HLB induce changes in the tree physiology of citron (Citrus medica L. var. sarcodactylis Swingle).

Huanglongbing (HLB) is a highly destructive disease that decreases the yield and quality of Citrus medica L. var. sarcodactylis Swingle (C. medica var. sarcodactylis) and poses a great threat to the development of the global citrus industry. To explore the influence of HLB infection on C. medica var. sarcodactylis, levels of photosynthetic pigments, malondialdehyde (MDA), and carbohydrates, as well as antioxidant enzyme activities, were measured. The results show that HLB infection decreased photosynthetic pigment content, increased MDA content and antioxidant enzyme activities, and caused various changes in carbohydrate levels in stem, fruit, and leaf tissues. Transcriptomic analysis of C. medica var. sarcodactylis was also used to identify key genes related to the carbohydrate metabolic synthesis pathway in C. medica var. sarcodactylis. The C. medica var. sarcodactylis ADP-glucose pyrophosphorylase1 (CmAGP1), CmAGP2, C. medica var. sarcodactylis Granule-bound starch synthase (CmGBSS), C. medica var. sarcodactylis Sucrose synthases1 (CmSUS1), CmSUS2, C. medica var. sarcodactylis Sucrose phosphate synthase (CmSPS), C. medica var. sarcodactylis alkaline/neutral invertase1 (CmNi1), CmNi2, CmNi3 and CmNi4 were successfully cloned and identified, and differential expression analysis showed that HLB infection significantly upregulated these genes in stems and leaves. In conclusion, HLB infection causes cellular damage, a reduction in photosynthetic capacity, decreased pathogen resistance, and severe disorders in carbohydrate metabolism in C. medica var. sarcodactylis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01129-z.

High-throughput screening of secondary metabolites by Sorbus pohuashanensis cells under environmental stress using UHPLC-QTOF combined with AntDAS.

Environment stress can promote the synthesis and accumulation of a series of secondary metabolites, which are important quality factors in medicinal plants. However, the data related to metabolites is often too large, making it difficult to screen quickly, accurately and comprehensively various differential compounds. In this study, a high-throughput screening method for differential secondary metabolites produced by medicinal plants under environmental stress has been developed based on ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) and automatic data analysis strategy. This work uses Sorbus pohuashanensis cells with biotic stress (Harpin protein) and abiotic stress (Cd2+ ) as potential environmental stress factors. The results showed that S. pohuashanensis cells could rapidly respond to both Harpin protein and Cd2+ within 24 h, and a significant positive correlation was observed between their concentration (within a certain range) and induction time. The proposed screening method can automatically screen the bulk UHPLC-QTOF metabolic data for differential compounds with high-throughput, and also perform preliminary identification of their possible structures. The screening results indicated that the stress response of S. pohuashanensis cells to Cd2+ was significantly higher than that of Harpin protein, and all of them could produce a series of biphenyls, terpenes, and other phytoalexins with stress-resistance and physiological functional properties. Overall, the screening method provides an efficient and powerful tool to study the response mechanisms of plants to environmental stress, to improve the resistance of medicinal plants and also to select and breed high-quality Chinese medicinal plants.

[Reference gene screening for Real-time quantitative PCR in Polygonum multiflorum].

