Liu Baoyou, Li Zhiwei, Du Jianfeng, Zhang Wei, Che Xiaozhi, Zhang Ziran, Chen Ping, Wang Yingzi, Li Yang, Wang Shaoli, Ding Xinhua
State Key Laboratory of Crop Biology, Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant Protection, Shandong Agricultural University, Tai'an 271018, China.
Institute of Plant Protection and Resource and Environment, Yantai Academy of Agricultural Sciences, Yantai 265500, China.
Pathogens. 2022 Oct 23;11(11):1221. doi: 10.3390/pathogens11111221.
Apple Alternaria blotch disease, caused by (Fr.) Keissl, is one of the most famous leaf diseases. When the disease is prevalent, it causes leaf abscission and influences the formation of flower buds and photosynthesis. Therefore, a simple, rapid, high-specificity and sensitivity method for monitoring infected leaves at early developmental stages is urgently needed, so that the occurrence and expansion of can be controlled in time. In our research, a rapid, specific and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect within 60 min. Six primers of LAMP detection can only specifically amplify the gene in but not in four other important fungi in apples. The gene encodes endopolygalacturonase in , and there are significant differences among different species. Thus, it was applied as the target for LAMP primers. Compared to conventional PCR detection, our LAMP method had the same sensitivity as that of detecting as little as 1 fg of pure genomic DNA of . When leaves were inoculated with conidia, LAMP detected 1 × 10 conidia/mL as the minimum concentration. However, the traditional tissue isolation and identification method only isolated from leaves inoculated with 1 × 10 and 1 × 10 conidia/mL, indicating that the LAMP method was more sensitive than the traditional tissue isolation and identification method for before symptoms. Further tests also indicated that LAMP detection was more accurate and sensitive than the traditional tissue isolation and identification method for in leaves with the Alternaria blotch symptom collected from the field. Our results showed that the LAMP-targeting the gene has the advantages of high sensitivity, specificity and simplicity and can be used for rapid detection and early monitoring of in the field. LAMP is instructive for us to effectively prevent and control apple Alternaria blotch disease.
苹果链格孢叶斑病由（弗里斯）凯斯勒引起，是最著名的叶部病害之一。病害流行时，会导致叶片脱落，影响花芽形成和光合作用。因此，迫切需要一种简单、快速、高特异性和高灵敏度的方法来在发育早期监测感染叶片，以便及时控制苹果链格孢叶斑病的发生和扩散。在我们的研究中，开发了一种快速、特异且高效的环介导等温扩增（LAMP）方法，可在60分钟内检测苹果链格孢。LAMP检测的六条引物只能特异性扩增苹果链格孢中的苹果链格孢基因，而不能扩增苹果中其他四种重要真菌的基因。苹果链格孢基因在苹果链格孢中编码内切多聚半乳糖醛酸酶，不同物种之间存在显著差异。因此，它被用作LAMP引物的靶标。与传统PCR检测相比，我们的LAMP方法具有与检测低至1 fg苹果链格孢纯基因组DNA相同的灵敏度。当叶片接种苹果链格孢分生孢子时，LAMP检测到的最低浓度为1×10分生孢子/mL。然而，传统的组织分离和鉴定方法仅从接种1×10和1×10分生孢子/mL的叶片中分离到苹果链格孢，这表明在症状出现前，LAMP方法对苹果链格孢的检测比传统组织分离和鉴定方法更灵敏。进一步的试验还表明，对于从田间采集的有链格孢叶斑症状的叶片中的苹果链格孢，LAMP检测比传统组织分离和鉴定方法更准确、灵敏。我们的结果表明，靶向苹果链格孢基因的LAMP具有灵敏度高、特异性强和操作简单的优点，可用于田间苹果链格孢的快速检测和早期监测。LAMP对我们有效预防和控制苹果链格孢叶斑病具有指导意义。
