Apple replant disease (ARD) is a common problem in major apple planting areas, and biological factors play a leading role in its etiology. Here, we isolated the bacterial strain QSB-6 from the rhizosphere soil of healthy apple trees in a replanted orchard using the serial dilution method. Strain QSB-6 was provisionally identified as Bacillus amyloliquefaciens based on its morphology, physiological and biochemical characteristics, carbon source utilization, and chemical sensitivity. Maximum likelihood analysis based on four gene sequences [16S ribosomal RNA gene (16S rDNA), DNA gyrase subunit A (gyrA), DNA gyrase subunit B (gyrB), and RNA polymerase subunit B (rpoB)] from QSB-6 and other strains indicated that it had 100% homology with B. amyloliquefaciens, thereby confirming its identification. Flat standoff tests showed that strain QSB-6 had a strong inhibitory effect on Fusarium proliferatum, Fusarium solani, Fusarium verticillioides, Fusarium oxysporum, Alternaria alternata, Aspergillus flavus, Phoma sp., Valsa mali, Rhizoctonia solani, Penicillium brasilianum, and Albifimbria verrucaria, and it had broad-spectrum antibacterial characteristics. Extracellular metabolites from strain QSB-6 showed a strong inhibitory effect on Fusarium hyphal growth and spore germination, causing irregular swelling, atrophy, rupture, and cytoplasmic leakage of fungal hyphae. Analysis of its metabolites showed that 1,2-benzenedicarboxylic acid and benzeneacetic acid, 3- hydroxy-, methyl ester had good inhibitory effects on Fusarium, and increased the length of primary roots and the number of lateral roots of Arabidopsis thaliana plantlet. Pot experiments demonstrated that a QSB-6 bacterial fertilizer treatment (T2) significantly improved the growth of Malus hupehensis Rehd. seedlings. It increased root length, surface area, tips, and forks, respiration rate, protective enzyme activities, and the number of soil bacteria while reducing the number of soil fungi. Fermentation broth from strain QSB-6 effectively prevented root damage from Fusarium. terminal restriction fragment length polymorphism (T-RFLP) and quantitative PCR (qPCR) assays showed that the T2 treatment significantly reduced the abundance of Fusarium in the soil and altered the soil fungal community structure. In summary, B. amyloliquefaciens QSB-6 has a good inhibitory effect on Fusarium in the soil and can significantly promote plant root growth. It has great potential as a biological control agent against ARD.

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.

In this study, an endophytic phlorizin-degrading Bacillus licheniformis XNRB-3 was isolated from the root tissue of healthy apple trees, and its control effect on apple replant disease (ARD) and how it alleviates the pathogen pressure via changes in soil microbiomes were studied. The addition of strain XNRB-3 in Fusarium infested soils significantly reduced the number of pathogens in the soil, thus resulting in a lower disease incidence, and the relative control effect on Fusarium oxysporum reached the highest of 66.11%. The fermentation broth can also protect the roots of the plants from Fusarium oxysporum, Fusarium moniliforme, Fusarium proliferatum, and Fusarium solani infection. These antagonistic effects were further validated using an in vitro assay in which the pathogen control was related to growth and spore germination inhibition via directly secreted antimicrobial substances and indirectly affecting the growth of pathogens. The secreted antimicrobial substances were identified using gas chromatography-mass spectrometry (GC-MS) technology. Among them, alpha-bisabolol and 2,4-di-tert-butylphenol had significant inhibitory effects on many planted pathogenic fungi. Butanedioic acid, monomethyl ester, and dibutyl phthalate promoted root development of Arabidopsis plants. Strain XNRB-3 has multifarious plant growth promoting traits and antagonistic potential. In pot and field experiments, the addition of strain XNRB-3 significantly promoted the growth of plants, and the activity of enzymes related to disease resistance [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] was also significantly enhanced. It also reduced the abundance of four species of Fusarium and the content of phenolic acids in the rhizosphere soil, improved soil microbial community structure and nutritional conditions, and increased soil microbial diversity and activity, as well as the soil enzyme activity. The above results indicated that B. licheniformis XNRB-3 could be developed into a promising biocontrol and plant-growth-promoting agent.

Background: Apple replant disease is a soilborne disease caused by Fusarium proliferatum f. sp. malus domestica strain MR5 (abbreviated hereafter as Fpmd MR5) in China. This pathogen causes root tissue rot and wilting leaves in apple seedlings, leading to plant death. A comparative transcriptome analysis was conducted using the Illumina Novaseq platform to identify the molecular defense mechanisms of the susceptible M.26 and the resistant M9T337 apple rootstocks to Fpmd MR5 infection. Results: Approximately 518.1 million high-quality reads were generated using RNA sequencing (RNA-seq). Comparative analysis between the mock-inoculated and Fpmd MR5 infected apple rootstocks revealed 28,196 significantly differentially expressed genes (DEGs), including 14,572 up-regulated and 13,624 down-regulated genes. Among them, the transcriptomes in the roots of the susceptible genotype M.26 were reflected by overrepresented DEGs. MapMan analysis indicated that a large number of DEGs were involved in the response of apple plants to Fpmd MR5 stress. The important functional groups identified via gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were responsible for fundamental biological regulation, secondary metabolism, plant-pathogen recognition, and plant hormone signal transduction (ethylene and jasmonate). Furthermore, the expression of 33 up-regulated candidate genes (12 related to WRKY DNA-binding proteins, one encoding endochitinase, two encoding beta-glucosidases, ten related to pathogenesis-related proteins, and eight encoding ethylene-responsive transcription factors) were validated by quantitative real-time PCR. Conclusion: RNA-seq profiling was performed for the first time to analyze response of apple root to Fpmd MR5 infection. We found that the production of antimicrobial compounds and antioxidants enhanced plant resistance to pathogens, and pathogenesis-related protein (PR10 homologs, chitinase, and beta-glucosidase) may play unique roles in the defense response. These results provide new insights into the mechanisms of the apple root response to Fpmd MR5 infection.

Apple replant disease (ARD) causes enormous economic loss and threatens the survival of apple industry worldwide. Fusarium solani is one of the pathogens that has been proven to cause ARD. Samples were collected at different time periods to investigate the mechanism of defense responses of apple to F. solani infection by monitoring the biomass, reactive oxygen species (ROS), and antioxidant enzyme activities of the apple rootstock ‘M.9T337’. In addition, the abundance of transcription of four pathogenesis-related (PR) proteins involved in antifungal defense was monitored. The results showed that the apple root system was normal and had small brown areas. However, there is a rapid burst of ROS during the early infection stage, and the activities of antioxidant enzymes and transcription of PRs increased during this period. With the extension in infection time, the infected root tissues displayed dark brown necrosis, and the activities of antioxidant enzymes and abundance of transcription of PRs decreased gradually after reaching their peak. Eventually, the plant biomass decreased, and the plant died. In conclusion, the levels of ROS and activities of antioxidant enzymes played an active role during the early stage of resistance of ‘M.9T337’ apples to infection by F. solani . Infection by F. solani can destroy the ROS scavenging system, causing oxidative damage and inhibiting the growth of apple rootstocks.