To select suitable references gene of Polygonum multiflorum for gene expression analysis in different tissues, five candidate reference genes like Actin,GAPDH,SAND,PP2A,TIP41 were selected from the transcriptome data of P. multiflorum, then the specific primers were designed. The expression stability of the five reference genes in different tissues of P. multiflorum was analyzed by Real-time quantitative PCR through avilable analysis methods such as geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, to ensure the reliability of the analysis results. The results showed that there were significant differences in the expression levels and stability of candidate genes in different tissues of P. multiflorum. Ct distribution analysis of the expression levels of candidate genes showed that the expression levels of Actin and GAPDH genes were relatively high in different tissues, while the expression levels of SAND, PP2A and TIP41 were lower. The stability of each candidate gene was analyzed by different methods. The results of geNorm analysis showed that the expression of PP2A and GAPDH was the most stable, the expression stability of SAND was the worst, the stability of PP2A was the highest in both NormFinder and Delta CT, the stability of SAND was the lowest, and the stability of Actin was the most stable in BestKeeper analysis. Through the comprehensive evaluation and analysis of the stability of candidate genes by RefFinder, it is concluded that the stability of PP2A gene is the highest, followed by GAPDH, Actin, TIP41, SAND, and SAND gene is the worst. Therefore, the PP2A gene is an ideal reference gene for the analysis of gene expression in different tissues of P. multiflorum.

[Screening of plant growth-promoting rhizobacteria and its effect on seed germination of Polygonum multiflorum].

In this study, the rhizobacteria and actinomycetes of Polygonum multiflorum were screened for the strains with indole acetic acid(IAA)-producing capacity by Salkowski method, the siderophore-producing strains by Chrome Azurol S(CAS) assay, and the strains with inorganic phosphorus-solubilizing capacity by PKO inorganic phosphorus medium. The strains were identified by morphological identification, physiological and biochemical characteristics, and 16 S rDNA sequences. Furthermore, the effect of growth-promoting strains on the seed germination and development of P. multiflorum was tested. The results showed that among 196 strains, two strains F17 and F42 were found to be capable of producing IAA and siderophore and solubilizing inorganic phosphorus simulta-neously. For F17 and F42, the results are listed below: 38.65 and 33.64 mg·L~(-1) for IAA production, 0.85 and 0.49 for siderophore-producing capacities(A_s/A_r), and 1.35 and 1.70 for inorganic phosphorus-solubilizing capacities(D/d), respectively. Comprehensive analysis revealed that strains F17 and F42 were identified as Pseudochrobactrum asacharolyticum and Bacillus aryabhattai, respectively, and both could significantly promote the seed germination of P. multiflorum.

[Preliminary study on memory function of Sorbus aucuparia suspension cell to biotic stress].

Plants have a memory function for the environmental stress they have suffered. When they are subjected to repeated environmental stress, they can quickly and better activate the response and adaptation mechanism to environmental stress, thus realizing long-term stable reproduction. However, most of the relevant studies are applied to crops and Arabidopsis thaliana rather than medicinal plants about the improvement of plant growth status and the effect on phytoalexin biosynthesis. In this study, yeast extract(YE) was used as an elicitor to simulate biotic stress, and the changes in biomass and the content of some secondary metabolites were measured by giving repeated stresses to Sorbus aucuparia suspension cell(SASC). The results showed that the accumulation levels of biomass and some secondary metabolites in SASC subjected to repeated stress are significantly increased at some time points compared with single stress. A phenomenon that SASC can memorize biotic stress is confirmed in this study and influences phytoalexin accumulation in SASC. Furthermore, the work laid the groundwork for research into the transgenerational stress memory mechanism of medicinal plant.

[Extraction and separation, structure analysis and biological activity of polysaccharides from Atractylodis Rhizoma].

Atractylodis Rhizoma(AR) is a traditional Chinese medicinal material for food and medicine, with the functions of eli-minating dampness, strengthening the spleen, expelling wind evil and dispersing cold. AR contains a variety of compounds, including sesquiterpenoids, alkynes, triterpenoids, aromatic glycosides, polysaccharides and so on. At present, the researches on AR mainly focus on volatile components, with relatively fewer researches on non-volatile components. Polysaccharide from Atractylodis Rhizoma(ARP) is an important material basis among non-volatile components for the efficacy. Due to its many biological activities such as immunomodulatory activity, anti-tumors, anti-virus and anti-oxidation, ARP has certain research value and potential. The diversity of the polysaccharide structure is the basis for biological functions, but it also increases the difficulty of carbohydrate research. The research on the extraction, separation, purification, structure and activity of ARP is in the preliminary exploration stage, still with many shortcomings. Herein, recent advancements in the extraction, purification, structural characteristics and biological activities of ARP were summarized in this article to provide scientific reference for the in-depth systematic research of ARP and the development of AR resources.

