Dissertation > Excellent graduate degree dissertation topics show

The Study on Identification of Main Fungal Pathogens and Establishment of Multiplex RT-PCR Detection System of Viral Pathogens on Ananas Comosus (L.) Merr. in Hainan Province

Author: LuoZhiWen
Tutor: HeFan
School: Hainan University
Course: Molecular Plant Pathology
Keywords: Ananas comosus (L.) Merr. identification of fungal pathogens detection of viruses biological characteristics amplification of rDNA-ITS RT-PCR multiplex RT-PCR system
CLC: S436.68
Type: Master's thesis
Year: 2011
Downloads: 35
Quote: 1
Read: Download Dissertation

Abstract

Pineapple (Ananas comosus (L.) Merr.) is one kind of evergreen perennial monocotyledonous herbaceous fruit plants, mainly cultivated in Guangdong, Hainan, Guangxi. Taiwan, Fujian, Yunnan in south China, which is a member of genus Ananas from family Bromeliaceae and one of the three large-scale tropical fruits in Hainan province. Although diseases played a greatest role in decreasing the growth and production of pineapple in Hainan all the time, we have not acquired much more comprehensive and intensive information about these because of the shortage of systemic investigation and research. Aiming at such situation, a more comprehensive and detailed study on the fungal and viral disease of pineapple all over the Hainan was accomplished in this program.The main conclusion of this thesis were as follows:1.The investigation showed that there were 9 kinds of main diseases having been found on pineapple, in which the Pineapple Mealybug Wilt(PMW), anthracnose caused by Colletotrichum gloeosporioides Penz., fruit rot caused by Alternaria alternata (Fr) Keissler and heart rot caused by Phytophthora parasitica Dast. were the most destructive, serious and widespread ones. Besides, the gray leaf spot(Exserohilum rostratum) and ring leaf spot(Chaetomium aureum) were first reported on food pineapple.2.The study on biological characteristics of C. gloeosporioides showed the optimum conditions for mycelial growth and spore germination:the temperature were 28℃(for mycelial growth, the same as follow) and 30℃(for spore germination, the same as follow) respectively; the PH were 7 and 6; the carbon sources were glucose, D-fructose, maltose(for both growth and germination) and sucrose(only for germination); the nitrogen sources were yeast extract (for both growth and germination) and beef extract, L-aspartic acid and peptone(only for germination); the full-darkness was more beneficial for mycelial growth than the other illuminations, and the spore germination rates under full-light and 12 h-light/12 h-darkness were extremely significantly higher than that in full-darkness; spores would germinate if RH≥90% and germinate most in water droplets; the lethal treatment for conidia was 55℃for 10 min.The related research on A. alternata showed the optimum conditions for mycelial growth and spore germination:the temperature were both 28℃; the PH were 9 and 8; the carbon sources were maltose(both for the growth and germination) and sucrose, D-fructose, D-xylose, D-galactose(only for the germination); the nitrogen sources were NH4NO3 and L-aspartic acid, yeast extract; the full-light and 12 h-light/12 h-darkness were more beneficial for mycelial growth and spore germination respectively than the other illumination conditions; spores would germinate if RH≥95% and germinate most in water droplets; the lethal treatment for conidia was 55℃for 20 min.Experiments on E. rostratum figured out the optimum conditions for mycelial growth and spore germination:the temperature were 28℃and 30℃respectively; the PH were 8 and 7～8; the carbon sources were sucrose, maltose(for the both) and D-galactose(only for growth), D-xylose(only for germination); the nitrogen sources were yeast extract, peptone, NH4NO3 and beef extract, yeast extract, peptone; the 12 h-light/12 h-darkness condition were more beneficial for mycelial growth and spore germination; spores would germinate if RH≥90% and germinate most in water droplets (72.50～96.33%); the lethal treatment for conidia was 55℃for 15 min.3.The PCR results of fungus rDNA-ITS amplification using universal primer pair ITS1/ITS4 suggested that:the sequences of the 2 suspected strains of A. alternata and E. rostratum were 569 bp and 602 bp separately, which were proved to be located in the rDNA-ITS of A. alternata and E. rostratum after blast and homology analysis. We confirmed that, as a result, the rDNA-ITS analysis should be a dependable feasible supplementary method for morphological identification to identify and classify the fungal pathogens.4.The RT-PCR detection showed that:These 4 fragment were sequenced as 520、542、473 and 730 bp in length, and classified as the partial sequences of PMWaV-1. PMWaV-2, PMWaV-3 HSP70h gene and PBCoV polyprotein gene respectively based on the homology analysis.Therefore, we considered that there’re at least 4 kinds of virus inhabiting in PMW plants, setting a remarkable mixed infection example.5.The series experiment on multiplex RT-PCR of PMWaV-1, PMWaV-2 and PBCoV suggested the optimal factors for this system were as below:the annealing temperature (Tm) was 53℃, the dNTPs was 100～200μM(reaction volume was 25μL in total, the same as below), the Taq polymerase was 0.5～1.0 U, the extension time was 60～90 s, the reaction cycles were 30～35.3 distinct electrophoretic bands in specific length of target viruses should be effectively and clearly present, simultaneous and rapid, under these conditions.The sensitivity tests told us≥10-2,10-5 and 10-3μg viruses-infected tissue were essential for the effective detection of PMWaV-1, PMWaV-2 and PBCoV respectively, and≥10-2μg of diseased tissue was required for detecting the 3 viruses in a same system simultaneously.

Related Dissertations

CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Horticultural Crops Pest and Disease Control > Fruit tree pests and diseases > Perennial herbaceous fruit pests and diseases
© 2012 www.DissertationTopic.Net Mobile