Discovery of Fusarium proliferatum f. sp. malus domestica Causing Apple Replant Disease in China

Apple replant disease (ARD) is the most serious threat facing the apple industry globally. ARD is mainly manifested as decreased plant growth, serious root rot disease, and considerable yield loss. Microbial factors are the dominant factors leading to the occurrence of ARD. Research on soil-borne pathogenic fungi leading to the occurrence of ARD in China is limited. In the present study, we selected 16 replanting orchards from the Northwest Loess region and around the Bohai Gulf. Diseased roots and rhizosphere soil from healthy apple trees and trees showing ARD symptoms were sampled at random. High-throughput sequencing was used to study the fungal communities in the rhizosphere soil, which showed that the composition of the rhizosphere soil fungal community of ARD-symptomatic and healthy apple trees was different. Nectriaceae at the family level and Fusarium at the genus level dominated the rhizosphere soil fungal community in the two regions, while for healthy apple trees, the relative abundance of Mortierella, Minimedusa, Tetracladium, and Chaetomium was higher. Tissue separation and serial dilution were used to separate fungi, and a total of 89 genera and 219 species were obtained, most of which were Fusarium. Fusarium was further confirmed to be the most abundant pathogen species leading to the occurrence of ARD in China through pathogenicity assays. A pathogenicity assay was carried out by the dip-and-cut technique using different host plants. It was found that Fusarium MR5 showed strong aggressiveness to apple rootstocks. Diseased seedlings specifically exhibited chlorosis of the leaves, browning from the edge of the leaf, followed by rolling and yellowing of the leaves, resulting in wilting and eventually death. Strain MR5 was preliminarily identified as F. proliferatum according to the morphological and cultural characteristics. A maximum likelihood analysis of identities based on six gene sequence (ITS, TUB2, IGS, mtSSU, RPB2, and the TEF gene) alignments between the MR5 strain and other strains showed 99 to 100% homology with F. proliferatum. Based on our test results, strain MR5 was identified as F. proliferatum f. sp. malus domestica, which is of great significance for finding new measures to control ARD in China.