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.
Pinellia/genetics* ; Fungicides, Industrial/pharmacology* ; Seedlings ; Bacillus subtilis ; Mycelium

The evaluation of germplasm resources is the prerequisite for the development, utilization, and conservation of Chinese medicinal resources. The selection of excellent germplasm is the key to the breeding and orderly production of Pinellia ternata. In this study, 21 germplasm materials of P. ternata from major production areas in China were collected and analyzed for population diversity after phenotypic preliminary screening. The results have revealed that the P. ternata population has abundant phenotypic variation, and the phenotypic changes could be divided into five phenotypes in terms of organ trait variation. Further analysis of variation in 20 quantitative traits of the population revealed that the coefficient of variation for adenosine content(339.05%) was the largest, while the coefficient of variation for the underground plant height(16.35%) was the smallest. Correlation analysis showed that there was a strong correlation among various traits, with 52 pairs of traits showing highly significant correlation(P<0.01) and 19 pairs of traits showing a significant correlation(P<0.05). The 21 germplasms in the test could be classified into three major clusters by cluster analysis, with Cluster Ⅱ having the highest number and content of nucleosides, making it suitable for the selection and breeding of P. ternata varieties with high content of nucleosides. The yield in Cluster Ⅲ was higher than that in other groups, making it suitable for the selection and breeding of P. ternata varieties with a high yield. All trait indicators could be simplified into five principal component factors through principal component analysis, and the cumulative contribution rate was up to 86.04%. Further, comprehensive analysis using membership function and stepwise regression analysis identified nine traits, such as plant height, main leaf length, and underground plant height as characteristic indicators for the comprehensive evaluation of germplasm resources of P. ternata. BX007, BX008, and BX005 were identified as germplasms with both high yield and high uridine content, with BX007 having the highest uridine content of 479.51 μg·g~(-1). It belonged to the germplasm of P. ternata with double bulbils and could be cultivated as a potential good variety. Based on the phenotypic classification of P. ternata, systematic resource evaluation was carried out in this study, which could lay a foundation for the excavation of genetic resources and the breeding of new varieties of P. ternata.
Plants, Medicinal ; Pinellia/genetics* ; Plant Breeding ; Phenotype ; Uridine

Pinellia ternata is an important medicinal plant, and its growth and development are easily threatened by high temperature. In this study, comprehensive research on physiological, cytological and transcriptional responses to different levels of heat stress were conducted on a typical phenotype of P. ternata. First, P. ternata exhibited tolerance to the increased temperature, which was supported by normal growing leaves, as well as decreased and sustained photosynthetic parameters. Severe stress aggravated the damages, and P. ternata displayed an obvious leaf senescence phenotype, with significantly increased SOD and POD activities (46% and 213%). In addition, mesophyll cells were seriously damaged, chloroplast thylakoid was fuzzy, grana lamellae and stroma lamellae were obviously broken, and grana thylakoids were stacked, resulting in a dramatically declined photosynthetic rate (74.6%). Moreover, a total of 16 808 genes were significantly differential expressed during this process, most of which were involved in photosynthesis, transmembrane transporter activity and plastid metabolism. The number of differentially expressed transcription factors in MYB and bHLH families was the largest, indicating that these genes might participate in heat stress response in P. ternata. These findings provide insight into the response to high temperature and facilitate the standardized cultivation of P. ternata.
Pinellia/genetics* ; Heat-Shock Response/genetics* ; Photosynthesis/genetics* ; Plants, Medicinal/genetics* ; Phenotype

OBJECTIVEIn order to clarify the genetic background of Pinellia ternata germplasm resources in China, the chromosomal constitution and cytogeographical distribution of P. ternata were investigated in 27 different populations among 16 provinces and regions in China systematically.

METHODCytological and cytogeographical methods were used in the study.

