Molecular Detection Of The Quarantine Phytophthora Pathogens Of The Customs-entry Fruits And Seedlings

Phytophthora spp. is one of the most destructive diseases of fruits and seedlings. The five Phytophthora, P. syringae, P. hibernalis, P. cambivora, P. fragariae and P. fragariae var. rubi, are China entry-forbidden quarantine fungal Phytophthora pathogens. Root rot, crown rot (gummosis), brown rot of fruit and ulcer are the main symptoms in fruits and seedlings caused by several Phytophthora species. They can cause the reduction in yield, lead to no harvest and even death. And they are epidemic and destructive. The traditional identification of Phytophthora is based on morphological and biological characteristics, but they are time-consuming and need tedious steps and can not meet the requirements of rapid detection at frontier ports. Thus, it is necessary to establish synchronous molecular detection methods which are specific, accurate and sensitive and to strengthen the efforts detecting the customs-entry fruits and seedlings, protecting the safety of the fruit industry in our country.The main detection methods of Phytophthora of damaged imported fruits and seedlings are conventional PCR and real-time PCR; however, multiplex-PCR and multiplex real-time PCR for the same purpose have been less reported at home and abroad. This research concerned with the molecular detection of five quarantine Phytophthora pathogens of four host plants, Citrus, Malus Miller, Prunus and strawberry fruits and seedlings. By alignment analyses of multiple genes, namely the 18S rRNA, ITS, Heat Shock Protein 90 (HSP 90), Ras-like Protein(Ypt1), nad 9 and Enolase of Phytophthora, universal primers and five pairs of specific primers for the five quarantine Phytophthora pathogens were designed. And the molecular detection methods for the quarantine Phytophthora pathogens of the fore-mentioned fruits and seedlings were established. The main results are as follows:1. Multiplex PCR detection methods of Citrus, Malus Miller, Prunus and the varieties of strawberry fruits and seedlings were established.The universal primers and five pairs of specific primers for the five Phytophthora species were designed based on alignment analyses of the 18S rRNA, ITS, HSP 90, Ypt 1 and nad 9 genes and using Primer Premier 5.0. Through the optimization of the reaction conditions, multiplex-PCR molecular detection methods for the quarantine Phytophthora pathogens of the four fruits and seedlings were established. By this method, the simulated carrier detection successfully detected the target bands simultaneously. There was no band amplified with negative control. The amplification products can be readily visualized by agarose gel electrophoresis and EB staining.2. Multiplex real-time PCR detection methods for the quarantine Phytophthora pathogens of Citrus, Malus Miller and Prunus fruits and seedlings were established.By alignment analyses of enolase, HSP 90 and Ypt 1 genes and using Prime Primer 3.0., three probes and three pairs of primers for P. hibernalis, P. syringae and P. cambivora were designed respectively. Through the optimization of the reaction conditions, the multiplex real-time PCR molecular detection methods for the three Phytophthora species of Citrus, Malus Miller and Prunus fruits and seedlings were established. By this method, the simulated carrier detection detected the specific PCR amplification curves simultaneously, and it showed that the multiplex real-time PCR could achieve the synchronous and specific detection of quarantine Phytophthora pathogens.3. Loop-mediated isothermal amplification (LAMP) detection methods for Phytophthora hibernalis, Phytophthora syringae, and Phytophthora cambivora were established.By using Primer Premier (https://primerexplorer.Jp/e/), three groups of LAMP primers based on Enolase, ITS and Ypt1 and for P. hibernalis, P. Syringae and P. cambivora respectively, were designed. Through the optimization of the reaction conditions, bands of DNA ladders could be observed by 2% agar gel electrophoresis in the positive samples, and the positive reaction appears as a white turbidity on visual inspection. The color of the positive sample changed from orange to light green after adding the SYBR Green I fluorescent dye, indicating that the established LAMP could achieve synchronous and specific detection of three quarantine Phytophthora pathogens.The established multiplex PCR, multiplex real time PCR and LAMP molecular detection methods in this research can be used for the direct detection of pathogens in fruits and seedlings. A single testing could be finished within 1 d. This research offers a variety of detecting methods for the surveillance at frontier ports and field monitoring of the pathogens and can be used effectively to promote the customs clearance of fruits and seedlings.