Evolution-guided multiomics provide insights into the strengthening of bioactive flavone biosynthesis in medicinal pummelo.

Pummelo (Citrus maxima or Citrus grandis) is a basic species and an important type for breeding in Citrus. Pummelo is used not only for fresh consumption but also for medicinal purposes. However, the molecular basis of medicinal traits is unclear. Here, compared with wild citrus species/Citrus-related genera, the content of 43 bioactive metabolites and their derivatives increased in the pummelo. Furthermore, we assembled the genome sequence of a variety for medicinal purposes with a long history, Citrus maxima 'Huazhouyou-tomentosa' (HZY-T), at the chromosome level with a genome size of 349.07 Mb. Comparative genomics showed that the expanded gene family in the pummelo genome was enriched in flavonoids-, terpenoid-, and phenylpropanoid biosynthesis. Using the metabolome and transcriptome of six developmental stages of HZY-T and Citrus maxima 'Huazhouyou-smooth' (HZY-S) fruit peel, we generated the regulatory networks of bioactive metabolites and their derivatives. We identified a novel MYB transcription factor, CmtMYB108, as an important regulator of flavone pathways. Both mutations and expression of CmtMYB108, which targets the genes PAL (phenylalanine ammonia-lyase) and FNS (flavone synthase), displayed differential expression between Citrus-related genera, wild citrus species and pummelo species. This study provides insights into the evolution-associated changes in bioactive metabolism during the origin process of pummelo.

Effects of phosphorus stress on the growth and secondary metabolism of Artemisia argyi.

Phosphorus is essential in critical plant processes such as signaling, photosynthesis, energy metabolism, and enzyme activity during respiration. Phosphorus stress therefore has a significant impact on plant growth and metabolism. Here, we characterized the biochemical responses of Artemisia argyi Level. et Vant to low phosphorus (LP) and high phosphorus (HP) stress. Plants were treated with 0 g (LP), 1.5 g (control), or 3 g (HP) P per 10 kg of soil. The results demonstrated that CK encouraged the most plant growth, as quantified by leaf size and plant biomass. We also found that the total amounts of phenolic and flavonoid compounds (such as chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cryptochlorogenic acid, neochlorogenic acid, hispidulin, jaceosidin, eupatilin, and casticin) were increased in the leaves of A. argyi plants exposed to LP stress compared to those raised under CK conditions. The levels of these compounds were inversely related to the amount of phosphorus added, and therefore peaked in plants treated with LP stress. Levels of terpenoids were also found to fluctuate under LP and HP stress compared to CK conditions. Furthermore, transcriptomic analyses showed up-regulation of several genes encoding key enzymes in the flavonoid and phenolic acid metabolic pathways under LP stress. There were also alterations in the expression levels of genes in the methylerythritol 4-phosphate and mevalonate pathways of terpene synthesis. This study contributes to a deeper understanding of the physiological and molecular mechanisms underlying phosphorus stress responses and their impacts on the growth and quality of the economically important species A. argyi.

Gene Cloning and Characterization of Transcription Factor FtNAC10 in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn.).

NAC transcription factors play a significant role in plant stress responses. In this study, an NAC transcription factor, with a CDS of 792 bp encoding 263 amino acids, was cloned from Fagopyrum tataricum (L.) Gaertn. (F. tataricum), a minor cereal crop, which is rich in flavonoids and highly stress resistant. The transcription factor was named FtNAC10 (NCBI accession number: MK614506.1) and characterized as a member of the NAP subgroup of NAC transcriptions factors. The gene exhibited a highly conserved N-terminal, encoding about 150 amino acids, and a highly specific C-terminal. The resulting protein was revealed to be hydrophilic, with strong transcriptional activation activity. FtNAC10 expression occurred in various F. tataricum tissues, most noticeably in the root, and was regulated differently under various stress treatments. The over-expression of FtNAC10 in transgenic Arabidopsis thaliana (A. thaliana) seeds inhibited germination, and the presence of FtNAC10 enhanced root elongation under saline and drought stress. According to phylogenetic analysis and previous reports, our experiments indicate that FtNAC10 may regulate the stress response or development of F. tataricum through ABA-signaling pathway, although the mechanism is not yet known. This study provides a reference for further analysis of the regulatory function of FtNAC10 and the mechanism that underlies stress responses in Tartary buckwheat.

Integrative Analysis of Metabolomic and Transcriptomic Data Reveals the Mechanism of Color Formation in Corms of Pinellia ternata.

