Biological Characteristics,Molecular Detection And Quarantine Treatment Of Sunflower Black Stem And Stem Canker Pathogens (Leptosphaeria Lindguistii And Diaporthe Helianthi)

Sunflower is one of the important oil crops of high nutritional value in the world. In recent years, sunflower seeds imported from abroad may carry a variety of dangerous diseases that would spread into our country and increase the risks. Leptosphaeria lindguistii and Diaporthe helianthi are two important quarantine fungal diseases among them. So far, sunflower black stem disease has been already observed occuring in our country while the stem canker has not. There is an urgent need for the establishment of a rapid molecular detection technique to detect these two fungal diseases.This paper first described the symptoms of Leptosphaeria lindguistii occurred in the field and the biological characteristics of both diseases. Symptoms of sunflower black stem disease at various periods were observed in the field; morphological observations of the colonies, pycnidium and conidia of the two pathogens and other sunflower parasitic pathogens were also observed and compared. The residues of sunflower plants with obvious symptoms collected by investigators from the fields of Ningxia and Xinjiang is quarantined and identified in the laboratory. Through the morphological and ITS sequence comparison, it was found that TXMGX, BXC1, XYHJ from Xinjiang sunflower was identified to be sunflower black stems disease Phoma macdonaldii. In addition, Thielavia hyalocarpa, Preussia sp, Alternaria helianth, Verticillium dahliae, and Nigrospora sphaerica were also found in the Xinjiang region and Alternaria helianthi and Alternaria alternate were found in the Ningxia region.In order to establish a rapid molecular detection method for Leptosphaeria lindguistii and Diaporthe helianthi, the strains of Leptosphaeria lindguistii and Diaporthe helianthi and other parasitic pathogens on sunflower and the standard strains were collected as the test materials; based on actin gene sequence of Leptosphaeria Hndquistii, the universal primers act F/act R was designed and used to amplify the mycelial DNAs of the experimental materials. The specific primer pairs, LLF/LLR and DHF/DHR, for Leptosphaeria lindguisti and Diaporthe helianthi respectively were designed based on their differences in actin gene. The universal primers of ITS1/TTS4and the specific primer pairs of LLF/LLR and DHF/DHR were used simultaneously for triplex PCR amplification of all the mycelial DNAs after system optimization. For Leptosphaeria lindguistii, triplex PCR detected a580bp band of the ITS conserved region and a255bp specific band of Leptosphaeria lindguistii; and for Diaporthe helianthi, triplex PCR detected a580bp band of the ITS conserved region and a384bp specific band of the actin gene. No nonspecific bands appeared on triplex PCR of the other tested strains. Thus, the established triplex PCR detection is a specific and operative method to detect the two quarantine fungal diseases, Leptosphaeria lindguistii and Diaporthe helianthi, of sunflower.Two germ-killing methods, bromomethane fumigating and heat treatment, were used for quarantine treatment of the mycelia of the two kinds of pathogens under different condition; and their effects on germination of sunflower seeds were also observed under the same processing conditions. Leptosphaeria lindguistii and Diaporthe helianthi were killed in entirety by32g/m3bromomethane fumigating for24h and no effect on germination of sunflower seed observed. Leptosphaeria lindguistii and Diaporthe helianthi were inactive through more than three minutes of52℃hot water treatment while no effect on the germination of sunflower seeds by hot water treating for15min, was observed.Therefore, sunflower seeds carrying Leptosphaeria lindguistii and Diaporthe helianthi can be quarantine treated by bromomethane fumigation and hot water treatment.