臺灣柑橘黃龍病之病菌原噬菌體類型、罹病率及複合感染柑橘萎縮病之現況調查

Abstract

柑橘黃龍病 (Citrus huanglongbing, HLB) 由Candidatus Liberibacter asiaticus (LAS) 引起，全球多數柑橘產區都遭受其巨大危害。此病在臺灣自1951年發現以來已經危害超過60年，田間發病普遍且常複合感染柑橘萎縮病毒 (Citrus tristeza virus, CTV)，造成柑桔產業莫大危害。由於LAS目前尚未能人工方式純培養，目前僅能以生物檢定法或分子資訊分析來推測其病菌系統和菌株分化；近年來其基因體中的原噬菌體基因備受關注，許多研究利用此基因的序列差異來探討LAS遺傳多樣性及菌系演化。本研究首次進行臺灣LAS原噬菌體類型之調查，探討原噬菌體的種類與分布特性，及其和柑橘品種、HLB病徵表現的關係，並輔以HLB及CTV田間發病情形之分析，以獲取嶄新且深入的疫情資訊，供防治策略擬定時之參考。本論文利用PCR及RT-PCR分子檢測技術為基礎，隨機採自全台27產區、115座果園的880個樣本，感染LAS「原噬菌體基因型第一型 (Type1, T1)」之情形最為優勢 (佔39.7%)、且在全台皆有出現；感染LAS「原噬菌體基因體第二型 (Type2, T2)」為10.3%、且發現越往南部之比例越高；兩型複合感染 (T1+T2) 占10.7%，同樣在南部較多；另各地皆有未檢出任一型原噬菌體之樣本約39.0%。比較各原噬菌體類型與HLB病徵的關係，推測T1的存在與HLB病徵表現可能相關，感染T1之LAS能被採集到較高比例具嚴重病徵的葉片；帶T1+T2的LAS與CTV複合感染的比例最低。品種方面，T1廣泛發現於多數品種，T2較多在柚類相關品種及甜橙上，T1+T2則集中柚類品種。整個調查結果顯示田間黃龍病 (HLB) 罹病率為31.9%、其中以柚類平均罹病率最高；萎縮病 (CTV) 罹病率42.4%，在寬皮柑雜交種及蜜柚、金柑、四季橘、甜橙類等皆有高罹病率；二病複合感染率14.4%、以蜜柚和桶柑最為明顯；地區分布上以北回歸線為界，有南部好發黃龍病、北部好發萎縮病的趨勢。此為臺灣第一份柑橘黃龍病菌原噬菌體基因類型與分布之研究，也是臺灣田間柑橘最新的HLB與CTV之疫情調查，期望能透過田間資料的建立，提供未來臺灣之LAS病菌系統及菌株演化研究參考，並協助HLB及CTV的防疫工作。

Citrus huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (LAS) is a destructive disease of citrus worldwide. It has already impacted the citrus industry in Taiwan for more than 60 years since 1951. Citrus tristeza virus (CTV) and HLB usually co-infect citrus plants in the field and lead more tremendous disaster. LAS has not been successfully cultured in vitro, bioassays and molecular analysis were frequently used to investigate strain-differentiation and pathogen diversity. The genomic sequences located in the prophage gene of LAS were recently selected for the categorization of LAS. This thesis is dedicated to investigate the genomic types of prophage of LAS in Taiwan and analyze the relationship among genomic types, citrus cultivars and the symptoms. Another target in this thesis is to survey the morbidity of HLB and CTV in the field of Taiwan. Totally 880 samples collected from 27 townships or cities were detected by the PCR assays for LAS and the RT-PCR assays for CTV. The results showed that T1 (Genomic type 1 of prophage gene of LAS) were detected in 39.7% of samples whereas T2 (Genomic type 2 of prophage gene of LAS) were detected in 10.3% of samples. The T2-positive samples collected from southern regions are more than those from northern regions. T1+T2 co-infection of LAS was 10.7% with a higher percentage in the southern region. The LAS-infected samples beyond T1 and T2 were 39.0%. The data of relation between the prophage types and the HLB symptoms indicated that T1 might be correlated to the symptom expression of HLB. T1 seemed to cause symptoms and the lower morbidity of co-infection with CTV. The results also showed that T1 were widely found in most citrus cultivars; T2 were more in the pummelo and sweet orange; and T1+T2 mostly appeared in pummelo. The infectious percentages of HLB, CTV and HLB+CTV were 31.9%, 42.4% and 14.4%. The pummelo presented higher infectious percentages of HLB. The mandarin, sweet orange and kumquat had higher ingectious percentages of CTV. Honey pomelo and tankan showed higher morbidities of HLB+CTV co-infection. More HLB-infected samples were collected from southern Taiwan (the tropical area), while more CTV-infected samples were collected from northern Taiwan (the subtropical area). This is the first study to survey the genomic types of prophage gene of LAS and their distribution in Taiwan. It also provided the update data associated with morbidities of HLB and CTV in the field of Taiwan. These data should be helpful to some information associated with the categorization and differentiation of the LAS strains in the future.