Sensitivity baseline establishment and resistance risk assessment of Botrytis cinerea from Panax ginseng to prochloraz / 中国中药杂志

This study aimed to establish the baseline sensitivity of Botrytis cinerea from Panax ginseng to prochloraz, and ensure the fitness of prochloraz-resistant mutants and the cross-resistance of B. cinerea to prochloraz and commonly used fungicides for the prevention and control of gray mold including boscalid, pyraclostrobin, iprodione, and pyrimethanil. The sensitivity of B. cinerea from P. ginseng to fungicides was determined by the mycelial growth rate method. The prochloraz-resistant mutants were screened out through fungicide domestication and ultraviolet(UV) induction. The fitness of resistant mutants was determined through the stability of subculture, mycelial growth rate, and pathogenicity test. The cross-resistance between prochloraz and the four fungicides was determined by Person correlation analysis. The results showed that all B. cinerea strains tested were sensitive to prochloraz, and the EC_(50) value ranged from 0.004 8 to 0.062 9 μg·mL~(-1), with an average of 0.022 μg·mL~(-1). The sensitivity frequency distribution diagram showed that 89 B. cinerea strains were located within the main peak with a continuous single peak curve, and the average EC_(50) value of 0.018 μg·mL~(-1) was taken as the baseline sensitivity of B. cinerea to prochloraz. The fungicide domestication and UV induction obtained 6 resistant mutants, among which 2 strains were unstable and the other 2 strains showed decreased resistance after multiple generations of culture. Furthermore, the mycelial growth rate and spore yield of all resistant mutants were lower than those of their parents, and the pathogenicity of most mutants was lower than that of their parents. In addition, prochloraz had no obvious cross-resistance with boscalid, pyraclostrobin, iprodione, and pyrimethanil. In conclusion, prochloraz has great potential for controlling gray mold in P. ginseng, and the resistance risk of B. cinerea to prochloraz is low.

Isolation and identification of pathogen causing damping off at seedling stage of Trollius chinensis / 中国中药杂志

Trollius chinensis is a traditional Chinese medicinal material in China, the wild resource of T. chinensis are now exhausted, and commercial medicinal T. chinensis mainly depends on artificial cultivation. As one of the most severely happened diseases at the seedling period, damping off has been a serious threaten to the breeding of T. chinensis seedlings. However, no related research have been reported so far. So, the authors collected damping-off samples of T. chinensis in 2018 from seedling breeding nursery in Guyuan, Hebei province, and carried out study on taxonomic identification of the pathogen. Damping off occurs in the T. chinensis production area from mid-May to late June every year. At the beginning, brown lesions were observed on the basal stem, then the lesions circumferential expanded and constricted, and finally resulted in the fall and death of T. chinensis seedlings. Pathogenic isolate was growing rapidly on the PDA medium, well developed aerial mycelia were grey white at first, then turned brown gradually, and a great number of small dark brown sclerotia were developed in the middle and periphery of the colony. Mycelial diameter of the pathogen was about 7 to 10 μm, near right angle or acute angle branches, near branches with septa, branches and septa with constriction. After the healthy T. chinensis seedlings were inoculated by pathogenic isolate, damping-off was observed soon, and the symptom was as same as those observed in the field. Through homogenous blast, the rDNA-ITS sequence of the pathogenic isolate shown 99.49% to 99.84% homology with Rhizoctonia solani, R. solani AG-1 IC mycelium anastomosis group and Thanatephorus cucumeris, the sexual type of Rhizoctonia. Furthermore, obvious mycelial anastomosis phenomena were observed when the pathogenic isolate and R. solani AG-1 IC strain were confronting cultured. Based on the results above, the pathogenic isolate causing damping off of T. chinensis was identified as R. solani AG-1 IC mycelial anastomosis group. RESULTS:: in the present work have important significance for further research on basic biology of the pathogen and integrated control of damping off causing by it on T. chinensis.

Isolation and identification of a new phytopathogen causing root rot of Rehmannia glutinosa / 中国中药杂志

