Effect of microwave heat treatment in eliminating "Candidatus Liberibacter asiaticus" on periwinkle infected with Citrus Huanglongbing

Citrus Huanglongbing (HLB) is one of the most serious bacterial diseases in the citrus industry. Starch and flavonoids are two organic materials that are associated with HLB affection. Thermotherapy can be effective in eliminating the “Candidatus Ldiberibacter asiaticus” (CLas), the putative pathogen of HLB. However, the previous thermotherapy cannot fully meet the large-scale production, due to the low efficiency, high energy consumption, or with high cost. In this study, a microwave heat treatment was proposed to control the citrus HLB. A systematic investigation was implemented to explore the inhibitory effect of microwave heat treatment on the physiological materials of HLB-affected periwinkle plants. Firstly, a batch of 30-day-old periwinkles seedlings were cultivated and then graft-inoculated in an insect-proof screen house. Secondly, a microwave thermotherapy platform was built preliminarily. A single-factor test was conducted on three microwave parameters, including the rotating speed of the turntable (RST), microwave power (MP), and number of magnetrons (NM). The parameter levels were determined after the orthogonal test, according to the degree of overheating. Thirdly, the orthogonal test of L9(3⁴) was carried out. Range and analysis of variance (ANOVA) were performed on the three indicators i.e., plant overheating degree, temperature range, and activity evaluation. The optimal combination of parameters was determined using the comprehensive balance. Finally, the optimized microwave treatment was used for thermotherapy on HLB-affected periwinkle plants. CLas titers were determined by real-time quantitative PCR. The starch and flavonoid concentrations of the leaves from the treated and untreated plants were detected within 90 days after heat treatment. The results show that: 1) The factor values for the L9(3⁴) orthogonal test were obtained after single factor testing: RST of 10, 15, and 20 r/min, MP of 150, 200, and 250 W, and NM of 2, 3, and 4. The range analysis showed that the MP was the most important influencing factor on heating, followed by NMs, and finally RSP. 2) Combined with the ANOVA, the optimal combination was determined to be MP of 150 W with 3 magnetrons, where the value range of RST was from 15 r/min. The Ct value of infected samples increased from 17.01±0.97 before treatment to 31.91±2.35 at 90 days post-treatment, with the titers of CLas decreased by 99.98% after treatment with the optimized. 3) The previously CLas-infected plants were recovered after heat treatment, compared with the untreated plants, with more new leaves and lateral branches. The contents of starch and flavonoid were gradually recovered to the levels in the healthy control plants. Comparatively, the starch content of healthy samples increased significantly on the day of heating but gradually recovered to normal level. While there was no significant difference in the total flavonoid content. The flavonoid content in the untreated HLB-affected control was lower than that of the healthy control. The flavonoid content continued to increase until died, as time went by. In conclusion, 1) most CLas in periwinkle plants were eliminated to recover the growth conditions of plants into the healthy control level after microwave heat treatment. 2) Microwave heat treatment gradually restored the starch and flavonoid contents of infected samples, compared with the healthy control and the untreated diseased plants. 3) The platform of microwave heat treatment can be effective for pot-growing periwinkle seedlings. The microwave heat treatment is also required for long-term verification with the different conditions in citrus trees in the future. 4) Microwave heat treatment performed the better inhibitory effect on CLas, with the low cost, short treatment cycle, low consumption and high efficiency. It is also expected to achieve large-scale promotion in the field.