The Identification Of The Pathogens Causing Tree Peony Red Spot And The Pathogens Causing Tree Peony Black Spot

The red spot andblack spot wereboth the most seriousdiseases which harmed peony tree. Thediseases not only harmed the leaves, but also caused serious damage to the flowers,seeds, stems, thediseases could also causelesions, perforation of peonyleaves. In severe cases,they even lead to the death of the entire plant.In this case, thediseases greatly reduced the ornamental value of peony and brought great difficulties to peony industrial production.Currently,there were less research on peony diseases at home and abroad,identification of tree peony red spot remained at the level of morphological identification,the study on peony black spot symptoms were only simple recordings and control researches, the pathogens of this diseaseremained unclear. We used molecular identification combined with traditional morphological identification and pathogenic identification to identify thepathogens of tree peony red spot and black spot in this study,which aimed to providethe reference for the rapid diagnosis and prevention of tree peony disease, and provide the information for phylogenetic and taxonomic identification of Cladosporium spp..1. The isolation and morphological identification of the pathogens causing tree peony redspotThe diseased leaves samples on different varieties of tree peony and Chinese herbaceous peony were collected from Forestry Experiment Station germplasm peony garden of Shandong Agricultural University. The pathogens were isolated by tissue separation,then cultured on PDA medium to obtain pure colonies, and saved for utilization.The pathogen mycelia and sporeswere observed under the microscope.By calculating the average of spore-length and spore-wide length,we could get the average of the length and the average of the width,which was between 9.4μm-9.8μm and 4.5μm-4.9μm. The result showed no difference between different host species, and itwas consistent with the description of Wei Jingchao for Cladosporium paeoniae.2.Molecular identification of pathogens causing tree peony red spotAfter separation and purification, the author PCR-amplify the ITS sequence of the rDNAof strains the pathogens.The result suggestedthat thehighest homology sequences in Genbank was GQ395812.1, homology is greater than 99%.By registering four nucleotide sequences ofof ITSobtained from different varieties of tree peony and Chinese herbaceouspeonyin Genbank,we could get theirlog numbers : KJ638030, KJ682319, KJ682320,KJ682321.The ITS phylogenetic trees weregeneratedwith the soft of Mega5.The results showed that theCladosporium spp.was divided into four groups. The four sample strains KJ638030, KJ682319, KJ682320, KJ682321 reached the highest homology with Cladosporium paeoniae,which were more than 99%.The four sample strains clustered into a group.By combing morphological identification and pathogenic identification,we could identifythe pathogens causing tree peony red spot was Cladosporium paeoniae. Four strains were isolated from different host species, which indicated that there is no genetic differences between Cladosporium paeoniaes caused tree peony red spot in Taian, and the differences of the host species didn’t affect the genetic characteristics of the strain.3.Pathogenic Identification of pathogens causing tree peony red spotIn order to inoculate the pathogen into five varieties of peony leaves,we used acupuncture mycelium block inoculation and mycelium block no wound inoculationin. From which we could observe no difference among these five species when used the same method.Byre- isolating the lesion,we found that the pathogen was consistent with the inoculants,which proved that the pathogen causing tree peony red spot was this fungus. The wound leaves and the intact ones were both infected by the pathogen. Butthe expansion rateof the disease spot on the wound leaves was faster than the intact ones.4.The identification of the pathogens causing tree peony black spotThe black spot was one of important diseases which harmed peony tree,the pathogens of this diseaseremained unclear. The aim of this research was to clarify its taxonomic status by identifying the pathogen of tree peony black spot.In this study,the pathogen was identifiedbased on morphology and molecular identification, the ITS phylogenetic trees was generated with the soft of Mega 5. The result suggested that the pathogen of tree peony black spot was Alternaria alternate(Accession No. KJ682317). The homology between the sample strain and Alternariaalternate was up to 100 percent.The wound leaves and the intact ones were both infected by the pathogen. Butthe expansion rateof the disease spot on the wound leaves was faster than the intact ones. The molecular identification and morphology identification researched the same results.