Evaluation of Virus-Free Chrysanthemum 'Hangju' Productivity and Response to Virus Reinfection in the Field: Molecular Insights into Virus-Host Interactions.

The shoot apical meristem culture has been used widely to produce virus-free plantlets which have the advantages of strong disease resistance, high yield, and prosperous growth potential. However, this virus-free plant will be naturally reinfected in the field. The physiological and metabolic responses in the reinfected plant are still unknown. The flower of chrysanthemum 'Hangju' is a traditional medicine which is unique to China. In this study, we found that the virus-free 'Hangju' (VFH) was reinfected with chrysanthemum virus B/R in the field. However, the reinfected VFH (RVFH) exhibited an increased yield and medicinal components compared with virus-infected 'Hangju' (VIH). Comparative analysis of transcriptomes was performed to explore the molecular response mechanisms of the RVFH to CVB infection. A total of 6223 differentially expressed genes (DEGs) were identified in the RVFH vs. the VIH. KEGG enrichment and physiological analyses indicated that treatment with the virus-free technology significantly mitigated the plants' lipid and galactose metabolic stress responses in the RVFH. Furthermore, GO enrichment showed that plant viral diseases affected salicylic acid (SA)-related processes in the RVFH. Specifically, we found that phenylalanine ammonia-lyase (PAL) genes played a major role in defense-related SA biosynthesis in 'Hangju'. These findings provided new insights into the molecular mechanisms underlying plant virus-host interactions and have implications for developing strategies to improve plant resistance against viruses.

First Report of Fusarium annulatum Causing Blight on Bletilla striata (Baiji) in China.

Bletilla striata (Thunb. ex A. Murray) Rchb (known as baiji in Chinese), a herbal plant distributed mainly in China, has become a focus of scientific attention recently due to its medicinal value (He et al. 2017). In May 2023, blight symptoms on leaves and stems were observed approximately 60% of Bletilla striata in Hangzhou, Zhejiang, China (29.80° N, 119.67° E). Brown spots initially appear on the infected leaves, which gradually decay as the spots expand. The wilting is accompanied with fading and yellowing, and eventually leading to defoliation. The infected stem initially appears brown spots, which gradually decay as the spots expand, resulting in the death of the whole plant, affecting the yield and quality of the herbs ultimately. To isolate the pathogen, small symptomatic leaves and stems (5×5 mm) were surface-disinfected with 75% ethanol for 30 s and 1% NaClO for 2 min, then rinsed in distilled water 3 times. Subsequently, the disinfected tissues were placed on PDA and incubated at 27 ℃ for 3 days. A total of 8 fungal isolates with similar morphological characteristics were obtained. The colony by single-spore purification was light purple to dark purple with abundant aerial mycelium. Macroconidia were relatively slender with a curve, mainly three to five septate and measuring 24.34 to 54.64 µm (average 40.29 µm) × 3.59 to 5.45 µm (average 4.49 µm) (n=30). Microconidia appeared obovoid to pyriform, with sizes of 5.31 to 8.43 µm (average 7.12 µm) × 2.30 to 4.29 µm (average 3.22 µm) (n=30). The morphological characteristics were consistent with Fusarium annulatum (Yilmaz et al. 2021). To further confirm the isolate's identification, the genomic DNA of isolates were extracted and identified by phylogenetic analyses of multilocus sequences of the RNA polymerase largest subunit (rpb1, primers Fa and G2R), RNA polymerase second largest subunit (rpb2, primers 7cf and 11ar) and the translation elongation factor 1-alpha (tef1, primers EF1 and EF2) (O'Donnell et al. 2022). The sequences were deposited in GenBank (rpb1: OR493933, OR493934, OR753402; rpb2: OR753398, OR753399, OR753400; tef1: OR493935, OR493936, OR753401). BLAST searches of the rpb1, rpb2, and tef1 sequences revealed 99.83% (1775/1778 nt), 99.79% (957/959 nt), and 98.98% (678/685 nt) homology with those of Fusarium annulatum CBS:258.54 from New Caledonia (rpb1: MT010944; rpb2: MT010983; tef1: MT010994). To confirm pathogenicity, one-year-old B. striata leaves and stems were disinfected with 75% ethanol, wounded with a sterile syringe on 3 healthy leaves and stems, inoculated with 5 × 5 mm mycelial discs of strain BJ-L1 and BJ-S1, respectively. And the control were treated similarly except that they were inoculated with PDA discs. The experiment was replicated 3 times. After 5 days, all inoculated leaves and stems showed similar symptoms to those initially observed on infected plants. The same pathogen was re-isolated and identified by morphological characterization and molecular analysis, confirming Koch's postulates. Thus, the pathogen causing blight of B. striata was determined to be F. annulatum. To our knowledge, this is the first report of F. annulatum causing blight on B. striata in China. F. annulatum has a wide range of hosts and has been reported to infect a wide range of crops, fruits and vegetables (Bacon et al. 1991). This study provides the basis for further research on this disease and is important for the management of this disease and the improvement of the economic benefits of B. striata.

