McWRKY43 Confers Cold Stress Tolerance in Michelia crassipes via Regulation of Flavonoid Biosynthesis.

WRKY transcription factor (TF) plays a crucial role in plant abiotic stress response, but it is rarely reported in Michelia crassipes. Our studies have found that the transcription factor McWRKY43, a member of the IIc subgroup, is strongly upregulated under cold stress. In this study, we cloned the full length of McWRKY43 to further investigate the function of McWRKY43 in resistance to cold stress and its possible regulatory pathways in M. crassipes. Under cold stress, the seed-germination rate of transgenic tobacco was significantly higher than that of the wild type, and the flavonoid content, antioxidant enzyme activities, and proline content of transgenic tobacco seedlings were significantly increased, which promoted the expression of flavonoid pathway structural genes. In addition, the transient transformation of McWRKY43 in the M. crassipes leaves also found the accumulation of flavonoid content and the transcription level of flavonoid structural genes, especially McLDOX, were significantly increased under cold stress. Yeast one-hybrid (Y1H) assay showed that McWRKY43 could bind to McLDOX promoter, and the transcription expression of McLDOX was promoted by McWRKY43 during cold stress treatment. Overall, our results indicated that McWRKY43 is involved in flavonoid biosynthetic pathway to regulate cold stress tolerance of M. crassipes, providing a basis for molecular mechanism of stress resistance in Michelia.

Metabolomics analysis of bioactive compositions of Michelia macclurei Dany and its antioxidant and enzyme inhibitory activities.

BACKGROUND: Michelia macclurei Dandy is a traditional Chinese medicinal plant, but little is understood about the bioactive compositions and biological potential of its different parts, limiting their applications. This study aims to identify the bioactive compositions and analyze differences in accumulation patterns from different parts of Michelia macclurei (heartwood, sapwood, bark, root, leaf, and fruit) using metabolomics. It also seeks to explore their biological potential and analyze the relationship between the bioactive compositions and biological potential. RESULTS: A total of 63 volatile metabolites (VMs) were identified by gas chromatography-mass spectrometry (GC-MS) in six parts, and the VMs in each part were dominated by sesquiterpenes and their derivatives (71.40-88.32%). Six parts of Michelia macclurei contained structurally diverse non-volatile metabolites (NVMs) with a total of 207 bioactive compounds, including 92 alkaloids, 30 flavonoids, 19 lignans, and 18 organic acids, utilizing ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Multivariate statistical analysis showed that the accumulation patterns of bioactive compositions differed significantly among the different parts, and the 25 VMs and 72 NVMs could be considered potential markers for distinguishing the different parts of Michelia macclurei. The excellent antioxidant and enzyme inhibitory capacity of extracts of all six parts was indicated by in vitro bioactivity assays. Pearson's correlation analysis showed that the bioactive compositions in the six parts were significantly correlated with antioxidant and enzyme inhibitory activities. CONCLUSION: This study offers helpful information on the distribution of bioactive compositions in different parts of Michelia macclurei and confirms the excellent antioxidant, and enzyme inhibitory potential of its extracts, which could provide scientific evidence for its potential applications in the pharmaceutical industry, cosmetics, and functional foods. © 2024 Society of Chemical Industry.

Using the Montgomery-Koyama-Smith equation to calculate the stomatal area per unit lamina area for 12 Magnoliaceae species.

BACKGROUND AND AIMS: The Montgomery-Koyama-Smith (MKS) equation predicts that total leaf area per shoot is proportional to the product of the sum of individual leaf widths and maximum individual leaf length, which has been validated for some herbaceous and woody plants. The equation is also predicted to be valid in describing the relationship between the total stomatal area per micrograph (AT) and the product of the sum of individual stomatal widths (denoted as LKS) and maximum individual stomatal length (denoted by WKS) in any particular micrograph. METHODS: To test the validity of the MKS equation, 69,931 stomata (from 720 stomatal micrographs from 12 Magnoliaceae species) were examined. The area of each stoma was calculated using empirical measurements of stomatal length and width multiplied by a constant. Six equations describing the relationships among AT, LKS, and WKS were compared. The root-mean-square (RMSE) and the Akaike information criterion (AIC) were used to measure the goodness of fit, and the trade-off between the goodness of fit and the structural complexity of each model, respectively. KEY RESULTS: Analyses supported the validity of the MKS equation and the power-law equation AT ∝ (LKS∙WKS)α, where a is a scaling exponent. The estimated values of α at the species level and for the pooled data were all statistically smaller than unity, which did not support the hypothesis that AT ∝ LTS∙WTS. The power-law equation had smaller RMSE and AIC values than the MKS equation for the data from the 12 individual species and the pooled data. CONCLUSIONS: These results indicate that AT tends to allometrically scale with LKS∙WKS, and that increases in AT do not keep pace with increases in LTS∙WTS. In addition, using the product of LKS and WKS is better than using only one of the two variables.

