Alternaria leaf spot seriously threatens the sustainable development of the global apple industry, causing significant losses and reducing fruit quality and yield. The causal agent Alternaria alternata f. sp. mali (Alternaria mali, ALT) produces various molecules to modulate infection, such as cell wall-degrading enzymes, toxins, and elicitor-like molecules. ALT produces the host-specific AM-toxin, an important pathogenicity factor. ALT also releases effectors into apple cells that modify host defense, but these proteins have not yet been described. Here, we identified the pathogenic fungal types responsible for early defoliation from diseased leaves of Fuji (Malus domestica cv. ‘Fuji’) apple collected from five districts in Shandong Province, China. The ALT isolates ALT2 to ALT7 were pathogenic to four apple cultivars, with ALT7 being the most aggressive. We extracted mycotoxins (AM-toxin-2 to AM-toxin-7) from each isolate and used them to treat different apple varieties, which led to leaf-spot symptoms and damaged chloroplasts and nuclear membranes, followed by cell death. AM-toxin-7 produced the most severe symptoms, but chloroplasts remained intact when the mycotoxin was inactivated. Mass spectrometry identified 134 secretory proteins in ALT7 exosomes, and three secreted proteins (AltABC, AltAO, and AltPDE) were confirmed to be involved in apple pathogenesis. Therefore, ALT secretes AM-toxin and secretory proteins as an infection strategy to promote fungal invasion and overcome the host defense system.

Nutrient acquisition through symbiotic ectomycorrhizal fungi is carbon (C) costly but fundamental for plant growth, community, and ecosystem functioning. Here, we examined the functions of roots and mycorrhiza with respect to nutrient uptake after artificially inducing C limitation-seven months after girdling of an ectomycorrhizal tree, Pinus taeda. Root physiological activity (measured as root nitrogen content and root exudation) declined after girdling and was accompanied with 110% and 340% increases in mycorrhizal colonization and extramatrical hyphal length, respectively. Fungi colonizing roots switched to a community characterized by higher C efficiency (lower C cost) of nutrient acquisition (CENA, the amount of nutrient acquisition per unit C cost) and lower network complexity, indicating a tradeoff between CENA and stability of the fungal community. Root transcriptome analysis suggested a shift in metabolic pathways from a tricarboxylic acid cycle decomposition of carbohydrate to lipid biosynthesis to maintain closer associations with mycorrhiza for nutrient cycling after the girdling. By integrating multi-level evidence, including root transcriptome, fungal composition, and network complexity data, we demonstrate an increased dependence on mycorrhiza for nutrient acquisition under the C limitation condition, which is likely due to a shift to fungal community with higher CENA at the cost of lower stability.

Apple (Malus domestica) is an economically important fruit crop, but its production is affected by Glomerella leaf spot, a devastating disease caused by the fungal pathogen Colletotrichum gloeosporioides. MicroRNA (miRNA) is a kind of non-coding RNA that plays an important role in the process of plant–pathogen interactions. However, little is known about the miRNAs that influence apple resistance against C. gloeosporioides. A novel miRNA, MIR396d-p3, was identified through small RNA sequencing (sRNA-seq). Functional analyses revealed that MIR396d-p3 negatively regulates apple resistance to C. gloeosporioides. In addition, MdUGT89A2 and MdRGA3 were confirmed as targets of MIR396d-p3 using 5′ RACE and heterologous expression assays. We further found that overexpressing MdUGT89A2 and MdRGA3 induce apple disease resistance to C. gloeosporioides, while silencing of MdUGT89A2 and MdRGA3 reduces resistance to C. gloeosporioides. These results indicate that MIR396d-p3 plays a role in the response to the infection of C. gloeosporioides through regulating the expressions of MdUGT89A2 and MdRGA3. This research provides a new perspective on the interaction between apples and C. gloeosporioides and offers possible targets for resistance breeding.