Cyclocarya paliurus (Batalin) Iljinsk. is a multiple function tree species distributed in subtropical areas, and its leaves have been used in medicine and nutraceutical foods in China. However, little information on the effects of nitrogen (N) forms and ratios on growth and secondary metabolite accumulation is available for C. paliurus. The impact of five NO₃⁻/NH₄⁺ ratios on biomass production, triterpenoid accumulation and related gene expression in C. paliurus seedlings was evaluated at the middle N nutrition supply. Significant differences in seedling growth, triterpenoid accumulation and relative gene expression were observed among the different NO₃⁻/NH₄⁺ ratio treatments. The highest triterpenoid content was achieved in a sole NO₃⁻ or NH₄⁺ nutrition, while the mixed N nutrition with equal ratio of NO₃⁻ to NH₄⁺ produced the highest biomass production in the seedlings. However, the highest triterpenoid accumulation was achieved at the treatment with the ratio of NO₃⁻/NH₄⁺ = 2.33. Therefore, the mixed N nutrition of NO₃⁻ and NH₄⁺ was beneficial to the triterpenoid accumulation per plant. The relative expression of seven genes that are involved in triterpenoid biosynthesis were all up-regulated under the sole NH₄⁺ or NO₃⁻ nutrition conditions, and significantly positive correlations between triterpenoid content and relative gene expression of key enzymes were detected in the leaves. Our results indicated that NO₃⁻ is the N nutrition preferred by C. paliurus, but the mixture of NO₃⁻ and NH₄⁺ at an appropriate ratio would improve the leaf triterpenoid yield per area.

Cyclocarya paliurus (Batal.) Iljinskaja is a rare and multifunctional tree species endemic to China. This study aimed to establish a feasible method for polyploid induction in C. paliurus with colchicine treatment, and the obtained polyploid plants were identified and characterized. In this study, natural diploid and tetraploid C. paliurus seedlings were treated with different concentrations and durations of colchicine to induce polyploidization. The results indicated that a colchicine concentration of 0.4% for 4 d was the most suitable induction protocol, respectively. Compared with diploid and tetraploid control plants, the induced polyploid plants were shorter and thinner. The number of compound leaves in induced polyploids were fewer, and the compound leaf areas became smaller. The stomatal density of induced polyploids decreased, but the stomata became larger and wider, with an increased number of chloroplasts in the guard cells. The induced polyploids exhibited higher levels of carotenoid, and the contents of soluble sugar, soluble starch, and soluble protein were higher than those of controls. The polyploid plants exhibited an increase in the contents of growth-inhibiting hormones (JA) and a decrease in those of growth-promoting hormones (IAA, GA₃, BR). In summary, the induced polyploids with a dwarfing effect would provide superior germplasm resources for leaf-harvesting plantation. As C. paliurus is endemic to China, our results have a rather local effect. Overall, the established polyploid induction method of C. paliurus will provide technical support for creating superior C. paliurus germplasm resources and subsequent plant breeding research.

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.

This study aimed to provide foundational research for the biological control of postharvest avocado fruits anthracnose and establish a microbial system of postharvest avocado fruits. The high-throughput sequencing of avocado fruits from the anthracnose-infected and healthy groups was performed using Illumina NovaSeq second-generation sequencing technology. The results revealed that, except for Colletotrichum sp. strain 38#, there were differences in the bacterial community structure of avocados before and after infection, as determined through alpha and beta diversity analysis. Additionally, there were significant differences in the endophytic fungal community structure, allowing clear differentiation between the infected and healthy avocados. The endophytic bacterial community was primarily composed of 4 phyla and 10 genera, with the Bacteroidota phylum and Chryseobacterium genus demonstrating sensitivity to anthracnose pathogens, as evidenced by a decrease in their relative abundance after infection. The endophytic fungal community was characterized by 3 phyla and 10 genera. After infection, the relative abundance of 2 phyla (Anthophyta and Basidiomycota) and 7 genera (Eucalyptus, Candida, Kluyveromyces, Talaromyces, Oidiodendron, Nigrospora, and Pestalotiopsis) decreased, whereas the relative abundance of the Colletotrichum genus increased dramatically. The LEfSe (Linear discriminant analysis Effect Size) analysis indicated that significant biomarkers were more prevalent in endophytic bacteria than in endophytic fungi in the avocados. In endophytic bacteria, the key biomarkers included the Firmicutes phylum (Bacilli class), Proteobacteria phylum (Gammaproteobacteria class, Pseudomonadales order, Pseudomonadaceae family, and Pseudomonas genus), Flavobacteriales order, Weeksellaceae family, and Chryseobacterium genus. In endophytic fungi, the important biomarkers were Saccharomycetes class (Saccharomycetales order), Glomerellales order (Glomerellaceae family and Colletotrichum genus), and Botryosphaeriales order (Botryosphaeriaceae family and Lasiodiplodia genus). These results may provide a theoretical basis for the development of future biological agents for avocado anthracnose.

Background: Avocado is an important economic fruit tree that requires a lot of nitrogen (N) to support growth and development. Nitrate transporter (NRT) gene family plays an essential role in N uptake and use in plants. However, no systematic identification of the NRT gene family has been reported in avocado. Methods: Bioinformatic analysis was used to identify and characterize the NRT gene family in avocado. The five N additions (29.75, 59.50, 119.00, 178.50, and 238.00 mg/L N) were used to identify the N requirement of avocado seedlings based on physiological indexes, while RNA-seq was conducted to analyze the response of PaNRTs under low-N and high-N conditions. Results: Sixty-one members of the NRT gene family were identified and dispersed on 12 chromosomes in avocado. Many cis-regulatory elements (CREs) related to phytohormonal and stress response were found in the PaNRTs promoter regions. The avocado leaves in N3 have the highest activities of N-assimilating enzymes and N content as well as the lowest activities of antioxidant enzymes. Thus, 29.75 mg/L and 119.00 mg/L were chosen as low-N supply and normal-N supply for transcriptome analysis. The transcriptome analysis showed that PaNRT1.11, PaNRT1.22, PaNRT1.32, PaNRT1.33, PaNRT1.38, and PaNRT1.52 and PaNRT1.56 among PaNRT1 members were up-regulated under normal-N condition in the leaves or roots, suggesting that these genes might affect N absorption under nitrate-sufficient conditions in avocado. RT-qPCR analysis found the relative expression patterns of selected genes among four samples were consistent with transcriptome data, suggesting that transcriptome data were reliable. Conclusions: This study would provide valuable information for identifying the functions of the NRT gene family in avocado.

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.