The Etiology And Molecular Detection Of A Leaf Spot On Pepper In China

The newly occurred pepper disease, pepper leaf spot, observed in greenhouse of Liaoning Province has become more and more serious in China in recent years. Because limited knowledgement is known on the etiology, pathogenic mechanism, occurrence regularity and molecular detection of the disease, the effective control strategies have not been established by now. In order to solve these problems, firstly the pathogen was identified in this study. In the aspect of etiology research, biological characteristics, pathogenicity, genetic diversity and infection characteristics were studied systematicly. Then a preliminary study on pathogenic mechanism of the pathogen was carried on. Besides, a molecular technigue for early diagnosis was established.1. The First Identification of Pepper Leaf Spot Pathogen in ChinaIn June2009, for the first time, leaf spotting was observed on pepper leaves in Wafangdian County of Liaoning Province. During2009to2012, a comprehensive survey of the occurrence and damage of pepper leaf spot was conducted in pepper cultivation areas of Liaoning Province. By tissue isolation and morphological identification,22strains of the pathogenic were collected. The pathogen was identified as Cladosporium oxysporum according to morphology and Koch’s postulates testing, in combination with rDNA-ITS, β-tubulin, Actin genes sequences data sets.2. Systematic Determination of Biological Characteristics of C. oxysporumThe results of effect factors on mycelium growth and conidium germination of the pathogen showed that the best medium for mycelium growth was V8juice. The pathogen could effectively use of sugar and nitrogen sources. Sorbitol and malt extract were used as better carbon and nitrogen sources for mycelium growth, respectively. The optimum temperature for mycelium growth was20to25℃and the optimum pH was6to8. Light could promote mycelium growth. The mycelium lethal temperature was77℃. Water was best for conidium germination without special carbon sources. But nitrogen sources seriously inhibited conidium germination. The optimum temperature for conidium germination was20to30℃and the optimum pH was7. Light had no obvious promoting effect on conidium germination. The conidium lethal temperature was48℃.3. Determination of the Pathogenicity of C. oxysporum from Different Geographic Origins in Liaoning ProvincePathogenicity was determined of C. oxysporum from different geographic origins in Liaoning Province on four pepper cultivars (Xunchi,4th Shen pepper,1st Hang pepper and New golden horn) by artifical inoculation. The results showed22strains could be classified into three pathogenic types, which were strong pathogenic type, moderate pathogenic type and weak pathogenic type. The C. oxysporum strains showed significant difference in pathogenicity, while there was no significant correlation between the pathogenic types of strains and their geographic origins. The strains from the same geographic origin also exhibited different levels of pathogenicity.4. Aanlysis of the Genetic Diversity of C. oxysporumIn order to explore heredity variation and evolution of the pathogen on molecular level, genetic diversity of all C. oxysporum strains was analyzed by ISSR and SRAP technique. The phylogenetic tree based on ISSR markers revealed that similarity coefficient of22strains was from0.56to0.93, and the strains were divided into four clusters at the threshold of genetic similar coefficient0.65by UPGMA. The phylogenetic tree based on SRAP markers revealed that similarity coefficient of22strains was from0.59to0.90, and the strains were divided into three clusters at the threshold of genetic similar coefficient approximately0.63by UPGMA. Both ISSR and SRAP analysis showed that there were abundant genetic diversity and remarkable genetic differentiation among C. oxysporum strains. The genetic clusters of C. oxysporum strains were associated with their geographic origins, but there was no significant correlation between the genetic relationships and pathogenicity of the strains. 5. Preliminary Study on the Infection Cycle of Pepper Leaf SpotThe results showed that C. oxysporum adhering to plant residues could overwinter successfully and become primary infection sources of the disease. Temperature and humidity were two main factors on disease occurring. The optimum temperature for infection was20to25℃when incubation period was7to8days. Under optimum temperature, keeping moist increased incidence of the disease. The infection process of C. oxysporum on pepper leaves was studied micrologically and ultramicrologically. It showed that the pathogen was able to penetrate host through two paths such as epidermis and stomata. The infectious stage started with conidium germination8h after inoculation, subsequently germinated hyphae grew over the tissue and conidiophores emerged through the stomata which produced conidia. Conidia fell off naturally after matured, then repeated infection. A large number of mycelia expanded rapidly within leaf tissue, and infected cells were damaged and died finally.6. Preliminary Study on the Pathogenic Mechanism of C. oxysporumThe pathogen could produce toxin in fluid media. The biological activity assay of the crude toxin showed that it could induce the characteristic symptom of pepper leaf spot, cause seeding wilting and inhibit seed germination and radicle growth. The optimum conditions of producing toxin were Richard culture filtrate with pH7and sustained oscillation culture for14d in dark at25℃. A series of cell wall degrading enzymes (CWDEs) including pectinase (PG, PMG, PGTE and PMTE) and cellulase (Cx and β-glucosidase) were found in the pathogen. Pectinase produced from the pathogen showed higher activities as compared with cellulase in vitro CWDEs. Activities of pectinase were significantly improved after inoculation and reached the peak in3-4d after inoculation. Perhaps pectinase firstly play a role in pathogenic process. Activities of cellulase were gradually increased after inoculation. Maybe cellulase was conducive to the expansion of pathogen in the host tissues.7. Establishment of the Nested PCR Detection System of C。 oxysporumBased on the rDNA-ITS conservative sequence of C. oxysporum, one pair of specific primers Clad-F/Clad-R was designed. Then intermal primers Clad-NF/Clad-NR were designed based on the regular PCR product amplified with the primers Clad-F/Clad-R as outer primers. Combined these two kinds of primers, a nested PCR protocol was established. The sensitivity of this assay was1fg DNA of C. oxysporum. The nested PCR detection results in artificially inoculated pepper leaves with different incubation period showed that pathogen could be detected on the second day after inoculation. For symptomless samples that randomly collected from fields, the efficiency of the nested PCR assay was compared to that of culture in vitro method. As a result, the detection rate of nested PCR was52%, while incidence of leaves culture in vitro was32%. These data showed that the nested PCR had higher sensitivity than traditional method. The nested PCR could be effective to diagnose disease in incubation period when pepper leaves without visible symptoms in field.