柑橘黃龍病之病菌系統演化、發病生態與 植物菌質相關性之探討

Abstract

柑橘黃龍病自1951年在台灣首次發現後至今已超過六十年，病菌在早期只感染椪柑、桶柑及柳橙，於1971年感染原本屬於抗病的柚類，由病勢發展推想在病害長年發生之下，黃龍病菌可能發生演化，出現不同病原性或毒性之病菌系統，於2005的分離株收集調查已有4種系統存在。本研究欲探討爾後黃龍病菌的繼續演化情形，採集挑選23個黃龍病菌代表分離株，經病原性和毒性的生物鑑別可得22株黃龍病菌系統II及1株系統III，系統II病菌仍然為田間優勢系統，和2005年前的調查結果相似，但田間的病菌目前已難分離到系統III和I。檢視2005年以前保留的52株黃龍病菌分離株，結果顯示以系統II最多，系統III少數存在，但系統I的黃龍病菌已分離不到。在保留的黃龍病菌中挑選11個來自不同柑橘品種的各系統代表性分離株測試病原性與毒性的差異，後代分離株有9株為系統II及2株屬系統III，依不同的毒性及潛伏期差異可將系統II再細分成強毒系統 (II/S)及弱毒系統 (II/M)。利用Tomimura等人 (2010)開發的duplex PCR偵測黃龍病菌的基因型 (genotype)，對2005年保留之系統分離株和2010-2014年採集的黃龍病菌分離株進行檢測，結果皆以genotype I為主要，genotype II少量存在，而扁實檸檬 (Citrus depressa)病株可偵測到最多數的genotype II，似為genotype之演化與柑橘品種及品系(cultivar)有關。由保存的黃龍病菌核酸，檢視東南亞等各國家的黃龍病菌基因型也有genotype II，而genotype I仍為主要。然而無法釐清黃龍病菌系統和基因型的關聯性。柚類 (C. grandis)普遍被認為是耐病品種，於11種國內外重要的柚類品種接種黃龍病菌強系統II以進行抗感性評估，結果以紅肉品種的台灣紅文旦及泰國Thong Dee為最耐病，病徵也最輕微。分析4種田間不同品種柑橘株的黃龍病菌分佈，以椪柑最罹病，病菌分佈全株並呈嚴重病徵而柳橙株的病菌分佈最不均病徵稍輕，相較之下CTV、CTLV或CEVd等病毒在柑株中的分佈均勻。柬埔寨及南越一帶原生的2種Cochin China atalantia (Atalantia citroides) (圓葉及長葉)，經人工接種及柑橘木蝨傳播試驗證實為黃龍病菌新中間寄主，屬永續性寄主 (persistent host)。木蘋果 (Feronia limonia) 之泰國、越南及柬埔寨等不同葉型品系，經HLB病菌嫁接接種証實病菌僅以微量且短暫性存在泰國種木蘋果，不久即消失，沒有成為中間寄主之潛力，屬短暫性寄主 (transient host)。以帶毒柑橘木蝨進行月橘的傳播試驗，結果顯示2種月橘 Murraya paniculata及M. exotica皆無法偵測到黃龍病菌，認為月橘為柑橘木蝨的良好寄主但非黃龍病菌之寄主，屬非寄主 (non-host)。由廣東檸檬及檸檬病株之果實種子長成的柑橘苗皆無偵測到黃龍病菌，雖其種皮中可測到病菌但不經種媒傳播。近年來多篇國際報告指出黃龍病與植物菌質體 (Candidatus Phytoplasma spp.)相關，本研究檢測多年來保存的黃龍病菌系統分離株均無菌質體混合存在，而2010-2013年採集從具有黃化斑駁病徵及無病徵之田間柑樹，共706個不同柑橘品種樣本，結果僅有5個柑株樣本偵測到植物菌質體，而其中1例由菌質體單獨感染為無病徵柚株，其餘4例皆由黄龍病菌與菌質體複合感染之病株。經16S rDNA部份序列定序命名為Taiwan citrus symptomless (TCS) phytoplasma (KJ847723)，經NCBI的資料庫比對發現TCS 菌質體與Echinacea purpurea witches' broom (EPWB) phytoplasma有100％ 序列相似度和PnWB菌質體有99.7％序列相似度，均屬於16SrII-A subgroup。以TCS菌質體為接種原進行人工接種發現多種柑株可被感染，但皆無病徵呈現。以PnWB菌質體及PLY菌質體 (16Sr I-B)經菟絲子接種於多種柑株，PnWB菌質體可感染柑橘株但不呈現病徵而PLY菌質體卻無法感染。根據以上的結果認定PnWB菌質體在田間對柑橘感染稀少且無病徵不至於為害柑樹，與黃龍病沒有相關性。利用Alkali extraction原理，以非有機溶劑之粗萃核酸萃取法用於黃龍病菌PCR的簡易檢測，可有效、簡便利用於檢疫，但其敏感度略差於一般有機溶劑萃取核酸的PCR檢測效果。

