Effects of climate warming and human activities on the distribution patterns of Fritillaria unibracteata in eastern Qinghai-Tibetan Plateau.

Fritillaria unibracteata is an endangered medicinal material species endemic to the Qinghai Tibet Plateau, and belongs to the national Class III endangered plant. In addition to expelling wind and removing damne, it also warms menstruation and relieves pain in clinic use of tranditional Chinese medicine. In recent years, affected by the destruction of shrubs and climate change, the habitat of F. unibracteata wild resources has been seriously damaged, indicating of great significance to predict its potential suitable habitat using MaxEnt model. The AUC values without human activities were 0.983 ± 0.013-0.988 ± 0.001, while it is 0.982 ± 0.015-0.989 ± 0.000 with human activities, justifying their applications for predicting the potential areas of F. unibracteata. Without human activities, there were 8.47 × 104 km2 of highly suitable habitats in northern Sichuan, southern Gansu and southeastern Qinghai. But the poorly, moderately and highly suitable areas of F. unibracteata have decreased to 33.8 × 104 km2, 9.66 × 104 km2 and 6.64 × 104 km2 due to human activities. Environmental variables affecting F. unibracteata distribution included the minimum temperature in the coldest month (-16.89--4.96 °C), annual precipitation (416.64-866.96 mm), temperature annual range (24.83-31.97 °C), elevation (2879.69-3981.82 m), human footprint (2.58-23.66) and mean UV-B of highest month (7381.92-8574.27 kJ/m2). In the 2050s and 2090s, human activities would significantly reduce the highly suitable habitats of F. unibracteata. Under SSP1-2.6, SSP2-4.5 and SSP5-8.5 scenarios, the centroid would move to the low latitude area from the current position first, and then to a high latitude area. Wild resources of F. unibracteata in China can be effectively conserved based on our results.

Assessment of Genetic Diversity and Genetic Structure of Saussurea medusa (Asteraceae), a "Sky Island" Plant in the Qinghai-Tibet Plateau, Using SRAP Markers.

Saussurea medusa Maxim. is a typical "sky island" species and one with the highest altitude distributions among flowering plants. The present study aimed at analyzing the genetic diversity and population structure of 300 S. medusa accessions collected from 20 populations in the Qilian Mountains in the northeastern Qinghai-Tibet Plateau (QTP), using sequence-related amplified polymorphism (SRAP) markers. A total of 14 SRAP primer combinations were employed to analyze genetic diversity and population structure across all accessions. Out of 511 amplified bands, 496 (97.06%) were polymorphic. The populations in the eastern Qilian Mountains had significantly higher genetic diversity than those in the central and western groups. Population structure analysis revealed greater genetic differentiation among populations with a Gst of 0.4926. UPGMA-based clustering classified the 300 S. medusa accessions into 3 major clusters, while the Bayesian STRUCTURE analysis categorized them into 2 groups. Correlation analyses showed that the genetic affinity of the populations was based on differences in geographical distance, moisture conditions, and photothermal conditions between the habitats. This study represents the first comprehensive genetic assessment of S. medusa and provides important genetic baseline data for the conservation of the species.

Culturable bacterial endophytes of Aconitum carmichaelii Debx. were diverse in phylogeny, plant growth promotion, and antifungal potential.

Medicinal plants harbor tremendously diverse bacterial endophytes that maintain plant growth and health. In the present study, a total of 124 culturable bacterial endophytes were isolated from healthy Aconitum carmichaelii Debx. plants. These strains were clustered into 10 genera based on full-length 16S rDNA sequences, among which Bacillus and Pseudomonas were the dominant genera. In addition, A. carmichaelii may capture 10 potential new bacterial species based on multi-locus sequence analysis of three housekeeping genes (gyrA, rpoB, and atpD). The majority of these bacterial endophytes exhibited plant growth-promoting ability through diverse actions including the production of either indole acetic acid and siderophore or hydrolytic enzymes (glucanase, cellulose, and protease) and solubilization of phosphate or potassium. A total of 20 strains inhibited hyphal growth of fungal pathogens Sclerotium rolfsii and Fusarium oxysporum in vitro on root slices of A. carmichaelii by the dual-culture method, among which Pseudomonas sp. SWUSTb-19 showed the best antagonistic activity. Field experiment confirmed that Pseudomonas sp. SWUSTb-19 significantly reduced the occurrence of southern blight and promoted plant biomass compared with non-inoculation treatment. The possible mode of actions for Pseudomonas sp. SWUSTb-19 to antagonize against S. rolfsii involved the production of glucanase, siderophore, lipopeptides, and antimicrobial volatile compounds. Altogether, this study revealed that A. carmichaelii harbored diverse plant growth-promoting bacterial endophytes, and Pseudomonas sp. SWUSTb-19 could be served as a potential biocontrol agent against southern blight.