First Report of Leaf Spot Caused by Botrytis cinerea on Strawberry in China.

In the winter of 2022, circular or irregular leaf spots were observed on strawberry (Fragaria × ananassa) planted in commercial fields (cultivar 'xuetu', 'mengzhifu') in Yinzhou, Ningbo, Zhejiang, China (N29°48'48″, E121°39'47″), with disease incidence ranging from 10 to 15% in a field approximately 0.67 ha in size. The estimated crop loss associated with this disease was ~10%. Symptoms included circular or irregular lesions with brown halos and wheel marks, which eventually developed into leaf blight and petiole decay, but spore masses were seldom found on the leaf surface. In severe cases, leaves withered and abscissed. To isolate the causal agent, ten diseased leaves from ten different plants were collected, surface-sterilized with 75% ethanol for 50 s, rinsed twice with sterile distilled water, cut into small pieces (0.5 cm × 0.5 cm), and plated on potato dextrose agar (PDA), then incubated at 25°C in darkness for 5 days. Isolates , which displayed one kind of colony morphology were consistently obtained from each of the ten samples, and 58 single-conidium isolates with the same colony morphology were obtained. The isolation frequency was 58 of 60 samples. The colonies that grew on PDA produced white mycelia, which sporulated after 1 week, producing typical Botrytis-like gray spores. Three isolates (NBCM-1, NBCM-2, NBCM-3) were selected for identification and pathogenicity assays. Conidia were round to ellipsoid, 9.2 to 14.3 µm long (n=50), and 6.4 to 9.2 µm wide (n=50). Sclerotia were not observed on PDA. Based on these characteristics, the pathogen was tentatively identified as Botrytis cinerea (Zhang 2001). PCR was conducted for each of the three isolates to amplify the G3PDH, HSP60, RPB2, NEP1, and NEP2 genes, which are typically used for molecular identification of Botrytis species (Staats et al. 2005; Liu et al. 2016). The resulting amplicons were sequenced, and the sequences were processed using BLAST in the National Center for Biotechnology Information. Sequences of the three isolates were deposited in GenBank (accession nos. OR052082 to OR052086, OR493405 to OR493414). BLASTn analyses showed that isolates were 99 to 100% identical to B.cinerea reported causing leaf spot on strawberry in California; accession numbers MK919496 (G3PDH, 883/883 bp), MK919494 (HSP60, 992/992 bp), and MK919495 (RPB2, 1081/1081 bp). The resulting concatenated data set of G3PDH-HSP60-RPB2-NEP1-NEP2 was used to conduct a multilocus phylogenetic analysis (MLSA) using the maximum likelihood method. The MLSA tree indicated that the three isolates belonged to Botrytis cinerea. To test for pathogenicity, three 1-month-old strawberry (cultivar 'xuetu') plants were inoculated with each isolate (NBCM-1, NBCM-2, NBCM-3). A noninoculated control (sterile water only) was also included. The strawberry plants were inoculated by spraying with conidia suspension (1.0 × 105/ml) until run-off. Inoculations with sterile water served as controls. All plants were kept at 28/25°C (day/night), under a 12:12-h light/dark photoperiod. All plants were covered with transparent plastic bags to maintain humidity for the first 48 h, after which the bags were removed. After 4 to 7 days, leaf spot symptoms similar to those observed in the field were observed in all inoculated plants, while the controls remained healthy. The experiment was repeated three times. The pathogen was reisolated from the inoculated leaves and again identified as B. cinerea, with the same methodology used for the initial identification. Leaf spot caused by B. cinerea on strawberry was recently reported in California (Mansouripour and Holmes 2020) and Florida (Marin and Peres 2022). To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in China. The pathogen is also the causal agent of Botrytis fruit rot on strawberry. Given the high variability of this pathogen (Marin and Peres 2022), further studies on its occurrence, spread, management, and control are required. The identification of this pathogen provides a basis for further research on its management and control.