Citrus huanglongbing (HLB), locally called “Likubin” was first found in 1951 in Taiwan. The causal agent, Candidatus Liberibacter asiaticus (Las) belonging to Asian form, is a Gram-negative bacterium, phloem limited, non-culturable and has submicroscopic, pleomorphic and walled bodies inhabiting in the plant sieve tubes. In the early phase (1960s), HLB caused severely damage to several citrus cultivars such as Ponkan mandarin, Tankan tangor and Liucheng sweet orange, but not affected Wentan pummelo. Wentan pummelo became infected by HLB in 1971. It was attributed to the change of host range due to the evolution of Las strain in pathogenicity and virulence. The 23 representative Las isolates without co-infection with the citrus viruses were isolated from the field in recent years for strain identification by pathogenicity assay. Strain II (S-II) which attacked all citrus cultivars was found being the dominating strain and occupying highest population in 22/23 (96％). Strain III was recovered seldom in 1/23 (4％) from LSO. No Strain I and IV were detected any more. On the other hand, further evolution of Las strains have been traced in serial generations of bud-grafted plants in differential citrus cultivars during 7 years since 2005 until 2012. The ultimate population of Las strains were laid as S-II in the most (96％), S-III, a fewer (4％) and non S-I surviving. By testing the pathogenicity and virulence, S-II isolates of Las were separated into II/S and II/M. The 52 Las strain isolates in Lab collection and 198 samples from HLB-affected citrus field trees were subjected to identification of genotypes by means of duplex PCR (Tomimura et al., 2010). The most isolates were identified belonging to genotype-I (G-I), some belonging to G-II and a few belonging to G-III. In field survey, G-II were commonly detected in most citrus trees of Hirami lemon (HL, C. depressa), the same as those in HLB-affected Shikuwasha trees (C. depressa) of Okinawa. The genotype II was also detected in some citrus cultivars, however, genotype I was commonly detected in the most HLB-diseased samples in Southeast Asia countries. However, the relationship between Las strains based on pathogenicity natures and genotypes based on genetic constitution, was discordant. The 11 different cultivars of pummelo were subjected resistance test with HLB-II strain by graft inoculation. The “Red Wentan” cultivar from Taiwan and “Thong Dee” cultivar from Thailand were the most tolerant cultivars with red inner meal by showing low titer of PCR detection of Las and mild symptom expression 8 months after inoculation. Las was examined uneven by distributing in four citrus cultivars while CTV, CTLV and CEVd showed quite even distribution in the citrus trees. The Cochin China atalantia plants (Atalantia citroides) locally called wild lime (WL) including elliptic leaf (WL-1) and elongated leaf (WL-2) types are growing wildly in Cambodia and Cochin China of Southern Vietnam. Through graft inoculation and psyllid transmission experiments, the Cochin China atalantia was found to be a new alternative host of Las belonging to “persistent host”. The different leaf-types of wood apple (Feronia limonia) including three types from Thailand, Vietnam and Cambodia, were subjected to graft-inoculation host with Las-II infected scion. The Las was found existing temporarily then disappearing in wood apple plants, belonging to transient host. The two kinds of jasmine orange plants including Murraya paniculata and M. exocortica were inoculated with citrus psyllids harboring Las in insect cages. The fact that no Las was detected by PCR assay during long period up to one year, concluded that jasmine orange belonging to “non-host” of Las. No Las was detected in the seedlings derived from seeds harvested from fruits born on Las-infected Rangpur lime and Eureka lemon trees. Accordingly, Las was not seed-born. In order to clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was conducted by using the standardized nested PCR assay. The HLB-diseased citrus trees with typical HLB symptoms showed a high detection of HLB-Las (89.7％, 322/359) and low detection of phytoplasma at 1.1％ (4/359) in field citrus trees including HLB-affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia, 3/53) those were co-infected with HLB-Las. The phytoplasma alone was also detected in a healthy-looking Wentan pummelo tree (1/60) at a low incidence within total examined (1/347, 0.3％). The citrus phytoplasma was nominated Taiwan citrus symptomless phytoplasma (KJ847723) belonging to PnWB phytoplasma (16SrII-A) according to sequence alignment. Peanut witches’ broom phytoplasma and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI-B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection by positive detection with the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. However, none of the phytoplasma-infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma via dodder into the healthy citrus plants demonstrated no infection. A simple PCR detection method has been developed by using crude extraction instead of complicate purification of Las-DNA. The crude method with two chemicals (250 mM NaOH and 2.5M acetic acid) and simple process is much more easily handling than the conventional method with hazardous solvents and phenol. It was adopted successfully to detect Las.