A high-quality haplotype genome of Michelia alba DC reveals differences in methylation patterns and flower characteristics.

Michelia alba DC is a highly valuable ornamental plant of the Magnoliaceae family. This evergreen tropical tree commonly grows in Southeast Asia and is adored for its delightful fragrance. Our study assembled the M. alba haplotype genome MC and MM by utilizing Nanopore ultralong reads, Pacbio Hifi long reads and parental second-generation data. Moreover, the first methylation map of Magnoliaceae was constructed based on the methylation site data obtained using Nanopore data. Metabolomic datasets were generated from the flowers of three different species to assess variations in pigment and volatile compound accumulation. Finally, transcriptome data were generated to link genomic, methylation, and morphological patterns to reveal the reasons underlying the differences between M. alba and its parental lines in petal color, flower shape, and fragrance. We found that the AP1 and AP2 genes are crucial in M. alba petal formation, while the 4CL, PAL, and C4H genes control petal color. The data generated in this study serve as a foundation for future physiological and biochemical research on M. alba, facilitate the targeted improvement of M. alba varieties, and offer a theoretical basis for molecular research on Michelia L.

Phenylethanoid glycosides from Michelia champaca leaves.

Chemical investigation on the leaves of Michelia champaca L. (Magnoliaceae) led to the isolation of five previously undescribed phenylethanoid glycosides (PhGs), 4-O-ß-d-glucopyranosyl-acteoside (1), 4‴-O-(6-O-E-caffeoyl)-ß-d-glucopyranosyl-acteoside (2), 4‴-O-(6-O-E-caffeoyl)-ß-d-glucopyranosyl-isoacteoside (3), 6""-O-E-feruloyl-echinacoside (4), and 6""-O-p-E-coumaroyl-echinacoside (5), together with eighteen known PhGs. Their structures were determined by spectroscopic and chemical methods. All the known PhGs except acteoside (8) were not previously reported in the genus. Twenty-one PhGs exhibited more potent DPPH radical scavenging activity and FRAP than l-ascorbic acid (l-AA), and twenty-two PhGs showed better ABTS radical cation scavenging activity than l-AA. In addition, twelve PhGs displayed more potent cellular reactive oxygen species scavenging activity than curcumin. The results revealed that the leaves of M. champaca are a rich source of phenylethanoid glycosides and antioxidants.

Prediction of cyanidin 3-rutinoside content in Michelia crassipes based on near-infrared spectroscopic techniques.

Currently the determination of cyanidin 3-rutinoside content in plant petals usually requires chemical assays or high performance liquid chromatography (HPLC), which are time-consuming and laborious. In this study, we aimed to develop a low-cost, high-throughput method to predict cyanidin 3-rutinoside content, and developed a cyanidin 3-rutinoside prediction model using near-infrared (NIR) spectroscopy combined with partial least squares regression (PLSR). We collected spectral data from Michelia crassipes (Magnoliaceae) tepals and used five different preprocessing methods and four variable selection algorithms to calibrate the PLSR model to determine the best prediction model. The results showed that (1) the PLSR model built by combining the blockScale (BS) preprocessing method and the Significance multivariate correlation (sMC) algorithm performed the best; (2) The model has a reliable prediction ability, with a coefficient of determination (R2) of 0.72, a root mean square error (RMSE) of 1.04%, and a residual prediction deviation (RPD) of 2.06. The model can be effectively used to predict the cyanidin 3-rutinoside content of the perianth slices of M. crassipes, providing an efficient method for the rapid determination of cyanidin 3-rutinoside content.

Flavonoid glycosides from the leaves of Michelia champaca.

Michelia champaca L. (Magnoliaceae) was cultivated in large scale for flowers as cosmetic raw materials, whereas the value of its leaves remains to be discovered. Our chemical study on the leaves yielded four new flavonol diglycosides, champaflavosides A-D (1-4), together with twenty-three known flavonoid glycosides (5-27). Their structures were determined by spectroscopic and chemical methods. Compounds 5-21 and 23-27 were not previously reported from the genus Michelia, and kaempferol 3-O-rutinoside (22) was obtained from this species for the first time. All the compounds were evaluated for antioxidant activity by four in vitro assays. Compounds 3-12 and 20 showed more potent 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity than l-ascorbic acid (l-AA). Compounds 2-23, 25, and 27 exhibited 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical cation scavenging activity superior to l-AA. The ferric reducing antioxidant powers (FRAP) of compounds 2-13, 17, and 19 were higher than l-AA. Further, eighteen compounds demonstrated cellular reactive oxygen species (ROS) scavenging activity, of which champaflavoside D (4), rhamnetin 3-O-neohesperidoside (8), quercetin 3-O-(6-O-E-p-coumaroyl)-neohesperidoside (9), and liquiritin (27) were more potent than curcumin. The results revealed that the renewable leaves of M. champaca are a rich source of flavonoids and antioxidants.

