Molecular Identification of LeptosphaeriamaculansandDetermination of AggressiveNewPathotypes Canola Phoma stem Canker in north Iran

Abstract

Introduction: Canola is one of the important oil crops in Iran and blackleg disease caused by Leptosphaeriamaculans is an economically important disease of rapeseed especially in the northern provinces of Iran. At the beginning of the season in early autumn, L. maculansareinitiatesby air-borne ascospores released from infected stubbles of previous crops. These ascospores germinate and produce leaf lesions. The fungus then grows systemically from the leaf lesions to stem where cankers are producedwhich can result in major yield loss. Blacklegiscaused by a complex of at least two species of Leptosphaeria: L. maculans and L. biglobosa. Of these two species whichL. maculanswasis much more prevalent and virulent than L. biglobosa and was reported from almost all oilseed rape growing regions of the world.A number ofmethods has been developed to differentiate isolates of thesetwo groupsthrough morphological, physiological, genetic and molecular markers. Among these virulence markers of group pathogenicity are most widely used for characterizing the pathogen population. The purpose of this research work was to investigate isolates identification based on colony morphology and molecular methods. Moreover, for management strategies to be effectiveit is important to recognize the population structure based on pathogenicity groups and an understanding of possible shift in fungus population in thenorth of Iran. Materials and Methods: Infected plants of oilseed rape were collected from the northeast Iranduring 2013-2016. Diseased plant organs with clearly defined symptoms of the disease were used for the isolation of the fungi. All isolates were analyzed using a combination of morphological and physiological. In order to confirm the identification of L. maculans from oilseed rape isolates, DNA was prepared using the standard method described previously. The ITS region of genome of all isolates was amplified using polymerase chain reaction with specific primers pair LmF, lmR. Some isolates were sequenced with ITS1 and ITS2 along with the 5.8S rRNAregion and then sequence data were compared with that of available L. maculans isolates in Gene Bank. Aggressive isolates of L. maculans collected in widely separated geographic regions were further divided into pathogenicity groups based on virulence of three differentBrassica napuscultivars. Results and Discussion: Morphological and physiological and molecular characterizations of 72 isolates were performed. Isolates grewslowly, the pycnidia of the fungus were black, globose to subglobose in shape and conidia were single-celled, hyaline. The most of isolates formed yellow and pigment formation was scored in six groups in PDB at 18°C in the dark on the shaker. The PCR detection showed that all isolates were amplified by L. maculans-specific primer pair and a 334-bp PCR product was reliably amplified from L. maculans. The sequence analysis of the ITS region revealed that the sequences had 99.7% sequence similarity with the ITS sequences of known L. maculansisolates in GenBank by NCBI Blast search. This isolates were registered with accession number KX649997 and KX792142 in gene bank and this confirmed that the pathogen exists in Brassica napus samples. The role of morphological identification in virulence is complex and the production of pigment and mycelial growth is not always correlated with virulence. Therefore, based on amplification with type specific primers the amplified product fragment specific for group A was found in all isolates. It is important to know which pathotype is present, because the pathotypes of L. maculans differ in the amount of damage they cause. Based on the reaction of isolates on differential cultivars all four group pathogenicity PG-2, PG-3, PG-4 and PGT were observed in aggressive isolates. The most isolates were classified to be a pathogenicity PG-4 virulent on three differentcultivars and for the first time wasidentified in Iran. Conclusions: In order to determine whether isolates belonging to the canker L. maculans are present in the north of Iran, initial species identification based on colony morphology was confirmed using molecular methods. Asthe pathogen causes considerable losses, thefast identification and pathotype determination are important for agriculture and successful management of blackleg disease. Our results showed that an understanding of possible shift in fungus populations of PG2 to PG4 will be of value in developing strategies for successful management of blackleg disease.

Keywords

• Blackleg stem canker
• Morphological
• Pathogenicity groups
• Specific primers