Molecular and Morphological Characteristics of Leaf Spot and Black Spike in Golestan Province, Iran

Black dot disease triggered by Dilophospora alopecuri causes considerable damage in some fields in Golestan province, North of Iran. D. alopecuri is its causal agent and carried in to wheat by wheat gall nematode. Extracellular appendages on the conidia adhere to the cuticule of the nematode juveniles. The disease was reported in united state of America, Canada, Germany, Ugoslavia, India and Pakistan.The fungi is classDothideomycetes and order Dothideales. The geographical distribution of disease was studied in South-Eastern Australia in the summer 1995.The disease in Iran has been reported by Bamdadian since 1973 from Baluchistan, Isfahan, Golestan, Khorasan, Kerman and Khuzestan. The disease agent causes considerable damage in some fields in Golestan province, North of Iran. Symptoms have been found during a survey of foliar disease of wheat in Golestan province. The disease incidence was very low but fungus interaction with seed gall nematode causes considerable damage. The symptoms start with yellow spindle-shaped flecks which develop and become tan brown with black border. This spots may occur on peduncle and heads. The pathogen survives as mycelium in host debris or as conidia on seeds. The purpose of this study was determining the geographical distribution, disease incidence (DI) and disease severity (DS). Then molecular identification of isolates with ITS rDNA in North of Iran, Golestan province.

 Wheat fields infected with the disease in the cities of Golestan province were visited and subjected to sampling during spring 2017-2018. 42 samples suspected to infection with the twist disease were gathered from seven cities of Golestan province. The rate of disease distribution from seven cities farms with disease symptoms, three farms were selected in each crop year and their geographical coordinates were recorded. In this research mapping the geographical distribution of disease was prepared by ArcGIS10.2 software. During 2018 and 2019, a survey was conducted to characterize the disease incidence (DI) and disease severity (DS). The D. alpoecuri leaf spot reading scales rate carried out in this study. No visible symptoms are observed and the leaf remain a health 0; A few chlorotic lesions are present and the infection site is a tan-spot colored 1: Necrosis and chlorosis both exist on the leaf 2: A few pycnidia are visible on the infected site and less than 30% of the leaf is occupied by pycnidia 3: The entire leaf is covered by pycnidial lesions scored 4. A significant difference was observed between the disease rates in the two cropping years. Seven D. alopecuri isolates were identified on the based on morphological and molecular parameters. After purification of fungal isolates, 50 conidia and pycnidia were selected in each isolate and the length and width of pycnidia, conidia cell number, conidia size (length and width), colony shape on the culture medium were also measured in the laboratory. After that identifying of the fungus isolates pathogenicity test was performed in the greenhouse. The D. alopecuri spore suspension with 1.75× 106 CFU concentration and for nematode population with 25000 larvae (L2) were used for inoculation to plants. After three months symptoms of leaf spot (twist) disease was appeared in tillering stage. Fungal DNA extraction from mycelium mass of selected isolates was performed by Murray and Thompson (1980) method and part of ITS region was amplified using ITS4 and ITS5 primers by polymerase chain reaction (PCR). The amplified PCR products of fragments were purified and sequenced at sequencing Microsense Company in Switzerland.

  Comparing of harvested fungal isolates which have been grown on PDA medium showed some variation in different characteristics such as the number of conidia walls, conidia appendages and pycnidia size. In both subsequent years, maximum disease incidence (DI) and disease severity (DS) was measured in Kalaleh by 3.6% to 3.9%, while for Bandar-Torkaman, the city with the lowest DS, were1.3% to 1.65%. The average size of conidia length was 4-8×1 μm in diameters.The pathogenicity test showed that the D.alopecuri is capable to produce twist  disease symptoms only  in the presence with seed gall nematode in the host. The nucleic acid sequences of Internal Spacer Transcribed (ITS) regions for Golestan isolates showed 100% similarity and had small genetic similarity with D. alopecuri MH859142.1 deposited in NCBI Genbank. The sequences belong to D.alopecuri from Gonbad, Aqala, Azadshahr, Maravehtapeh, Kalaleh, Minodasht cities were registered in NCBI Genbank with MW302360, MW291507, MW291561, MW303438, MW303517, MW303518 accession number, respectively.

This is the first study carried out on the morphological and molecular characteristics of twist disease agent isolates, disease severity and its distribution in Golestan province as a major wheat production area of Iran. Many of results especially molecular data and submitted sequences form Iranian isolates of D.alopecuri to databanks are new for Iran.