Transcriptome data analysis provides insights into the conservation of Michelia lacei, a plant species with extremely small populations distributed in Yunnan province, China.

BACKGROUND: Michelia lacei W.W.Smith (Magnoliaceae), was classified as a Plant Species with Extremely Small Populations (PSESP) by the Yunnan Provincial Government in both action plans of 2012 and 2021. This evergreen tree is known for its high ornamental and scientific value, but it faces significant threats due to its extremely small population size and narrow geographical distribution. The study aims to understand the genetic structure, diversity, and demographic history of this species to inform its conservation strategies. RESULTS: The analysis of transcriptome data from 64 individuals across seven populations of M. lacei identified three distinct genetic clusters and generated 104,616 single-nucleotide polymorphisms (SNPs). The KM ex-situ population, originating from Longling County, exhibited unique genetic features, suggesting limited gene flow. The genetic diversity was substantial, with significant differences between populations, particularly between the KM lineage and the OTHER lineage. Demographic history inferred from the data indicated population experienced three significant population declines during glaciations, followed by periods of recovery. We estimated the effective population size (Ne) of the KM and OTHER lineages 1,000 years ago were 85,851 and 416,622, respectively. Gene flow analysis suggested past gene flow between populations, but the KM ex-situ population showed no recent gene flow. A total of 805 outlier SNPs, associated with four environmental factors, suggest potential local adaptation and showcase the species' adaptive potential. Particularly, the BZ displayed 515 adaptive loci, highlighting its strong potential for adaptation within this group. CONCLUSIONS: The comprehensive genomic analysis of M. lacei provides valuable insights into its genetic background and highlights the urgent need for conservation efforts. The study underscores the importance of ex-situ conservation methods, such as seed collection and vegetative propagation, to safeguard genetic diversity and promote population restoration. The preservation of populations like MC and BZ is crucial for maintaining the species' genetic diversity. In-situ conservation measures, including the establishment of in-situ conservation sites and community engagement, are essential to enhance protection awareness and ensure the long-term survival of this threatened plant species.

Leaf Spot Disease of Michelia alba caused by Alternaria alternata in China.