Nigrospora oryzae Causing Leaf Spot Disease on Chrysanthemum × morifolium Ramat and Screening of Its Potential Antagonistic Bacteria.

Chrysanthemum × morifolium Ramat. is a famous perennial herb with medicinal, edible, and ornamental purposes, but the occurrence of plant diseases can reduce its value. A serious disease that caused leaf spots in C. morifolium appeared in 2022 in Tongxiang City, Zhejiang Province, China. The C. morifolium leaves with brown spots were collected and used for pathogen isolation. By completing Koch's postulates, it was proven that the isolate had pathogenicity to infect C. morifolium. It was determined that the pathogen isolated from chrysanthemum leaves was Nigrospora oryzae, through morphology and a multilocus sequence analysis method using a combination of the internal transcribed spacer gene (ITS), beta-tubulin gene (TUB2), and translation elongation factor 1-alpha gene (TEF1-α). This is the first report of C. morifolium disease caused by N. oryzae in the world. Through dual culture assay on PDA plates, 12 strains of bacteria with antagonistic effects were selected from 231 strains from the C. morifolium phyllosphere, among which Bacillus siamensis D65 had the best inhibitory effect on N. oryzae growth. In addition, the components of a strain D65 fermentation broth were profiled by SPME-GC-Q-TOF analysis, providing a foundation for further application and research of biological control.

Colletotrichum siamense Strain LVY 9 Causing Spot Anthracnose on Winterberry Holly in China.

Winterberry holly (Ilex verticillata) is an economically valuable landscaping ornamental plant. Serious outbreaks have been reported, in its leaf tips curl upward, irregular black brown spots appear on leaves, and extensive defoliation is commonly observed. The incidence in Hangzhou was estimated at 50% and resulted in large economic losses for growers in 2018. Samples were collected from the main cultivation area in Zhejiang Province. In total, 11 fungal isolates were obtained from diseased leaves through a single-spore purification method, and isolate LVY 9 exhibited strong pathogenicity. Based on morphology and molecular phylogenetic analyses based on multilocus sequence typing of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), and chitin synthase (CHS-1) genes, we identified the pathogen as Colletotrichum siamense, causative agent of anthracnose of winterberry holly.

Characterization and Control of Dendrobium officinale Bud Blight Disease.

Dendrobium officinale is an important traditional Chinese medicine (TCM). A disease causing bud blight in D. officinale appeared in 2021 in Yueqing city, Zhejiang Province, China. In this paper, 127 isolates were obtained from 61 plants. The isolates were grouped into 13 groups based on collected areas and morphological observations. Four loci (ITS, LSU, tub2 and rpb2) of 13 representative isolates were sequenced and the isolates were identified by constructing phylogenetic trees with the multi-locus sequence analysis (MLSA) method. We found the disease to be associated with three strains: Ectophoma multirostrata, Alternaria arborescens and Stagonosporopsis pogostemonis, with isolates frequencies of 71.6%, 21.3% and 7.1%, respectively. All three strains are pathogenic to D. officinale. A. arborescens and S. pogostemonis isolated from D. officinale were reported for the first time. Iprodione (50%), 33.5% oxine-copper and Meitian (containing 75 g/L pydiflumetofen and 125 g/L difenoconazole) were chosen to control the dominant pathogen E. multirostrata, with an EC50 value of 2.10, 1.78 and 0.09 mg/L, respectively. All three fungicides exhibited an effective inhibition of activities to the growth of the dominant pathogen E. multirostrata on potato dextrose agar (PDA) plates, with Meitian showing the strongest inhibitory effect. We further found that Meitian can effectively control D. officinale bud blight disease in pot trial.

Regenerating Saffron (Crocus sativus L.) from Corm Lateral Buds via Indirect Somatic Embryogenesis.