Pinellia ternata (Thunb.) Breit. (P. ternata) is a very important plant that is commonly used in traditional Chinese medicine. Its corms can be used as medicine and function to alleviate cough, headache, and phlegm. The epidermis of P. ternata corms is often light yellow to yellow in color; however, within the range of P. ternata found in JingZhou City in Hubei Province, China, there is a form of P. ternata in which the epidermis of the corm is red. We found that the total flavonoid content of red P. ternata corms is significantly higher than that of yellow P. ternata corms. The objective of this study was to understand the molecular mechanisms behind the difference in epidermal color between the two forms of P. ternata. The results showed that a high content of anthocyanidin was responsible for the red epidermal color in P. ternata, and 15 metabolites, including cyanidin-3-O-rutinoside-5-O-glucoside, cyanidin-3-O-glucoside, and cyanidin-3-O-rutinoside, were screened as potential color markers in P. ternata through metabolomic analysis. Based on an analysis of the transcriptome, seven genes, including PtCHS1, PtCHS2, PtCHI1, PtDFR5, PtANS, PtUPD-GT2, and PtUPD-GT3, were found to have important effects on the biosynthesis of anthocyanins in the P. ternata corm epidermis. Furthermore, two transcription factors (TFs), bHLH1 and bHLH2, may have regulatory functions in the biosynthesis of anthocyanins in red P. ternata corms. Using an integrative analysis of the metabolomic and transcriptomic data, we identified five genes, PtCHI, PtDFR2, PtUPD-GT1, PtUPD-GT2, and PtUPD-GT3, that may play important roles in the presence of the red epidermis color in P. ternata corms.

[Prediction analysis of quality markers and resource evaluation of Artemisiae Argyi Folium based on chemical composition and network pharmacology].

This study is based on ultra-high-performance liquid chromatography(UPLC), gas chromatography-mass spectrometry(GC-MS), and network pharmacology methods to analyze and predict potential quality markers(Q-markers) of Artemisiae Argyi Folium. First, UPLC and GC-MS techniques were used to analyze the content of 12 non-volatile components and 8 volatile components in the leaves of 33 Artemisia argyi germplasm resources as candidate Q-markers. Subsequently, network pharmacology was employed to construct a "component-target-pathway-efficacy" network to screen out core Q-markers, and the biological activity of the markers was validated using molecular docking. Finally, cluster analysis and principal component analysis were performed on the content of Q-markers in the 33 A. argyi germplasm resources. The results showed that 18 candidate components, 60 targets, and 185 relationships were identified, which were associated with 72 pathways related to the treatment of 11 diseases and exhibited 5 other effects. Based on the combination of freedom and component specificity, six components, including eupatilin, cineole, ß-caryophyllene, dinatin, jaceosidin, and caryophyllene oxide were selected as potential Q-markers for Artemisiae Argyi Folium. According to the content of these six markers, cluster analysis divided the 33 A. argyi germplasm resources into three groups, and principal component analysis identified S14 as having the highest overall quality. This study provides a reference for exploring Q-markers of Artemisiae Argyi Folium, establishing a quality evaluation system, further studying its pharmacological mechanisms, and breeding new varieties.

[Effect of apigenin in combination with oxymatrine on non-small cell lung cancer and mechanism].

This study explores the effect of apigenin(APG), oxymatrine(OMT), and APG+OMT on the proliferation of non-small cell lung cancer cell lines and the underlying mechanisms. Cell counting kit-8(CCK-8) assay was used to detect the vitality of A549 and NCI-H1975 cells, and colony formation assay to evaluate the colony formation ability of the cells. EdU assay was employed to examine the proliferation of NCI-H1975 cells. RT-qPCR and Western blot were performed to detect the mRNA and protein expression of PLOD2. Molecular docking was carried out to explore the direct action ability and action sites between APG/OMT and PLOD2/EGFR. Western blot was used to study the expression of related proteins in EGFR pathway. The viability of A549 and NCI-H1975 cells was inhibited by APG and APG+OMT at 20, 40, and 80 µmol·L~(-1) in a dose-dependent manner. The colony formation ability of NCI-H1975 cells was significantly suppressed by APG and APG+OMT. The mRNA and protein expression of PLOD2 was significantly inhibited by APG and APG+OMT. In addition, APG and OMT had strong binding activity with PLOD2 and EGFR. In APG and APG+OMT groups, the expression of EGFR and proteins in its downstream signaling pathways was significantly down-regulated. It is concluded that APG in combination with OMT could inhibit non-small lung cancer, and the mechanism may be related to EGFR and its downstream signaling pathways. This study lays a new theoretical basis for the clinical treatment of non-small cell lung cancer with APG in combination with OMT and provides a reference for further research on the anti-tumor mechanism of APG in combination with OMT.

