In China, citrus Huanglongbing is a destructive disease associated with ‘Candidatus Liberibacter asiaticus’ (CLas), which currently threatening the sustainable development of citrus industry. Previous studies had identified three types of prophage sequences in the genome of CLas strains. Prophage group typing analysis revealed that the Type 2 prophage strains and Type 1+3 prophage strains dominated in Guangdong Province. However, the difference in propagation capacity of these two strains of CLas in Asian citri psyllid (Diaphorina citri) and pathogenicity of these two strains leading by insect transmission remains unknown.

The objective of this study is to assess the difference in proliferation capacity between Type-2-CLas and Type-1+3-CLas in D. citri and the pathogenicity variations between them on Citrus reticulata Blanco cv. Shatangju.

The buds with different strains of CLas were grafted to healthy Shatangju. Fifty nymphal and fifty adult D. citri were caged on the young shoots of infected Shatangju to acquire different strains of CLas for 6, 12, and 18 days, respectively. Real-time quantitative PCR (qPCR) was used to detect and analyze the difference in the acquisition rate and quantity of Type-2-CLas and Type-1+3-CLas between the nymphal stage and the adult stage of D. citri. Further, twenty adult D. citri infected with Type-2-CLas or Type-1+3-CLas were placed on the young shoots of healthy Shatangju to inoculate for two weeks, respectively. The D. citri were collected and detected by qPCR. The general information of infection rate and quantity of CLas was collected. Symptom development of Shatangju and titers of CLas in leaves were monitored each month. Morphological change of leaves phloem and parenchyma cell was visualized by light microscopy 360 days after inoculation.

There was no significant difference between acquisition rate and quantity of Type-2-CLas and Type-1+3-CLas detected in D. citri that fed on the infected plant at the nymphal stage. In contrast to fed as nymphs, the acquisition rate and quantity of Type-2-CLas were significantly higher than Type-1+3-CLas when D. citri fed on infected plant at the adult stage. The leaves presented more severe mottled symptom after inoculation with Type-2-CLas at 120 d after D. citri removal. The immature leaves at young shoots had an obstacle on turning green, presenting leathery, small size, and yellowing which was infected with Type-2-CLas. The symptoms on Type-1+3-CLas infected plants were presented as typical mottled leaves and leathery. Anatomical analyses indicated that Type-2-CLas caused more severe damage to phloem and accumulated more starch in parenchyma cell even if the titer of Type-1+3-CLas was closed to Type-2-CLas.

Compared to Type-1+3-CLas, Type-2-CLas was more capable of invading and proliferating in adult D. citri, accompanied by a higher titer of CLas which indirectly proved the high contagious capacity of Type-2-CLas. The young leaves infected with Type-2-CLas presented symptoms of uniform yellowing, small size, while infected with Type-1+3-CLas showed lightly mottled and leathery. Type-2-CLas destroyed more phloem cells and caused severe starch accumulation, indicating that Type-2-CLas had stronger pathogenicity to citrus than Type-1+3-CLas.

Citrus Huanglongbing (HLB) is a citrus disease caused by ‘Candidatus Liberibacter asiaticus’ (CLas). The main approaches to control HLB include plant quarantine, establishing disease-free nurseries, removing disease trees, and concentrating on large area joint control of citrus psyllids (Diaphorina citri). The first three methods all rely on accurate HLB diagnosis techniques.

The objective of this study is to establishment of a rapid and handy field/laboratory nucleic acid detection method of CLas using loop-mediated isothermal amplification (LAMP) combined with membrane adsorption rapid DNA extraction and Gelgreen fluorescence dye visualization.

The LAMP primers were designed using the β-operon and the prophage DNA polymerase gene of CLas as templates, including outer primer F3/B3, inner primer FIP/BIP, loop primer LoopF/LoopB and stem primer StemF/StemB. The LAMP primer set was optimized by setting different dosage combinations for loop primers and stem primers to determine the appropriate primer concentration. A total of 188 field citrus leaves were detected using the optimized LAMP primer set, and the receiver operating characteristic (ROC) curves were constructed to analyze the accuracy of real-time fluorescent LAMP (qLAMP) for CLas detection. The qLAMP premixed reaction solution was dried in two steps at room temperature, storage temperature (4, 25 and 35℃) and storage time (1, 2 and 4 weeks) were also set to assess the enzyme activity stability of the dry LAMP reagent. Using dry LAMP reagent, combined with membrane adsorption rapid DNA extraction technique in this study, 71 citrus leaf samples and 35 citrus fruit samples collected in the field were detected, while the detection results of real-time fluorescent quantitative (qPCR) were used as controls to compare the coincidence rates of the two detection methods.

The addition of loop primer, stem primer or increasing their concentrations in LAMP reaction could promote the increase of reaction rate, and the addition of both loop primer and stem primer at a final concentration of 1.6 μmol·L^-1 could further improve the reaction rate. The reaction activity of LAMP premix could be maintained unchanged by two-step drying at different temperatures for 1-4 weeks, indicating that the two-step drying LAMP reagent prepared in this experiment had good detection performance and fair stability at low and room temperatures, and only at 35℃ storage would slightly increase the reaction time of LAMP reagent. Using 0.1 μm pore size nylon membrane instead of cellulose filter paper as nucleic acid adsorption material could improve the sensitivity of rapid diagnostic techniques. The overall accuracy of rapid DNA diagnosis for HLB established by combining rapid DNA extraction and visual LAMP was high, and the lowest detectable plasmid concentration was 10² copies/μL. The diagnostic results of this method were not significantly different from those of qPCR by paired Chi-square test. The visual LAMP rapid detection was less cost and time-consuming than routine detection, and visual LAMP rapid detection required no expensive instruments such as centrifuges and PCR instruments, requiring only a 65℃ thermostatic device.

The rapid DNA detection method for CLas established in this study has low cost and can observe detection results in 30 min, easy to operate and high accuracy, which can replace qPCR for rapid identification of HLB in the field.