Plant immune responses can be induced by plant growth-promoting rhizobacteria (PGPRs), but the exact compounds that induce resistance are poorly understood. Here, we identified the novel natural elicitor 3,4-dihydroxy-3-methyl-2-pentanone from the PGPR Bacillus subtilis HN09, which dominates HN09-induced systemic resistance (ISR). The HN09 strain, as a rhizobacterium that promotes plant growth, can induce systemic resistance of Arabidopsis thaliana plants against Pseudomonas syringae pv. tomato DC3000, and the underlying role of its metabolite 3,4-dihydroxy-3-methyl-2-pentanone in this induced resistance mechanism was explored in this study. The stereoisomers of 3,4-dihydroxy-3-methyl-2-pentanone exhibited differential bioactivity of resistance induction in A. thaliana. B16, a 1:1 mixture of the threo-isomers (3R,4S) and (3S,4R), was significantly superior to B17, a similar mixture of the erythro-isomers (3R,4R) and (3S,4S). Moreover, B16 induced more expeditious and stronger callose deposition than B17 when challenged with the pathogen DC3000. RT-qPCR and RNA-seq results showed that B16 and B17 induced systemic resistance via JA/ET and SA signalling pathways. B16 and B17 activated different but overlapping signalling pathways, and these compounds have the same chemical structure but subtle differences in stereo configuration. Our results indicate that 3,4-dihydroxy-3-methyl-2-pentanone is an excellent immune elicitor in plants. This compound is of great importance to the systemic resistance induced by HN09. Its threo-isomers (3R,4S) and (3S,4R) are much better than erythro-isomers (3R,4R) and (3S,4S). This process involves SA and JA/ET signalling pathways.

Chinese olive (Canarium album Raeusch.) is a traditional Chinese medicinal plant, mainly cultivated in Guangdong and Fujian provinces in China (Lai et al. 2022). In October 2023, Chinese olive fruit spots were observed in all the Chinese olive orchards surveyed in Chaozhou city (23.75°N, 116.67°E) of Guangdong, with an incidence up to 15%. Early disease symptoms on fruits appeared as circular or irregular, dark brown to black spots with yellowish lesions, and later the spots slowly coalesced to form large necrotic areas, which seriously affected the fruit marketability. To isolate the causal agent, small pieces (~0.3 mm2) of fruit tissue were excised from the lesion margins, and surface-disinfested with 75% (v/v) ethanol for 1 min, followed by 1% NaClO for 3 min, and rinsed three times with sterile water. The pieces were then placed on potato-dextrose-agar (PDA) and incubated at 27°C. Ultimately, four fungal isolates were obtained with similar morphology phenotypes, colonies initially appeared white with irregular margins and after 4-6 days turned dark gray gradually with dense aerial myceliu. Microscopy revealed conidia were single-celled, hyaline, aseptate, fusiform to subclavate, and measured 18.1-22.5 μm × 6.4-9.3 μm (19.8 × 7.4 m on average, n = 30), which were consistent with those descriptions of Botryosphaeria dothidea (Vasić et al. 2013; Zhang et al. 2023). To further identity the isolates, partial sequences of ribosomal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α), and β-tubulin (TUB2) genes were amplified using primers ITS1/ITS5, TEF-F/R, TUB2-F/R, respectively (Xu et al., 2023; Hong et al. 2006). The sequences of four isolates were identical, and those of representative strain GDCZ-1 were deposited in GenBank (ITS, OR584295; TEF1-α, OR685157; TUB2, OR685158). Using Neighbor-Joining algorithm, phylogenetic tree based on concatenated sequences of ITS, TEF1-α, and TUB2 showed that GDCZ-1 clustered with B. dothidea. To fulfill Koch's postulates, pathogenicity tests were performed on healthy Chinese olive fruits using the needle-prick inoculation method. The fruits were wounded with a sterile needle at the equatorial area (depth of 3-4 mm), and inoculated with 10 µL of spore suspension (106 /mL). The control fruits were inoculated with sterile water. Inoculated fruits were placed in sterile plastic containers to maintain high relative humidity (almost 100%) and incubated at 27°C. After 4 days, the inoculated fruits showed similar symptoms with those observed in the field infected fruits, while the control remained asymptomatic. Pathogen re-isolated from the inoculated fruits showed identical morphological characteristics to the original isolate GDCZ-1. As far as we know, fruit rot caused by Alternaria alternata has been recently reported on C. album in China (Shao et al. 2024). To our knowledge, this is the first report of B. dothidea causing fruit rot disease on C. album in Guangdong. Our report will provide crucial information for studying the epidemiology and management of this disease.