[Quality of moxa from Artemisia argyi and A. stolonifera in different storage years based on simultaneous thermal analysis].

The quality of moxa is an important factor affecting moxibustion therapy, and traditionally, 3-year moxa is considered optimal, although scientific data are lacking. This study focused on 1-year and 3-year moxa from Artemisia stolonifera and A. argyi(leaf-to-moxa ratio of 10∶1) as research objects. Scanning electron microscopy(SEM), Van Soest method, and simultaneous thermal analysis were used to investigate the differences in the combustion heat quality of 1-year and 3-year moxa and their influencing factors. The results showed that the combustion of A. stolonifera moxa exhibited a balanced heat release pattern. The 3-year moxa released a concentrated heat of 9 998.84 mJ·mg~(-1)(accounting for 54% of the total heat release) in the temperature range of 140-302 ℃, with a heat production efficiency of 122 mW·mg~(-1). It further released 7 512.51 mJ·mg~(-1)(accounting for 41% of the total heat release) in the temperature range of 302-519 ℃. The combustion of A. argyi moxa showed a rapid heat release pattern. The 3-year moxa released a heat of 16 695.28 mJ·mg~(-1)(accounting for 70% of the total heat release) in the temperature range of 140-311 ℃, with an instantaneous power output of 218 mW·mg~(-1). It further released 5 996.95 mJ·mg~(-1)(accounting for 25% of the total heat release) in the temperature range of 311-483 ℃. Combustion parameters such as-R_p,-R_v, D_i, C, and D_b indicated that the combustion heat quality of 3-year moxa was superior to that of 1-year moxa. It exhibited greater combustion heat, heat production efficiency, flammability, mild and sustained burning, and higher instantaneous combustion efficiency. This study utilized scientific data to demonstrate that A. stolonifera could be used as excellent moxa, and the quality of 3-year moxa surpassed that of 1-year moxa. The research results provide a scientific basis for the in-depth development of A. stolonifera moxa and the improvement of moxa quality standards.

[Anti-inflammatory material basis and mechanism of Artemisia stolonifera based on UPLC-Q-TOF-MS combined with network pharmacology and molecular docking].

This study aimed to explore the anti-inflammatory material basis and molecular mechanism of Artemisia stolonifera based on the analysis of the chemical components in different extracted fractions of A. stolonifera and their antioxidant and anti-inflammatory effects in combination with network pharmacology and molecular docking. Thirty-two chemical components were identified from A. stolonifera by ultra-performance liquid chromatography coupled to tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS). Among them, there were 7, 21 and 22 compounds in water, n-butanol and ethyl acetate fractions, respectively. The antio-xidant capacity of different extracted fractions was evaluated by measuring their scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl(DPPH) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) free radicals and total antioxidant capacity [ferric reducing antioxidant power(FRAP) assay]. The inflammatory model of RAW264.7 cells was induced by lipopolysaccharide(LPS), and the levels of nitrite oxide(NO), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) in the supernatant and the mRNA expression of related inflammatory factors in cells were used to evaluate the anti-inflammatory effects. The results revealed that ethyl acetate fraction of A. stolonifera was the optimal antioxidant and anti-inflammatory fraction. By network pharmacology, it was found that flavonoids such as rhamnazin, eupatilin, jaceosidin, luteolin and nepetin could act on key targets such as TNF, serine/threonine protein kinase 1(AKT1), tumor protein p53(TP53), caspase-3(CASP3) and epidermal growth factor receptor(EGFR), and regulate the phosphatidylinositol-3-kinase-protein kinase B(PI3K-AKT) and mitogen-activated protein kinase(MAPK) signaling pathways to exert the anti-inflammatory effects. Molecular docking further indicated excellent binding properties between the above core components and core targets. This study preliminarily clarified the anti-inflammatory material basis and mechanism of ethyl acetate fraction of A. stolonifera, providing a basis for the follow-up clinical application of A. stolonifera and drug development.

[Effects of shading intensity on growth and quality of Artemisia stolonifera].

