柑橘黃龍病菌在柑橘寄主與媒介木蝨體內的定性與定量偵測

Abstract

柑橘黃龍病(citrus huanglongbing，世界上慣稱citrus greening)，為柑橘的重要病害之一。目前發現主要分布於亞洲、非洲及美洲等地區。罹病時會導致葉片葉脈間黃化、落葉、果實轉色異常、果皮變厚等病徵，得病植株容易矮化衰弱，嚴重時死亡，使得果園產量減少、樹齡短於10年，因此造成柑橘產業的重大損害。此病由特殊的薄壁細菌所引起，目前病菌暫定的學名為Candidatus Liberibacter sp.，具韌皮部侷限性，寄生於維管束之篩管細胞，至今尚未能人工培養，主要經由接穗嫁接以及媒介昆蟲來傳播。台灣的黃龍病菌屬於亞洲種(Candidatus Liberibacter asiaticus，簡稱Las)，可藉由亞洲柑橘木蝨(Diaphorina citri Kuwayama)以永續性方式來傳播，造成此病之蔓延。由於黃龍病菌通常在柑橘寄主體內濃度低且分布不均勻，因此病菌偵測不易。目前的偵測方法中以 PCR檢測法較為敏感而廣被使用，但尚無法進行病菌定量追蹤。本論文旨應用更先進的Real-time PCR的技術去偵測柑橘寄主與柑橘木蝨體內的黃龍病菌，藉由建構定量的標準曲線去估算其寄主體內所帶有的黃龍病菌套數(copies)，達到定性兼定量偵測病菌的目標，進而有助於追蹤病菌在柑橘寄主與媒介木蝨的增殖動態，並能深入探討柑橘木蝨的帶菌能力與媒介特性。根據本論文在椪柑、柳橙、檸檬及文旦四種品種的嫁接感染試驗中，利用Real-time PCR法做病菌追蹤，發現椪柑於感染3週後即可被偵測到病菌，柳橙則約晚2週才被偵測到；但柳橙自第8週後病菌增殖速度加快，椪柑則約自第12週後增殖才加快；到第16週以後椪柑內的病菌濃度明顯超越柳橙，而兩者約在嫁接感染4個月後開始於上位葉發現黃化病徵。檸檬及文旦則是感染約7~8週後才可被偵測到，嫁接24週後帶菌量仍低且尚無出現明顯病徵。同時比較田間不同柑橘品種的黃龍病株Real-time PCR之檢測結果得知，不同柑橘寄主體內病菌含量有其明顯差異性，樣本中以茂谷柑測到的菌量最高，其次為椪柑、柳橙、四季橘以及文旦。在木蝨傳菌試驗中，發現與嫁接感染試驗的結果有所差異，木蝨傳菌三週後即可同時於椪柑及柳橙植株內偵測到，但後續的病菌增殖緩慢，至14週後病菌數量皆尚未有明顯增加的趨勢。根據木蝨成蟲族群獲菌試驗的結果發現，獲菌時間的增長並無法使族群帶菌率明顯提升，帶菌率皆分布於40~60%；單隻成蟲木蝨體內可測到約數百至數千copies的菌量。相較於田間帶菌蟲的檢測結果得知，單隻田間成蟲帶菌量遠高於成蟲獲菌試驗所得的菌量，由此可知成蟲獲菌不易，即使獲菌成功也較難增殖；進而推論田間帶菌量高的木蝨主要藉由若蟲期來獲菌，增殖至成蟲期後可達到較大量的黃龍病菌進行有效之傳播。此外，由若蟲的檢測結果得知，若蟲需至4齡以上帶菌率才會較顯著提高，且發現單隻若蟲帶菌量相較於成蟲低許多，再次證明若蟲至成蟲之發育期也是病菌重要增殖期。本論文結果顯示黃龍病菌的傳播以嫁接方式較蟲傳方式效率佳，嫁接感染者病菌增殖較快，因此防治黃龍病以建立健康種苗制度為首要。其次，在媒介昆蟲木蝨方面，應特別注意病株上寄生的若蟲，其為主要的帶菌毒蟲源，若能落實砍除病株並盡量降低木蝨族群數量，將可有效降低木蝨傳播效率，防止黃龍病的流行。

Citrus huanglongbing (HLB), also called citrus greening, is a destructive disease of citrus, which has been found in Asia, African and America. The disease is caused by a phloem-limited bacterium, tentatively named Candidatus Liberibacter sp., which can not be cultured in vitro so far. The pathogen can be transmitted by psyllids and bud-wood grafting. In Taiwan, the pathogen was categorized into Candidadus Liberibacter asiaticus (Las) and its vector is the Asia citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae). Las inhabits its host plants with low concentration and uneven distribution, the detection of Las used to be not easy. The PCR-based assay was considered to be a more reliable and adoptable method for Las detection, but it can not be applied in quantitative monitoring of Las. This thesis was dedicated to apply the real-time PCR technique for the quantitative detection of Las. This newly devised method could be also used to monitor the multiplicative fluctuation of Las in the graft-inoculation and psyllid-transmission tests and to study the relationship between psyllids and Las. In the graft-inoculation tests, the result demonstrated that the multiplicative fluctuation of Las was different among four citrus hosts such as Ponkan mandarins (PM), Liu-Cheng sweet oranges (LC), Wentan pomeloes (WP) and Eureka lemons (EL). Based on the developed real-time PCR assays, Las could be detected in PM 3 weeks post-inoculation (wpi) whereas in LC 5 wpi. The multiplicative rate of Las was significantly increased in LC 8 wpi whereas in PM 12 wpi. However, the concentration of Las in PM was higher than that in LC 16 wpi. Both PM and LC started to show yellowing symptoms on leaves 4 months after graft-inoculation. Las could be detected in EL and WP 7~8 wpi, and their concentration of Las kept low level even 24 wpi. The quantitative difference was also showed in the detection of various citrus cultivars infected by Las with the real-time PCR assay, and the results indicated the comparative concentration was Mucot＞PM＞LC＞Calamondin＞WP. Las could be detected in both PM and LC 3 wpi in the psyllid-transmission tests, but the amount of Las did not significantly increase even 14 wpi. Obviously, the graft-inoculation had a better efficiency for replication of Las than the psyllid-transmission. In the acquisition tests, the data revealed that the extension of acquisition-access time did not elevate the Las-carrying percentages (keeping a range at 40~60%) for psyllid populations. The amounts of Las were approximately hundreds to thousands of copies in individual infected adult in this acquisition test, which were less than those collected from the field. This result hints that acquisition of Las is not easy for adult psyllids, and most adults with high concentration of Las should originate from Las-carrying nymphs. The nymph tests also showed that the quantity of Las was abundant after 4-instar, and adults carried more Las than nymphs, which suggests that the nymphal stages are importantly multiplicative periods for Las in psyllids. Our results demonstrated that the transmission efficiency of Las with graft-inoculation was better than that with psyllid-inoculation. Thus, establishment of Las-free nursery foundation should be considered to be the most important measure for the control of HLB. For avoidance of vector-transmission, elimination of diseased plants and decrease of psyllid vectors in the field should be seriously conducted to prevent the production of Las-carrying psyllids and retard the spreading of HLB.