Chinese olive (Canarium album) is a well-known plant used both as food and medicine, extensively cultivated in southern China (Kuo et al., 2016). Its production is challenged by various fungal diseases. From July to October 2023, Chinese olive plants in an approximately 400 hectare conventional hillside orchard in Chaozhou, Guangdong province, China (23°39'45.36″N, 116°37'45.48″E), where sustainable Chinese olive farming has been practiced for over a decade, exhibited leaf blotch symptoms with a disease incidence of 40% (n = 100 plants investigated). Symptoms include yellow or brown leaf blotches, followed by drying, curling, and eventual leaf drop. To identify the causal agent, seven symptomatic leaves from seven trees were collected. The symptomatic leaves were cut into pieces of about 0.3 mm2 at the necrotic lesion edges and surface disinfected. The pieces were placed on potato dextrose agar (PDA), incubated in the dark at 28°C for 5 days, then subcultured for 7 days. Four fungal isolates were obtained with an isolation frequency of 57%. Genomic DNA was extracted from the isolates. Morphological analysis and internal transcribed spacer (ITS) region sequencing revealed no observable differences among isolates. The representative isolate (LB-1) cultured on PDA was initially white, turning light grey with dense aerial mycelium after 7 days. The transparent hyphae displayed intricate branching patterns and multiple cellular structures. The conidia were single-celled, hyaline, fusiform structures measuring 3.6-7.8 × 1.17-2.83 μm (n = 50). Morphologically, the fungal isolates resembled Phyllosticta capitalensis (Wikee et al. 2013). Primers ITS1/ITS5, ACT-512F/ACT-783R, and EF1/EF2 were used to amplify the ITS region, the actin (ACT) gene, and the transcription elongation factor (TEF), respectively (Wang et al. 2023). The sequences obtained from the LB-1 isolate have been deposited in NCBI GenBank (accession nos. PP922193; PP960207 and PP960206). A phylogenetic tree was constructed based on aligned sequences (Wang et al. 2023). BLAST analysis showed LB-1 isolate shares 99.4%-100% similarity with sequences KY855645, KY855590, and KY855919 from P. capitalensis CPC27084. Based on morphological and molecular analysis, isolate LB-1 was identified as P. capitalensis. Pathogenicity assays of the LB-1 isolate were conducted on 2-year-old Canarium album seedlings. A 10⁶ spores/mL LB-1 suspension was applied to 3 mm spots on the leaves of four plants for inoculation. Controls leaves was treated with sterile water. The inoculated plants were placed in a greenhouse at 28°C with 80% humidity. The experiment was repeated 3 times. Leaves inoculated with LB-1 isolates showed blotch symptoms similar to those in the field, the controls remained healthy. Reisolation of P. capitalensis was performed confirmed by morphology and DNA sequencing, fulfilling Koch's postulates. Previous studies have shown that P. capitalensis causes fungal diseases on fruit trees such as Citrus lalifolia and Mangifera indica (Wikee et al. 2013). To our knowledge, this is the first report of Phyllosticta capitalensis causing leaf blotch on Chinese olive in China. Preliminary observations suggest it may be present in other olive-producing regions of Guangdong. It causes significant leaf wilting and shedding, potentially reduces fruit quality, and lowers overall yield. This discovery will offer valuable insights for creating effective control measures against this recently emerged severe disease.