Root rot was occurred widely in the production area of Rehmannia glutinosa, and which result in serious influence on the yield and quality of R. glutinosa. In the present work, a new phytopathogen was isolated from roots with root rot symptom in the production area of R. glutinosa. The colony of the pathogen growing on PDA medium was gray-black, the structure of hyphae was compact, the aerial hyphae was less developed, and the back of the colony was black. The hyphae of the pathogen were uneven in size, about 2 to 3 μm in diameter and twined with each other, the conidia of the pathogen were small, nearly round and about 1 μm in diameter. The healthy roots of R. glutinosa were inoculated with the pathogen in vitro, black-brown rot was observed at the inoculate sites after a few days' incubation. The rhizosphere soil of healthy R. glutinosa seedlings were inoculated in vivo, the leaves were wilted and the roots were black-brown rotted after several days' normal culture, the symptoms were consistent with those observed in the field. The genomic DNA of the pathogen was amplified by fungus rDNA-ITS universal primer ITS1/ITS4 and homologous analyzed, the pathogen was in a branch with Heterophoma sp., Phoma sp., P. novae-verbascicola and P. herbarum with the nuclear acid homology of 99.21% to 99.43%. The pathogen shown 97.00% to 98.02% nuclear acid homology with H. verbascicola, H. novae-verbascicola, H. poolensis, P. herbarum, H. sylvatica, H. verbascicola and H. verbasci-densiflori when amplified by the tub2 gene special primer Btub2 fd/Btub4 rd, and H. novae-verbascicola was the highest. The pathogen was in a branch with H. novae-verbascicola when amplified by the lsu gene special primer LR0 R/LR7. Based on the morphological characteristics, nucleotide sequence analysis and Koch's test results, the isolated pathogen causing root rot of R. glutinosa was identified as H. novae-verbascicola. This study is of great significance for the further theoretical research on root rot of R. glutinosa and root rot control in field.

Molecular identification of cucumber mosaic virus in woad (Isatis tinctoria) with mosaic disease in Beijing / 中国中药杂志

To detect possible pathogenic virus(es) in woad (Isatis tinctoria) cultivated at Institute of Medicinal Plant Development in Beijing, reverse transcription(RT)-PCR was performed using total RNA of symptomatic woad leaves with primers for poty-, polero-, tobamovirus, broad bean wilt virus 2(BBWV2) and cucumber mosaic virus (CMV). A 657 bp fragment was amplified from symptomatic woad using CMV primers. Sequencing and BLAST analysis indicated that this fragment shared 99% nucleotide identity and 100% amino acid identity with CMV-Vi isolate. The isolate was named CMV-Isatis tinctorial (CMV-It). Phylogenetic analysis based on nucleotide sequences of CP genes showed that CMV-It clustered with CMV-K and belonged to subgroup I. To our knowledge, this is first identification of CMV in woad by RT-PCR and the CP gene was analyzed. This work provided data for research and control of woad mosaic disease.

Gene cloning and expression analysis of benzoic acid stress responsive transcription factor WRKY7 in Panax ginseng / 中国中药杂志

WRKY transcription factor is one of the most important transcription factor families widely existing in higher plants, which playing critical role in plant morphogenesis, development, biotic (including phytopathogens, pests etc.) and abiotic (drought, salt, chilling, high temperature, etc.) stress. In the present work, primers used to amplify full-length gene encoding WRKY transcription factor were designed based on the transcriptome data of P. ginseng that induced by benzoic acid, one of the most important autotoxins identified from root exudates and rhizosphere soil of P. ginseng. Then, a WRKY gene, temporarily named as WRKY7, was confirmed by RT-RCR. Furthermore, sequencing and sequence analysis of WRKY7 was conducted. Results indicated that, the full length cDNA of WRKY7 was 1 216 bp, the open reading frame (ORF) of which was 1 014 bp, encodes 337 amino acids. Homologous analysis and phylogenetic tree showed that, WRKY7 belonged to the Ⅲ category of WRKY families, which showing 87% similarity to WRKY6 in P. quinquefolius. Real-time PCR results showed that the expression of WRKY7 in P. ginseng induced by benzoic acid was up-regulated markedly than the control, so we speculated that WRKY7 was involved in the response to benzoic acid stress, which will be helpful for further research on the molecular mechanism of ginseng plant response to benzoic acid stress.

Study on standard of safe application of thiamethoxam on GAP of Lonicera japonica / 中国中药杂志

The paper is aimed to establish a method of residue analysis for thiamethoxam and to study its degradation dynamic and final residue and its standard of safe application of thiamethoxam on Lonicera japonica. Samples extracted with methanol by ultrasonication were purified with dichloromethane by liquid-liquid extraction and SPE column and analysed by HPLC-UV. The results showed that average rate was 84.91%-94.44% and RSD 1.74%-4.96% with addition of thiamethoxam in respectively diverse concentration, which meets inspection requirement of pesticide residue. Two kinds of dosages of thiamethoxam were treated- varying from recommended dosage (90 g x hm(-2)) to high dosage (135 g x hm(-2)), Results of two years test showed that thiamethoxam was degraded more than 90% seven days after application and the half - life period of thiamethoxam was 1.54-1.66 d. The digestion rate of thiamethoxam was fast in the L. japonica. The recommended MRL of thiamethoxam in the L. japonica is 0.1 mg x kg(-1), the dosage of 25% thiamethoxam WDG from 90-135 g x hm(-2) is sprayed less than three times a year on L. japonica and 14 days is proposed for the safety interval of the last pesticide application's and harvest's date.

