First report of molecular detection of Leveillula taurica associated with powdery mildew of linseed (Linum usitatissimum) from India.

Abstract

Linseed (Linum usitatissimum L.) is an important oil yielding crop cultivated widely in India. During the field survey, 2018-19 (Rabi season; January-February), characteristic powdery mildew disease symptoms were observed in around 90 hectares of linseed in Northern Karnataka (Agricultural Research Station, Raichur). The incidence of the disease was estimated, and it was found in the range up to 96 per cent. Initially, the symptoms were appeared as tan lesions, which later turned into small, circular chlorotic lesions and yellowing of the abaxial leaf surface with white fungal mycelial growth on the abaxial leaf surface. As the disease progress, white fungal mycelial growth was also noticed on the adaxial leaf surface. In severe infection, drying and premature defoliation of infected leaves was observed (Figure S1 A-D). A total of 12 samples were collected and subjected to morphological and molecular identification. Microscopic examination revealed the presence of conidia borne on conidiophores (simple, sometimes branched). Conidiophores were measured 83.17 – 116.45 µm (n=90). Conidia were single-celled, hyaline, dimorphic (pyriform and cylindrical), and 70.2 - 82.7μm × 15.1 - 26.3 μm (n=100) (Figure S1 E&F). A reference specimen was deposited at Agharkar Research Institute’s (ARI) Ajrekar Mycological Herbarium (AMH 2015-07/AKC 2231-03/SKS/DKM). Since the conidial morphology and characters were very similar to Leveillula taurica the fungus was identified as L. taurica (Lév.) G. Arnaud. (Braun and Cook, 2012). Further, to confirm the identity based on ITS-rDNA barcoding, genomic DNA was isolated from conidial suspension harvested from powdery mildew affected linseed samples. ITS was amplified using powdery mildew-specific ITS universal primer pair PN23/PN34 and sequenced directly (Chen et al., 2008). The ITS regions were amplified at ~700bp. nBLAST analysis revealed that the ITS sequence shared 100% similarity with reference sequence AB045148.1 respectively. The representative sequences were deposited in GenBank with accession numbers OK036584 (LuLtR1) and OK036585 (LuLtR2). Further amplification of L1/L2 primer pair from ITS region were PCR amplified (Reddy et al., 2013) and an expected amplicon of 374bp was analyzed. Though, the ITS-rDNA and L1/L2 specific region analyzed, they are not specific exclusively to the L. taurica. However, combination of morphology and molecular sequence was used to confirm the identity. Further to prove Koch’s postulates, pathogenicity tests were conducted on healthy linseed plants grown under greenhouse condition (28±2° C; >80% Relative Humidity). Conidia were harvested from infected leaves and the suspension was sprayed on 30 (40 days old) linseed plants. The development of powdery mildew symptoms was recorded on 22 plants 10 days after inoculation. Control plants inoculated with sterile water remained healthy, and no powdery mildew symptoms were observed. The identity was confirmed upon microscopic observation of spores from artificially inoculated plants, thus confirming the association. Leveillula taurica is known to cause powdery mildew on tomato, chilli and other crops. Amano (1986) and Saharan and Saharan (1994) reported the occurrence of L. taurica on linseed, however, no further details are available on the association, as well as molecular confirmation of L. taurica associated with linseed from India (Farr and Rossman, 2021). To the best of our knowledge, this is the first report of L. taurica associated with powdery mildew of linseed in India. Since it is the first confirmed record through molecular sequence analysis, it is important to know its geographical distribution and impact on linseed production to chalk the management strategies.