CYP76B74 Catalyzes the 3''-Hydroxylation of Geranylhydroquinone in Shikonin Biosynthesis.

Shikonin and its derivatives are the most abundant naphthoquinone pigments formed in species of the medicinally and economically valuable Boraginaceae. A key step in the shikonin biosynthetic pathway, namely the C-3'' hydroxylation of the prenylated phenolic intermediate geranylhydroquinone, is expected to be catalyzed by a cytochrome P450. To identify cytochrome P450 candidates with transcription profiles similar to those of genes known to be involved in shikonin biosynthesis, we carried out coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines and examined the spatial expression of candidate genes in different organs of Arnebia euchroma In biochemical assays using geranylhydroquinone as the substrate, CYP76B74 exhibited geranylhydroquinone 3''-hydroxylase activity and produced 3''-hydroxy-geranylhydroquinone. In CYP76B74 RNA interference A. euchroma hairy roots, shikonin derivative accumulation decreased dramatically, which demonstrated that CYP76B74 is required for shikonin biosynthesis in the plant. Phylogenetic analysis confirmed that CYP76B74 belonged to the CYP76B subfamily and was most likely derived from an ancestral geraniol 10-hydroxylase. In a subcellular localization analysis, a GFP-CYP76B74 fusion localized to endoplasmic reticulum membranes. Our results demonstrate that CYP76B74 catalyzes the key hydroxylation step in shikonin biosynthesis with high efficiency. The characterization of the CYP76B74 described here paves the way for further exploration of the ring closure reactions generating the naphthoquinone skeleton as well as for the alternative metabolism of geranylhydroquinone 3''-hydroxylase to dihydroechinofuran.

[Analysis of bacteria distribution characteristics in different layers of agarwood based on Hiseq sequencing].

To explore the diversity of bacterial community structure between different layers of agarwood, Hiseq(high-throughput sequencing) was used to analyze the bacterial community structure of samples from different layers of agarwood. Our results showed that 1 150 096 optimized sequences and 9 690 OTUs were obtained from 15 samples of 5 layers of agarwood, which belonged to 28 bacterial phyla, 61 classes, 110 orders, 212 families and 384 genera. Further analysis revealed that the normal layer(NL) had the lowest bacterial species richness and the smallest number of OTUs. And the total number of OTUs of the agarwood layer(AL) and NL was zero, which was quite different.At the same time, there were significant differences in bacterial community structure and species diversity between NL and the other four layers. While there were some common dominant bacterial genera in both transition layer(TL) and NL. The similarity of bacterial distribution in 4 non-NL layers was relatively high, which had four common genera, such as Acidibacter, Bradyrhizobium, Acidothemus and Sphingomonas. While Acidibacter, Bradyrhizobium and Acidothemus were the dominant bacterial genus of DA and AL, and all of these layers contained volatile oil. In addition, the Bradyrhizobium was the most abundant in agarwood layer. Our results showed that bacterial community diversity and abundance were decreasing from DL to AL, and different layers showed significant differences in bacterial enrichment. It provided the clues to investigate how bacteria participate in the formation of agarwood.

[Strategies for medicinal plants adapting environmental stress and "simulative habitat cultivation" of Dao-di herbs].

