Mulberry (Morus) is an economically important woody tree that is suitable for use in sericulture as forage and in medicine. However, this broad-leaved tree is facing multiple threats ranging from phytopathogens to insect pests. Here, a Gram-positive, endospore-forming bacterium (ZJU1) was frequently isolated from healthy mulberry plants by screening for foliar endophytes showing antagonism against pathogens and pests. Whole-genome sequencing and annotation resulted in a genome size of 4.06 Mb and classified the bacterium as a novel strain of Bacillus amyloliquefaciens that has rarely been identified from tree leaves. An integrative approach combining traditional natural product chemistry, activity bioassays, and high-resolution mass spectrometry confirmed that strain ZJU1 uses a blend of antimicrobials including peptides and volatile organic compounds to oppose Botrytis cinerea, a major phytopathogenic fungus causing mulberry gray mold disease. We showed that the inoculation of endophyte-free plants with ZJU1 significantly decreased both leaf necrosis and mortality under field conditions. In addition to the direct interactions of endophytes with foliar pathogens, in planta studies suggested that the inoculation of endophytes also induced plant systemic defense, including high expression levels of mulberry disease resistance genes. Moreover, when applied to the generalist herbivore Spodoptera litura, ZJU1 was sufficient to reduce the pest survival rate below 50%. A previously undiscovered crystal toxin (Cry10Aa) could contribute to this insecticidal effect against notorious lepidopteran pests. These unique traits clearly demonstrate that B. amyloliquefaciens ZJU1 is promising for the development of successful strategies for biocontrol applications. The search for new plant-beneficial microbes and engineering microbiomes is therefore of great significance for sustainably improving plant performance.

The samples were diluted in methanol:water (1:1, dilution 1/10, UHPLC-grade CHEMSOLUTE^®), and 1 μL was injected into a C-18 column (Kinetex, 100 × 2.1 mm, 2.6 μm, Thermo Accucore) and analyzed with a UHPLC-HR-MS (UltiMate 3000 UHPLC, Dionex, USA) coupled to a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). The mass spectrometry analysis was performed in both positive- and negative-ion mode with a scan range of m/z 100–1,500 for both the full-scan and the targeted data-dependent mass spectrometry experiment (ddMS, Top N). The peak resolutions were set at 70,000 and 17,500 for the full-scan and ddMS analyses, respectively. The MS/MS experiments were conducted with an isolation window of 0.4 m/z at a peak resolution of 35,000 (average NCE 15, 30, 45). The maximum ion time was set to 200 ms, and the AGC target was set to 3e6. The analytical standard was prepared in methanol:water (1:1), and a 10 μL aliquot was injected under similar conditions to those mentioned above.