The purpose of this study was to analyze the effects of shading intensity on the growth, yield, and quality of Artemisia stolonifera so as to provide references for the artificial cultivation of A. stolonifera. The seedlings of A. stolonifera with consistent growth underwent shading treatment at four shading intensity levels(0, 55%, 85%, and 95%) with different layers of black shading nets. The agronomic indexes, yield, moxa yield, total ash, quality characteristics of moxa during combustion and pyrolysis, main volatile components, flavonoids, and phenolic acids were measured. The results showed that under shading conditions, the stem diameter, leaf width, 5-leaf spacing, branch number, and yield of A. stolonifera decreased significantly, while the plant height, leaf length, leaf number, chlorophyll content, and moxa yield increased first and then decreased with the increase in shading intensity. The burning performance of moxa under natural light was better than that under moderate and severe shading conditions. The content of eucalyptol first increased and then decreased with the increase in shading intensity. The humulene content was negatively correlated with shading intensity. Other major volatile components showed no significant difference under various shading conditions. The content of neochlorogenic acid, cryptochlorogenic acid, isoschaftoside, and isochlorogenic acid B was positively correlated with shading intensity, while the content of chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C decreased first and then increased with the increase in shading intensity. To sum up, A. stolonifera is a light-loving plant, and shading can greatly reduce the yield, the content of internal components, and the burning performance of moxa. It is the main reason why A. stolonifera is mainly distributed in the forest edge, open forest, roadside, and wasteland grass in the middle and high mountains in the wild. For artificial domestication and cultivation of A. stolonifera, it is better to select plots with sufficient light.

[Comparison of growth and quality of wild and cultivated Artemisia stolonifera].

This paper aims to compare the difference of growth and quality between wild and cultivated Artemisia stolonifera, thereby providing references for further development and utilization of A. stolonifera. The wild and cultivated A. stolonifera from different altitudes were collected, and the agronomic characters, moxa yield, volatile components, flavonoids, and phenolic acids were determined. The results showed that the cultivated species were taller and stronger, with more leaves and branches, than the wild species. The moxa yield and combustion quality of wild products were higher than those of cultivated products. The content of main volatile components in cultivated products was higher than that in wild products. The content of flavonoids and phenolic acids in wild products was higher than that in cultivated products. At high altitude, the ignition performance, combustion persistence, comprehensive combustion performance, and heat release during combustion of the wild and cultivated A. stolonifera. were optimal. At middle altitude, the content of main characteristic volatile components and flavone phenolic acids in the leaves of the cultivated and wild A. stolonifera were the highest. At low altitude, the combustion quality and the content of the above components of the cultivated A. stolonifera decrease significantly. Considering the combustion quality and the content of the internal components of the leaf lint, the middle and high altitude areas are suitable for the artificial cultivation of A. stolonifera.

[Distinguishing between Artemisia stolonifera and A. argyi by specific PCR of leaves and non-glandular trichomes].

Artemisia stolonifera is a relative of A. argyi. The two species are difficult to be distinguished due to the similarity in leaf shape and have even less distinctive features after processing. This study aims to establish a method to quickly distinguish between them. At the same time, we examined the reasonability and applicability of the specific polymerase chain reaction(PCR) method. The C/T single nucleotide polymorphism was detected at the position 202 of the sequence, based on which specific primers were designed to identify these two species. The PCR with the specific primer JNC-F and the universal primer ITS3R produced a specific band at 218 bp for A. argyi and no band for A. stolonifera, which can be used to detect at least 3% of A. argyi samples mixed in A. stolonifera samples. The PCR with the specific primer KY-F and the universal primer ITS3R produced a specific band at 218 bp for A. stolonifera and no band for A. argyi, which can be used to detect at least 5% of A. stolonifera samples mixed with A. argyi. The limit of detection of the established method was 5 ng DNA. The established PCR method can accurately distinguish between A. stolonifera and A. argyi, which provides an experimental basis for the quality control of A. stolonifera and determines whether the herbs are adulterated.

[Corythucha marmorata affects growth and quality of Artemisia argyi].

