The Botrytis bunch mold, Botrytis cinerea pathogen is a necrotrophic ascomycete that infects hundreds of plant species, including hazelnut. B. cinerea produces toxins that induce cell wall degrading enzymes. In the current research work, we used eight hazelnut varieties and recorded their resistance levels in response to B. cinerea infection. Results showed that different varieties respond differently to B. cinerea infection. Disease index analysis revealed the resistance level of eight hazelnut varieties in the order of DW > OZ > L3 > PZ > XD1 > YZ > L1 > QX. Moreover, cell membrane permeability as well as the activities of cell wall degrading enzymes were measured. The increased level of cell wall degrading enzymes facilitates the fungal pathogens’ ability to colonize plants and cause infection. According to the results obtained through enzyme analysis, the hazelnut varieties L1 and QX, which were proved to be highly susceptible against B. cinerea, had the highest cell wall degrading enzyme production. DW and OZ, which were revealed to be resistant varieties through disease index data, also showed relatively lower activity of degrading enzymes as compared to other varieties. Our comparison analysis between the disease index and enzyme production confirms that disease occurrence and plant susceptibility strongly depend upon cell wall permeability. Our enzyme activity results validated the resistance order revealed by disease index assessment results (DW > OZ > L3 > PZ > XD1 > YZ > L1 > QX), and varieties DW and OZ were found to be the most resistant, while QX and L1 were found to be the most susceptible varieties against B. cinerea infection. Our study lays the foundation to further explore other factors involved in grey mold resistance in hazelnut.

Background: Hazelnut (Corylus), a significant woody oil tree species in economic forests, faces production constraints due to biotic stresses, with Hazelnut Husk Brown Rot, caused by the pathogenic necrotrophic fungus Botrytis cinerea (B. cinerea), being the most severe. To date, limited information is available regarding the resistance of hazelnuts to B. cinerea. To better understand the mechanisms of resistance to B. cinerea. in hazelnut, we conducted metabolomics and WGCNA analyses of a B. cinerea-resistant Ping’ou hybrid hazelnut variety (Dawei; DW) and a susceptible variety (Qiuxiang; QX). Methods: In this study, metabolomics and weighted gene co-expression network analysis (WGCNA, weighted correlation network analysis) were applied to elucidate the resistance mechanisms underlying different hazelnut varieties to B. cinerea. Our study focused on the metabolome profiles of DW and QX plants after 72 h of B. cinerea infection. Results: Venn analysis of QX_0 vs. DW_0 and QX_72 vs. DW_72 revealed 120 differential accumulation metabolites (DAMs) that were upregulated. Among these metabolites, the concentrations of flavonoids and phenolic acids in DW were significantly higher than those in QX, respectively, suggesting that the elevated levels of these compounds contribute substantially to the resistance of hazelnut against B. cinerea. 3,4-hydroxyphenyllactic acid and phloretin were significantly more abundant in accumulation in DW than in QX after infection by B. cinerea. Conclusions: This study provides that the elevated levels of these compounds (flavonoids and phenolic acids) contribute substantially to the resistance of hazelnut against B. cinerea. Furthermore, 3,4-hydroxyphenyllactic acid and phloretin were identified as pivotal metabolites in modulating the resistance of hazelnut to B. cinerea. Through WGCNA analyses, we identified four transcription factors (WRKY19, HSFC1, ERF071, and RAP2-1) that are most likely to regulate the synthesis of 3,4-dihydroxyphenyllactic acid and phloretin. This study provides crucial insights for further investigation into the regulatory network of metabolites associated with hazelnut resistance to B. cinerea.

