LAMP Detection Of Bacterial Canker Of Tomato Pathogen And Screening Of Antagonistic Strains

Bacterial canker caused by Clavibacter michiganensis subsp. Michiganensis(Cmm) is a potentially serious disease for tomato. In 1909, it was discovered in a Michigan greenhouse of America for the first time. Then it spread to most sixty tomato growing regions of the world. In China, Bacterial canker was first reported in 1954. Some documents showed that it can cause 25% ~75% loss of tomato production in Beijing, Jilin, Liaoning, Heilongjiang, Xinjiang, etc. In recent years, the epidemic of the disease is rapidly increasing, which seriously limited the development of seed production and tomato processing.This organism is introduced primarily via infected seed or transplants into plantings. Then, the pathogen spreads within plantings by splashing rain and by human activity as well. Tomato plants of all ages are susceptible to bacterial canker. This infectious disease is capable of spreading rapidly, resulting in the devastating losses. Indeed, it is a particularly difficult disease to manage. In order to control its spread in the world, many countries considered it as a quarantine pathogen. Establishing a simple, rapid, sensitive detection technique and finding a biological control are therefore important to reduce the incidence and spread of this disease.In this study, PCR and LAMP methods for detecting Cmm were established by employing three specific genes of mic A,cyt C and tom A. Several strains which have strong antagonistic effect against Cmm were screened from the soil samples surrounding the root of potato plants. The significant results were summarized as follows:1. A PCR method was established for detection of Cmm by employing three specific genes of mic A, cyt C, and tom A. The results indicated that the sensitivity of the detection method using tom A gene as test target could reach 5copy/?L or 103cfu/m L, which is more sensitive than the other two methods by employing mic A and cyt C gene, respectively.2.Using two specific genes cyt C and tom A of Cmm as detection target, two sets of LAMP primers { cyt C-49, cyt C-170 } and {tom A-107,tom A-8}as well as the corresponding loop primer were designed. The experimental results from the primer specificity and sensitivity test demonstrated that the primer tom A-107 is the best one among them. The detection sensitivity of the primer tom A-107 can achieve 5×102copy/?L for Cmm. The Cmm LAMP detection approach established in this study by using tom A gene as test target is able to fulfill a whole detection process in 50 minutes, and importantly thereof the result is visible. This approach is thus quite useful for the spot rapid diagnosis of Cmm bacterial strain.3.We successfully seperated 260 different biocontrol bacteria from collected fourteen tomato rhizosphere soil samples. By using plate confrontation method for the antagonistic screening, 35 strains were screened out. They represented strong antagonistic effect against Cmm. In light of morphology and molecular biology, these 35 strains were thus categorized into three types: Bacillus, Pseudomonas, and Streptomyces.