This study aims to investigate the impact of the invasive pest Corythucha marmorata on the growth and quality of Artemi-sia argyi. The signs of insect damage at the cultivation base of A. argyi in Huanggang, Hubei were observed. The pests were identified based on morphological and molecular evidence. The pest occurrence pattern and damage mechanism were investigated. Electron microscopy, gas chromatography-mass spectrometry(GC-MS), and high performance liquid chromatography(HPLC) were employed to analyze the microstructure, volatile oils, and flavonoid content of the pest-infested leaves. C. marmorata can cause destructive damage to A. argyi. Small decoloring spots appeared on the leaf surface at the initial stage of infestation. As the damage progressed, the spots spread along the leaf veins and aggregated into patches, causing yellowish leaves and even brownish yellow in the severely affected areas. The insect frequently appeared in summer because it thrives in hot dry conditions. After occurrence on the leaves, microscopic examination revealed that the front of the leaves gradually developed decoloring spots, with black oily stains formed by the black excrement attaching to the glandular hairs. The leaf flesh was also severely damaged, and the non-glandular hairs were broken, disor-ganized, and sticky. The content of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acids A and B, hispidulin, jaceosidin, and eupatilin at the early stage of infestation was significantly higher than that at the middle stage, and the content decreased at the last stage of infestation. The content of eucalyptol, borneol, terpinyl, and caryophyllin decreased in the moderately damaged leaves and increased in the severely damaged leaves. C. marmorata was discovered for the first time on A. argyi leaves in this study, and its prevention and control deserves special attention. The germplasm materials resistant to this pest can be used to breed C. marmorata-resis-tant A. argyi varieties.

Artemisia argyi allelopathy: a generalist compromises hormone balance, element absorption, and photosynthesis of receptor plants.

BACKGROUND: Allelopathy is expressed through the release of plant chemicals and is considered a natural alternative for sustainable weed management. Artemisia argyi (A. argyi) is widely distributed throughout Asia, and often dominates fields due to its strong allelopathy. However, the mechanism of A. argyi allelopathy is largely unknown and need to be elucidated at the physiological and molecular levels. RESULTS: In this study, we used electron microscopy, ionomics analysis, phytohormone profiling, and transcriptome analysis to investigate the physiological and molecular mechanisms of A. argyi allelopathy using the model plant rice (Oryza sativa) as receptor plants. A. argyi water extract (AAWE)-treated rice plants grow poorly and display root morphological anomalies and leaf yellowing. We found that AAWE significantly inhibits rice growth by destroying the root and leaf system in multiple ways, including the integrity of ultrastructure, reactive oxygen species (ROS) homeostasis, and the accumulation of soluble sugar and chlorophyll synthesis. Further detection of the hormone contents suggests that AAWE leads to indole-3-acetic acid (IAA) accumulation in roots. Moreover, ionomics analysis shows that AAWE inhibits the absorption and transportation of photosynthesis-essential mineral elements, especially Mg, Fe, and Mn. In addition, the results of transcriptome analysis revealed that AAWE affects a series of crucial primary metabolic processes comprising photosynthesis in rice plants. CONCLUSIONS: This study indicates that A. argyi realizes its strongly allelopathy through comprehensive effects on recipient plants including large-scale IAA synthesis and accumulation, ROS explosion, damaging the membrane system and organelles, and obstructing ion absorption and transport, photosynthesis and other pivotal primary metabolic processes of plants. Therefore, AAWE could potentially be developed as an environmentally friendly botanical herbicide due to its strong allelopathic effects.

Morphogenesis, ultrastructure, and chemical profiling of trichomes in Artemisia argyi H. Lév. & Vaniot (Asteraceae).

MAIN CONCLUSION: Glandular trichomes of Artemisia argyi H. Lév. & Vaniot are the key tissues for the production of flavonoid and terpenoid metabolites. Artemisia argyi H. Lév. & Vaniot is an herbaceous perennial plant that has been widely used in traditional medicine for thousands of years. Glandular trichomes (GTs) and nonglandular trichomes (NGTs) have been reported on the leaf surface of A. argyi. The aim of this study was to elucidate the morphogenetic process and to analyze the metabolites of trichomes in A. argyi. The morphogenesis of GTs and NGTs was characterized using light, scanning, and transmission electron microscopy. The constituents of GTs were analyzed using laser microdissection combined with gas and liquid chromatography-mass spectrometry. Five developmental stages of two types of GTs and four developmental stages of one type of NGTs were observed. Two types of mature GT and one type of NGT were composed of 10, 5, and 4-6 cells, respectively. A large storage cavity was detected between the cuticle and cell walls in the first type of mature GT. Large nuclei, nucleoli, and mitochondria were observed in the basal and intermediate cells of the second type of GT. In addition, large vacuoles were observed in the basal and apical cells, and large nuclei were observed in the middle cells of NGTs. One monoterpene and seven flavonoids were identified in GTs of A. argyi. We suggest that GTs are the key tissues for the production of bioactive metabolites in A. argyi. This study provides an important theoretical basis and technical approach for clarifying the regulatory mechanisms for trichome development and bioactive metabolite biosynthesis in A. argyi.

