Quantitative and Qualification Detection of Candidatus Liberibacter asiaticus in Citrus Hosts and the Asian Citrus Psyllid (Diaphorina citri Kuwayama)
Date Issued
2009
Date
2009
Author(s)
Tien, Hsing-Ju
Abstract
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.
Subjects
Citrus huanglongbing
citrus greening
HLB
Real-time PCR
Diaphorina citri
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