Influence of Chrysanthemum morifolium-maize intercropping pattern on yield, quality, soil condition, and rhizosphere soil microbial communities of C. morifolium.

Introduction: Chrysanthemum morifolium Ramat. is a perennial herb in the Compositae family, often employed in traditional Chinese medicine due to its medicinal value. The planting of C. morifolium faces the challenges of continuous cropping, and intercropping is able to somewhat overcome the obstacles of continuous cropping. Methods: In our study, we designed two different C. morifolium-maize intercropping patterns, including C. morifolium-maize narrow-wide row planting (IS) and C. morifolium-maize middle row planting (IM). Compared with monoculture, the agronomic traits, yield, active ingredients, soil physicochemical properties, soil enzyme activities, and rhizosphere soil microbial communities of C. morifolium and maize were measured under the two C. morifolium-maize intercropping patterns. Results: The findings indicated that (1) Intercropping elevated the agronomic traits, yield, and active ingredients of C. morifolium, especially in C. morifolium-maize narrow-wide row planting pattern, which indicating that interspecific distance played an important role in intercropping system; (2) Intercropping enhanced soil physicochemical properties and enzyme activities of C. morifolium and maize; (3) Intercropping altered rhizosphere soil microbial communities of C. morifolium and maize, making microbial interrelationships more complex. (4) Intercropping could recruit a large number of beneficial microorganisms enrich in the soil, including Bacillus, Sphingomonas, Burkholderia-Caballeronia-Paraburkholderia, Chaetomium, and Ceratorhiza, which may increase the content of AN, NN, AvK, ExCa, AvCu, AvZn and other nutrients in soil and promoted the growth and quality of C. morifolium. Discussion: In summary, intercropping with maize could promote the accumulation of beneficial microorganisms in the soil, thus improving the overall growing environment, and finally realizing the growth and improvement of C. morifolium.

The water-soluble subfraction from Artemisia argyi alleviates LPS-induced inflammatory responses via multiple pathways and targets in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine, Artemisia argyi has been used medicinally and eaten for more than 2000 years in China. It is widely reported in treating inflammatory diseases such as eczema, dermatitis, arthritis, allergic asthma and colitis. Although several studies claim that its volatile oil and organic reagent extracts have certain anti-inflammatory effects, the water-soluble fractions and molecular mechanisms have not been studied. AIM OF THE STUDY: To evaluate the therapeutic effect of A. argyi water extract (AAWE) on lipopolysaccharide (LPS)-induced inflammatory responses and to identify the most effective water-soluble subfractions. Moreover, the relevant pharmacological and molecular mechanisms by which the active subfraction mitigates inflammation were further investigated. MATERIALS AND METHODS: Firstly, RAW 264.7 cells stimulated with LPS were treated with AAWE (50, 100, and 200 µg/mL) or the water-soluble subfractions separated by D101 macroporous resin (AAWE1-AAWE4, 100 µg/mL), and NO production and mRNA levels of inflammatory genes were evaluated to determine the most effective water-soluble subfractions. Secondly, the chemical components of the active subfraction (AAWE4) were analyzed by UPLC-QTOF-MS. Thirdly, transcriptome and network pharmacology analysis, RT-qPCR and Western blotting assays were conducted to explore the underlying anti-inflammatory mechanism and active compounds of AAWE4. Subsequently, the binding ability of the potential active components in AAWE4 to the core targets was further determined by molecular docking. Eventually, the in vivo anti-inflammatory activity of AAWE4 (1.17, 2.34 and 4.68 g/kg, administered per day for 7 d) was evaluated in mice with LPS-induced systemic inflammation. RESULTS: In this study, AAWE showed excellent anti-inflammatory effects, and its water-soluble subfraction AAWE4 exhibited the strongest inhibitory effect on NO concentration and inflammatory gene mRNA expression after LPS stimulation, indicating that it was the most effective subfraction. Thereafter, four main compounds in AAWE4 were confirmed or tentatively identified by UPLC-QTOF-MS, including three flavonoid glycosides and one phenolic acid. Furthermore, the transcriptome and network pharmacology analysis showed that AAWE4 inhibited inflammation via multiple pathways and multiple targets. Based on the RT-qPCR and Western blotting results, AAWE4 downregulated not only the p38, PI3K, CCL5, MMP9, AP-1, and BCL3 mRNA expression levels activated by LPS but also their upstream and downstream protein expression levels and protein phosphorylation (p-AKT/AKT, p-p38/p38, p-ERK/ERK, p-JNK/JNK). Moreover, four identified compounds (isochlorogenic acid A, vicenin-2, schaftoside and isoschaftoside) could significantly inhibit NO content and the overexpression of inflammatory factors TNF-α, IL-1ß, iNOS and COX-2 mRNA induced by LPS, and the molecular docking confirmed the high binding activity of four active compounds with selected core targets (p38, AKT1, MMP9, and CCL5). In addition, the mRNA expression and immunohistochemical analysis showed that AAWE44 could inhibit lung inflammation via multiple pathways and multiple targets in vivo. CONCLUSIONS: The findings of this study suggest that the water-soluble subfraction AAWE4 from A. argyi ameliorated the inflammation caused by LPS through multiple pathways and multiple targets in vitro and in vivo, providing scientific support for the medicinal use of A. argyi. Importantly, it shows that the A. argyi subfraction AAWE4 can be developed as an anti-inflammatory drug.