Genome sequencing reveals chromosome fusion and extensive expansion of genes related to secondary metabolism in Artemisia argyi.

Artemisia argyi, as famous as Artemisia annua, is a medicinal plant with huge economic value in the genus of Artemisia and has been widely used in the world for about 3000 years. However, a lack of the reference genome severely hinders the understanding of genetic basis for the active ingredient synthesis of A. argyi. Here, we firstly report a complex chromosome-level genome assembly of A. argyi with a large size of 8.03 Gb, with features of high heterozygosity (2.36%), high repetitive sequences (73.59%) and a huge number of protein-coding genes (279 294 in total). The assembly reveals at least three rounds of whole-genome duplication (WGD) events, including a recent WGD event in the A. argyi genome, and a recent burst of transposable element, which may contribute to its large genome size. The genomic data and karyotype analyses confirmed that A. argyi is an allotetraploid with 34 chromosomes. Intragenome synteny analysis revealed that chromosomes fusion event occurred in the A. argyi genome, which elucidates the changes in basic chromosome numbers in Artemisia genus. Significant expansion of genes related to photosynthesis, DNA replication, stress responses and secondary metabolism were identified in A. argyi, explaining the extensive environmental adaptability and rapid growth characteristics. In addition, we analysed genes involved in the biosynthesis pathways of flavonoids and terpenoids, and found that extensive gene amplification and tandem duplication contributed to the high contents of metabolites in A. argyi. Overall, the reference genome assembly provides scientific support for evolutionary biology, functional genomics and breeding in A. argyi and other Artemisia species.

The role of antifungal activity of ethyl acetate extract from Artemisia argyi on Verticillium dahliae.

AIMS: This study investigated the antifungal activity and mechanisms of ethyl acetate extract of Artemisia argyi (EAAA) against Verticillium dahliae. METHODS AND RESULTS: Optical and scanning electron microscopy observation showed that 2.0 mg ml-1 EAAA treatment reduced spore germination rate to 4.56%. Histochemical staining showed that 2.0 mg ml-1 EAAA treatment increased reactive oxygen species (ROS) by more than two times. Physiological test showed that EAAA treatment decreased the contents of soluble proteins and sugars, and reduced the activities of malate dehydrogenase and succinate dehydrogenase by nearly half. Transcriptome analysis showed that EAAA treatment down-regulated the expression of genes involved in primary metabolic pathways of V. dahliae. CONCLUSIONS: Our results revealed that EAAA inhibited the growth and development of V. dahliae from multiple levels and multiple targets, including inhibiting the germination and development of V. dahliae spores, destroying the structure of cell membranes, inducing ROS burst, reducing the activities of respiratory-related enzymes and down-regulating the expression of genes in primary metabolic pathways. SIGNIFICANCE AND IMPACT OF THE STUDY: The mechanism of the multitarget effects of EAAA against V. dahliae may limit the potential of fungus developing resistance and provide the efficient methods to control verticillium wilt disease in the future.

Genome Resequencing and Transcriptome Analysis Reveal the Genetic Diversity of Wolfiporia cocos Germplasm and Genes Related to High Yield.

Wolfiporia cocos is a saprophytic fungus belonging to the phylum Basidiomycota. The dried sclerotium of this organism has been widely used in traditional Chinese medicine for several thousand years and it is prescribed in many formulations. The W. cocos germplasm resources are complex and diverse, and the molecular mechanisms underlying the growth and development of its sclerotia are unclear. In this study, we used genome resequencing and transcriptome analysis to evaluate the genetic diversity of W. cocos germplasm resources in China and the mechanism of sclerotium growth and development. Phylogenetic and population structure analyses revealed that all the 39 tested strains were divided into three major groups. Most of the strains were clustered into one group, and the remaining strains were clustered into the other two groups. There may be a shared origin of cultivated W. cocos in the main production areas. Transcriptome analysis and quantitative reverse transcription-polymerase chain reaction confirmed that candidate genes related to the yield of W. cocos were mainly enriched in oxidation-reduction and carbohydrate metabolism and highly expressed in the ShenChuan strain, which had the highest comprehensive cultivation score. The findings will be helpful for further understanding the evolution and population structure of W. cocos and determining the functional genes that contribute to the high yield of sclerotia.

First report of Botrytis cinerea causing gray mold on Prunella vulgaris in Hubei province, China.