Identification of bacterial strain ge15 and its controlling effect on ginseng diseases / 中国中药杂志

Based on previous results of 16S rDNA sequence homologuous and results of physic-biochemical indexes and morphological characteristics in the present work, bacterial strain ge15 isolated from roots of ginseng plants was identified as Stenotrophomonas rhizophila. Confronting incubation results showed that, strain ge15 inhibited the growth of Alternaria panax, Phytophthora cactorum, and Cylindrocapon destructans significantly, and the width of inhibition zone was 13.3, 24.0, 12.0 mm, respectively. Further results showed that the emergence rate and seedling survive rate of ge15 treatment was significantly higher than those of the control, and which was similar to pesticide carbendazol treatment. The ge15 strain has good application potential in ginseng diseases control without contamination.

Mitigative effect of micribial degradation on autotoxicity of Panax ginseng / 中国中药杂志

Continuously cropping obstacle restricts ginseng production and rational use of land resource severely, and autotoxicity is one of the most important factors. In our previous work, ginseng autotoxin degrading bacteria were isolated, in the present re- search, plate culturing method and traditional physiological and biochemical method were used to analyze biological indices and protective enzyme activities, in order to elucidate the mitigative effect of autotoxin degrading bacteria on autotoxicity of P. ginseng. Results indicated that, except for palmitic acid, autotoxicity of benzonic acid, diisobutyl phthalate, diisobutyl succinate, and 2,2-bis (4- hydroxyphenyl) propane on the growth of ginseng seeds was significantly alleviated after autotoxins degrading bacteria was inoculated, and which have no evident difference with control. Except for benzoic acid, enzyme activity of SOD, POD and CAT in other autotoxin degrading treatments decreased significantly. The present research showed that, microbial degradation could alleviate the autotoxicity of autotoxins on ginseng seeds effectively, and which will be helpful for the resolution of ginseng continuously cropping obstacle problem.

Fermentation of Bacillus subtilis ge25 strain and preliminary study on its antagonistic substances / 中国中药杂志

Panax ginseng is one of the most important traditional Chinese herbal medicine, soil borne diseases influenced the yield and quality severely. In our previous work, endophytic Bacillus subtilis ge25 strain was isolated from ginseng root, and which showed significant antagonistic activity against several most destructive ginseng phytopathogens. In the present work, crude protein and lipopeptid extracts were prepared from LB and Landy supernate by salting out, acid precipitation methods respectively. The antagonistic activity of crude extracts and stability to temperature and protease digestion were examined by ginseng phytopathogen Alternaria panax. Results showed that, the antagonistic activity of crude protein extracts from LB culture was complete and partially lost when treated by high temperature and proteinase K. However, crude lipopeptid from Landy culture showed significant stabile antagonistic activity to them. Acid-hydrolyzation and TLC-bioautography analysis showed, that the crude lipopeptide contained at least one cyclic lipopeptide. In consideration of the stability and perfect antagonistic activity of ge25, further researches will promote the biocontrol of ginseng diseases in the field.

Isolation and characteristics of Panax ginseng autotoxin-degrading bacterial strains / 中国中药杂志

In this study, traditional plate culturing method was used to isolate autotoxin-degrading microbial strains, and which were then identified by 16S rDNA homological analysis and morphological characteristics. Furthermore, the growth and autotoxin-degrading efficiency of them were analyzed by liquid culturing method and GC-MS to illustrate their autotoxin-degradation characteristics. As a result, five bacterial strains having autotoxin-degrading activity were isolated from 6-years ginseng nonrhizospheric soil successfully, and which can growth successfully by taking autotoxins added artificially as carbon source in liquid culturing condition. Results indicated that it was feasible to isolate autotoxin-degrading bacteria from ginseng nonrhizospheric soil, and the isolated bacterial strains can be used to degrade autotoxins in soils once planted Panax ginseng.

