Basic information on the evolution of HLB strains in terms of pathogenicity and disease resistance is of primary importance in formulating adequate measures for controlling the HLB disease. This two-year FFTC/NIFTS international collaboration project focused on the investigation of pathological and epidemiological natures of HLB pathogen strains in Taiwan and in other countries in the ASPAC region. Under this collaborative project, the following researches were performed at the Virus Laboratory of the Department of Plant Pathology, National Taiwan University (NTU):
In the preliminary test, Taiwan cultivars of Murraya paniculata showed immunity to HLB. Most HLB isolates collected from important citrus cultivars such as Ponkan mandarin, Tungkan tangor, Liuchen (LC) orange, and Wentan pummelo (WT) were identified to belong to strain II. Meanwhile, the wild strain I, which causes typical greening symptom on mandarin and orange seedling, was seldom found. This indicates that strain I has evolved into strain II, which has dominated over the citrus areas all over Taiwan.
A survey was also conducted in the citrus growing areas of northern (Hai Phong, Hanoi and Ha Tay provinces) and central (Ha Tinh, Nghe An and Nam Dinh provinces) of Vietnam. Dried citrus samples collected were confirmed to have HLB infection by PCR analysis. The HLB DNA was prepared for molecular characterization of HLB strains. It was assumed that as in Taiwan, strain II attacking mandarin and pummelo plants has evolved and dominated in the north and central Vietnam. All samples collected from Vietnam were indexed to be free from citrus tatter leaf virus, while citrus tristeza virus was found commonly infecting mandarin and orange trees.
In the second year project implementation, a survey team visited several citrus-growing areas in Vietnam and Cambodia to gather citrus samples for disease indexing, which will serve as benchmark information in developing integrated pest management (IPM) to control the citrus HLB problem.
Vietnam. The survey team visited the Plant Protection Research Institute (PPRI) in Hanoi to get a better understanding of the institute's programs on pathogen-free (PF) citrus foundation and seedling nursery. The number of insect-proof screenhouses has increased considerably from the initial citrus foundation screenhouse donated by FFTC/RDF in 1996, in order to meet the rapid increase in demand for PF seedling production in northern Vietnam.
Collection of additional major cultivars such as red mandarin, king mandarin, Xadoi sweet orange, Boe Gien, and Phuc-trach pummelo has been done through shoot-tip micrografting. The conservation of PF citrus germplasm has been managed well, while extension and cooperation with other institutes and with all citrus industry stakeholders in the area has been intensified.
The survey team also visited citrus growing areas in northern Vietnam, as well as the Van Giang Experimental Station of Agricultural Genetic Institute. A suvey of the institute's experimental areas showed that common infection of HLB (greening) was present on citrus trees of Xa Doi sweet orange and Cam Canh mandarin. Several trees showing typical HLB symptoms were found to be infected with HLB pathogen. The establishment of pathogen-free nursery system is of primary importance in controlling the heavy incidence of HLB infection.
The samples collected from the citrus orchards of Van Giang Station were subjected to HLB and virus indexing using ELISA, PCR, RT-PCR and iodine test at the Virology Laboratory of NTU. HLB pathogen was detected in all citrus cultivars with greening symptoms by PCR and iodine tests. CTV was commonly detected in Xa Doi sweet orange and Cam Canh mandarin trees in the ELISA test. Citrus tatter leaf virus was also detected by RT-PCR in samples of Xa Doi sweet orange and Cam Canh mandarin.
In southern Vietnam, the survey team visited the Southern Fruit Research Institute (SOFRI). Intercropping citrus with guava trees was found to be a promising measure in controlling HLB infection in recent field trials at the institute. It was observed that only a few incidences of HLB infection occurred in the king mandarin test orchard intercropped with guava trees, possibly due to the latter's insecticidal properties against vector psyllids in addition to serving as effective barrier for psyllid migration.
Some pummelo trees growing near the test orchard showed some HLB symptom, which was confirmed by using the iodine test. Further test of the collected samples were done in NTU by PCR test in NTU, which proved positive as well. While no HLB infection was detected on King mandarin tree by iodine and PCR tests, CTV and CTLV were found to be present. Further studies were recommended on the effectiveness of guava intercropping as a preventive measure for HLB and other virus diseases.
Cambodia. The survey team donated some CTV/HLB diagnostic reagents and iodine kits for rapid diagnosis of HLB and CTV to the Royal University of Agriculture (RUA). During the discussion on the progress of collaboration between RUA and FFTC, the need to establish an indexing laboratory was emphasized. Construction of an insect-proof screenhouse is expected to be completed soon for the production of pathogen-free citrus foundation seedlings for field demonstration by the end of the year. Researchers will also be trained on graft techniques for rapid propagation of PF citrus seedlings, and techniques for rapid diagnosis of CTV and HLB detection.
In a survey of some citrus nurseries near Phenom Penh, some pummelo seedlings showed HLB or chlorosis symptoms. Leaf samples were collected from the nursery for disease indexing. Mexican lime trees grown in the RUA campus were found to exhibit mottling symptoms. The citrus samples collected were subjected to iodine test in the RUA laboratory, and some dried materials were brought back to NTU for PCR and ELISA tests. Two samples of pummelo and king mandarin with mottle symptom were found to be infected with HLB by using the iodine and PCR tests. This indicates that HLB has been commonly spreading via infected citrus seedlings in Cambodia.
This two-year (April 2005 - March 2007) special project was carried out in Taiwan ROC, Japan, Vietnam, and other southeast Asian countries.
Co-sponsor: National Institute of Fruit Tree Science (NIFTS), Japan
Figure 1 Visit to the Pathogen-Free Citrus Foundation Screenhouse at Ppri, Hanoi, Vietnam Donated by FFTC/RDF in 1996.
Figure 2 FFTC Consultant and Project Coordinator Prof. Hong-Ji Su Demonstrates HLB Rapid Detection Using Iodine Test Kit to Ppri Laboratory Staff.
Figure 3 Positive Reaction of HLB-Infected Leaf of Xa Doi Tree Using the Iodine Test Kit at the Van Giang Experiment Station in Northern Vietnam.
Figure 4 The Survey Team Visited the Guava-Intercropped King Mandarin Orchard of Sofri, Southern Vietnam.
Figure 5 Laboratory Exercise on Grafting Technique for the Rapid Propagation of Pathogen-Free Seedlings at Rua, Cambodia.
Download the PDF. of this document, 300,774 bytes (294 KB).