Prunella vulgaris L. is a perennial herb plant of the Lamiaceae family, and its dried spicas have been widely used as medicine, health-promoting food or tea around the world. P. vulgaris is distributed all over the world, such as Europe, Asia, northwestern Africa and North America, as well as the Huaihe River Basin and the middle and lower Yangtze River Basin in China. In February 2022, a serious disease like gray mold occurred in planting fields of P. vulgaris in Wuhan, Hubei (N30°27'07″, E114°15'49″), causing approximately 20% of plants were diseased in the field. Early symptoms were characterized by small, round gray-brown lesions on the leaves of P. vulgaris. Later, a large number of stems and leaves are wilted or necrotic, associated with wet rot and waterlogged spots and covered with light gray or grayish white flocculent mildew layer. To determine the causal agent of disease, 10 plants with the typical symptoms were collected from fields. The stems and leaves of diseased plants were cut into pieces (2 to 3 mm×5 mm), disinfected with 75% ethanol for 3 minutes, rinsed 3 times with sterile water. Each lesion sample was isolated and purified using separate PDA petri dishes at 25°C, and ultimately all samples yielded morphologically consistent pure strain colonies. From the 10 isolates obtained, XKC-1 was chosen as a representative isolate for further study. XKC-1 colonies showed gray aerial mycelia, which were fast-growing and grew over the whole plate (9 cm) after 4 days. In addition, some black and hard sclerotia (1.88±0.94 mm, n=50) with round or irregular shape developed on the colonies after approximately 10 days of incubation at 25°C (Fig. 2A, B). XKC-1 showed branched conidiophores with enlarged apical cells and numerous conidia (Fig. 2C). Unicellular conidia were colorless or gray, ellipsoid or ovoid, smooth and 7.91-12.38 µm × 10.08-13.82 µm (n=30) in size (Fig. 2D). Based on morphological characteristics, the isolate was initially identified as Botrytis sp. (Ellis 1971). To further identify the species, the genomic DNA of XKC-1 was extracted, and the ITS, LSU and G3PDH genes were amplified with the primers ITS1/ITS4, LROR/LR5 (Zhou et al. 2022) and G3PDH-F/G3PDH-R (Jin et al. 2022), respectively. The results indicated that the ITS (ON090404), LSU (ON090417), and G3PDH (ON169893) sequences were 99.80%, 100% and 99.46% identical to the sequences of Botrytis cinerea Pers. strain (MK370693.1, MN148533.1, MN630267.1), respectively. A phylogenetic tree constructed based on a concatenated sequence (ITS, LSU, G3PDH) using the neighbor-joining method in MEGA7 (Tamura et al. 2013) revealed that XKC-1 grouped with concatenated sequences from three representative B. cinerea isolates in GenBank. Based on the morphological characteristics and molecular identification, the strain XKC-1 was identified as Botrytis cinerea. For pathogenicity tests on detached leaves, 5 mm PDA cakes prepared from XKC-1 were placed on the leaves obtained from healthy P. vulgaris after wounding with a needle (n=10), while PDA medium without mycelia were used as control (25 ± 2°C) (Li et al.2020). Mycelia began to germinate and infect plant tissues at 1 dpi. A large part of the leaves showed water soaked spots covered with mycelia on the surface at 4 dpi. For whole plant inoculations, stem bases of five P. vulgaris seedlings were pierced with sterile needle, and then inoculated with three XKC-1 mycelium PDA cakes. Five plants were inoculated with three PDA cakes without mycelia as a control. After 2-4 days, lesions appeared on the leaves and covered with a gray-white mycelial layer, similar to those observed in the field. However, controls remained symptom free. The pathogen was reisolated from the diseased tissues, the colonies, microscopic characteristics and molecular identification were consistent with those of XKC-1. To our knowledge, this is a first report of B. cinerea causing gray mold on P. vulgaris in Hubei, China. This report would provide resources and reference for controlling of the increased incidence and economic losses of gray mold on P. vulgaris.

First report of seedling blight caused by Fusarium tricinctum on Cynanchum stauntonii in China.