This paper analyzed life form, habitats and environmental stresses of medicinal plants and algal fungi collected in Chinese Pharmacopoeia(2015). ①It was found that only 0.94% of the medicinal plants mainly cultivated in field. The most common habitats of medicinal plants are divided into two types: those whose natural habitats are forest margins/undergrowth(about 42.53%) and those whose natural habitats are roadside, hillside, wasteland/sand(about 43.78%). The former mainly faces environmental stresses such as weak light, pests and diseases; the latter often faces the main environmental stresses of drought, strong light, ultraviolet radiation, high temperature, low temperature(day and night or annual temperature difference is large), nutrient deficiency, pests and so on. ②Based on analyzing the strategies of medicinal plants to adapt to environmental stresses, it is pointed out that the synthesis and accumulation of secondary metabolites are the most important strategies of medicinal plants to protect against environmental stresses. In the process of long-term adaptation to specific stress, the accumulation of relevant genetic variation and epigenetic inheritance has become an important condition for the formation of quality of medicinal plants. ③It is proposed that "simulative habitat cultivation" has obvious advantages in balancing growth and secondary metabolism and guaranting the quality of traditional Chinese medicinal materials.

[Land use strategy of ecological agriculture of Chinese materia medica in future development].

With the rapid development of comprehensive health industry, the demand for Chinese medicinal materials is increasing. There is also a growing demand for land for the cultivation of Chinese medicinal materials.Based on the analysis of the demand characteristics of planting habitats for Chinese medicinal herbs, this paper finds that compared with the cultivated environment, the wild environment is more conducive to the improvement of the quality and stress resistance of medicinal plants. The eco-planting for Chinese medicinal materials is the only way to achieve high quality, efficient and sustainable production of traditional Chinese medicine. Therefore, according to the habitat distribution characteristics of wild medicinal plants, combined with the current situation of land resource utilization in China and the increasing demand for land for Chinese herbal medicine cultivation, the land use strategy of Chinese herbal medicine ecological agriculture was proposed. ① To vigorously develop underwood planting and change the existing field cultivation mode. ② To make full use of mountainous areas and barren slopes to carry out wild planta tending or planting imitates wild condition. ③ According to the development law and biological characteristics of medicinal plants, the land resources should be developed and used rationally according to local conditions.This can not only meet the requirements of the specific growth environment of Chinese medicinal materials, realize the sustainable development of the Chinese medicinal materials industry, but also increase the economic income of people in mountainous areas, provide scientific and effective solutions for the land use of Chinese medicinal materials, and also have important significance for the protection of wild Chinese medicinal materials.

[Jasmonate signal pathway involved in phytoalexin biosynthesis in Sorbus aucuparia cell elicited by Hpa1].

The Harpin protein Hpa1 can induce defense responses in plant. This study aimed at investigating the role of jasmonate (JA) signal pathway in the process of biosynthesis of secondary metabolite in Sorbus aucuparia cell eliciting by Hpa1 crude extract (Hpa1 CE). The results showed that Hpa1 crude extract (Hpa1 CE) could induce phytoalexin synthesis in S. aucuparia cell， most of which was noraucuparin and its glycosides. Meanwhile Hpa1 CE treatment resulted in methyl jasmonate (MeJA) production increased and noraucuparin was de novo synthesized in large quantities. Combination of Hpa1 CE and salicylhydroxamic acid (SHAM, JA signaling inhibitor) caused the decreased MeJA and noraucuparin in the S. aucuparia cell compared with that in Hpa1 CE group. Real-time PCR results indicated that Hpa1 CE treatment caused down-regulation of JAZ and up-regulation of mcy2 in transcription level. Therefore Hpa1 CE elicited defense mechanism and JA signaling pathway involved in phytoalexin biosynthesis in S. aucuparia cell. It presented information to elucidate the role of JA signal pathway in stress response in the perspective of secondary metabolism of plant.

[Research and application progress of Lepidium meyenii（maca）].

