Detection of Huanglongbing (Citrus Greening) Disease by Nucleic Acid Spot Hybridization

Polymerase chain reaction (PCR) amplification with primers specific to the rDNA region successfully amplified the 1160-bp DNA fragment from a Huanglongbing (HLB)-infected sweet orange sample with mottling symptoms leaves, but not from healthy sweet orange plants. The PCR product of 1160-bp was used as probe labeled with biotin for detection of the HLB pathogen in the nucleic acid spot hybridization (NASH) test. It was found that the HLB pathogen could be detected up to 1:100 dilution in HLB-infected tissue. Total DNA extracted from HLB-infected tissue was diluted 2-fold as 900 ng in TE buffer and spotted on a nitrocellulose membrane. Strong signals were observed up to 225 ng of DNA dilution, whereas a moderate signal was recorded at 112 ng. No hybridization signal was observed in the healthy samples, while strong signals were observed in the positive control

Management of Whiteflies, Bugs, and Leafminers on Fresh Market Tomatoes, Fall 1996

Transplants were set 4 Sep, 18 inches apart on raised beds on EauGallie fine sand covered with black polyethylene mulch. Plots were 3-21 -ft-long rows on 5-ft centers and were irrigated by a seepage subirrigation system. Insecticidal treatments were replicated 4 times in a RCB design. Spray treatments were applied with a high-clearance, self-propelled sprayer on 13, 18, 23, 30 Sept, 9, 14, 21, 28 Oct, 4, 18, 25 Nov, 2 and 9 Dec. The sprayer was operated at 200 psi and 3.4 mph and was outfitted with orange Albuz™ ceramic nozzles. The number of nozzles per row was increased from 4 to 8 to increase gallonage as the plants grew. Thus, 60 gpa was applied for the first six sprays (four nozzles), 90 gpa was applied for the seventh spray (six nozzles) and 120 gpa was applied for the remaining six sprays (eight nozzles). Admire treatments were applied in 4 oz water/plant on 4 Sep. All plots were sprayed weekly with Bacillus thuringiensis for control of army worm larvae. The terminal leaflet was collected from the 7-8th leaf from the top of one branch of each of 10 plants in the middle row of each plot on 30 Oct, 22 Nov, and 16 Dec. Numbers of crawlers, sessile nymphs, and pupae of SLWF were counted and the data were averaged over all dates for analysis. All of the plants in the two outer rows of each plot were examined weekly for symptoms of virus. There are two viruses present which have nearly indistinguishable symptoms: tomato mottle virus (ToMoV), a geminivirus vectored by the SLWF, and potato virus Y (PVY), a potyvirus vectored by several species of aphids. All red ripe fruits were harvested from the middle 10 plants of the middle row of each plot on 12 Oct, 20 Nov, 4 and 17 Dec. The fruits were separated by the presence or absence of thrips and damage by the bugs SGS + LF. These fruits were counted and weighed. Each fruit was also rated 1-4 for increasing severity of irregular ripening (IRR), a disorder caused by SLWF feeding. The numbers of leafmines by LM were counted during a 2-min search of the middle row of each plot on 17 Dec. At the end of the experiment, a sample of three terminal leaflets was collected from each plant with ToMoV symptoms in the checks. The samples were frozen for later determination of the presence of ToMoV using nucleic acid spot hybridization and the presence of PVY using ELIS A.