Development Of Multiplex Pcr And DNA Microarray For Tree Canker Disease And Application In Ecology

Fungal pathogens causing tree canker diseases are cosmopolitan in species, occurring on awide range of hosts. Anamorphs of these pathogens are diverse and they are overlapping inmorphology; Teleomorphs are hardly discovered in nature. Furthermore, the identification ofthese pathogens is usually limited by geography, host and taxonomic knowledge. Therefore, aculture-independent method to determine pathogens is expected to be developed for fielddiagnostics. Through amplifying, sequencing and analyzing on multiplex nucleic acidtemplates and genetic marked target sequences, a multiplex PCR has been established and usedto directly detect various environmental samples. The multiplex PCR can amplify multiplextemplates or sites. Simultaneously, the DNA microarrays offer the potential for simultaneousdetection of many pathogens. The combination of Multiplex PCR and DNA microarrays candetect multiple species parallelly and rapidly. At present, multiplex PCR-DNA Microarray(ArrayTube) technology is not used in the canker pathogens detection.The purpose of this study is to establish a multiplex PCR-ArrayTube technologyplatform，which adapt to the research of microbial biological populations and the applicationsbaesd on the biological populations analysis and detection. This can effectively solve problemsof accurate identification of different forest microbial analysis and lay the foundation forpathogen ecology and epidemiology research of trees disease.In this research, through the comparision of pathogens, the importance of the disease andthe nucleic acid information, we choose the Botryosphaeria and related Val sa, Leucostoma andEntoleuca as the main materials. Using Internal Transcribed Spacer (ITS) and ElongationFactor-1α (EF-1α) as the target sequences, a total of26probes were designed(ITS:12; EF-1α:14). Through Bioedit software, we established the target nucleic acid sequence database ofcanker fungal pathogens and developed Arraystrips. Firstly, we collected12strains, inculdingBotryosphaeria dothidea, Botryosphaeria rhodina, Valsa sordida, Ascochyta sp.,Botryosphaeria berengeriana, Leucostoma sp., Valsa ambiens, Entoleuca mammata, Leptographium terebrantis(Ascomycetes); Phytophthora cactorum (Oomycete); Agaricusbisporus(Basidiomycetes); Cunninghamella bertholletiae (Zygomycetes). These strains wereactivated and extracted, amplified for ITS gene and EF-1α gene by biotin-labeled single PCRand multiplex PCR, then the PCR products were hybridized for the verification of thesensitivity and specificity of the ArrayTube. Secondly, the multiplex PCR-ArrayTubetechnology was used in the environmental samples, a total24poplar samples (12infectedsamples,12asymptomatic samples) which collected from12regions of Beijing were used asmaterials, environmental samples DNA were extracted directly, then the products of multiplexPCR amplification of total DNA were used for microarray hybridization. Meanwhile, clonewas used for the validation of hybridization results. Simultaneously the collected samples wereisolated, cultured and sequenced to study the the diversity of the pathogen population andrelated endophytes.Our result showed that the multiplex PCR could successfully amplify the target gene andidentify the canker pathogens from environmental samples in the condition of an annealingtemperature of55.6℃and primers final concentration of0.2μM. The multiplex PCR couldamplify the target at the concentration level of approximately1ng of genomic DNA. ExceptE.mammata, there were total11strains that were used to hybridization, target strainsB.dothidea，V.sordida，B.rhodina(L.peudotheobromae) could hybridized to specific probes.Except the Ascochyta sp., the hybridization results of the control strains(L.terebrantis，P.cactorum， A.bisporus， C.bertholletiae) were negative, other strains were non-specifichybridization. By diluting multiplex PCR products for hybridization showed that sensitivity ofArraytube was0.1ng. The24testing samples showed that the ArrayTube could directly detectthe B.dothidea and V.sordida from the diseased and healthy samples, which consisted to theclones results. The study showed that DNA microarray could achieve the desired goals. Bymorphology and sequencing we got the ascomycetes and basidiomycetes from environmentalsamples, Botryosphaeria spp.and Valsa spp.were the dominant populations, and other genussuch as Coniothyrium spp., Fusarium spp., Peyronellaea spp., Hyalodendriella spp., Alternariaspp., Lecanicillium lecanii, Leucostoma niveum, Meyerozyma guilliermondii, Colletotrichum gloeosporioides, Schizophyllum commune and Funalia trogii were also found in samples. Someof these were endophytes. This provides theoretical basis for the development of multiplexPCR-ArrayTube used in the research of the interactions among host, pathogens and endophytesrelationship and the forest microbial ecology.