Survey of morbidity of citrus huanglongbing and tristeza diseases infecting several important citrus cultivars in Taiwan and improvement of their diagnostic method

Citrus is one of the most important fruit crops worldwide. Citrus huanglongbing (HLB) and Citrus Tristeza Virus (CTV) are two of the most serious and destructive diseases for citrus. Because of lacking high effective bactericides and resistant citrus varieties for the diseases, many countries mainly relies on uprooting and burning diseased trees from quarantine areas; or enhancement of quarantine regulations in non-quarantine area to prevent the invasion of both diseases.
The cultivated citrus trees infected by HLB bacteria (HLBB) were commonly discovered in the field of Taiwan. Many citrus trees were infected simultaneously with HLB bacteria and Citrus tristeza virus (CTV). In this study, we aimed at surveying of morbidity of citrus huanglongbing and tristeza diseases on several important citrus cultivars all over Taiwan and improving their diagnostic method. In the field survey, about 34% of citrus trees showing yellowing symptoms were infected by HLB , about 33% were infected by CTV and approximate 8% were co-infected with both pathogens. Sweet orange samples collected from Yunling area had a highest percentages of both HLB-infection at 71% and 79% infection for CTV. As a result, approximate 58% of the HLB bacteria-infected sweet orange were co-infected with CTV. Ponkan samples collected from Taichung had a lower percentage of HLB-infection at 4% , 33% infection for CTV and none co-infection. 38% of Eureka lemon samples collected from Pintung were infected by HLB bacteria, 46 % of pummelo samples from Tainan were infected and both of them tolerated by CTV infection. In Chiayi, the main citrus industry in Taiwan, sweet orange samples had a high percentage of CTV-infection at 88%, 33% for HLB-infection and 25% for co-infection; Ponkan samples were infected by HLB bacteria at 29%, 54% by CTV and approximate 17% for co-infection; 63% of pummelo samples were infected by HLB bacteria and none by CTV.
In this study, we aimed to improve the diagnostic method. We developed a multiplex polymerase chain reaction detection system using unique primer sets specific for Candidatus Liberibacter asiaticus (Las) and also Citrus Tristeza Virus. The two fragments ( 226 for Las and 655 for CTV) were simultaneously amplified using a single PCR reaction and Reverse Transcript-PCR. Consequently, the results of the multiplex PCR were compared with simplex PCR and RT-PCR for detection of each pathogen respectively, it matched. We reported the discovery of co-infection of Las and CTV in the field citrus plants in Pingtung, Tainan, Chiayi, Yunlin, Taichung, Taiwan. Our rapid, sensitive , specific duplex assay should be useful for diagnosis of HLB and CTV especially for quarantine purpose, identification of Las and CTV, and certification and plant improvement program for obtaining HLB-free or CTV-free planting materials. This method should be applicable for confirmation of visual method upon routine symptom.

Keywords: Candidatus Liberibacter asiaticus (Las), Citrus Tristeza Virus, Multiplex PCR. Detection, HLB, CTV