Microwave thermotherapy can be expected to prevent the citrus Huanglongbing (HLB) in recent years. Yet the excessively long experimental cycles can rely mainly on many seedlings during large-scale application. It is often required to accurately characterize the dynamic distribution in the internal temperature field of the citrus seedlings during microwave heating. In this study, an efficient simulation model was developed for microwave heat treatment. A dielectric inversion model was constructed and then validated for the microwave thermotherapy of citrus seedlings. Firstly, the morphological data were captured from the citrus seedlings. A parameterized three-dimensional (3D) geometric model of the seedlings was constructed using SolidWorks 2025, in order to accurately reproduce the structural features of the leaves, branches, and their topological connections. Subsequently, a coupled "microwave electromagnetic-solid heat transfer" physical field was established to simulate the microwave heating in COMSOL Multiphysics 6.3. The key dielectric properties of the citrus seedling components (leaves and branches) were determined to optimize the microwave energy absorption. The Interior Point OPTimizer (IPOPT) algorithm was also employed for the inverse optimization. The real and imaginary parts of the complex dielectric parameters were iteratively adjusted from the pre-set initial values. The better performance was achieved by minimizing the root mean square error (RMSE) between the simulated temperature curves and the experimentally measured ones. Single-component experiments also validated the reliability of the inverse optimization. In both leaves and branches, the coefficient of determination (R²) between the simulated temperatures and the measurements exceeded 98.4%, while the RMSE was below 3% of the average measured temperature, indicating a high degree of consistency between the simulation and the actual heating. The whole-plant experiments were conducted under different microwave power conditions (150, 250, 350, 450, and 550 W), in order to further verify the applicability of the model to intact citrus seedlings. Temperature monitoring points were arranged at the top, middle, and bottom of the seedlings. The dynamic temperature was then recorded during monitoring. The results showed that the high prediction accuracy was maintained in the power range of 150-450 W: the R² for all monitoring positions was ≥ 98.5%, and the RMSE was below 3% of the average measured temperature. The actual microwave heating of the whole citrus seedlings was accurately simulated in the power range. Thereby, the temperature range (48-54°C) was required for the HLB pathogen inactivation. Once the microwave power increased to 550 W, significant deviations were observed between the simulated and measured temperatures. There was a rapid rise in the internal temperature of the seedling tissues under high power. Specifically, the molecular thermal motion was intensified to shorten the polarization relaxation time, leading to the nonlinear variations in the real and imaginary parts of the dielectric constant in the current model. Fixed dielectric properties failed to fully capture during this time. The key findings were as follows: 1) The IPOPT inversion of the dielectric parameters exhibited excellent stability, with the relative variation less than 0.5% over three repeated experiments, indicating the reliable input parameters of the model. 2) The coupled multi-physics modeling significantly reduced the consumption of the experimental seedlings. Only 3 seedlings were required at the initial construction stage, where the seedling usage was reduced by more than 90%. The efficiency of parameter optimization was improved with the cost savings, compared with the conventional method. 3) A power-adaptive range (150–450 W) was defined to stably and accurately predict the temperature distribution of citrus seedlings. A precise operational window was provided for the practical application of microwave thermotherapy in HLB control. In summary, this simulation platform can accurately reproduce the dynamic microwave heating of the citrus seedlings. A resource-efficient and scalable tool can offer to optimize the parameters of HLB microwave thermotherapy. The dependency on physical seedlings can be effectively reduced to provide technical support for the green and large-scale promotion of HLB control technologies. Future research should focus on the nonlinear variation of dielectric properties under high power. The high applicability and prediction accuracy can also be required to consider the microstructural heterogeneity (e.g., vascular bundles in leaves and branches).

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.

Huanglongbing (HLB), which is mainly associated with “Candidatus Liberibacter asiaticus” (CLas), is currently threatening citrus production around the world. This destructive century-old disease results in callose deposition in phloem plasmodesmata and starch overaccumulation in leaf chloroplasts. Heat treatment with high humidity has proven to be effective in eliminating the CLas titers in potted citrus trees. This study explored the curative mechanisms and impact of heat treatment (from 32 to 48 ℃ with 75% ± 5% relative humidity, 14 h in light and 10 h in dark for four days) on mandarin trees (Citrus reticulata Blanco) that were severely infected by CLas using Solexa/Illumina's digital gene expression (DGE) profiling. A total of 838 differentially expressed genes (DEGs) were identified, of which 510 were downregulated. Protein-protein interaction analysis of the DEGs indicated that the thermotherapy of HLB trees downregulated the circadian clock related genes in chloroplasts and upregulated the RNA-editing process in the mitochondria, characterized by 141 genes encoding pentatricopeptide repeat-containing proteins (PPR) being all upregulated. Furthermore, the expression of phenylpropanoid and flavonoid biosynthesis genes, and auxin-induced genes were suppressed. The defense response pathway of the thermotherapy treated diseased trees was apparently disrupted, as indicated by the downregulation of LRR receptor-like serine/threonine-protein kinase FLS2 gene. This study improves our understanding of the curative mechanisms of heat treatment on HLB trees and CLas biological regulation. Importantly, this provides new insights on the involvement of RNA modification by PPR genes and changes in the circadian clock after the heat stress treatment of CLas-infected citrus.