Isolation And Identification Of The Pathogen Of Sunflower Black Stem And Its Fast Molecular Detection

Phoma macdonaldii causeed lesions on the stems, leaves, heads of diseased sunflowers,resulting reduction in oil content of seeds and thousand seed weight. It was a destructivedisease. In the Ili river valley, Sinkiang, China, a damaged sunflower disease axpandedrapidly, resulting death of a large area of sunflowers as well as giving rise to severe loss.Therefore, we carried out the research on isolating and identifying the pathogens of blackstem of sunflowers. Meanwhile, we established the fast molecular detection system of P.macdonaldii.1. Isolate and identify the pathogens of black stem of sunflowersThe diseased stems, leaves and faceplates of sunflower with black stem symptom weresampled in Xinjiang Autonomous Region, China.20fungal isolates were finally isolated fromthe materials. After pathogenicity test isolates XJ011and XJ111were found to be pathogenicto sunflower plants. The internal transcription spacer (ITS) region of isolates XJ011andXJ111was amplified and sequenced by ITS1/ITS4universal primers. The length of sequenceof XJ011and XJ111were523bp and525bp (Accession number in GenBank were AB690462and AB690463). Compared with the sequences in the NCBI database, these sequences had asimilarity of99%-100%with Leptosphaeria lindquistii (anamorphic: Phoma macdonaldii).Meanwhile, in the Phylogenetic tree based on sequences of rDNA ITS region, the stains ofXJ011and XJ111as well as Leptosphaeria lindquistii formed a subclade with a bootstrapvalue of100%, indicating their close affinity. The colony of XJ011and XJ111were greywhite, with irregular edge. Pycnidia were scattered, gathered, semi-buried, with a papilla. Apink or milk white gelatinous substance was exuded from ostiole of pycnidia, containingmassive conidiospore. Based on the morphological characteristics, sequences analysis andpathogenicity test, these isolates isolating from Xinjiang Autonomous Region and leading toserious disease, were identified as Phoma macdonaldii Boerema.2. Build a fast molecular detection system of P. macdonaldii In the experiment, we aligned the nucleotide sequences of rDNA-ITS regions ofpathogens(containing Sclerotinia sclerotiorum, Sphaerotheca fuliginea, Puccinia helianthi,Verticillium dahliae, Verticillium albo-atrum, Verticillium nigrescens, Alternaria helianthi,Leptosphaeria dryadis, Phomopsis lindquistii, Leptosphaeria lindquistii, Leptosphaerialindquistii, XJ011, XJ111). Meanwhile, a pair of specific primers320FOR/320REV weredesigned for rDNA-ITS of P. macdonaldii according to polymorphism region, it can be usedto detect the P. macdonaldii specifically from the pathogens causing sunflower commondiseases, saprophytes and relative species of P. macdonaldii with highly sensitivity of1fgconcentration.