A field study of the efficacy of PVC film cover in controlling Huanglongbing

Abstract: 【Objective】Huanglongbing(HLB) is one of the most destructive diseases in citrus worldwide.No effective control measures against the causal pathogen- Candidatus Liberibacter asiaticus(Las) or resistant(or tolerant) citrus cultivars are available so far. Current HLB control strategies focus on controlling the vector insect. We demonstrated that heat treatment could significantly reduce Las population in HLBaffected plants under laboratory condition and that humidity was one of the most important factors affecting the efficacy of heat treatment in the field. The objective of this study was to test the efficacy of heat treatment under field conditions for HLB control.【Methods】The study was carried out during August and September, 2012 and 2013, in Shunchang County, Fujian Province. Forty 5-to-8-year-old Las-positive Citrus reticulate trees, confirmed by nested polymerase chain reaction(PCR), were subject to the heat treatment each year. The plants were randomly divided into two groups- the treated group and the untreat-ed control, with five plants per replicate(rep) and four reps per treatment. The treated plants were completely covered by polyvinyl chloride(PVC) film from 10:00 to 17:00. The PVC film cover was supported by two crossing bamboo poles. The top of the cover was about 30-50 cm above the canopy. Ten holes of 2cm in diameter were made on the top of each cover. The plants were treated three times at intervals in each year. The plants in the control group were not covered by PVC film. Three HOBO temperature sensors(U23-001) were separately placed 1.25 m above the ground on the north side of the trunk of three trees(i.e. one sensor/tree), and another three sensors attached to three untreated control trees in the same way. The sensors recorded temperature data every five min. HLB symptom evaluation was made prior to treatment, 30 days after heat treatment(DAT) and 90 DAT. A total of 20 plants, ten from the treated group and ten from the untreated group, were used for the evaluation. Ten mature leaves were randomly selected from each tested plants for recording disease severity and calculating disease index. Leaf samples were washed with tap water and air dried. Leaf midribs(approximately 400 mg) were obtained, cut into segments of 1-2 mm, and ground with a mill after freeze drying. Total genomic DNA was extracted by CTAB(Cetyltrimethyl ammonium bromide) extraction method. The yield and purity of DNA samples were estimated by measuring OD260 nm/280 nm(optical density) and OD260 nm/230 nmwith a spectrophotometer. The DNA was used as a template for PCR assays after the concentrations were adjusted to 50 mg·L-1. The Las titer was detected by quantitative real-time PCR(QRT-PCR).【Results】The result indicated that the minimum and average temperatures inside the PVC cover(the treated group) were 39.0 and 40.4, 44.8 and 45.2 ℃in 2012 and 2013, respectively. The minimum temperatures were higher than that effective to inhibit or kill Las. The average temperatures of the treated group were 16.7 and 16.6 ℃ higher than the untreated group in 2012 and 2013, respectively. HLB disease symptoms in the treated group were significantly mitigated in both years. New flushes were abundant 90 DAT, bearing healthy green leaves without typical HLB symptoms such as yellowing or mottles. Disease index was reduced significantly from 67.00 and68.33 to 34.00 and 33.33 at 90 DAT in 2012 and 2013, respectively. HLB symptoms became more severe in the untreated control plants. HLB disease index at 90 DAT increased significantly from 64.33 and65.67 to 89.00 and 87.00 in 2012 and 2013, respectively. The Las titer was decreased by 71.77 and49.22% at 30 DAT in the treated group in 2012 and 2013, respectively, and Las titer in 19 plants was decreased by more than 90%. The mean Ct value for the treated plants before treatment was 26.75 and 26.53 in 2012 and 2013, respectively. They were changed into 30.84 and 31.40 at 90 DAT. The Las titer had been decreased by 85.36 and 81.67% by 90 DAT in 2012 and 2013, respectively, with a total of 27 plants having a Las reduction of more than 90%. The mean Ct value for the control plants before treatment was28.86 and 28.85 in 2012 and 2013, respectively, and changed into 25.32 and 25.68 at 90 DAT. The Las titer had increased 22.86 and 25.50 times by 90 DAT in 2012 and 2013, respectively, with the highest reduction of 95 folds.【Conclusion】The result of this study demonstrated that PVC film cover can significantly reduce Las titer in affected plants. HLB disease symptoms were also dramatically reduced by the treatment of PVC film covering, which provides an effective option for HLB disease management.