Saffron (Crocus sativus L.), being one of the distinguished commercial spice crops in the world, is in demand for its culinary, colorant, and pharmaceutical benefits. In this study, a novel indirect somatic embryogenesis (SE) system was, thus, established for the study of this plant. To this end, firstly, the lateral buds were cultured. Then, the cultures were transformed using Murashige and Skoog (MS) medium supplemented with either 6-benzyladenine (BA: 5 and 10 mg/L), naphthalene acetic acid (NAA: 0, 1, and 2 mg/L), or trans-zeatin (tZ: 0, 0.5, and 1.0 mg/L), before being classified into four structures: white globular (WG), yellow compact nodular (YCN), yellow-brown fragile (YBF), and dark-brown porous (DBP). As soon as BA (10 mg/L) and NAA (2 mg/L) were added, elevated percentages of white globular calli (56.8%) and white globular calli (31.5%) structures were induced. Additionally, 6-benzyladenine (5 mg/L) and naphthalene acetic acid (1 mg/L) allowed the formation of yellow-brown fragile structures, and the combination of 6-benzyladenine (10 mg/L) with trans-zeatin (1 mg/L) formed the DBP structures. After three months, the white globular calli were incubated using the MS basal medium, before being augmented with thidiazuron (TDZ: 1 mg/L) and picloram (PIC: 2 mg/L), from which 60% of the cases matured into shoots and, ultimately, cormlets. Morphoanatomical analyses also showed that the white globular calli cells were closely arranged, as they had a dense cytoplasm, a significant vascular differentiation, and embryoids. Furthermore, the yellow compact nodular structures were characterized by a strong differentiation capacity and contained many meristematic cells with high caryomitosis centers. We observed that the yellow-brown fragile calli had looser cell arrangements, with a vascular structure located on the protoderm edge, while there was no obvious cellular arrangement in the dark-brown porous structures. The induction of the adventitious buds in vivo on the MS medium that was supplemented with thidiazuron and picloram accordingly demonstrated the highest rates (60%) of white globular calli.

First Report of Pantoea ananatis Causing Crown Necrobiosis on Strawberry in China.

Strawberry (Fragaria × ananassa) is an economically important crop in Zhejiang, China. In the autumn of 2021, crown necrobiosis and angular leaf spot was observed in commercial strawberry fields (cultivar 'fenyu') in Cixi, Ningbo, Zhejiang, China (N30°9'55″, E121°21'13″). The disease incidence ranged from 5 to 8 % in the field, but could reach 50 to 60 % in some heavily affected plastic tunnels. In the affected field, this disease could reduce strawberry production by 50%. Early symptoms were water-soaked lesions around the vein of the abaxial leaves; subsequently, reddish-brown irregular spots and coalesced lesions developed. In humid conditions, a sticky bacterial ooze exuding from lesions was observed. Finally, the crown of the diseased plant was necrotized, and several pockets were observed inside the crown after dissection. To isolate the causal agent, the infected leaves and crown tissues from six different plants were surface-sterilized with 75% ethanol for 1 min, rinsed twice with sterile distilled water, cut into small pieces, and soaked in 5 ml of sterile distilled water for 20 min. The supernatant from the cut-up pieces was serially diluted and spread on nutrient agar medium. After 2 to 3 days at 28℃, several yellow colonies were grown on the medium. The colonies from five infected plants were gram-negative, anaerobic rods, yellow, viscous, and gloss, which are typical characteristics of Erwinia anana (Wells et al. 1986). To confirm the identity of the causal bacteria, PCR was conducted for six randomly selected colonies to amplify 16S rRNA (Monciardini et al. 2002), fusA, and gyrB (Stice et al. 2002). The amplicons were sequenced and blasted, and the results showed that the six colonies were identical. The 16S rRNA, fusA, gyrB sequences of the isolate CM3 were deposited in GenBank with accession number ON754076.1, OP587277, and OP587278; BLAST search showed 99.93% (1445 bp out of 1446 bp), 100% (746 bp out of 746 bp), 99.64% (1371 bp out of 1376 bp) similarity with strains of Pantoea ananatis (KT741001.1, MH015093.1 and CP066803.1 accessions, respectively). The resulting concatenated data set of 16S rRNA-fusA-gyrB was used to build a multilocus phylogenetic analysis (MLSA) by maximum likelihood criteria. The MLSA tree indicated that the isolate CM3 belonged to Pantoea ananatis. The isolate's identity was further confirmed by P. ananatis-specific primers pagyrB-F/R (Xiao et al. 2022). Thus, this isolate was designated as P. ananatis CM3. To fulfill Koch's postulates, two old leaves were broken off each of the ten 2-month-old strawberry (cultivar 'fenyu') plants to create wounds, each plants was sprayed with a cell suspension of P. ananatis (107CFU/ml, 0.5 ml) on the stem base. Ten plants were sprayed with water to serve as a control. All plants were kept at 28/25°C (day/night) under a 12-h/12-h photoperiod. All plants were covered with transparent plastic bags to maintain humidity. After 48 h, the bags were removed. After 2 weeks, water-soaked lesions on some leaves were observed similar to those in the field . Three to five weeks after inoculation, the crown of the inoculated plants was necrotized, which was similar to the symptoms in the field. No symptoms were observed in the control plants. The experiment was repeated three times. The bacteria were successfully reisolated from the inoculated crown tissues and leaves and confirmed as CM3 according to the same methodologies used for the initial identification. Bacterial leaf blight in strawberry caused by Pantoea ananatis has been reported in Nova Scotia, Canada, and Egypt (Bajpai et al. 2019; Abdel-Gaied et al. 2022). To our knowledge, this is the first report of Pantoea ananatis causing crown necrobiosis on strawberry in China. This report provides a basis for further research on this disease and its management and control.

