Sichuan Agricultural Department, Plant Quarantine Station, No. 4 Wuhoci Street, Chengdu, Sichuan 610041, China; e-mail: [email protected]
This paper outlines the present status of pest inspection operations in China and summarizes recent development of several rapid detection methods for invasive pests. It introduces the main speed detection techniques for particular citrus diseases. Application of these speed detection methods in China is not perfect. It is restricted by some deficiencies in technique, equipment, funding and quantity.
Phytosanitary procedures such as pest detection and identification rely on technology. With the rapid development of science, it is necessary for national plant protection organizations to keep informed and to make diagnoses more sensitive and accurate. Previous general pest detection methods frequently have limitations for the diagnosis of quarantine pests. In the past ten years, China has conducted considerable research into how to improve its diagnostic measures and methods.
Traditional determination of morphology by microscope is used mostly to detect fungi and adult insects. The morphological method may be used, for example, in the separate identification of wheat dwarf bunt (Tilletia controversa), wheat karnal bunt (T. indica) and wheat common bunt (T. tritici). On the other hand, it is hard to detect seed-borne pests using the morphological method.
Isolation culture is also used to detect fungi and bacteria in China. It is an effective method for detecting seed-borne disease or systemic infection. However, it takes a long time (more than 1 - 2 weeks).
Inoculating the indicated plant (a healthy plant that is a host for the suspected pest organism) is used mainly to identify viruses. This was the principal and time-consuming detection method used before immunological and molecular biological methods were developed. Because viruses are very important kinds of quarantine pests of young plants, effective and rapid methods for detection were urgently needed.
Examples of the development of improved pest detection methods in China include those used to identify Tobacco ring spot virus (TRSV), Prunus necrotic ring spot virus (PNRSV), Curvularia lunata and Bursaphelenchus xylophilus. Polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) form the basis of the detection methods.
TRSV is a very important alien quarantine pest in China. We reported the detection results using a reverse transcription PCR method in 1995. This method allowed direct detection of TRSV from the infected sap. Gold immunochromatography assay was developed in 2002, allowing results to be obtained within one to two minutes of testing the sap of infected plants. In the same year, a staphylococcal protein A ELISA kit was developed for detecting TRSV, in which alkaline phosphatase was conjugated with protein A.
PNRSV is a key import quarantine pest for China. Its host range is extensive and its symptoms are variable. For rapid detection of the virus from plant tissue, a pair of PNRSV-specific primers were designed for use in the reverse transcription PCR method. This proved successful: the target fragment was amplified in a sample from the infected plant, but not from healthy tissue.
Maize leaf spot, caused by Curvularia lunata, is a very important seed-borne disease which has occurred in the north of China. Research into detection of C. lunata in maize using different ELISA techniques has indicated that the sensitivity of avidin-biotin system ELISA is two to five times greater than that of indirect ELISA.
Bursaphelenchus xylophilus, the pathogen causing the destructive pine wilt disease, is a quarantine organism in many countries. It is very similar to B. mucroratus in morphology. To overcome the limitation of the traditional morphological identification of these two organisms, our study developed a PCR method using two specially designed primers. This method proved to be very practical for quarantine purposes, allowing quick determination of individual samples.
Citrus plays an important role in China. The countrys citrus production is the third highest in the world, exceeded only by that of Brazil and the United States. Citrus canker (Xanthomonas axonopodis pv. citri) and citrus huanglongbing disease (caused by the phloem-limited bacterium, Candidatus Liberibacter asiaticus) are important quarantine diseases both globally and in China. According to the current plant quarantine laws and rules of China, plants and trees that have been infected must be removed and eradicated. The economic cost is very high. The Agriculture Ministry of China and Chongqing University have developed three rapid detection kits for these two diseases. Such diagnostic methods are useful for building a citrus production industry and for establishing citrus pest free areas.
For detection of citrus canker, two kits are available. An immunological detection kit is based on traditional ELISA methods and uses a pyroxylin film, which turns blue only if the citrus canker bacterium is present. This method has proved practical in terms of its sensitivity, storage life and cost. The second method uses PCR with specially designed primers. The sample is easy to prepare using this method, sensitivity is very high and all strains of citrus canker are detected.
Another PCR kit has been specifically designed for rapid detection of citrus huanglongbing disease. It offers similar advantages to those described above.
At present, there are many studies on speed detection measures and methods in China. Few are applied, for the following main reasons:
a shortage of ideal and stable technology
There remains a gap between developing a method in the research laboratory and its practical application. However, most detecting measures and methods are being improved and studied to meet the need of quarantine in practice.
lack of funding
Because the cost of detection is very high and there is a shortage of investment, some optimum measures can not be put in practice. They are applied only in scientific research groups rather than by local users.
small lots and quantities of detecting
Because the user demand for pest detection products are often very limited (for example TRSV), the production of detection kits may decrease or even stop. This causes inconvenience for quarantine detection when some plant quarantine institutes have a need for the products.
limited technology and equipment.
PCR and ELISA are applied only in scientific research groups, academies and some provincial plant quarantine laboratories. Other laboratories do not have enough equipment and the necessary skill level. Extensive use of these detection methods will depend on the methods being simple and easy.