The perennial deciduous tree Michelia alba is a widely cultivated street plant in China. In June 2021 and March 2022, M. alba trees with leaf spots were found in the green belt of the approximately 200,000 m2 community (32.62°N, 116.98°E) of Tianjia'an District, Huainan, Anhui, China, where approximately half of the M. alba trees had brown leaf spots surrounded with irregular yellow halos ranging from 2 to 6 mm in diameter (Fig S1A). The leaves of M. alba trees with multiple lesions became blighted. To isolate the potential pathogens causing leaf spot symptoms in M. alba trees, twenty fragments (2 cm2) were excised from the margin of the necrosis on symptomatic leaves, immersed in 1% sodium hypochlorite for 45 s, and then washed three times in sterile water. The fragments were plated and incubated on potato dextrose agar (PDA) at 25 °C and 15 dark green fungal colonies were obtained 5 days later. Single-spore isolates of the fungal colonies plated on potato carrot agar (PCA, Simmons 2007) produced gray, floccose colonies, which reached 71 mm after 7 days at 25 °C were obtained 5 days later (Fig S1C). Optical microscopy analysis showed that single-spore isolates formed sparsely branched chains with pale brown conidiophores on PCA after incubation at 25 °C in darkness for 7 days. The conidia were ellipsoidal, inverted rod, or ovoid, light brown, and 10.0 to 52.5 × 4.5 to 22.7 µm, with zero to four longitudinal or oblique and zero to eight transverse septa (n = 50). Partial conidia are 2.5 to 27.5 × 0.6 to 3.7 µm with cylindrical light brown beaks (n = 50) (Fig S1D, E). The cultural and morphological characteristics of the isolated fungi were consistent with the description of Alternaria alternata (Woudenberg et al. 2015). To further characterize the isolated fungi, the genomic DNA of three representative strains (BYL-1, BYL-2 and BYL-3) were extracted from their mycelia, respectively. ITS region and housekeeping genes GPD, and TEF, were amplified and sequenced using ITS4/ITS5 (White et al. 1990), Gpd1/Gpd2 (Berbee et al. 1999), and EF1-728F/EF1-986R (Carbone and Kohn 1999), primer pairs, respectively. BLAST analysis showed that the isolates BYL-1 (GenBank accession nos. OP325693, OP405008, and OP405009), BYL-2 (GenBank accession nos. PP057859, PP138442, and PP138444), and BYL-3 (GenBank accession nos. PP057860, PP138443, and PP138445) shared 99 to 100% identity with Alternaria alternata (GenBank accession nos. AF347032.1, AY278809.1, KC584693.1), which suggested that all the three isolates belong to A. alternata. The identifications were further confirmed by phylogenetic analysis based on combined DNA sequences data of ITS, GPD, and TEF. As showed in Fig S2, the strains of BYL-1 , BYL-2 and BYL-3 formed a robust clade with A. alternata CBS918.96. Taken together, the morphology and molecular assays suggest that strain BYL-1 is A. alternata. To test pathogenicity, the isolate BYL-1 was cultured on PCA for 7 days to prepare conidial suspensions, and the spore concentration was adjust to a final concentration of 105 spores/ml. The leaves of 3-5-leaf stage of six 5-years-old natural planting M. alba plants were sprayed with conidial suspensions and sterile distilled water, respectively. The petiole of each inoculated leaves of M. alba were secured with sterile wet cotton, and covered with plastic bags to prevent moisture evaporation after incubation. After a 3- to 5- day of inoculation, necrotic lesions appeared on the leaves inoculated with conidial suspensions, whereas no necrotic lesion was observed in the control leaves inoculated with sterile distilled water (Fig S1B). To fulfill the Koch,s postulates, fungi were re-isolated from the margin of necrotic lesions and identified as A. alternata by DNA sequencing the ITS gene. To our knowledge, this is the first report of A. alternata causing leaf spot on M. alba. Because the disease could cause damage to the foliage influencing the greening and ornamental effects of these trees, control measures may need to be implemented during daily management.

Octadecadienoate derivatives from Michelia champaca seed extract as potential larvicide and pupicide against Dengue vector Aedes albopictus.

The present study was designed aiming at finding novel botanicals for controlling the vector population. Objective was to evaluate the larvicidal and pupicidal efficacies of crude and solvent extracts of Michelia champaca seed against the notorious dengue vector Aedes albopictus. 0.5% concentration of the crude extractive and 40 ppm concentration of ethyl acetate extractive were enough to execute 100% of larval mortality of all the instars after 72 h of exposure and the LC50 and LC90values (95% confidence level) of ethyl acetate extractive were 0.9880 ppm and 36.0491 ppm. In case of pupicidal bioassay, 100% mortality was observed at 200 ppm of ethyl acetate extract. Through TLC techniques, the bioactive compounds were isolated, which caused remarkable larval toxicity at 15 ppm concentration. Three-way factorial ANOVA analysis showed different concentrations, time intervals, and instars revealed a significant difference in larval death. FT-IR analysis revealed the presence several important functional groups. Presence of methyl 5,12-octadecadienoate and ethyl 9cis,11trans-octadecadienoate were ascertained by GC-MS analysis. The said bioactive compounds showed very low toxicity in non-target organisms such as damselfly (Ischnura sp.) and water bug (Diplonychus sp.) Thus, proclaiming the potentialities of Michelia champaca seed extracts as larvicidal and pupicidal agents against Ae. albopictus.

Comparison of Composition, Free-Radical-Scavenging Capacity, and Antibiosis of Fresh and Dry Leave Aqueous Extract from Michelia shiluensis.

