Deciphering the role of fungal calmodulin gene using Host-mediated gene silencing approach in apple

Premature leaf fall caused by Marssonina coronaria is one of the most destructive diseases of apple in India. In this study, host induced gene silencing approach was exploited to develop resistance to this disease in an apple cultivar ‘Red Chief’. Calmodulin gene (CaM) having its role in fungal differentiation, development and pathogenicity was selected as target. hpRNAi construct was prepared from the conserved off target free partial gene sequence of CaM and used for transformation trials. Upto 6% kanamycin resistant shoots were obtained on selective medium having 5–6 mg/l kan after 7 weeks of coculture. In PCR analysis of 13 RNAi putative transformants, 10 lines were found positive with CaM and nptII gene specific primers and six lines showed hybridization signal. Semi qRT-PCR revealed variable levels of transgene expression among RNAi lines which seems to be related to copy number of integrated gene. In vitro detached leaf assay revealed lesion development and disease progression in wild type after 5 dpi but not visible in five CaM RNAi lines. Microscopic examination of infected control leaves showed fully developed, septate mycelium, and conidia along with necrosis of whole tissue while three transformants showed reduced growth and differentiation of fungus and in rest three, hyphal development and necrosis were strongly restricted. We conclude that trafficking of dsRNA/ siRNA from apple plants to pathogen might have triggered the down regulation of fungal CaM gene which confirms that deciphering the role of CaM through HIGS lead to resistance to Marssonina blotch in apple.

First report of Fusarium proliferatum causing Sheath Rot Disease of Rice in Eastern India

Sheath rot is one of the most devastating diseases of rice because of its ability to reduce the yield significantly in all rice cultivating areas of the world (Bigirimana et al., 2015). Sheath rot disease is associated with various pathogens such as Sarocladium oryza, Fusarium fujikuroi complex and Pseudomonas fuscovaginae (Bigirimana et al., 2015). Hence, this disease has become more complex in nature and added more seriousness. From September to December 2018, plants were observed with typical sheath rot symptoms in research farm of ICAR-National Rice Research Institute and ten farmer’s fields of Cuttack district, Odisha, Eastern India. About 25 to 37% of sheath rot disease severity was recorded in the infected field. Diseased plants were observed with symptoms such as brownish or reddish brown irregular lesions, which were later, got enlarged with grayish centers. Further, rotting of the topmost leaf sheaths that surround the young panicle was observed. At the severe stages, the young panicle was partially emerged from sheath or completely rotted within the sheath. The white to pinkish powdery growth observed inside the infected sheath leading to chaffy and discolored grains. The sheath rot symptomatic plants were collected from the infected fields. To isolate the causal pathogen, infected sheath tissues were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed three times in sterile distilled water, and placed on potato dextrose agar medium (PDA) (HiMedia). Plates were incubated at 27 ± 1° C for 3 d. Further, fungal pathogen colonies were sub-cultured and purified to perform the pathogenicity test. On PDA, the colonies produced abundant white aerial mycelium with violet to pink pigmentation and hyphae were hyaline with septation. Abundant single celled, oval shaped microcondia (5.5-9 × 1.5-2 μm) were produced, whereas macrocondia were not produced and the fungal pathogen was tentatively identified as Fusarium sp. In order to characterize the pathogen at molecular level, ITS, alpha elongation factor gene (EF1-α), RNA polymerase II largest-subunit gene (RPB2), calmodulin gene (cld) were amplified using the primer pair of ITS1/ITS4, EF1/EF2, 5F/7CR and CLPRO1/CLPRO2 respectively and PCR amplicons were subjected to sequencing (White et al. 1990; O’Donnell et al. 1998; Chang et al. 2015). Furthermore, a species-specific primer Fp3-F/Fp4-R was used to identify the pathogen (Jurado et al., 2006). The resulting sequences were confirmed by BLAST analysis and the FUSARIUM-ID database (http://isolate.fusariumdb.org). BLASTn search showed 100% similarity between the query sequence and ITS, EF1-α, RPB2, Calmodulin gene sequences of F. proliferatum available in the Genbank. The following GenBank accession numbers were obtained; MT394055 for ITS; MT439867 for EF1-α; MT790774 for calmodulin; MT940224 for RPB2 and MT801050 for species-specific to F. proliferatum. To confirm the pathogenicity under glass house conditions, fungus grown on sterilized chaffy grains were placed in between boot leaf sheath and panicle and covered with moist cotton (Saravanakumar et al., 2009). After 15 days post inoculation (dpi), rotting symptoms were observed and these were similar to that of field symptoms. Pathogen was constantly re-isolated from symptomatic tissue, satisfying Koch’s postulates. Disease symptoms were not observed on un-inoculated plants. Morphological characters, pathogenicity test and molecular characterization have identified the pathogen as F. proliferatum. To the best of our knowledge, this is the first confirmed report of F. proliferatum causing sheath rot disease on rice from Eastern India.