Screening strains for Trichoderma spp. for strong antagonism against ginseng root pathogens and study on their biological characters / 中国中药杂志

<p><b>OBJECTIVE</b>To screen the Trichodenna spp. for strong antagonist against ginseng root pathogens.</p><p><b>METHOD</b>The biological characters of ten Trichoderma strains were compared by culturing on different media. And their antagonistic activity against Phytophthora cactorum, Cylindrocarpon destructans and Rhizoctonia solani were measured on PDA.</p><p><b>RESULT AND CONCLUSION</b>Tv04-2 and Th3080 showed a good growth on soil solution medium and PDA, and also showed high inhibitory efficacy to the three pathogens. The two Trichoderma strains showed different growth rate under light conditions and pH. Trichoderma strains were sensitive to most fungicides used in ginseng root disease controlling, however Tv04-2 was not sensitive to the fungicide Junchong Jueba.</p>

Progress on the control of medicinal plants soil-borne disease by anti-microorganism / 中国中药杂志

Much success had been achieved in the following aspect: the filtration of anti-microorganism, the action mechanisms, the inhibitory action in the field and so on. Though the narrow object and the unstable effect really exist now, it still has a broad developing future for it's advantage in keeping ecological balance and in fitting the requirement of GAP.

Applied studies of biological control techniques on healthful Chinese crude drugs / 中国中药杂志

Medicinal plants always suffer from diseases and pests in their cultivation and growing, and the yield and quality are reduced. At present, chemical pesticide is mainly used to control the diseases and pests. The long-term use and over-use of pesticide result in pesticide residue, pests resistance to pesticide, water and soil pollution and other environmental contamination. It is necrssary to provide the integrated pests management, including new technology of biological control for developing green drug. This project makes research on the following aspects: 1. Applied studies on bethyloid wasp (Scleroderma guani) control for stem borer; 2. To develop Trichoderma spp. and use it to control the diseases of medicinal plants; 3. Applying Agro-antibiotics 120 to control phytophthora root rot of ginseng; 4. Researches on application of entomopathogenic nematodes against ten-spotted lema (Lema decempuntata) et al.

Biological effect of seed-coating in Carthamus tinctorins / 中国中药杂志

<p><b>OBJECTIVE</b>To study the Biological effect of seed-coating in Carthamus tinctorins.</p><p><b>METHOD</b>Two kinds of seedcoating chemicals SCF1 and SCF2 were used in this experiment, the seed YM-99 and 27981-99 were coated by three kinds of ratio of seedcoating chemicals to seed. It was investigated that the germination energy and germination percentage in the room and the emergence rate, seedling stage growing, pest in the field.</p><p><b>RESULT</b>Seedoating can improve the emergence rate and seedling stage growing, it also can effectively control aphid, rust and virosis during the growing period in C. tinctorins.</p><p><b>CONCLUSION</b>Seedcoating has significant biological effect in C. tinctorins.</p>

Presearch on preventing the medicinal plant diseases with Trichoderma harzianum preparation / 中国中药杂志

<p><b>OBJECTIVE</b>To control the medicinal plant diseases with the preparation of Trichoderma harzianum.</p><p><b>METHOD</b>Antagonistic action of the preparation to the pathogens of the medicinal plants in vitro, and controling effects of the preparation on these diseases in greenhouse and in the field were tested.</p><p><b>RESULT</b>The test in vitro showed that Trichoderma harzianum, used as a biocontrol factor, had stronger antagonistic action to Fusarium equiseti, Sclerotinia sp. and Rhizoctonia solani which were the medicinal plant pathogens of Astragalus membranaceus, Glehnia littoralis and Panax quinquefolium respectively. Biological controling effects on sclerotium root rot of Glehnia littoralis were 83.6% and 72.5% respectively in greenhouse and in the field with the preparation of Trichoderma harzianum. And controling effects on root rot of Astragalus membranaceus and seedling damping-off of Panax quinquefolium were 80% and 60% respectively in the field. The dosage of the preparation used in the field was 10 g.m-2.</p><p><b>CONCLUSION</b>The preparation of Trichoderma harzianum can be used as a substitute for such chemicals as Carbendazim. Using the preparation to control medicinal plant diseases provides a technical safeguard for the good agricultural practice of medicinal plants.</p>

Study on the pollination characteristic of Cistanche deserticola / 中国中药杂志

<p><b>OBJECTIVE</b>To study the Pollination Characteristic of C. deserticola.</p><p><b>METHOD</b>Four pollination methods were used, cross pollination coming different plant, cross pollination in one plant, wind pollination and no-pollination.</p><p><b>RESULT</b>Cross pollinations coming different plant are fructiferous. The average fructication rate of cross pollination coming different plant is 94.1%; the average fructication rate of cross pollination in one plant is 52.2%; The seed qulitity of cross pollination coming different plant is better than the cross pollination in one plant.</p><p><b>CONCLUSION</b>Cross pollinations coming different plant have a high quantity and quality of seeds of C. deserticola.</p>