Study On Etiology Of Kiwifruit Soft Rot Disease

Sichuan Province is a main kiwifruit （Actinidia chinensis Planch.） growing area of China, where the production of 2011 is about 130,000 tons. Kiwifruit soft rot is the most serious disease in the storage period, and the disease develops rapidly and is difficult to control. At present, the research of kiwifruit soft rot is still in its infancy. The pathogen species, pathogenesis and physiological changes of diseased kiwifruit were rarely reported. For these reasons, this thesis systematically researches on the pathogen of kiwifruit soft rot, and obtained some results in the following.In total,135 isolates of species in the Botryosphaeriaceae family were obtained from rotten fruit of kiwifruit from Cangxi, Dujiangyan, Mingshan, Pengzhou, Qionglai, and Shuangliu regions of Sichuan Province. Based on morphological characteristics and comparisons of DNA sequences of the internal transcribed spacer, transcription elongation factor 1-a, and β-tubulin genes, the pathogens of kiwifruit rot disease were identified as Botryosphaeria dothidea, Lasiodiplodia theobromae, and Neofusicoccum parvum. This is the first report of N. parvum and L. theobromae causing kiwifruit rot disease. The research result shown that B. dothidea could over winter as pycnidia and pseudothecia. Same as the Botryosphaeria dothidea、Lasiodiplodia theobromae and Neofusicoccum parvum pathogens which were isolated from rotten kiwifruit, the over winter conidia and ascospores of B. dothidea also could cause disease symptom on fruit, leaves and shoots of kiwifruit. A large number of pycnidia commonly developed on leaves and shoots after inoculation with B. dothidea, L. theobromae, and N. parvum. These results indicate that the spores of B. dothidea from the over-wintered pycnidia and pseudothecia could as the primary inoculum sources of kiwifruit rot disease. The Botryosphaeria dothidea, Lasiodiplodia theobromae and Neofusicoccum parvum mycelial growth and spore germination optimum temperature is 25-30℃. Within the range of pH=4-10, the mycelial growth optimum pH value is 5-8, and spore germination optimum pH value is 6-8. For Neofusicoccum parvum, the optimum carbon source is Xylose; The glucose is the prefered carbon source of Botryosphaeria dothidea and Lasiodiplodia theobromae. The nitrogen source utilization research results shown that the optimum nitrogen source of B. dothidea and N. parvum is NH4C1. while L. theobromae prefer Glutamate.A total of 52 isolates were used for pathogenicity determination and analysis of genetic diversity, which includes 37 isolates of Botryosphaeria dothidea,2 isolates of Lasiodiplodia theobromae and 13 isolates of Neofusicoccum parvum. The results shown that they could cause kiwifruit rot, but there were significant differences in virulence. For B. dothidea species, the weak virulent isolates accounted for 29.73%（11 isolates）, the moderate virulent isolates accounted for 43.24%（16 isolates）, highly virulent isolates accounted for 27.03%（10 isolates）. Similarly, the moderate virulent moderate of N. parvum were the highest proportion, reaching 53.85%. And 2 isolates of L. theobromae had highly virulence. Inter-simple sequence repeats （ISSR） and Sequence Related Amplified Polymorphism （SRAP） were used for the genetic diversity analysis of these 52 isolates.9 ISSR primers were used for PCR amplification, and 128 bands were obtained, including 86 polymorphism bands which accounted for 67.19% of the total number of bands, and each primer obtained an average of 9.56 bands.8 pairs of SRAP primers combinations amplified 114 bands, among them, the polymorphic bands were 78, accounted for 68.42% of the total number of bands, and each primer pair obtained an average of 9.75 bands. At about 0.645 genetic distance,9 ISSR markers could divide the pathogens of the kiwifruit soft rot into three groups:all isolates of B. dothidea （37 isolates） as group Ⅰ; All N. parvum isolates （13 isolates） were included as group Ⅱ; group Ⅲ only had two isolates, and both were L. theobromae. At 0.798 genetic distance, group Ⅰ was divided into five subgroups. At 0.708 genetic distance, group