Aralia elata (Miq.) Seem., is grown for its medicinal and nutritional properties in northeastern China. The tender shoots are used as wild vegetables. The plant saponin components have antioxidant and neuroprotective activities, and are used for the treatment of chronic disease (Xia et al. 2021). In July 2021, root rot disease was observed in five-year-old A. elata plants in Qingyuan County (41°91' N, 124°59' E), Liaoning Province, China. The incidence of roots rot was approximately 50% in old fields, with the leaves of the infected plants appearing chlorotic and wilting. The lesions on the taproots were dark brown and soft, with degraded internal organization. Leading edge of necrotic tissue from symptomatic roots was cut 5×5×3 mm, placed in 75% ethanol for 30 s, and then in 3% sodium hypochlorite for 2 min. After three rinses in sterile distilled water, the samples were dried on sterile filter paper before plating on potato dextrose agar (PDA) and incubation at 25℃. Monosporic cultures were obtained by the collection of single spores from individual isolates. After 7 days on PDA, mycelia in the colonies appeared cottony and pink, white, or purple in color, while their undersides were pink and white. Spore characteristics were evaluated after transfer to carnation leaf agar (CLA) and incubation for 20 days (Zhang et al. 2021). The macroconidia were falciform, slightly curved or straight, two to five septate, and 20.57 to 33.75 × 3.62 to 6.11 μm (n=40). The microconidia were ovoid or oval, zero to one septate, and 5.12 to 13.53 × 3.04 to 4.79 μm (n=40). Chlamydospores were globose to subglobose, intercalary or terminal, with an average diameter of 13.76 μm (n=40).To identify the pathogen, the internal transcribed spacer (ITS) region, large subunit (LSU) ribosomal DNA, and translation elongation factor 1 alpha (TEF-1α) gene were amplified using the respective primer pairs ITS1/ITS4, LR0R/LR7, and EF1-728F/EF1-986R (Cheng et al. 2020; Fu et al. 2019). Comparisons with GenBank, the sequences of ITS, LSU, and TEF-1 had 99 to 100% homology with Fusarium oxysporum (accessions numbers- MH707084, OQ380519, and GU250609, respectively). The sequences were deposited in GenBank: OP482273 (ITS), OP491955 (LSU), and OP503498 (TEF-1α). Maximum likelihood phylogeny of the identified sequences using MEGA-X software indicated that the isolate represented F. oxysporum. The taproots of 30 one-year-old A. elata were washed and inoculated with 1×106/ml of the conidial suspension for two hours, and another 30 used as controls with sterile water. After planting in sterilized forest soil in flowerpots (36×30 cm), the plants were grown in a greenhouse for two weeks at 25℃ with 14 h of light. It was found that 50% of the roots showed typical root rot symptoms, while the controls were asymptomatic. The pathogenicity test was repeated three times, and reisolation of F. oxysporum from the roots fulfilled Koch's postulates. This is the first report of root rot in A. elata caused by F. oxysporum in China and indicates the necessity for suitable management strategies to protect A. elata production. References: Cheng, Y., et al. 2020. Plant Dis. 104:3072. Fu, R., et al. 2019. Plant Dis. 103:1426. Xia, W., et al. 2021. Mini-Rev Med Chem. 21:2567. Zhang, X. M., et al. 2021. Plant Dis. 105:1223.

Botrytis cinerea, a pathogenic fungus that causes necrosis in plants, is one of the most destructive pathogens of hazelnuts. This fungus is responsible for causing Husk Brown Rot, a significant threat to hazelnut production. The plant’s defense mechanism against this pathogen, as well as other pathogens, is a complex biological process that involves changes at molecular, biochemical, and physiological levels. To better understand the molecular responses of hazelnut plants to B. cinerea infection, we conducted a comparative transcriptome profiling study between a B. cinerea-resistant Ping’ou hybrid hazelnut variety (Dawei; DW) and a susceptible variety (Qiuxiang; QX). Our study focused on the transcriptome profiles of DW and QX plants after three days of B. cinerea infection. The results of our study showed moderate changes in the defense strategies of both DW and QX plants in response to B. cinerea infection. Specifically, we observed that the expression of 14 disease-resistant genes was significantly different between DW and QX. Our comparative analysis revealed that DW had a higher number and expression of immunity-related differentially expressed genes compared to QX, which indicates that these genes play a crucial role in inducing innate resistance in DW plants against B. cinerea infection. This study highlights that plant resistance to pathogens like B. cinerea is a complex process that is controlled by multiple genes and biological pathways, each playing a specific role. Our findings provide new insights into the development of hazelnut varieties that are resistant to B. cinerea infection. By using the candidate genes identified in this study, it may be possible to enhance the resistance of hazelnut plants to B. cinerea and reduce the impact of Husk Brown Rot on hazelnut production.