Dendrobium Multi-Omics Reveal Lipid Remodeling in Response to Freezing.

Freezing damage is a common phenomenon responsible for reduced yields of economic crops. Regulation of lipid metabolism plays an important role in plant growth and adaptation during freezing. We previously carried out transcriptome and untargeted metabolome analyses to determine the regulation of flavonol and anthocyanin biosynthesis during freezing treatment (FT) and post-freezing recovery (FR) in Dendrobium catenatum. However, changes in lipid levels are hard to confirm by untargeted metabolomics analysis alone. Regulation of lipid metabolism in response to freezing is largely unknown in Dendrobium. In this study, a multi-omics strategy was used to offer a better means of studying metabolic flow during FT and FR. To this end, 6976 proteins were identified by the 4D_label-free proteome, including 5343 quantified proteins. For each of the two conditions, we enriched differentially accumulated proteins (DAPs) into 15 gene ontology (GO) terms, including primary metabolism, lipid metabolism, and photosynthesis processes. We also identified 7 lipid categories and 3672 lipid species using lipidome assays. We found significant remodeling occurring in the phospholipid category during FT and FR. We also found that most sphingolipids were significantly upregulated. An integrated multi-omics analysis revealed significant changes in the expression levels of 141 mRNAs and encoding proteins under both FT and FR conditions. During FT, phospholipase A (PLA) and phospholipase D (PLD) were associated with phospholipid editing and galactolipid remodeling. These results provide valuable new insights into how the freezing tolerance of D. catenatum might be improved by genetic engineering.

Identification of Two GDSL-Type Esterase/Lipase Genes Related to Tissue-Specific Lipolysis in Dendrobium catenatum by Multi-Omics Analysis.

Dendrobium catenatum is an important herb and widely cultivated in China. GDSL-Type Esterase/Lipase proteins (GELPs) are widely distributed in plants and play crucial roles in stress responses, plant growth, and development. However, no identification or functional analysis of GELPs was reported in D. catenatum. This study identifies 52 GELPs in D. catenatum genome, which is classified into four groups by phylogenetic analysis. Four conservative blocks (Ser-Gly-Asn-His) are found in most GELP domains. Transcriptome analysis reveals the expression profiles of GELPs in different organs and flowering phases. Co-expression analysis of the transcriptome and lipidome identifies a GELP gene, Dca016600, that positively correlates with 23 lipids. The purified Dca016600 protein shows the optimum pH is active from 8.0 to 8.5, and the optimum temperature is active from 30 °C to 40 °C. The kinetic study provides Vmax (233.43 µmol·min-1·mg-1) and Km (1.49 mM) for substrate p-nitrophenyl palmitate (p-NPP). Integrated analysis of the transcriptome and proteome identifies a GELP gene, Dca005399, which is specially induced by freezing. Interestingly, Dca005399 shows high expression in symbiotic germination seeds and sepals. This study provides new insights into the function of D. catenatum GELPs in plant development and stress tolerance.

Fusarium spp. Associated with Dendrobium officinale Dieback Disease in China.

A rare plant species of the Orchidaceae family, Dendrobium officinale is considered among the top ten Chinese medicinal herbs for its polysaccharide. Since 2021, when the dieback disease of D. officinale was first reported in Yueqing City, Zhejiang Province, China, Fusarium isolates (number = 152) were obtained from 70 plants in commercial greenhouses. The disease incidence ranged from 40% to 60% in the surveyed areas. Multilocus sequence analysis (MLSA) coupled with morphological characterization revealed that the collected isolates belonged to five species (sp.), viz., Fusarium concentricum, F. fujikuroi, F. nirenbergiae, F. curvatum, and F. stilboides, with isolation frequencies of 34.6%, 22.3%, 18.4%, 13.8%, and 10.5%, respectively. Notably, at least two Fusarium species were simultaneously isolated and identified from the infected plants. Finally, the pathogenicity test results demonstrated that such species were responsible for the dieback disease of D. officinale. However, F. concentricum and F. fujikuroi were more invasive compared to the other species in this study. To the best of the authors' knowledge, this study was the first report of F. concentricum, F. curvatum, F. fujikuroi, F. nirenbergiae, and F. stilboides causing the dieback disease of D. officinale in China and worldwide. This work provides valuable data about the diversity and pathogenicity of Fusarium populations, which will help in formulating effective strategies and policies for better control of the dieback disease.