Artemisia argyi extract subfraction exerts an antifungal effect against dermatophytes by disrupting mitochondrial morphology and function.

Artemisia argyi (A. argyi), a plant with a longstanding history as a raw material for traditional medicine and functional diets in Asia, has been used traditionally to bathe and soak feet for its disinfectant and itch-relieving properties. Despite its widespread use, scientific evidence validating the antifungal efficacy of A. argyi water extract (AAWE) against dermatophytes, particularly Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum, remains limited. This study aimed to substantiate the scientific basis of the folkloric use of A. argyi by evaluating the antifungal effects and the underlying molecular mechanisms of its active subfraction against dermatophytes. The results indicated that AAWE exhibited excellent antifungal effects against the three aforementioned dermatophyte species. The subfraction AAWE6, isolated using D101 macroporous resin, emerged as the most potent subfraction. The minimum inhibitory concentrations (MICs) of AAWE6 against T. rubrum, M. gypseum, and T. mentagrophytes were 312.5, 312.5, and 625 µg·mL-1, respectively. Transmission electron microscopy (TEM) results and assays of enzymes linked to cell wall integrity and cell membrane function indicated that AAWE6 could penetrate the external protective barrier of T. rubrum, creating breaches ("small holes"), and disrupt the internal mitochondrial structure ("granary"). Furthermore, transcriptome data, quantitative real-time PCR (RT-qPCR), and biochemical assays corroborated the severe disruption of mitochondrial function, evidenced by inhibited tricarboxylic acid (TCA) cycle and energy metabolism. Additionally, chemical characterization and molecular docking analyses identified flavonoids, primarily eupatilin (131.16 ± 4.52 mg·g-1) and jaceosidin (4.17 ± 0.18 mg·g-1), as the active components of AAWE6. In conclusion, the subfraction AAWE6 from A. argyi exerts antifungal effects against dermatophytes by disrupting mitochondrial morphology and function. This research validates the traditional use of A. argyi and provides scientific support for its anti-dermatophytic applications, as recognized in the Chinese patent (No. ZL202111161301.9).

[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.

[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.

Mechanisms governing the impact of nitrogen stress on the formation of secondary metabolites in Artemisia argyi leaves.