RESULTP. ternata in China is a polyploid complex, which contains septuploid (2n = 7x = 91) , octoploid (2n = 8x = 104) , nonuploid (2n = 9x = 117) and decaploid (2n = 10x = 130). Meanwhile the aneuploid series (2n = 92, 103, 105, 115) of a minority of P. ternata were also found.

CONCLUSIONThe genetic differentiation and the phenomenon of ploidy miscellany commonly exist in the species of P. ternata in China, both for natural populations and cultivated populations. Toxicity and chemical components of different ploidy P. ternata should be clarified before the superior multiploid is selected for normalized plantation of the plant.

Aneuploidy ; China ; Chromosomes, Plant ; genetics ; Ecosystem ; Genetic Variation ; Pinellia ; genetics ; Plants, Medicinal ; genetics ; Polyploidy

OBJECTIVETo study the genetic diversity of pollen grains morphologies of Pinellia ternata in different populations and supply palynological evidences for further research.

METHODPollen exine ultrastructure were compared for 18 populations growing under in the same environment by means of scanning electron microscope(SEM).

RESULT AND CONCLUSIONBig difference was found in pollen size among populations of P. ternata: the mean value of seventeen populations of P. ternata with bulbils was 17.42 microm x 14.86 microm, the biggest of populations originated from Jangyan city is 20.43 micromx 18.16 microm, but the least originated from Peixian County is only 15.23 microm x 12.82 microm; the size of populations without bulbils originated from Linguo Temple side was only 13.23 microm x 11.98 microm and far less than of all populations with bulbils. The pollen surface of all populations sticks out with spines, varying in size and density, the base and top part of spinules was different among populations. The pollen grains patterns of germination apertures of P. ternata was likely to have colpate cover.

China ; Genetic Variation ; Pinellia ; genetics ; ultrastructure ; Plants, Medicinal ; genetics ; ultrastructure ; Pollen ; genetics ; ultrastructure

According to the data of Pinellia ternate transcriptome,two calmodulin genes were cloned and named as Pt Ca M1 and PtCa M2 respectively. The results of bioinformatics analysis showed that Pt Ca Ms genes contained a 450 bp open reading frame,encoding149 amino acids.The identity of the coding sequences was 80%,and the identity of amino acids sequence was 91%. Pt Ca Ms genes contained EF-hand structure domain,belonging to the Ca M families. The Real-time PCR analysed the expression patterns of Pt Ca Ms in different tissues and different treatments. RESULTS:: showed that Pt Ca M1 and Pt Ca M2 gene were the highest expression level in tuber. Under Ca Cl2 treatment,the expressions of Pt Ca Ms were significantly higher than the control. Under EGTA,La Cl3 and TFP treatments,the expression level of Pt Ca Ms decreased gradually. In this study,the Pt Ca Ms gene were successfully cloned from P. ternate,which laid a foundation for the functional characteristic of Pt Ca Ms gene and the synthesis of alkaloids from P. ternata for further study.
Calmodulin ; genetics ; Cloning, Molecular ; Genes, Plant ; Pinellia ; genetics ; Plant Tubers ; genetics

OBJECTIVETo develop microsatellite primers with the method of isolation of genomic microsatellite in Pinellia ternata by magnesphere.

METHODBy taking advantage of the high binding affinity of biotin to streptavidin, microsatellite probe of the 5' end biotin was combined with magnesphere paramagnetic particles, and then combinations were hybridized with the digested P. ternata DNA fragments in which both ends of them connected with special adaptor, the other DNA fragments were eluted out. The microsatellite library was established. The crossed fragments were used as the template to conduct PCR amplification with the adapter sequences as primers, the products were cloned directly, subsequently screened by bacterium liquid PCR, and DNA sequencing was carried out.

RESULTFifteen microsatellites of P. ternata were obtained, development efficiency was 93.75%.

CONCLUSIONThe result demonstrates that magnesphere is a fast and efficient method to develop microsatellite.