Numerous plants of medicinal value grow on Hainan Island (China). Given the lack of knowledge on the phytochemical and pharmacological properties of Michelia shiluensis Chun and Y. F. Wu (M. shiluensis), the application of natural antioxidants and antimicrobials in the food industry has attracted increasing interest. This study aimed to compare the chemical composition, free-radical-scavenging capacity, and antibiosis of aqueous extracts of the fresh and dried leaves of M. shiluensis. The aqueous extract of the leaves of M. shiluensis was obtained using steam distillation, and its chemical components were separated and identified via gas chromatography-mass spectrometry (GC-MS). The free-radical-scavenging capacity and antibiosis were determined. Further, 28 and 20 compounds were isolated from the fresh leaf aqueous extract of M. shiluensis (MSFLAE) and dried leaf aqueous extract of M. shiluensis (MSDLAE), respectively. The free-radical-scavenging capacity of MSFLAE and MSDLAE was determined by the 2,2-diphenyl-1 picrylhydrazyl (DPPH) method, which was 43.43% and 38.74%, respectively. The scavenging capacity of MSFLAE and MSDLAE determined by the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS)) method was 46.90% and 25.99%, respectively. The iron ion reduction capacity of MSFLAE and MSDLAE was determined by the ferric-reducing antioxidant power (FRAP) method as 94.7 and 62.9 µmol Fe2⁺/L, respectively. This indicated that the two leaf aqueous extracts had a certain free-radical-scavenging capacity, and the capacity of MSFLAE was higher than that of MSDLAE. The antibiosis of the two leaf aqueous extracts on the three foodborne pathogenic bacteria was low, but the antimicrobial effects on Gram-positive bacteria were better than those on Gram-negative bacteria. The antibiosis of MSFLAE on Escherichia coli and Staphylococcus aureus was greater than that of MSDLAE. Finally, MSFLAE and MSDLAE both had certain free-radical-scavenging capacities and antibiosis, confirming that the use of this plant in the research and development of natural antioxidants and antibacterial agents was reasonable. Plant aqueous extracts are an essential source of related phytochemistry and have immense pharmacological potential.

First Report of Leaf blight Caused by Colletotrichum karstii on Banana Shrub (Michelia figo) in Sichuan, China.

Banana Shrub (Michelia figo (Lour.) Spreng.) is widely cultivated in most of southern China (Wu et al, 2008). It can be used to make essential oil and flower tea（Ma et al, 2012; Li et al, 2010）.The first symptoms were observed in Sept. 2020 at a grower's field in Banana shrub seedlings (0.6 ha), Ya'an city (29°30'N, 102°38'E), Hanyuan county. The symptoms re-occurred in May-June of 2021 and became prevalent from August to September. the incidence rate and the disease index were 40% and 22%, respectively. Initially, purplish-brown necrotic lesions appeared at the leaf tip with dark-brown edges. Progressively, necrosis spread, to the middle of the leaves, and the older area turned gray-white. Dark sunken lesions appeared in the necrotic areas and orange conidial masses were visible under humid conditions. Ten isolates were obtained on potato dextrose agar (PDA) from 10 leaf samples using previously described tissue isolation method (Fang et al. 1998). All the 10 isolates exhibited similar morphological characteristics. Grey to white aerial mycelium at the center and in dispersed tufts, with numerous dark conidiomata scattered over the surface, reverse was pale orange with numerous dark flecks corresponding to the ascomata, orange conidial masses were formed from mature conidiomata. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical, apex round, the contents appearing granular 14.8 to 17.2 × 4.2 to 6.4 µm (average: 16.26 × 4.84 µm, n=30) as Colletotrichum spp. (Damm et al. 2012). For molecular identification, DNA was extracted from a representative isolate HXcjA using a plant genomic DNA extraction kit (Solarbio, Beijing). and the partial sequences of internal transcribed spacer region (ITS, OQ641677), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, OL614009), actin (ACT, OL614007), beta-tubulin (TUB2, OL614011), histone3 (HIS3, OL614010), and calmodulin (CAL, OL614008) were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT-512F/ACT-783R, CAL 228F/CAL 737R (Carbone et al. 1999), TUB1F/Bt2bR, CYLH3F/CYLH3R (Crous et al. 2004), respectively. BLASTn analysis for ITS, GAPDH, CAL, ACT, TUB2 and HIS3 sequences showed ≥99.7% identity to C. Karstii, namely, NR_144790 (532/532 bp), MK963048 (252/252 bp), MK390726 (431/431 bp), MG602039 (761/763 bp), (KJ954424, 294/294 bp), (KJ813519, 389/389 bp), respectively. The fungus was identified as C. karstii based on morphology and a multigene phylogeny. The conidial suspension (1 × 107 conidia/mL) with 0.05% Tween 80 buffer was used for pathogenicity test, by spraying 2-year-old Banana Shrub plants. Ten plants were inoculated with spore suspensions (approximately 2ml per plant). An equal number of plants were sprayed with 0.05% Tween 80 buffer to serve as a control. Fifteen days later, the inoculated plants showed similar symptoms as the original diseased plants but the controls remained asymptomatic. C. karstii was re-isolated from the infected leaves and identified by morphology and a multigene phylogeny. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of Banana Shrub leaf blight caused by C. karstii in China. This disease reduces the ornamental and economic value of Banana Shrub, and this work will provide a basis for the prevention and treatment of the disease in the future.

