Tuwo Mustika, Kuswinanti Tutik, Nasruddin Andi, Tambaru Elis
Doctoral Program of Agricultural Science, Graduate School, Universitas Hasanuddin, Makassar 90245, South Sulawesi, Indonesia; Department of Biology, Faculty of Mathematics and Natural Science, Universitas Hasanuddin, Makassar 90245, South Sulawesi, Indonesia.
Department of Plant Pest and Disease, Faculty of Agriculture, Universitas Hasanuddin, Makassar 90245, South Sulawesi, Indonesia.
J Genet Eng Biotechnol. 2024 Mar;22(1):100332. doi: 10.1016/j.jgeb.2023.100332. Epub 2024 Feb 27.
The citrus vein phloem degeneration (CVPD) disease is one of important diseases that infects citrus plants and threatens global citrus production and quality due to its severe symptoms and rapid spread. In the 2000s, South Sulawesi Province as one of the citrus producers in Indonesia reported CVPD infection. To date, it is still uncertain as to whether the citrus production center has already been rid of the CVPD infection, keeping in mind the low prevalence of certified citrus saplings use and sub-optimal management of plantations by farmers.
Field observation results revealed varied chlorosis symptoms from young to productive cultivation, which certainly makes it appealing to find out the presence of the causative bacterium, as it has yet to be known whether all the leaves with positive chlorosis symptoms carry the bacterium Candidatus Liberibacter asiaticus. Citrus saplings that appear healthy may carry CVPD pathogens as the incubation period of CVPD pathogens in the host plant spans three to five months. Thus, it is necessary to find the right, rapid way to detect the presence of CVPD pathogens in the citrus plant. The most effective method to use is PCR as the bacterium Candidatus L. asiaticus is non-culturable in vitro, but it is detectable using 16S rDNA. Sampling of leaves with CVPD symptoms was conducted purposively from eight varieties in five citrus cultivation locations, i.e., Pangkep, Sidrap, Bantaeng, Luwu Utara, and Kepulauan Selayar Regencies. DNA isolation was carried out following the Genomic DNA Kit (Geneaid) procedure, followed by detection using the specific pathogenic primer pair OI1 (5' GCG CGT ATG CAA TAC GAG CGG C 3') and OI2c (5' GCC TCG CGA CTT CGC AAC CCA T 3').
The PCR visualization result shows seven positive samples with DNA fragments measuring 1160 bp. The seven samples were samples of the Key lime, tangerine, Mandarin (cv. batu 55), and Mandarin (cv. selayar), each being derived from Sidrap, Luwu Utara, and Bantaeng. The average disease incidence rate was 66.56 %. Based on the field observation results, the insect vector Diaphorina citri was nowhere to be found in the five citrus cultivation locations in South Sulawesi.
柑橘静脉韧皮部退化（CVPD）病是感染柑橘类植物的重要病害之一，因其症状严重且传播迅速，威胁着全球柑橘生产和质量。在21世纪，印度尼西亚的柑橘生产省份之一南苏拉威西省报告了CVPD感染情况。鉴于认证柑橘树苗使用率低以及农民对种植园管理欠佳，目前仍不确定柑橘生产中心是否已摆脱CVPD感染。
实地观察结果显示，从幼龄到盛产期的种植都出现了不同程度的黄化症状，这无疑促使人们去探寻致病细菌的存在情况，因为目前尚不清楚所有出现阳性黄化症状的叶片是否都携带亚洲韧皮杆菌。看似健康的柑橘树苗可能携带CVPD病原体，因为CVPD病原体在寄主植物中的潜伏期为三到五个月。因此，有必要找到合适、快速的方法来检测柑橘植物中CVPD病原体的存在。最有效的方法是聚合酶链反应（PCR），因为亚洲韧皮杆菌在体外不可培养，但可通过16S核糖体DNA进行检测。从南苏拉威西省五个柑橘种植地点（即庞克普、锡德拉普、班唐、北卢武和塞拉亚尔群岛摄政区）的八个品种中，有针对性地采集了出现CVPD症状的叶片样本。按照基因组DNA试剂盒（Geneaid）的步骤进行DNA提取，随后使用特异性致病引物对OI1（5' GCG CGT ATG CAA TAC GAG CGG C 3'）和OI2c（5' GCC TCG CGA CTT CGC AAC CCA T 3'）进行检测。
PCR可视化结果显示有七个阳性样本，其DNA片段大小为1160 bp。这七个样本分别是绿檬、橘子、普通话（cv. batu 55）和普通话（cv. selayar）的样本，分别来自锡德拉普、北卢武和班唐。平均发病率为66.56%。根据实地观察结果，在南苏拉威西省的五个柑橘种植地点均未发现昆虫传播媒介柑橘木虱。
