Advances in the identification of pathogens associated with apple mosaic disease of apple trees in China

Abstract: Apple (Malus domestica) is one of the most economically important fruit crops. China is the largest apple producer all over the world, accounting for about 50% of apple productions and harvested areas worldwide. Apple mosaic disease is the most common viral disease in apple cultivation and is widely distributed in major apple-producing regions in China, which is a constant challenge to the de- velopment of apple industry. Apple mosaic disease was first reported in Europe and was one of the viral diseases that could be transmissible by budding or grafting. The mosaic-diseased apple leaves show pale yellow to bright cream-colored irregular spots, rings or brownish necrotic spots. Bands and/or line patterns along the main veins were observed with uneven distribution on apple leaves. Apple mosaic vi- rus (ApMV) is believed to be the only causal agent causing apple mosaic disease for a long period of time. However, for nearly a decade, ApMV has not been detected in our and other researchers’surveys on apple mosaic disease in China although the symptoms are similar to those caused by ApMV. In re- cent years, prunus necrotic ringspot virus (PNRSV) has also been considered as an important pathogen causing apple mosaic disease in China. PNRSV is a worldwide viral pathogen, which infects many eco- nomic fruit trees, such as peach, cherry, plum, hazelnut, apricot, apple and other ornamental plants. PNRSV can cause chlorotic ringspots, yellow rings, green line pattern, mosaic, systemic necrosis or se- vere stunting in the diseased fruit trees. Apple necrotic mosaic virus (ApNMV), a newly identified ilar- virus isolated from the mosaic-diseased apple trees, was clustered in subgroup 3 of genus Ilarvirus to- gether with PNRSV and ApMV, but showed relatively low nucleotide identities (49.2%-64.3%) with these two ilarviruses. ApNMV has shown a high correlation at a very high percentage (i.e., 92.1%, 268 out of 291) with mosaic-diseased trees being infected with ApNMV but not with ApMV in China, sug- gesting that ApNMV might be the main pathogen of apple mosaic disease in China. So far, crabapple (Malus spp., Rosaceae) and hawthorn (Crataegus spp., Rosaceae) have also been reported to be the nat- ural hosts for ApNMV in China. Here, we systematically reviewed the relationship between ApNMV and apple mosaic disease in China, and analyzed the roles of ApMV and PNRSV in apple samples with mosaic disease in China. Based on the previous researches, we found the primer sets ILAR 1/2, which were initially designed for the simultaneous detection and identification of ApMV and PNRSV from fruit trees, were usually used for the diagnose of ApMV in apple trees when no PNRSV was found to in- fect apple trees in China. Later on, PNRSV was identified from apple trees showing mosaic symptoms by next generation sequencing (NGS). And then the primer sets PNRSV-F/R were used to detect the in- cidence of PNRSV in mosaic-diseased apple leaves. After ApNMV was identified and isolated from mo- saic leaves, multiple sequence alignment of ApNMV with ILAR 1/2 and PNRSV-F/R were conducted. Results showed high nucleotide sequence identities between ApNMV and ILAR 1/2 or PNRSV-F/R. RT- PCR and clone sequencing confirmed that both ILAR 1/2 and PNRSV-F/R could be also used to detect ApNMV, indicating these two primers were not specific for the diagnosis of ApMV or PNRSV. In addi- tion, NGS data of the above mosaic-diseased leaf sample were deeply mined again. Total 32 assembled contigs (30-99 nt in length) were matched with ApNMV genome with 93-100% nucleotide identities while only 1 contig was mapped to genomes of PNRSV (21 nt in length) and ApMV (23 nt in length) with 76% and 83% identities, respectively. These results suggested ApNMV should be the main causal agent of apple mosaic disease in China. However, further studies are needed to fulfill the testing of Koch’s postulates on apple trees. Although the full-length cDNA clones of ApNMV have been con- structed and tested infectious in N. benthamiana and cucumbers, inoculation of the infectious clones in- to perennial woody plants (apple trees) remains very difficult. New inoculation strategies should be de- veloped and the proper conditions inducing apple mosaic disease should be studied. Furthermore, host range of ApNMV in woody tree species needs to be determined except for apple, hawthorn and crab ap- ple. Generally, the construction of infectious full-length cDNAs of ApNMV would be a powerful strate- gy to study functional genomics and molecular mechanisms of pathogenicity and host interactions with ApNMV. The infectious clones are also used to evaluate and screen out apple germplasm resources with the anti-ApNMV or durable ApNMV resistance due to the high incidence and damage of ApNMV in apple trees in fields. Taken together, our review article may provide a comprehensive understanding of the pathogen of apple mosaic disease in China, and lay a helpful foundation for the further study on its mo- lecular pathogenesis and control.