A Study of the Chemical Composition and Biological Activity of Michelia macclurei Dandy Heartwood: New Sources of Natural Antioxidants, Enzyme Inhibitors and Bacterial Inhibitors.

The wood of Michelia macclurei Dandy (MD) is an excellent material that is widely used in the furniture, handicraft, and construction industries. However, less research has been conducted on the chemical composition and biological activity of heartwood, which is the main valuable part of the wood. This study aimed to investigate the chemical composition and biological activities of the heartwood of Michelia macclurei Dandy (MDHW) and to confirm the active ingredients. Triple quadrupole gas chromatography-mass spectrometry (GC-MS) was used to characterize the volatile components of MDHW, while ultra-performance liquid chromatography-mass spectrometry was used to analyze the non-volatile components (UPLC-MS). The total reducing power, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging assays, acetylcholinesterase and α-glucosidase inhibition assays, and an antimicrobial test of 4 gram bacteria were used to describe the in vitro bioactivities. The GC-MS analysis showed that the volatile components of MDHW were mainly fatty compounds and terpenoids, with sesquiterpenes and their derivatives dominating the terpene composition. ß-elemene was the main terpene component in the steam distillation (11.88%) and ultrasonic extraction (8.2%) methods. A total of 67 compounds, comprising 45 alkaloids, 9 flavonoids, 6 lignans, and others, were found by UPLC-MS analysis. The primary structural kinds of the non-volatile components were 35 isoquinoline alkaloids. Alkaloids were the predominant active constituent in all MDHW extracts, including crude extracts, alkaloid fractions, and non-alkaloid fractions. These extracts all demonstrate some biological effects in terms of antioxidant, enzyme inhibition, and bacterial inhibition. The findings of this study show that MDHW is abundant in chemical structure types, has great bioactivity assessment, and has the potential to be used to create natural antioxidants, products that postpone Alzheimer's disease and lower blood sugar levels and antibacterial agents.

Transcriptomic and metabolomic analyses unveil the growth advantage mechanism conferred by heterosis of Michelia 'Zhongshanhanxiao'.

Michelia compressa (Maxim.) Sarg. is one of the important timber trees in Taiwan province, P. R. China. Michelia 'Zhongshanhanxiao' is a group of variants found among the progeny of M. compressa that exhibit higher growth rates compared with normal individuals, with a significantly increased stem diameter and height, as well as enlarged leaves and flowers. However, the molecular mechanisms fostering the growth advantage and morphological variations are unknown and deserve further study. Through analysing the transcriptome, metabolome and physiological processes of leaves, we identified remarkable differences in gene expression and metabolic profiles between Michelia 'Zhongshanhanxiao' and both the maternal M. compressa and its normal progeny. These differences were widely associated with a plant-pathogen interaction, phenylpropanoid biosynthesis, cyanoamino acid metabolism, carbon fixation in photosynthetic organisms and plant hormone signal transduction. Additionally, physiological measurements showed that Michelia 'Zhongshanhanxiao' possesses stronger photosynthetic capacity and higher plant hormone content. These results suggest that the heterosis of Michelia 'Zhongshanhanxiao' is regulated by candidates related to cell division, resistance to pathogens and the accumulation of organic compounds. The findings of this study provide crucial information on the molecular mechanisms underlying the growth advantages conferred by heterosis in trees.

Transcriptome and metabolome analyses reveal novel insights into the seed germination of Michelia chapensis, an endangered species in China.

Michelia chapensis Dandy, a well-known medicinal woody plant endemic to China, is endangered and seriously constricted by seed dormancy-induced low-regeneration in natural conditions. Cold stratification can effectively reduce seed dormancy and promote the seed germination of M. chapensis. However, the molecular events and systematic changes that occurred during seed germination in M. chapensis remain largely unknown. In this study, we carried out transcriptomic and metabolomic analyses to elucidate the potential molecular mechanisms underlying seed germination in M. chapensis under cold stratification. The results showed that the embryo cells became bigger and looser with increasing stratification time. Moreover, the endosperm appeared reduced due to the consumption of nutrients. Seventeen phytohormones were examined by the metabolome targeted for hormones. Compared with the ES (no stratification), the levels of indole-3-acetic acid (IAA) and gibberellin A3 (GA3) were increased in the MS (stratification for 45 days), while the abscisic acid (ABA) was downregulated in both MS and LS (stratification for 90 days). The transcriptome profiling identified 24975 differentially expressed genes (DEGs) in the seeds during germination. The seed germination of M. chapensis was mainly regulated by the biological pathways of plant hormone signal transduction, energy supply, secondary metabolite biosynthesis, photosynthesis-related metabolism, and transcriptional regulation. This study reveals the biological evidence of seed germination at the transcriptional level and provides a foundation for unraveling molecular mechanisms regulating the seed germination of M. chapensis.