Cynanchum stauntonii is a perennial herb plant of the Asclepiadaceae family. The dried roots and rhizomes have been used as medicine in China for 1500 years and are considered a remedy for cough and phlegm. In recent years, the wild C. stauntonii resources have not been sufficient for market demand, therefore, a large artificial cultivation area was established in Xinzhou, Tuanfeng and Macheng in Hubei province. In March and April 2022, serious outbreaks of seedling blight were observed in C. stauntonii in Xinzhou county (N30°48'12″, E114°49'24″), and the disease occurred on 10 to 15% of plants in five C. stauntonii nursery beds. Early symptoms included withered tips, chlorosis, stunting, yellow leaves and leaf drop, and later, seedlings die in patches. To determine the causal agent of disease, pieces (5 mm × 5 mm) of diseased tissue at the junction of disease and healthy tissue were surface disinfected by soaking in 75% ethanol for 3 min, rinsed three times with sterilized water, and pieces were placed on PDA at 25°C. Fungal isolates obtained were yellow-brown at the center and pink to white toward the periphery, and dark red pigments were observed in the agar. Isolates were cultured in synthetic low nutrient agar (SNA) and carnation leaf agar to observe the spore morphology. The macroconidia were sickle-shaped with 3-4 septate, with sizes of 30.26±2.36×3.77±0.53 µm on SNA and 33.52±2.20×3.81±0.48 µm on carnation leaf agar (n=30). Morphological characteristics of the isolates were consistent with those of Fusarium sp in the Fusarium Laboratory Manual (Leslie et al. 2006). Furthermore, the genomic DNA from a representative isolate BQ-2 was extracted, the ITS, TEF-1α, RPB1 and RPB2 genes were amplified with primers ITS1/ITS4, EF1/EF2, Fa/G2R and 5F2/7cr, respectively (Zhang et al. 2022). BLAST analysis showed that the ITS (ON935780.1), TEF-1α (OP985126.1), RPB1 (OP985125.1) and RPB2 (OP985124.1) amplicon sequence were 99.44%, 98.94%, 99.88% and 100% identical to the sequences of F. tricinctum strain (KU350724.1, AB674264.1, LC701712.1, MW474678.1), respectively. A phylogenetic tree constructed based on a concatenated sequence (ITS, TEF-1α, RPB1, RPB2) using the neighbor-joining and maximum likelihood method in MEGA7 revealed that BQ-2 grouped with concatenated sequences from four representative F. tricinctum isolates in GenBank. Based on the morphological characteristics and molecular identification, the strain BQ-2 was identified as F. tricinctum. For pathogenicity tests, 5 mm pieces of a BQ-2 colony on PDA were placed on excised leaves of healthy C. stauntonii wounded with a needle (n=5) and kept at 25±2℃. Leaves treated PDA were used as a control (Li et al.2020). After three days inoculation, the mycelia proliferated and began to infect leaf tissues. Ten days later, large parts of the detached leaves were extensively infested with the pathogen and brown. For live plant inoculation, stem bases of five healthy seedlings were punctured with sterile needle and then inoculated with BQ-2 mycelia from PDA. Controls were treated with only PDA. The seedlings began wilting after three days and at five days showed typical disease symptoms, similar to those observed in the field. The controls were asymptomatic. The pathogen was reisolated from the diseased tissues, and the colonies and microscopic characteristics were similar to those of BQ-2. To the best of our knowledge, this is the first report of F. tricinctum causing seedling blight on C. stauntonii in China. This report will provide resources and reference for controlling the increased incidence and economic losses of seedling blight on C. stauntonii.

[Identification, biological characteristics, and fungicide screening of pathogen of black spot in Aconitum carmichaelii].

In Hezhang county, Guizhou province, black spot tends to occur to Aconitum carmichaelii in the hot rainy summer, with the incidence up to 50%-70%, seriously impacting the yield and quality of the medicinal material. Thus, this study aims to clarify the pathogen and the occurrence characteristics. To be specific, the pathogen was isolated and identified according to Koch's postulates and the pathogenicity and biological characteristics were determined. In addition, the sensitivity of the pathogen to four microbial fungicides, four botanical fungicides, and five chemical fungicides was determined with the mycelium growth rate method for the purpose of screening out optimal fungicides. The pathogen was identified as Alternaria alternate, as evidenced by the similar colony morphology and microscopic characteristics and 99.55%-100% similarity in sequences of rDNA-ITS, LSU, 18S, and TEF of the two. The optimum growth conditions for A. alternata were 28 ℃, pH 8, and continuous darkness. Bacillus subtilis had strong inhibitory effect on the pathogen, and the inhibition rate was more than 90% when the concentration was 1 mg·L~(-1). In addition, difenoconazole and quinoline copper can also control the pathogen, with median effective concentration(EC_(50)) of 2.92 and 9.02 mg·L~(-1), respectively. This study lays a theoretical basis for the field control of black spot in A. carmichaelii.