Aralia elata (Miq.) Seem, is an important cash crop in northeastern China. The tender shoots are rich in amino acids, vitamins, and trace elements, and the saponins of leaves and roots have antioxidant and immune-boosting properties. In August of 2023, root rot disease was observed in 4-year-old A. elata plants in Dongzhou County (41.79 °N, 124.11 °E), Liaoning Province, China, with an overall incidence of approximately 40%. While previous studies have described root rot in A. elata caused by Fusarium oxysporum (Zheng et al. 2024), this new root rot showed different symptoms. The leaves of the diseased plants were water-soaked and wilted but not chlorotic. The internal tissue of the roots was disintegrated and soft, while the xylem appeared brown and empty with only the epidermis remaining. Diseased root tissues were sterilized with 75% ethanol for 30 s, treated with 2% sodium hypochlorite for 2 min, and rinsed twice in sterile distilled water. Twelve isolates with similar morphological characteristics were obtained (isolation frequency of 80%), the isolates were plated on PDA medium and incubated at 25℃. Single-zoospore cultures were obtained and transferred to soybean agar medium (SA). After growth on SA for 7 days, the formation of rounded colonies of white aerial mycelia was observed. The sporangia were ovoid or ellipsoid, papillate, terminally borne, with an average length and width of 30.24 (18.35 to 45.39) × 24.16 (14.55 to 37.64) μm. The isolates were homothallic, produced plerotic oogonia with smooth walls with an average diameter of 27.97 (21.06 to 35.90 μm), and yielded one paragynous or anohiginous antheridium, sized 12.21 (10.11 to 14.72) μm × 9.75 (8.39 to 11.31) μm. Based on these morphological characteristics, the isolates were identified as part of the Phytophthora genus (Montealegre et al. 2016). The internal transcribed spacer (ITS) region and cytochrome oxidase subunit Ⅱ (COXⅡ) gene were amplified using the respective primers ITS1/ITS4 and FM66/FM58 (Wiseman et al. 2018). The ITS and COXⅡ sequences were compared with sequences in GenBank, showing 99.83% to 100% sequence identity with Phytophthora cactorum (accession nos. OR347848 and OR351295, respectively). The sequences have been deposited in GenBank with the accession numbers PP479774 (ITS) and PP489221 (COXⅡ). Construction of a maximum-likelihood phylogenetic tree of the identified sequences using MEGA-X indicated that the isolate could be identified as P. cactorum. To confirm pathogenicity, shallow wounds were made in the taproots of 10 one-year-old A. elata and were inoculated with four 9-mm diameter agar plugs from one-week-old oomycete cultures on SA agar per root, and the pathogenicity test was repeated twice. Another 10 wounded plants inoculated with agar plugs without oomycete served as the controls. The plants were planted in flowerpots filled with sterilized forest soil and cultured in a greenhouse at 25℃ with 14 h of light per day. After 20 days, the inoculated plants showed similar symptoms to those initially observed in the field. No symptoms of root rot were observed in the controls. The P. cactorum was re-isolated from the inoculated plants, and identified based on the morphology and ITS and COXⅡ sequence, fulfilling Koch's postulates. P. cactorum has been reported to cause root rot in A. elata in Korea and Japan (Lee et al. 2005; Uchida et al. 1984). To our knowledge, this is the first report of P. cactorum causing root rot in A. elata in China. P. cactorum is a serious threat to A. elata cultivation, and disease management strategies need to be developed.