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Phytophthora hibernalis, P. syringaeand P. cambivoraare devastating quarantine pathogens to many economically important plants worldwide. They often co-infect the same plant, causing rots and leading to great loss in yield and even to the death of the plant. Conventionally, the detection of the three pathogens has been based on time-consuming and laborious morphological methods and, although PCR-based detection is much better, it is still thermal cycler- and electrophoresis-dependent. A high throughput, rapid, convenient and cost-effective detection is required for quarantine detection at the port of entry. In this study, based on the novel LAMP technique, three sets of four LAMP primers, PH-F3/PH-B3/PH-FIP/PH-BIP, PS-F3/PS-B3/PS-FIP/PS-BIP and PC-F3/PC-B3/PC-FIP/PC-BIP, designed on the sequences of enolase (Enol), internal transcribed spacers (ITS) and ras-like protein Ypt1genes of P. hibernalis, P. syringaeand P. cambivora, were designed. Through the optimization of the reaction system and conditions, a highly specific and extremely sensitive LAMP detection system was developed for P. hibernalis, P. syringaeand P. cambivora. The LAMP products showed the typical DNA ladders upon agarose gel electrophoresis, the visual turbidity of the reaction solution and the white precipitate of byproduct magnesium pyrophosphate upon centrifugation were observed; the LAMP solution turned light green from orange upon the addition of SYBR Green I. No cross reactions with the other closely related Phytophthoraspecies were found, whereas specificity and sensitivity were very high, for the detection of 0.02 fg of genomic DNA was more than 105times higher than the corresponding conventional PCR. To the best of our knowledge, this represents the first detection of the three quarantine Phytophthorapathogens by LAMP, a technique with a great potential for the identification of other Phytophthorapathogens.

In order to develop a simultaneous and specific molecular detection of the three quarantine Phytophthorapathogens, Phytophthora hibernalis, Phytophthora cambivoraand Phytophthora syringaethat infect Malus Miller, three pairs of real-time PCR primers (PH-F/PH-R, PC-F/PC-R and PS-F/PS-R) and three probes (PH-Pr, PC-Pr and PS-Pr) labeled with HEX, FAM and ROX, respectively, were designed for P. hibernalis, P. cambivoraand P. syringaeby alignment analyses of enolase (Enol), ras-like protein Ypt1and HSP90gene sequences with other Phytophthoraspp. Black Hole Quencher 1 (BHQ1) was used for P. hibernalisand P. cambivoraand BHQ2 for P. syringae. Through the optimization of the reaction conditions, a triplex real-time PCR simultaneous detection for the three Phytophthoraspecies infecting Malus Millerwas developed. It could achieve simultaneous and specific detection with a sensitivity of 2 × 10−4, 2 × 10−4and 2 × 10−2ng/μL genomic DNA, respectively, for P. hibernalis, P. cambivoraand P. syringae.