Nitrogen is a key factor in various physiological and metabolic processes in plants. Providing an adequate supply of nitrogen is essential for improving the total yield and quality of the medicinal plant Artemisia argyi (A. argyi), but the underlying mechanisms of how this nutrient alters the crop remains unclear. In this study, we conducted a series of pot experiments to investigate the agronomic traits and active components in the leaves of A. argyi plants under low and high nitrogen stress. Additionally, we used transcriptome analysis and RT-qPCR to explore the molecular pathways associated with nitrogen stress. Our results demonstrate a dramatic increase in the accumulation of phenolic acids and flavonoids in the low nitrogen (LN) stress group compared to the control (CK), with increases of 40.00% and 79.49%, respectively. Interestingly, plants in the high nitrogen (HN) stress group exhibited enhanced plant growth with larger leaves, thicker stems, and a 3% increase in volatile oil content compared to the CK. Moreover, A. argyi in the HN group displayed a 66% increase in volatile oil concentration compared to the LN group. Our combined transcriptome and q-PCR results indicate that LN stress promotes the expression of genes involved in flavonoid synthesis, while HN stress promotes the expression of genes related to terpene skeleton production and photosynthesis. Taken together, these findings suggest that different gene expression levels under LN and HN stress contribute to the photosynthesis capacity and the accumulation of active ingredients in A. argyi leaves. Our results elucidate the physiological and molecular mechanisms of nitrogen stress on A. argyi secondary metabolites and guide fertilization strategies for plant cultivation.

[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.

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.

Intraspecific variation in genome size in Artemisia argyi determined using flow cytometry and a genome survey.

Different collections and accessions of Artemisia argyi (Chinese mugwort) harbour considerable diversity in morphology and bioactive compounds, but no mechanisms have been reported that explain these variations. We studied genome size in A. argyi accessions from different regions of China by flow cytometry. Genome size was significantly distinct among origins of these 42 Chinese mugwort accessions, ranging from 8.428 to 11.717 pg. There were no significant intraspecific differences among the 42 accessions from the five regions of China. The clustering analysis showed that these 42 A. argyi accessions could be divided into three groups, which had no significant relationship with geographical location. In a genome survey, the total genome size of A. argyi (A15) was estimated to be 7.852 Gb (or 8.029 pg) by K-mer analysis. This indicated that the results from the two independent methods are consistent, and that the genome survey can be used as an adjunct to flow cytometry to compensate for its deficiencies. In addition, genome survey can provide the information about heterozygosity, repeat sequences, GC content and ploidy of A. argyi genome. The nuclear DNA contents determined here provide a new reference for intraspecific variation in genome size in A. argyi, and may also be a potential resource for the study of genetic diversity and for breeding new cultivar.

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 characterization, and fungicide screening of pathogens causing leaf spot of Belamcanda chinensis].

The leaf spot of Belamcanda chinensis often appears in May to June and spreads rapidly during the flowering stage(July to September) in the cultivation fields, seriously affecting the yield and quality of B. chinensis. To identify and characterize the pathogens of the leaf spot, we isolated two species of Alternaria, identified them according to Koch's postulates, and tested their pathogenicity and biological characteristics. Furthermore, we determined the inhibitory effects of 6 chemical fungicides, 1 plant fungicide, and 3 microbial fungicides on the pathogens by using mycelial growth rate and plate confrontation method to select the appropriate control agents. The results showed that the two pathogens causing B. chinensis leaf spot were Alternaria tenuissima and A. alternata. The conidia of A. tenuissima often formed long chains with no or a few branches, while those of A. alternata often formed short branched chains. The optimum growth temperature of both A. tenuissima and A. alternata was 25 ℃. The two pathogens grew well in alkaline environment. The indoor fungicide screening experiments showed that 40% flusilazole had good inhibitory effects on the two pathogens, with the EC_(50) values of 12.42 mg·L~(-1) and 12.78 mg·L~(-1) for A. tenuissima and A. alternata, respectively. The results of this study provide a theoretical basis for the subsequent theoretical research and field control of B. chinensis leaf spot.

[Identification, biological characteristics, and control of pathogen causing southern blight of Pinellia ternata].