Metabolic Profiling of Terpene Diversity and the Response of Prenylsynthase-Terpene Synthase Genes during Biotic and Abiotic Stresses in Dendrobium catenatum.

Dendrobium catenatum is a widely cultivated Chinese orchid herb rich in abundant secondary metabolites, such as terpenes. However, terpene distribution and characterization of terpene biosynthesis-related genes remain unknown in D. catenatum. In this study, metabolic profiling was performed to analyze terpene distribution in the root, stem, leaf, and flower of D. catenatum. A total of 74 terpene compounds were identified and classified. Clustering analysis revealed that terpene compounds exhibited a tissue-specific accumulation, including monoterpenes in the flowers, sesquiterpenes in the stems, and triterpenes in the roots. Transcriptome analysis revealed that the 'terpenoid backbone biosynthesis' pathway was only significantly enriched in root vs. flower. The expression of terpene biosynthesis-related genes was spatiotemporal in the flowers. Prenylsynthase-terpene synthases (PS-TPSs) are the largest and core enzymes for generating terpene diversity. By systematic sequence analysis of six species, 318 PS-TPSs were classified into 10 groups and 51 DcaPS-TPSs were found in eight of them. Eighteen DcaPS-TPSs were regulated by circadian rhythm under drought stress. Most of the DcaPS-TPSs were influenced by cold stress and fungi infection. The cis-element of the majority of the DcaPS-TPS promoters was related to abiotic stress and plant development. Methyl jasmonate levels were significantly associated with DcaTPSs expression and terpene biosynthesis. These results provide insight into further functional investigation of DcaPS-TPSs and the regulation of terpene biosynthesis in Dendrobium.

Molecular and Pathogenic Characterization of Fusarium Species Associated with Corm Rot Disease in Saffron from China.

Saffron (Crocus sativus L.) is a commercial spice crop well-known throughout the world, valued for culinary, colorant, and pharmaceutical purposes. In China, Fusarium nirenbergiae was detected as causative agent of saffron corm rot, the most pervasive disease for the first time in 2020. In the present study, 261 Fusarium-like isolates were recovered from 120 rotted corms in four saffron producing fields at Zhejiang, Shanghai, and Yunnan provinces, China, in 2021. A combination of morpho-cultural features and multilocus sequence analysis (MLSA) of the concatenated rpb2 (DNA-directed RNA polymerase II largest subunit) and tef1 (translation elongation factor 1-α) partial sequences showed that the isolates from saffron belong to Fusarium nirenbergiae as well as F. commune, and F. annulatum with isolation frequencies of 58.2%, 26.8%, and 14.9%, respectively. Notably, F. commune was more prevalent than F. annulatum in the collected samples. Pathogenicity tests confirmed that both species were pathogenic on saffron corm. This is the first report of F. annulatum and F. commune causing corm rot of saffron, globally. Outcomes of the current research demonstrate that Fusarium spp. associated with saffron corm rot are more diverse than previously reported. Furthermore, some plants were infected by two or more Fusarium species. Our findings broaden knowledge about Fusarium spp. that inflict corm rot and assist the development of control measures.

Analysis method development and health risk assessment of pesticide and heavy metal residues in Dendrobium Candidum.

Herbal medicines that are widely used worldwide are easily contaminated by pesticides and heavy metals, threatening human health. In this study, a modified QuEChERS pre-treatment method combined with HPLC/GC-MS/MS was established for the determination of 24 pesticide residues in Dendrobium candidum. The average recoveries of 24 pesticides in D. candidum were 76.9-110.0% with the relative standard deviation (RSD) of 0.28-11.40%, and their limits of detection (LOD) and limits of quantitation (LOQ) were 0.005-10 and 0.011-22 µg kg-1, respectively. The results showed that 83.33% of all samples had detected pesticide residues with the concentrations of 0.06-312.83 µg kg-1. Meanwhile, microwave digestion combined with ICP-MS was used to detect the residues of 8 heavy metals in D. candidum. The average recoveries of 8 heavy metals were 82.7-108.1% with an RSD of 1.4-8.0%, and their LOD and LOQ were 0.0001-0.05 mg kg-1 and 0.0003-0.2 mg kg-1, respectively. The results indicated that 8 heavy metals were all detected in all samples, and the highest concentration of Zn was 11.97 mg kg-1. Furthermore, the health risk assessment showed that the risk of the detected pesticides and heavy metals in samples to humans, specifically to the general population including adults and children, was acceptable.