Root endophytic bacterial community composition of Aconitum carmichaelii debx. from three main producing areas in China.

Aconitum carmichaelii Debx. is famous for the bioactive aconitum alkaloids as traditional Chinese medicine. Endophytic bacteria play vital roles in plant growth, health, and the production of secondary metabolites such as alkaloids. In this study, we employed 16 S rRNA amplicon high-throughput sequencing to determine the root endophytic bacterial community of A. carmichaelii Debx. collected from three main producing areas including the geo-authentic area in China, high performance liquid chromatography to measure the contents of six bioactive alkaloids and correlation analysis to explore the relationship among environmental factors, alkaloids contents, and endophytic bacterial community. The results indicated that the root core microbiota of A. carmichaelii Debx. was dominated by Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Root endophytic bacterial community in the geo-authentic area was distinct from the other two regions. Soil nitrogen contents, organic matter, and temperature were the main factors contributing to the endophytic bacterial community structure. Significant correlation was found between alkaloids contents and some bacterial genera. Particularly, the abundance of Lactobacillus was positively correlated with the contents of benzoyl-mesaconitine and benzoyl-aconine. This study provided the first insight into the root endophytic bacterial community composition of A. carmichaelii Debx., and can direct further isolation of functional bacterial strains.

Characterization of a Root-Knot Nematode Infecting Aconitum carmichaelii in Southwest China.

Growth of the Chinese herbal medicine industry has resulted in several new pests and diseases. China is one of the world largest producers of monkshood (Aconitum carmichaelii Debx.), but an unidentified root-knot nematode has become a significant pest in the southwestern provinces of Yunnan and Sichuan. Morphological characteristics and the ribosomal DNA-internal transcribed spacer and D2-D3 region of the 28S ribosomal RNA gene sequences were used to identify the nematode as Meloidogyne hapla. Through investigation, this is the first report of M. hapla infecting monkshood in Yunnan and Sichuan Provinces.

Biocontrol and plant growth promotion potential of endophytic Bacillus subtilis JY-7-2L on Aconitum carmichaelii Debx.

Aconitum carmichaelii Debx. is a famous medicinal plant rich in alkaloids and widely used to treat various human diseases in Asian countries. However, southern blight caused by Sclerotium rolfsii severely hampered the yield of A. carmichaelii. Beneficial microbe-based biological control is becoming a promising alternative and an environmentally friendly approach for the management of plant diseases. In this study, we evaluated the biocontrol potential of an endophytic bacterial strain JY-7-2L, which was isolated from the leaves of A. carmichaelii, against southern blight in vitro and by a series of field experiments. JY-7-2L was identified as Bacillus subtilis based on multi-locus sequence analysis. JY-7-2L showed strong antagonistic activity against S. rolfsii in vitro and on A. carmichaelii root slices by dual-culture assay. Cell-free culture filtrate of JY-7-2L significantly inhibited the hyphal growth, sclerotia formation, and germination of S. rolfsii. In addition, volatile compounds produced by JY-7-2L completely and directly inhibited the growth of S. rolfsii. Furthermore, JY-7-2L was proved to produce hydrolytic enzymes including glucanase, cellulase, protease, indole acetic acid, and siderophore. The presence of bacA, fenA, fenB, fenD, srfAA, and baeA genes by PCR amplification indicated that JY-7-2L was able to produce antifungal lipopeptides and polyketides. Field trials indicated that application of the JY-7-2L fermentation culture significantly reduced southern blight disease severity by up to 30% with a long-acting duration of up to 62 days. Meanwhile, JY-7-2L significantly promoted the fresh and dry weights of the stem, main root, and lateral roots of A. carmichaelii compared to non-inoculation and/or commercial B. subtilis product treatments. Taken together, JY-7-2L can be used as a promising biocontrol agent for the control of southern blight in A. carmichaelii.

First report of Meloidogyne arenaria infecting Maidong (Ophiopogon japonicus) in China.

