples of leafhoppers that were only crushed and boiled in buffer. Amplification of the target DNA was confirmed by digestion of the PCR product with Mbo I which yielded predicted sized fragments for all MLO strains except Bradford AY and eastern AY. Sequencing the PCR product from elm yellows and eastern AY-MLOs revealed greater than 90% homology, and the failure to restrict the PCR product with Mbo I was due to a single base change in the restriction endonuclease site. The ability of the assay to detect a wide range of MLOs with minimal sample preparation and high sensitivity will be useful in epidemiological studies of MLO-caused diseases.

Geminivirus-related extrachromosomal DNAs of the X-clade phytoplasmas share high sequence similarity

“…One group includes the EC-DNAs of the AY-clade phytoplasmas. Goodwin et al (1994) detected very high similarities of the EC-DNAs associated with the AY phytoplasma and the elm yellows phytoplasma, which belongs to a phylogenetic group not closely related to the AY-clade. A second group, which was the subject of the present study, appears to have a narrower host range, being associated only with phytoplasmas belonging to the X-clade.…”

“…EC-DNA molecules associated with phytoplasmas have been postulated to be plasmids because virus particles were not observed by electron microscopy (Kuske & Kirkpatrick, 1990;Kuske et al, 1991;Goodwin et al, 1994). Rolling circle replication (RCR)-type plasmids are widespread among Gram-positive bacteria and a plasmid of the pMV158 family (pKMK1) has been reported to occur in Mycopfasma mycoides subsp.…”

Confirmation on Status of Chaetocnema basalis (Coleoptera: Chrysomellidae) as A Vector of Stewart Wilt Disease

“…Symptomatic leaf samples were aseptically cut into 10 mm×10 mm pieces and put into NA medium, incubated over night to allow release of bacteria into the medium. The bacterial suspension was then transferred and purified on YDC, the DNA of 3-days pured cultures were then extracted using CTAB method of Goodwin et al (1994). Extracted pellet was checked to find out the presence of DNA by mixing the pellet with loading dye in 1:4 ratio, and then put into well of agarose 1% and electrophoreted (50 Volt for 40 min).…”

“…direct plant extraction and isolation of Nutrient Agar (NA) medium. DNA extraction was performed using method developed by Goodwin et al (1994). Leaf samples were washed with running water, surface sterilized with 70% ethanol for 1 min, rinsed 3 times with sterile water, and dried on sterile paper towels.…”

The detection of Melissococcus pluton in honey bees (Apis mellifera) and their products using a hemi-nested PCR

“…To perform this extraction individual samples were placed in an eppendorf tube that contained 200 mL of 2% hexadecyltrimethylammonium bromide (CTAB), (1.4 M NaCl, 1% polyvinylpyrrolidone, 0.02 M ethylenediaminetetraacetic acid, 0,1 M Tris-HCl, pH 8.0) and incubated for 5 min at 65 o C in a dry block heater (Xtron, Australia). CTAB extracts were purified using chloroform:isoamyl alcohol (24:1, v/v) (Goodwin et al, 1994). The nucleic acids were precipitated with 25 mL of sodium acetate (3 M, pH 5.2) and 400 mL of ice-cold ethanol (100%).…”