Identification and Characterization of Fusarium nirenbergiae Associated with Saffron Corm Rot Disease.

Saffron (Crocus sativus L.) is the most expensive spice plant and is distributed widely around the world. However, its production is limited by corm rot, a disastrous disease, attributed to Fusarium oxysporum in many regions of the world. In 2020, extensive surveys were carried out in Zhejiang, Shanghai, Anhui, and Guizhou provinces as saffron growing areas of China. Fourteen single-spore isolates were obtained from rotted corms and identified as F. nirenbergiae according to morphological appearance and multilocus phylogenetic analysis with translation elongation factor 1-α (tef1), DNA-directed RNA polymerase II largest subunit (rpb2), and ß-tubulin (tub2). Results of the pathogenicity assay supported the conclusion that F. nirenbergiae is the pathogen responsible for corm rot. In this study, we obtained the whole genome sequence of two highly virulent F. nirenbergiae strains via the Illumina HiSeq platform. Genome sequence assemblies of approximately 52.7 and 52.2 Mb were generated for isolates WY5 and SH1, respectively. To the best of our knowledge, this is the first report of F. nirenbergiae causing C. sativus corm rot in China and indeed worldwide. Results from this research contribute to our understanding of genetic diversity, genomic information, and host determination, which will enable researchers to design appropriate management measures for this hazardous disease.

Production of Virus-Free Chrysanthemum (Chrysanthemum morifolium Ramat) by Tissue Culture Techniques.

Almost all plants in their natural environment are commonly infected by viruses. These viral infections can cause devastating diseases and result in severe yield and economic losses, making viral diseases an important limiting factor for agricultural production and sustainable development. However, these losses can be effectively reduced through the productions and applications of virus-free plantlets. In vitro culture techniques are the most successful approaches for efficient eradication of various viruses from almost all the most economically important crops. Techniques for producing virus-free plantlets include meristem tip culture, somatic embryogenesis, chemotherapy, thermotherapy, electrotherapy, shoot tip cryotherapy, and micrografting. Among them, meristem tip culture is currently the most widely used. Here, we describe a detailed protocol for producing virus-free plantlets of Chrysanthemum morifolium Ramat using tissue culture techniques.

Identification and Expression Profiling of Nonphosphorus Glycerolipid Synthase Genes in Response to Abiotic Stresses in Dendrobium catenatum.

Dendrobium catenatum, a valuable Chinese herb, frequently experiences abiotic stresses, such as cold and drought, under natural conditions. Nonphosphorus glycerolipid synthase (NGLS) genes are closely linked to the homeostasis of membrane lipids under abiotic stress in plants. However, there is limited information on NGLS genes in D. catenatum. In this study, a total of eight DcaNGLS genes were identified from the D. catenatum genome; these included three monogalactosyldiacylglycerol synthase (DcaMGD1, 2, 3) genes, two digalactosyldiacylglycerol synthase (DcaDGD1, 2) genes, and three sulfoquinovosyldiacylglycerol synthase (DcaSQD1, 2.1, 2.2) genes. The gene structures and conserved motifs in the DcaNGLSs showed a high conservation during their evolution. Gene expression profiling showed that the DcaNGLSs were highly expressed in specific tissues and during rapid growth stages. Furthermore, most DcaNGLSs were strongly induced by freezing and post-freezing recovery. DcaMGD1 and DcaSQDs were greatly induced by salt stress in leaves, while DcaDGDs were primarily induced by salt stress in roots. Under drought stress, most DcaNGLSs were regulated by circadian rhythms, and DcaSQD2 was closely associated with drought recovery. Transcriptome analysis also revealed that MYB might be regulated by circadian rhythm and co-expressed with DcaNGLSs under drought stress. These results provide insight for the further functional investigation of NGLS and the regulation of nonphosphorus glycerolipid biosynthesis in Dendrobium.