Maidong (Ophiopogon japonicus) is a perennial evergreen plant of the Asparagaceae, occurring mainly in China, Japan, Vietnam, and India. It grows in the damp place on the hillside below 2000 meters above sea level, under the forest or beside the stream;It has been widely cultivated in the Sichuan ofhina for medicinal uses; and it is included in the Chinese Pharmacopoeia. During April 2019, Maidong plants exhibiting symptoms of stunting, leaf wilting, and multiple galls in the roots associated with root-knot nematode (Meloidogyne sp.) were detected in a commercial field in near the city of Mianyang (N105°42', E30°93'), Sichuan, China. The second-stage juveniles (J2) were collected from the soil in the root zone, and adult females were dissected from roots. Population densities of J2 ranged from 190 to 255 per 100 cm3. Subsequently, individual females (n=20) were extracted from root samples and submitted to Meloidogyne species identification by perineal pattern morphological analysis (n=20), and morphometric measurements of second stage juveniles (J2) (n = 20). The J2 showed the following morphometric characters：body length = 475.5 ± 24.2 µm, tail length = 55.2 ± 6.43µm, stylet length = 12.4 ± 1.56 µm and distance from dorsal esophageal gland opening to the stylet knot (DGO) = 2.97 ± 0.44 µm; perineal patterns of females showed a low dorsal arch, with lateral field marked by forked and broken striae, no punctate markings between anus and tail terminus were observed. These morphological characteristics are consistent with Meloidogyne arenaria (Neves et al. 2016). In addition, to confirm species identification, DNA was extracted from females (Blok, et al. 1997) and D2/D3 fragments of the 28S rRNA was amplified using the universal primers D2A/D3B. The DNA fragment obtained showed a 754 bp length (GenBank accession no. MW965614) that was sequenced and analyzed, sequences were 99.8% identical to the MH359158, KX151138 and EU364889 M. arenaria sequences. Furthermore, species-specific SCAR primers Far/Rar were used as described by Zijlstra et al. 2000. The PCR produced approximately 420 bp sequences, which was identical to that previously reported for M. arenaria (Zijlstra et al. 2000). Morphological and molecular characterization supports the identification of the isolate found on Ophiopogon japonicus as M. arenaria. To verify the nematode pathogenicity on Maidong plants, Maidong seed were planted in 20-cm diameter, 10-cm deep plastic pots containing 1000 cm3 sterilized soil and infested with 2000 M. arenaria J2 per seedling, using a sterilized micropipette. Plants were maintained at 20-25°C in a greenhouse. Control plants received sterile water, and the pathogenicity test was repeated three times. After 60 days, all inoculated plants showed reduced growth compared with control. The symptoms were similar to those observed in the field, a large number of galls (38.5 ± 2.4) and egg masses (18.5 ± 0.2) were found on each root system. Maidong was considered a good host for M. arenaria in Mianyang. M. arenaria is one of the most important plant parasitic nematode with a wide geographic distribution and causes great losses in many crops around the world (Perry et al. 2009). Through investigation, this is the first report worldwide of M. arenaria infecting Ophiopogon japonicus.

[Study on wavelength overlapping HPLC fingerprint and multi-component content determination of Danshen].

This project was launched to and establish the wavelength overlapping HPLC fingerprint for Danshen and determine the contents of 7 component in Danshen. The chromatographic fingerprints were built by using Agilent Eclipse Plus C_(18)( 4. 6 mm×100 mm,3. 5 µm) as stationary phase and 0. 1% formic acid solution( A)-acetonitrile( B) as mobile phase with gradient elution( 0-5 min,10%-20% B; 5-20 min,20%-30% B; 20-25 min,30%-50% B; 25-40 min,50%-65% B; 40-45 min,65%-80% B; 45-46 min,80%-10% B; 46-50 min,10% B) at a flow rate of 1 mL·min~(-1). The column temperature was maintained at 30 ℃ and the detection wavelength was set at 250,280,310 and 340 nm. The technique of wavelength overlapping fingerprint was established and the contents of 7 indicative compounds have been determined in this method. The results of wavelength overlapping HPLC fingerprint showed all-around information of the fingerprints at 250,280,310 and 340 nm,and the similarity among 17 batches of Danshen was over 0. 828-0. 936. In wavelength overlapping HPLC fingerprint,15 common peaks were selected as the common peaks,and 7 contents of them were indentified as rosmarinic acid,salvianolic acid B,salvianolic acid A,phenolic acid,dihydrotanshinone Ⅰ,tanshinone Ⅰ and tanshinone ⅡA. The results of methodological study demonstrated that the method met the requirements of the determination. The method established in this study is simple,convenient and durable,which can provide a scientific basis for the quality control of Danshen.

Transcriptome analysis of maize leaf systemic symptom infected by Bipolaris zeicola.

Bipolaris zeicola is a fungal pathogen that causes Northern corn leaf spot (NCLS), which is a serious foliar disease in maize and one of the most significant pathogens affecting global food security. Here, we report a genome-wide transcriptional profile analysis using next-generation sequencing (NGS) of maize leaf development after inoculation with B. zeicola. We performed High-Throughput Digital Gene Expression analysis to identify differentially expressed genes (DEGs) in resistant inbred Mo17 lines after infection with B. zeicola at four successive disease development stages--CP (contact period), PP (penetration period), IP (incubation period), and DP (disease period); the expression of the genes was compared with those in a CK (mock-treatment) control. In addition, a sensitive maize line (Zheng58) was used for the comparisons with the Mo17. Among all tested genes, 466 differentially expressed genes were identified in all libraries, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these genes suggested that they are involved in many biological processes related to systemic symptom development, such as plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis and photosynthesis. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in fungal-infected plants. This information will help in furthering our understanding of the detailed mechanisms of plant responses to fungal infection.