Genomic and Horticultural Approaches to Manage Blackline Disease in Walnuts

This multiyear project is aimed at developing a practical solution to managing blackline disease of walnuts in California orchards. Blackline disease in English walnuts is caused by a Cherry leaf roll virus (CLRV) infection of English walnuts (Juglans regia) grafted on black walnuts (J. hindsii) and ‘Paradox’ hybrid (J. hindsii x J. regia) rootstocks. The HR is due to the resistance of black walnuts to infection by CLRV (Mircetich and Rowhani, 1984). Here we report on the progress of the project on developing RNAi-mediated resistance in walnuts. A walnut strain of CLRV was mechanically inoculated on to Chenopodium quinoa leaves and double stranded RNA (dsRNA) was extracted from symptomatic systemically infected leaves. The dsRNA was converted into cDNA and a library was prepared and sequenced by next generation sequencing (NGS) technology. Sequence contigs were subjected to bioinformatics analysis and found representing RNA-1 and RNA-2 of CLRV. The sequence spans almost 90% of the genome of the virus and the sequence generated by NGS is being verified by RT-PCR analysis of individual products of 1 to 1.5 kb regions of the two genomic RNAs. The 5’-end of the genomic RNAs is also being determined. A 700 bp product from a highly conserved untranslated region of the genomic RNAs was cloned in sense and antisense orientation and a binary vector containing this chimeric region has been mobilized into Agrobacterium tumefaciens strain EHA 105 for transformation of walnuts.