Anti-inflammatory effects of Ganoderma lucidum sterols via attenuation of the p38 MAPK and NF-κB pathways in LPS-induced RAW 264.7 macrophages.

Ganoderma lucidum exhibits pronounced anti-inflammatory effects, polysaccharides and triterpenoids are regarded as major constituents displaying the anti-inflammatory activities, whether sterols contribute to this activity remains unclear. Herein Ganoderma lucidum sterols (GLS) were innovatively isolated by a single-step procedure, the profile of GLS was characterized by HPLC-ELSD and shown similar to that of sterols separated by a traditional method, but much higher in content. Furthermore, GLS inhibited inflammation in macrophages by significantly attenuating LPS-induced cell polarization as well as releases and mRNA expressions of pro-inflammatory mediators NO, TNF-α, IL-1ß and IL-6. Moreover, the anti-inflammatory activity of GLS was mediated by MAPK and NF-κB pathways, GLS suppressed MAPK pathways by blocking phosphorylation of p38 but not ERK and JNK, which is complementary with inhibitory effects of Ganoderma polysaccharides and triterpenes on JNK and ERK, indicating Ganoderma sterols may exert synergistic anti-inflammatory effect with polysaccharides and triterpenes. GLS also inhibited NF-κB pathways by restraining phosphorylation and degradation of IκB-α and blocking phosphorylation of NF-κB p65. Molecular docking confirmed that sterols of GLS were directly bound to active sites of p38 and p65 to suppress their activation. Therefore, our findings suggest GLS as natural and safe anti-inflammatory agents to prevent and treat inflammatory diseases.

Determination and Dietary Intake Risk Assessment of Pesticide Residues in Fritillariae Thunbergii Bulbs and Cultivated Soils.

BACKGROUND: Pesticide residues in traditional Chinese medicines pose a potential risk to human health. However, little is known about the characteristics of pesticide residues in the fritillariae thunbergii bulbs (FTB). OBJECTIVE: This study aims to establish a method for the determination of pesticide multi-residues in FTB and then measured their residual levels in the FTB collected from nine cultivation regions. METHODS: A modified QuEChERS method coupled with GC/UPLC was used to determine the residues of 24 pesticides in the FTB and soil samples. RESULTS: The recoveries of these pesticides at three concentrations were 72.17-112.48% in the FTB and 70.92-113.74% in the soil with RSD < 11.83%, and the LOD and LOQ ranged from 0.005-10 µg/kg and 0.011-22 µg/kg, respectively. A total of 13 pesticide residues were detected in the FTB samples with the residual levels of 0.0011-509.63 µg/kg, which were all below the referred MRLs in other Chinese herbs or food. Meanwhile, dietary intake risk assessment showed that the risk of pesticide residues in the FTB was acceptable to consumers. CONCLUSIONS: This study developed a method for the determination of pesticide multi-residues in the FTB and cultivated soil samples, and furthermore the dietary intake risk of pesticide residues in the FTB is safe. HIGHLIGHTS: A modified QuEChERS method was established for the determination of 24 pesticides in the FTB with high sensitivity, accuracy, and precision.

Metabolomic and transcriptomic analyses reveal the regulation of pigmentation in the purple variety of Dendrobium officinale.

We performed an integrated analysis of the transcriptome and metabolome from purple (Pr) and normal cultivated varieties (CK) of Dendrobium officinale to gain insights into the regulatory networks associated with phenylpropanoid metabolism and to identify the key regulatory genes of pigmentation. Metabolite and transcript profiling were conducted by ultra-performance liquid chromatography electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) and RNA sequencing. Pr had more flavonoids in the stem than did CK. Metabolome analyses showed that 148 differential metabolites are involved in the biosynthesis of phenylpropanoids, amino acids, purines, and organic acids. Among them, the delphinidin and quercetin derivatives were significantly higher in Pr. A total of 4927 differentially expressed genes (DEGs) were significantly enriched (p ≤ 0.01) in 50 Gene Ontology (GO) terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed significantly enriched phenylpropanoid biosynthesis and phytohormone signal transduction in Pr versus CK. The expression levels of flavanone 3-hydroxylase (F3H) and leucoanthocyanidin dioxygenase (LDOX) affected the flux of dihydroflavonol, which led to a color change in Pr. Moreover, DEG enrichment and metabolite analyses reflected flavonoid accumulation in Pr related to brassinosteroid (BR) and auxin metabolism. The results of this study elucidate phenylpropanoid biosynthesis in D. officinale.