Lepidium meyenii（maca）was a herbaceous plant of the family Cruciferae. It is native to the andes region of South America where the local people had been growing and consuming maca for centuries. The unique chemical composition and physiological function of maca were widely concerned worldwide. It was introduced to China in 2002, and were cultivated successfully in Yunnan, Tibet, Sichuan, Jilin and other places with a certain size. Maca contained not only rich nutrition such as protein, vitamin and mineral matter, but also lots of secondary metabolites as maca alkaloids, glucosinolates, volatile oils, sterols polyphenols and macaenes. Numerous studies suggested that maca may serve effects in resisting oxidation, fatigue resistance, raising fertility, regulating endocrine, enhancing immunity, tumour suppression, treating osteoporosis, regulate blood sugar and protection of nervous system. Maca was approved by the Ministry of Health as a new resource food in 2011, and its related products include food, health foods, cosmetics, etc. Certain exploratory researches were carried to take better advantage of maca's medicinal value. This paper briefly reviewed the research and application progress of maca in recent years from the aspects of botany, chemical composition, function, resources situation and related products development, which was supposed to provide reference for scientific research and utilization of maca.

[Ecological agriculture: future of agriculture for Chinese material medica].

The ecological agriculture of traditional Chinese medicine (TCM) is generally acknowledged as the most advanced agricultural mode. However, it's still a doubt whether ecological agriculture could be widely applied in TCM agriculture. In this study, we first analyze both the differences and relationships between ecological and organic agriculture, which suggesting that ecological agriculture does not need all the inputs as traditional agriculture. After introducing the situation of ecological agriculture from all across the world, we analyze the differences and characteristics between ecological and chemical agricultures. Considered with the big challenge caused by chemical agriculture, we pointed out that ecological agriculture could definitely replace chemical agriculture. Last but not the least, combined with the situation and problems of Chinese agriculture, we analyze the distinctive advantages of TCM ecological agriculture from 3 aspects as its unique quality characteristics, its unique habitat requirements in production and its unique application and market characteristics, respectively. In conclusion, ecological agriculture is the straight way of TCM agriculture.

Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract.

Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphenyl phytoalexins formation after yeast extract (YE) treatment. An ultra-performance liquid chromatography (UPLC) method coupled with quadrupole time of flight mass spectrometry (Q-TOF-MS) LC-MS/MS was applied to determine the time course of these biphenyl biomarkers accumulation in YE-treated S. pohuashanensis suspension cells. The results of quantitative analyses show the content of Noraucuparin, 2'-Hydroxyaucuparin, and their glycosides initially increased, then decreased over time. The Noraucuparin content reached its highest (225.76 µg·g(-1)) at 18 h after treatment, 6 hours earlier than that of Noraucuparin 5-O-ß-d-glucopyranoside. The content of 2'-Hydroxyaucuparin reached its highest (422.75 µg·g(-1)) at 30 h after treatment, also earlier than that of its glycoside. The understanding of phytoalexin metabolism in this study may provide a basis for improving Maloideae resistance to pathogens.

[Discussion on present situation of study on pesticide residues in Chinese herbal medicines].

Pesticide residues in traditional Chinese medicine has attracted widespread attention at home and abroad. This paper analyzed the pollution present situation and existing problems of pesticide residue for Chinese herbal medicines, explicited the analytical methods of pesticide residues in Chinese herbal medicines. Meanwhile, the commonly used pesticide residue degradation and application in Chinese herbal medicines were discussed. Moreover, on the basis of analysis of pesticide residue standards, this paper proposed the necessity and urgency of the limit standard of pesticide residues in Chinese herbal medicines, and provided a scientific references for deepening research and developing safe, green medicines.

[Mitogen-activated protein kinase genes of Artemisia annua and their expression analysis under Cadmium stress].

In order to study Artemisia annua under cadmium stress, whether there are corresponding MAPK genes involved in transduction of the cadmium signal. 17 AaMAPK genes, named AaMAPK1-AaMAPK17 repectively, were finally obtained by using Trinity method for de novo assembly of transcripts from SRA database and BLAST search against AtMAPK genes and determing conserved domain using a series of bioinformatics tools. There exist 16 MAPK genes contained T[D/E]Y conserved domains among the obtained genes. The expressions of these genes were analyzed by Real-time PCR under cadmium stress. The results showed that the expressions level of AaMAPK3 and AaMAPK10 were down-regulated and MAPK7, MAPK9 and MAPK12 were up-regulated. These indicated that there exist corresponding MAPK genes involved in transduction of the cadmium signal.

