Pathogen Strains, Disease Ecology and Chemotherapy of Citrus Huanglongbing
Date Issued
2007
Date
2007
Author(s)
Tsai, Chia-Hsin
DOI
zh-TW
Abstract
(Huanglongbing = HLB) has been causing great damages to citrus industry in Taiwan since it first occurred in northern Taiwan in 1951. This psyllid-borne virus-like disease also known as Likubin, was proven to be caused by Candidatus Liberibacter asiaticus, an unculturable phloem-limited bacterium. In the previous time, this pathogen infected main citrus cultivars in Taiwan such as Ponkan mandarins, Tankan tangors and Liucheng sweet oranges whereas it did not infect pummelos before 1971. However, HLB has been able to infect all important citrus cultivars in the Southeast Asia countries including pummelos in the rent years. It implied that the evolution of pathogenicity occurred in these decades. This thesis was amed at differentiating the strains of HLB bacteria (HLBB) through the bioassays with four indicator hosts. Based on their different pathogenicity and virulence, four strains of HLBB were differtiated. The strain I showed pathogenicity on mandarins and sweet oranges by inducing typical HLB symptoms. Strain Ⅱ showed pathogenicity and high virulence on the all differential cultivars and multiplied fast in the all cultivars. Strain Ⅲ caused intermediate symptoms on mandarins and sweet oranges and mild symptoms on pummelos, and did not infect Eureka lemons. The strain Ⅳ is a mild strain, which could infecte mandarins and sweet oranges without symptoms. Strain Ⅱ commonly infected all citrus cultivars with wild distribution in Taiwan, and dominated over the strains Ⅲ and I in the field.
The cultivated citrus trees infected by HLBB were commonly discovered in the field of Taiwan. Many citrus trees were infected simultaneously with HLBB, Citrus tristeza virus (CTV) and Citrus tatter leaf virus (CTLV). In the field survey, about 30% of the samples of citrus trees showing yellows symptoms were infected by HLB over the island. Eureka lemon samples collected from Pingtung area had a high percentage of HLB-infection at 52.8%. Approximately 40% of the HLBB-infected lemons were co-infected with CTLV and none CTV. Yellows pummelo samples collected from Yunlin and Hualien counties were infected at 30~45% by HLB and approximately 40% of HLBB-infected pummelos were co-infected with CTLV. Among HLBB-infected pummelo samples including Wentan, Peiyu, and Kao Pan, only 3~8% trees were co-infected by CTV. In HLBB-infected grapefruits and Minneola tangelos, 90% of samples were co-infection with CTV. Approximately 10% of yellows samples of kumquat collected from Yilan county were examined positive of HLBB, and 60% among the HLBB-infected kumquats were co-infected with CTV or CTLV. Therefore, HLB infection and complex infection with the viruses were significantly different among citrus cultivars. In latent infection of HLBB, only 1.9% healthy-looking pummelos and lemons were infected by HLBB. In general, HLBB-infection was closely correlated with yellowing symptoms.
The studies on effect of CTV/CTLV on revealed that development of symptoms caused by HLBB, Mix-infection of the two pathogens (HLBB+CTV or HBB+CTLV) had a synergistic effect to induce most severe leaf symptoms and dwarfing. The interaction betwen HLBB and CTV/CTLV were found that HLBB interfered with the replication of CTV, but not CTLV.
In the study of resistance/susceptibility of citrus cultivars to Huanglongbing disease, the seedlings of Sunki rootstock grafted with voriou virus-free scions graft-inoculated with HLBB-infected buds, Ponkan and Tonkan were the most susceptible for HLB produced most severe symptoms. Valenica sweet orange showed moderate susceptibility, and Wentan, Eureka lemon and kumquat were tolerant cultivars. According the disease index, the rootstock cultivars were categorized into 4 type including (1) susceptible type: Sunki, Calamondin and Kau Tou sour orange (C. aurantium) rootstocks were susceptible for HLB by producing mild HLB-symptoms and PCR detection 4 months after inoculation; (2) moderate tolerant type: Volkamer Lemon, Rangpur Lime and Rough Lemon. Citrus macrophylla and severinia boxifolia showing mild chlorosis six month later; (3) highly tolerant: Troyer citrange, Swingle citrumelo showing mild stunt and low PCR signal; (4) resistant: woody apple (Limonia acidissima), no symptom and PCR signal.
The distribution of HLBB in infected trees of pommelo, tangor and sweet orange in the field. The population of HLBB was most highly in the fruit; mature leaves came to the second; roots and young leaves harbored less; the bark of trunk was found no HLBB. Seasonal dynamic of HLBB population was high in late spring (April to May) and autumn (September and October), and low population in hot summer and cold winter. In general, the highest transmission rate of HLBB by vector psyllides was laid in spring (April and May), and increased at double times yearly. The pathogen-free citrus seedlings transplanted in an orchards near by HLB-diseased Eureka lemon orchard became infected by HLBB at 1.9％ in the first year, starting from the trees grown in edge of new orchard close to the inoculum source. The transmission rate increased to 5.5％ by the end of second year, and 12.7％ after the 3rd year coming to log phase. The HLB transmission by psyllid in PF-citrus trees revealed S curve in gathering pattein. In order to facilitate in disease management and IPM of psyllid, on iodine kit for HLBB detection with 80％ accuracy was developed. The standardized method was as follows: scratch the upper surface of disease leaf with a piece of sand paper (160~200C), then put the sand-paper piece, into a small PE bug, add 1 ml of pure water and rub the sand-paper piece in water for washing off the tissue debris into water. Finally a drop of iodine solution (KI, 2.5％＋I2, 1.25％) was dropping into the suspension in bug for coloring reaction with starch color-turning to purple black within one minute showed positive of HLBB infection. This method was cheap and simple, so that it is easy to be used by farmers.
The real-time PCR and simple method of Nucleic acid extraction were applied to improvement of PCR method for HLBB detection: (1) Simplified extraction of HLBB-DNA with two buffer solation was adopted successfully to detect HLBB by short time and cost. (2) Real-time PCR was used for quntily detection of HLBB content in citrus plants i.e. highest content of HLB in most susceptible Ponkan tree and lower cont in Tankan, Valencia and Wentan Pummelo in decending order, and lowest content in Eureka lemon resistant to the pathogen.
In order to improve the current transfusion method of tetracycline for chemotherapy of HLB since 1972 (Su and Leu), infusion devise was designed for screening antifiotics, and transfusion trials of antibiotic formula and application numbers in the field were made. Three applications (Autum 2, Spring 1) of 1000 ppm achromycin with air-pressured injector at 80 lbs. showed best curing efficacy of disease pummelo trees. No occurrence of HLB appeared in the recovered pummel trees which produced nomal fruits with good qulity. Pruning upper die-back improved the therapeutic efficacy of treated trees. This improved therapy procedure caused temporary phytotoxity including mild vein browning, slender leaves, leaf scoaching and defotiation soon followed by nomal growth.
Subjects
柑橘黃龍病
病原性
柑橘黃龍病菌系統
抗生素治療
柑橘品種
Huanglongbing
pathogenicity
HLBB strains
chemotherapy
citrus cultivars
Type
other
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