In summer in 2020, Pinellia ternata in many planting areas in Hubei suffered from serious southern blight, as manifested by the yellowing and wilted leaves and rotten tubers. This study aims to identify the pathogen, clarify the biological characteristics of the pathogen, and screen fungicides. To be specific, the pathogen was isolated, purified, and identified, and the pathogenicity was detected according to the Koch's postulates. Moreover, the biological characteristics of the pathogen were analyzed. Furthermore, PDA plates and seedlings were used to determine the most effective fungicides. The results showed that the mycelia of the pathogen were white and villous with silk luster, which produced a large number of white to black brown sclerotia. The pathogen was identified as Athelia rolfsii by morphological observation and molecular identification based on LSU and TEF gene sequences. The optimum growth conditions for A. rolfsii were 30 ℃ and pH 5-8, and the optimum conditions for the germination of sclerotia were 25 ℃ and pH 7-9. Bacillus subtilis, difenoconazole, and flusilazole were identified as effective fungicides with PDA, and their half maximal effective concentration(EC_(50)) was all less than 5 mg·L~(-1). The effective fungicides screened with the seedlings were hymexazol and difenoconazole. Based on the screening experiments, difenoconazole can be used as the main agent for the prevention and treatment of southern blight.

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.

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.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

Sequence Characteristics and Phylogenetic Analysis of the Artemisia argyi Chloroplast Genome.

Artemisia argyi Levl. et Van is an important Asteraceae species with a high medicinal value. There are abundant A. argyi germplasm resources in Asia, especially in China, but the evolutionary relationships of these varieties and the systematic localization of A. argyi in the family Asteraceae are still unclear. In this study, the chloroplast (cp) genomes of 72 A. argyi varieties were systematically analyzed. The 72 varieties originated from 47 regions in China at different longitudes, latitudes and altitudes, and included both wild and cultivated varieties. The A. argyi cp genome was found to be ∼151 kb in size and to contain 114 genes, including 82 protein-coding, 28 tRNA, and 4 rRNA genes. The number of short sequence repeats (SSRs) in A. argyi cp genomes ranged from 35 to 42, and most of them were mononucleotide A/T repeats. A total of 196 polymorphic sites were detected in the cp genomes of the 72 varieties. Phylogenetic analysis demonstrated that the genetic relationship between A. argyi varieties had a weak relationship with their geographical distribution. Furthermore, inverted repeat (IR) boundaries of 10 Artemisia species were found to be significantly different. A sequence divergence analysis of Asteraceae cp genomes showed that the variable regions were mostly located in single-copy (SC) regions and that the coding regions were more conserved than the non-coding regions. A phylogenetic tree was constructed using 43 protein-coding genes common to 67 Asteraceae species. The resulting tree was consistent with the traditional classification system; Artemisia species were clustered into one group, and A. argyi was shown to be closely related to Artemisia lactiflora and Artemisia montana. In summary, this study systematically analyzed the cp genome characteristics of A. argyi and compared cp genomes of Asteraceae species. The results provide valuable information for the definitive identification of A. argyi varieties and for the understanding of the evolutionary relationships between Asteraceae species.

[Assessment of soil fertility in Artemisia argyi planting areas in Qichun county based on minimum data set].

This study aims to explore the soil fertility in the main Artemisia argyi planting areas in Qichun county.To be specific, the soil physical and chemical properties in the main planting areas of A.argyi in Qichun county were analyzed.On this basis, 12 indexes were selected for principal component analysis(PCA) which was then combined with the norm value of each index and the correlation coefficients between the indexes to establish the minimum data set(MDS).The radar map was plotted to directly demonstrate the level of each index and the comprehensive level of the sampling sites.The comprehensive index model was used to calculate the soil fertility quality index(SFQI) of the total data set(TDS) and MDS(SFQI-TDS and SFQI-MDS, respectively), and linear regression of the two was performed.The results showed that the indexes that made up the MDS for soil fertility evaluation were pH, available potas-sium, available iron, available zinc, available manganese, available copper, and available magnesium.The radar map suggested the greatest difference in soil organic matter and smallest difference in available nitrogen among the 14 sampling sites.Moreover, the overall content of available phosphorus and available iron was high, while that of available nitrogen was the lowest.The SFQI-MDS which was yielded based on the weight of each index in MDS calculated with the norm value was more sensitive and the SFQI had stronger correlation with TDS-SFQI, which can better represent TDS-SFQI.SFQI-MDS was in the range of 0.298-0.784, with the average of 0.565 and variation coefficient of 18.38%.Caohe Town had the highest average SFQI-MDS.Clustering of SFQI-MDS value suggested that the soil fertility can be classified into 4 levels: level Ⅰ(SFQI ≥ 0.677) indicated excellent soil fertility, which accounted for 11.24%(mainly in Qingshi town, Balihu, and Zhangbang town); level Ⅱ(0.571≤SFQI≤0.680) meant good fertility, which made up 43.82%(mainly in Caohe town, Hengche town, and Pengsi town); level Ⅲ(0.466≤SFQI≤0.557) indicated average fertility, which took up 32.58%(mainly in Chidong town and Zhulin town); level Ⅳ(SFQI≤0.421) suggested poor fertility, which accounted for 12.36%(mainly in Guanyao town).It is recommended that nitrogen, potassium, magnesium, and calcium fertilizers should be increased and organic ferti-lizer should be applied for the cultivation of A.argyi in Qichun county to improve soil fertility and soil physical and chemical properties.