DNA, Plant ; genetics ; isolation & purification ; Genetic Techniques ; Genome, Plant ; Magnetics ; Microsatellite Repeats ; Pinellia ; genetics

Pinellia ternata belongs to the Araceae family and is a medicinal herb. The tuber is the medicinal organ with antitussive, antiemetic and anti-tumor activities. It is easy to encounter high temperature environment during the growth periods, leading to decrease of tuber production. At present, the mechanism of response to high temperature stress in P. ternata is still unknown. DNA methylation plays a vital role in plant protection against adversity stress as a way of epigenetic regulation. In this study, P. ternata was used as material for treatment of high temperature stress at 0 h, 6 h and 80 h, and methylation sensitive amplification polymorphism(MSAP) analysis was conducted on the changes of DNA methylation in its genome. The results showed that 20 pairs of MSAP primers were selected from 100 MSAP primers with multiple clear and uniform bands, and 353, 355 and 342 loci were amplified from materials of P. ternata treated in the high temperature stress 0 h, 6 h and 80 h, respectively. Cytosine methylation levels of CCGG context in the above materials were characterized as 60.91%, 44.79% and 44.74%, respectively. And the full methylation ratios were 16.71%, 22.25% and 29.24, respectively. It demonstrated that high temperature stress significantly induced the down-regulation of DNA methylation level and up-regulation of the full methylation rate in P. ternata genome. This study provides a preliminary theoretical reference for analyzing the mechanism of P. ternata responding to high temperature stress from the epigenetic perspective.
DNA Methylation ; Epigenesis, Genetic ; Hot Temperature ; Pinellia/genetics* ; Plants, Medicinal/genetics*

OBJECTIVETo extract RNA from Pinellia ternata and lay a foundation for studying the formation mechanism of P. ternata.

METHODBy modifying the method recommended by Guanidinium for extracting total RNA from plant tissues rich in phenolic and polysaccharidic compounds, a simple and convenient method for extraction of total RNA from the tubers, stems and leaves of P. ternate containing abundant polyphenols and polysaccharides was established. High concentrated p-mercaptoethanol was added in the RNA extracted buffer to remove polyphenols, phenol and chloroform were used to eliminate proteins, and isopropanol and sodium acetate were used to precipitate polysaccharides.

RESULTThe A260/A230 value of RNA extracted with improved method were all over 2.0 and the values of A260/A280 were between 1.7 and 2.0. The electrophoresis bands were cleared on agarosegel and integrity of RNA was good.

CONCLUSIONThe results showed that RNA obtained from the tubers, stems and leaves of P. ternate with this method had high purity and quality and could be used in molecular biological research, as DDRT-PCR and reverse Northern blotting analysis directly. This method is simple, economic, stable performance, and has a good repeatability as well as is suitable for extracting total RNA of medicinal plants with high concentrations of phenolics and polysaccharides.

Blotting, Northern ; Pinellia ; genetics ; Plant Leaves ; genetics ; Plant Stems ; genetics ; Plant Tubers ; genetics ; Polymerase Chain Reaction ; RNA, Plant ; isolation & purification

OBJECTIVETo assess population genetic diversity of Pinellia ternata with different phenotype and from different habitat by sequence-related amplified polymorphism (SRAP) technique.

METHODFourteen appropriate primer pairs were selected out from a total of 80 pairs for SRAP PCR amplification. A Jaccard's genetic similarity matrix and a dendrogram were established using SPSS 16.0 software.

RESULTThe 14 primer pairs could amplify 171 bands, and the percentage of polymorphic bands was 15.8%. Based on the dendrogram, P. ternata in the same habitat clustered in a clade.

CONCLUSIONThese results suggested that SRAP markers could be used as an effective molecular technique for the diversity study of P. ternata and the habitat was more important than the phenotype in identification of P. ternata germplasm resource.

DNA Fingerprinting ; Ecosystem ; Genetic Variation ; Geography ; Phylogeny ; Pinellia ; classification ; genetics ; Polymerase Chain Reaction