The complete chloroplast genome sequence of Michelia macclurei (Dandy, 1928) (Magnoliaceae), an important fire-resistant tree species.

Michelia macclurei (Dandy, 1928) is an evergreen broad-leaved tree species native to South China. This species has great ecological and economic importance. However, the genomic study of M. macclurei has lagged far behind. Here, we reported the complete chloroplast genome sequence of M. macclurei. The chloroplast genome size of M. macclurei was 160,139 bp, consisting of a pair of inverted repeat (IR) regions (26,575 bp), which was separated by a large single copy (LSC) region (88,167 bp) and a small single copy (SSC) region (18,822 bp). A total of 113 unique genes were annotated, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The overall GC content was 39.2%. Phylogenetic analysis based on 16 whole chloroplast genome sequences of Michelia species suggested that M. macclurei and M. maudiae are sister to each other, and jointly sister to M. chapensis.

Volatile Profiling of Magnolia champaca Accessions by Gas Chromatography Mass Spectrometry Coupled with Chemometrics.

Magnolia champaca (L.) Baill. ex Pierre of family Magnoliaceae, is a perennial tree with aromatic, ethnobotanical, and medicinal uses. The M. champaca leaf is reported to have a myriad of therapeutic activities, however, there are limited reports available on the chemical composition of the leaf essential oil of M. champaca. The present study explored the variation in the yield and chemical composition of leaf essential oil isolated from 52 accessions of M. champaca. Through hydrodistillation, essential oil yield was obtained, varied in the range of 0.06 ± 0.003% and 0.31 ± 0.015% (v/w) on a fresh weight basis. GC-MS analysis identified a total of 65 phytoconstituents accounting for 90.23 to 98.90% of the total oil. Sesquiterpene hydrocarbons (52.83 to 65.63%) constituted the major fraction followed by sesquiterpene alcohols (14.71 to 22.45%). The essential oils were found to be rich in ß-elemene (6.64 to 38.80%), γ-muurolene (4.63 to 22.50%), and ß-caryophyllene (1.10 to 20.74%). Chemometrics analyses such as PCA, PLS-DA, sPLS-DA, and cluster analyses such as hierarchical clustering, i.e., dendrogram and partitional clustering, i.e., K-means classified the essential oils of M. champaca populations into three different chemotypes: chemotype I (ß-elemene), chemotype II (γ-muurolene) and chemotype III (ß-caryophyllene). The chemical polymorphism analyzed in the studied populations would facilitate the selection of chemotypes with specific compounds. The chemotypes identified in the M. champaca populations could be developed as promising bio-resources for conservation and pharmaceutical application and further improvement of the taxa.

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In order to understand the interspecific and ecological relationships of Michelia odora (extremely small population) community and strengthen the protection of wild M. odora resources in Junzifeng Nature Reserve, we studied the niche characteristics and interspecific associations of dominant tree species. The results showed that M. odora, Machilus chekiangensis, Schima superba, and Alniphyllum fortunei had obvious niche breadth advantages, which were the constructive species of the community. Among the 190 groups of species pairs among the 20 dominant tree species, 50.5% of species pairs had niche overlap value greater than 0.5. The degree of ecological niche differentiation among species was general. M. odora had large niche overlap with other 19 species, indicating a competitive risk when resources were insufficient. The overall associations of dominant tree species were significantly positive, indicating the community was at the late stage of relatively stable succession. The results ofχ2 test, asso-ciation coefficient, and Pearson correlation coefficient showed that all the significance ratios of interspecific association were lower, and that the independence among species was relatively strong. There was a positive correlation between interspecific association and niche overlap. The M. odora community was relatively mature, with full utilization of resources and stable interspecific relationship. To promote the rejuvenation and create a good habitat of M. odora population, the population size with large overlap with M. odora niche and significant negative association could be appropriately limited, while that with positive interaction could be increased.

First report of Colletotrichum siamense causing leaf spot on Michelia macclurei in China.