[Identification, biological characteristics, and screening of effective fungicides of Phoma rhei causing leaf spot on Polygonum cuspidatum].

Severe leaf spot on Polygonum cuspidatum was found in the planting base of P. cuspidatum in Fangxian county, Shiyan of Hubei province. To clarify the types of pathogens and their pathogenesis, the present study isolated and purified the pathogen of leaf spot disease of P. cuspidatum according to Koch's postulates, determined the pathogenicity of the pathogen, and investigated its biological characteristics. Meanwhile, the inhibitory effects of 11 types of fungicides on the bacteria were determined according to the mycelium growth rate, and suitable prevention and control drugs were selected. The results showed that the pathogen isolated from the diseased leaves of P. cuspidatum was Phoma rhei by morphological and molecular identification. The colony morphology and microscopic characteristics were the same as those of Ph. rhei. The homology of rDNA-ITS and TEF gene sequences with Ph. rhei reached 99.96% and 99.43%, respectively. The optimal growth temperature of Ph. rhei was 25 ℃, and the optimal pH was 7-10. Furthermore, Ph. rhei grew faster under dark or light conditions. In fungicides, 0.3% eugenol, 250 g·L~(-1) propiconazole, and 33.5% quinoline copper had significant inhibitory effects on the pathogen with EC_(50) values of 57.54, 59.58, 88.69 µg·mL~(-1), respectively. Eugenol is a botanical fungicide, which can be used as a green and environmentally friendly fungicide in the prevention and control of P. cuspidatum. This study reported for the first time that the pathogen of P. cuspidatum leaf spot was Ph. rhei. investigated the biological characteristics of the pathogen, and screened the indoor chemicals, which provided a theoretical basis for the prevention and control of P. cuspidatum leaf spot in production.

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.

[Identification, biological characterization, and fungicide screening of pathogen causing southern blight in Aster tataricus].

This study aims to clarify the species and biological characteristics of the pathogen causing southern blight in Aster tataricus and screen out effective fungicides for the prevention and control of this disease. We collected the typical diseased plants and sclerotia on soil surface for the isolation of the pathogen, and identified the pathogen based on morphological characteristics, molecular biological characteristics, and pathogenicity. Further, we evaluated the inhibitory effects of 12 fungicides on the pathogen by plate growth inhibition assay. In the diseased plants, watery brown spots first appeared at the stem base and then spread upward, which were covered with white mycelia and surrounded by white to yellow-brown sclerotia. From the diseased plants, 15 strains with consistent traits were isolated. The pathogen was identified as Athelia rolfsii based on morphological characteristics and ITS and TEF sequences. The pathogenicity test was carried out according to Koch's rule, which showed the disease symptoms consistent with those in the field. The pathogen presented the optimum growth at 28-30 ℃, pH 5-8, and full darkness. The preliminary indoor screening demonstrated that four chemical fungicides(taifujin, hymexazol, flusilazole, and lime-sulphur-synthelic-solution), two botanical fungiticides(ethylicin and garlic oil), and a microbial agent(Bacillus subtilis) had good inhibitory effects on A. rolfsii. The results of gradient inhibition experiments showed that B. subtilis, flusilazole, and ethylicin had stronger inhibitory activity. The further in vivo screening indicated that ethylicin can be used as the main fungicide for the prevention and treatment of southern blight in A. tataricus.