Ⅱ was divided into three subgroups. At 0.996 genetic distance, groups Ⅱ lwas divided into two subgroups. The pathogens of the kiwifruit soft rot could be divided into two groups by 8 pairs of SRAP molecular markers at about 0.608 genetic distance:group Ⅰ contained all B. dothidea isolates （37 isolates）; group Ⅱ contained 13 N. parvum isolates and 2 isolates of L theobromae strains. At 0.779 genetic distance, group I was divided into seven subgroups. At 0.77genetic distance, group Ⅱ was divided into six subgroups, subgroups from the 1st to the 5th were all N. parvum isolates, and the 6th group has two L. theobromae isolates. Genetic diversity, pathogenicity and geographic of kiwifruit soft rot pathogens have no significant correlation.Botryosphaeria dothidea toxin could be produced in PDA, Fries and Czapek medium, but toxin which produced in Fries and Czapek medium had stronger activity. The B. dothidea toxin produced on the condition of shaking with light （12 h） and dark （12 h） alternately had stronger activity than toxin produced in the dark with shaking condition. The toxin extracted by Acetone had higher activity than extracted by Methanol+Chloroform. The result of pathogenicity test shown that toxins, pectinase and cellulase of Botryosphaeria dothidea, Lasiodiplodia theobromae and Neofusicoccum parvum could cause disease spots on fruit and leaves of kiwifruit.The specific primers of three housekeeping genes including ubiquitin conjugating enzyme （ubcB）, beta-tubulin （fl-tub） and RNA polymerase iii transcription primers factor （TFC1）, and seven cellulase genes of Neofusicoccum parvum were designed for detecting the expression levels of seven cellulase genes during disease process. The results shown that the expression levels of the seven cellulase genes were higher using TFC1 as the reference than using other two housekeeping genes. During six days of disease process, the gene expression trend of UCRNP2₂427 and UCRNP2₇122 followed the W-type trend, changing from decline to rise to decline, finally to rise. The trend of expression of UCRNP2₂356, UCRNP2₅067 and UCRNP2₇847 were U-shaped, which were declined following by rose. The trend of expression of UCRNP2₃883 was inverted V-shape, which rose following by declined. On the 1st day, the minimum of UCRNP2₂427 and the maximum of UCRNP2₂356 were detected; UCRNP2₅067 expressed in minimum level on the 2nd,3rd, and 4th day, and UCRNP2₃883 expressed in minimum level on 2nd day, and UCRNP2₂356 expressed in the maximum level on 3rd and 4th day; the same and lowest levels of expression were detected on UCRNP2₂427 and UCRNP2₅067 on the 5th days, while the highest levels was detected on UCRNP2₂356 on the same day; UCRNP2₂356 expressed the highest on the 6th day, but, UCRNP2₃883 expressed the lowest on the same day.A primer pairs （BZY-1/ZY-2） was designed to specifically amplify the Botryosphaeria dothidea DNA according to the ITS sequence of B. dothidea. The result shown that, the primer pairs can’t produce any DNA band from other 25 genus fungal DNA except B. dothidea when PCR. The primer pairs could produce specific band from the DNA which were extracted from rotten kiwifruit （inoculated by B. dothidea or naturally infected）, but could not produce any band from the DNA of healthy fruit.Two elicitors （salicylic acid and sterile culture filtrates of Lecythophora luteoviridis） could delay the kiwifruit soft rot disease occurrence. The change of superoxide dismutase （SOD）, peroxidase （POD）, catalase （CAT）, proline （Pro）, malondialdehyde （MDA） of kiwifruit which were induced disease resistance were measured in 6 successive days. The results shown that these 5 substances had different change trends in different treatments. But in most cases, kiwifruit which were inoculated by Botryosphaeria dothidea, Lasiodiplodia theobromae and Neofusicoccum parvum pathogens had higher CAT, POD, SOD, Pro and MDA contents than other kiwifruit which were not inoculated by pathogens. Kiwifruit which were soaked in distilled water and then inoculated by pathegens had higher CAT, POD, SOD, Pro and MDA contents than other kiwifruit which were treated by others methods.