[Ecological agriculture: future of Good Agriculture Practice of Chinese materia medica].

Based on the ecological and economic problems in Good Agriculture Practice (GAP) of Chinese material medica, we introduced the origin, concept, features and operative technology of eco-agriculture worldwide, emphasizing its modes on different biological levels of landscape, ecosystem, community, population, individual and gene in China. And on this basis, we analyzed the background and current situation of eco-agriculture of Chinese materia medica, and proposed its development ideas and key tasks, including: (1) Analysis and planning of the production pattern of Chinese material medica national wide. (2) Typical features extraction of regional agriculture of Chinese materia medica. (3) Investigation of the interaction and its mechanism between typical Chinese materia medica in each region and the micro-ecology of rhizosphere soil. (4) Study on technology of eco-agriculture of Chinese materia medica. (5) Extraction and solidification of eco-agriculture modes of Chinese materia medica. (6) Study on the theory of eco-agriculture of Chinese materia medica. Also we pointed out that GAP and eco-agriculture of Chinese material medica are both different and relative, but they are not contradictory with their own features. It is an irresistible trend to promote eco-agriculture in the GAP of Chinese material medica and coordinate ecological and economic development.

[Bioaccessibility of heavy metal in wild Artemisia annua and its health risk assessment].

In this study, we investigate the bioaccessibility of heavy metals (Cu, Pb, As, Cd and Hg) in wild Artemisia annua and use target hazard quotients (THQ) proposed by US Environmental Protection Agency to assess the health risk under the heavy metal exposure. The results showed that the bioaccessibility of Cu, Pb, As, Cd and Hg in A. annua are 0.77, 0.66, 0.46, 0.68 and 0, respectively, and that the value of THQ for adults and children were 0.030 and 0.025 calculated by risk assessment model. The results indicated that the heavy metals in A. annua were not able to be completely absorbed by human body and that their contents were in a safe range. In this study, by combining the bioavailability of heavy metal and health risk assessment, we assessed the security of heavy metals of wild A. annua, which will provide reference for the standard of heavy metals for medicinal materials.

[Uses of fresh herbs with antioxidant effect and prospect for population ecology reconstruction in Macau].

The average life expectancy in Macau is ranking the second in the world, the consumption of fresh medicinal plants is a profound culture in Macau. The paper focus on the distribution of the antioxidant herbs, a comprehensive investigation and analysis the amount of the plant resources was carried out. The antioxidant activity of alcohol extracts was determined by using the DPPH method, and six kinds fresh herbs with high antioxidant free radical activity were screened out. Reference to adult daily dose of vitamin C, it is calculated that the daily dose amount of fresh herbs is less than 200 g. For the expected shortage of resources and the ecological status of Macau, we give some suggestions of herbal introduction in population ecology reconstruction.

[In silico cloning and bioinformatics analysis of an AP2/EFR family gene from Arnebia euchroma].

A cDNA sequence of Arnebia euchroma AP2/ERF named AeAP2/ERF was cloned by in silico cloning in this study, using ACX71873 sequence from Lithospermum erythrorhizon as the probe sequence. Some characters of the AP2/ERF gene and encoded protein sequences were predicted and analyzed by the bioinformatics methods, including general physical and chemical properties, hydrophobieity, signal peptide, secondary structure, localization sites in cells. Results showed that the 876 bp long gene included a 1 077 bp ORF and encoding 205 amino acid. The AeAP2/ERF protein had no signal peptide, it was a hydrophilic proteins located in nucleus. The function of the AP2/ERF protein was mainly involved with metabolism controlling and signal transduction.