Michelia macclurei Dandy is an excellent timber and ornamental tree native to South China (Lan et al. 2010). In May 2020, a leaf spot disease of M. macclurei was found on the campus of Jiangxi Agricultural University (N28°45'56″, E115°50'21″). Approximately 25% (9 out of 35) of 32-year-old M. macclurei trees showed the leaf spot disease. On average, 40% of the leaves per individual tree were affected. The symptoms began as small dark brown lesions formed along the leaf margins and tips. The lesions' center was sunken with a dark brown border as the disease developed. Thirty pieces (5 × 5 mm) from the lesion margins were surface sterilized in 70% ethanol (30 s), then in 3% NaOCl (1 min), and finally rinsed three times with sterile water. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Sixteen strains with similar morphological characterizations were isolated, and three representative isolates (HX-1, HX-2, HX-3) were used for morphological and molecular characterization. The three isolates were white, cottony, and light gray on the reverse, producing dark-green pigmentation near the center. The conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 12.8-17.5 × 4.5-5.7 µm (14.7 ± 1.2 × 4.8 ± 0.2 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, and ranged from 5.9-8.8 × 4.4-6.7 µm (7.1 ± 0.6 × 5.6 ± 0.6 µm, n=100). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin2 (TUB2) were sequenced using the primers ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R, CL1/CL2, CHS-79F/CHS-345R, GDF/GDR, and T1/Bt2b, respectively (Weir et al. 2012). The sequences were deposited into GenBank (Accession Nos.: MZ323328, MZ323329, MW581269 for ITS, MZ889002, MZ889003, MW661166 for ACT, MZ889004, MZ889005, MW661167 for CAL, MZ889006, MZ889007, MW661168 for CHS-1, MZ889008, MZ889009, MW661169 for GAPDH, MZ889010, MZ889011, MW661170 for TUB2). A maximum likelihood and Bayesian posterior probability-based analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, and TUB2 ) placed three isolates in the clade of Colletotrichum siamense Prihastuti, L. Cai & K. D. Hyde. Based on the morphological characteristics and molecular data, three isolates were identified as C. siamense (Fu et al. 2019).The pathogenicity of each isolate was tested on potted 2-yr-old seedlings of M. macclurei grown in a greenhouse at 25 ℃, 70% relative humidity with a 12-h photoperiod. Twenty healthy leaves on 10 M. macclurei plants were inoculated with 10 µL of spore suspension (106 conidia/mL). Another 20 healthy leaves were inoculated with sterile water as the control. All leaves were wounded with a sterile needle (Φ=0.5 mm). The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic for 8 days after inoculation. C. siamense was consistently re-isolated only from the diseased leaves, fulfilling Koch's postulates. C. siamense can cause leaf diseases in a variety of hosts, such as Salix matsudana (Zhang et al. 2021), Liriodendron chinense [Hemsl.] Sarg. × tulipifera L. (Zhu et al. 2019) and Magnolia grandiflora (Zhou et al. 2022). This is the first report of C. siamense associated with leaf spot disease on M. macclurei in China, and its potential threat should be evaluated in the future. These results will help to develop effective strategies for appropriately managing this newly emerging disease.

Fingerprints of climatic changes through the late Cenozoic in southern Asian flora: Magnolia section Michelia (Magnoliaceae).

BACKGROUND AND AIMS: Ongoing global warming is a challenge for humankind. A series of drastic climatic changes have been proven to have occurred throughout the Cenozoic based on a variety of geological evidence, which helps to better understand our planet's future climate. Notably, extant biomes have recorded drastic environmental shifts. The climate in southern Asia, which hosts high biodiversity, is deeply impacted by the Asian monsoon. The origins and evolutionary dynamics of biomes occurring between the tropics and sub-tropics in southern Asia have probably been deeply impacted by climatic changes; however, these aspects remain poorly studied. We tested whether the evolutionary dynamics of the above biomes have recorded the drastic, late Cenozoic environmental shifts, by focusing on Magnolia section Michelia of the family Magnoliaceae. METHODS: We established a fine time-calibrated phylogeny of M. section Michelia based on complete plastid genomes and inferred its ancestral ranges. Finally, we estimated the evolutionary dynamics of this section through time, determining its diversification rate and the dispersal events that occurred between tropical and sub-tropical areas. KEY RESULTS: The tropical origin of M. section Michelia was dated to the late Oligocene; however, the diversification of its core group (i.e. M. section Michelia subsection Michelia) has occurred mainly from the late Miocene onward. Two key evolutionary shifts (dated approx. 8 and approx. 3 million years ago, respectively) were identified, each of them probably in response to drastic climatic changes. CONCLUSION: Here, we inferred the underlying evolutionary dynamics of biomes in southern Asia, which probably reflect late Cenozoic climatic changes. The occurrence of modern Asian monsoons was probably fundamental for the origin of M. section Michelia; moreover, the occurrence of asymmetric dispersal events between the tropics and sub-tropics hint at an adaptation strategy of M. section Michelia to global cooling, in agreement with the tropical conservatism hypothesis.