Secretive Expression of Insect Antifungal Peptide-Encoded Genes in Pichia pastoris and Activity Assay of the Products

The antifungal peptides, drosomycin （Drs） and its isoform drosomycin-like C （Drs-lC） from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.     

苍耳提取液与植物内生真菌抗菌活性研究(英文)

[Objective] The study was aimed to provide theoretical foundation for developing a new fungicide with high activity,low toxicity and low residua.[Method] Based on the plant growth rate and inhibition zone methods,the antifungal activity of extracts of every organ of Xanthium sibiricum,the fermentation liquid of endophytic fungi MC4-23,and the extract of mycelium were tested with six kinds of pathogenic fungi and three kinds of pathogenic bacteria respectively.[Result] The extract of every organ of Xanthium sibiricum,the fermentation liquid of endophyic fungi MC4-23,and the extract of mycelium all had antifungal activities.The acetone extracts of Xanthium sibiricum had stronger antifungal activities than the ethanol extracts of Xanthium sibiricum.The extracts of leaf have the strongest antifungal activities against fungi and bacteria.The inhibition rates of the six pathogenic fungi were all above 47.8%,and the inhibition diameters of the three pathogenic bacteria were all above 0.5 centimeter.The fermentation liquid of endophytic fungi also had strong activities against the fungi and bacteria.The inhibition rates of the six pathogenic fungi were all above 68.1%.The extracts of root of Xanthium sibiricum and the mycelium of endophytic fungi were all had antifungal activities.[Conclusion] Xanthium sibiricum and endophyic fungi MC4-23 had great value of development and utilization.     

Preliminary Studies on Antifungal Activity of Xanthium sibiricum and the Endophytic Fungi

[Objective] The study was aimed to provide theoretical foundation for developing a new fungicide with high activity,low toxicity and low residua.[Method] Based on the plant growth rate and inhibition zone methods,the antifungal activity of extracts of every organ of Xanthium sibiricum,the fermentation liquid of endophytic fungi MC4-23,and the extract of mycelium were tested with six kinds of pathogenic fungi and three kinds of pathogenic bacteria respectively.[Result] The extract of every organ of Xanthium sibiricum,the fermentation liquid of endophyic fungi MC4-23,and the extract of mycelium all had antifungal activities.The acetone extracts of Xanthium sibiricum had stronger antifungal activities than the ethanol extracts of Xanthium sibiricum.The extracts of leaf have the strongest antifungal activities against fungi and bacteria.The inhibition rates of the six pathogenic fungi were all above 47.8%,and the inhibition diameters of the three pathogenic bacteria were all above 0.5 centimeter.The fermentation liquid of endophytic fungi also had strong activities against the fungi and bacteria.The inhibition rates of the six pathogenic fungi were all above 68.1%.The extracts of root of Xanthium sibiricum and the mycelium of endophytic fungi were all had antifungal activities.[Conclusion] Xanthium sibiricum and endophyic fungi MC4-23 had great value of development and utilization.     

苍耳提取液与植物内生真菌抗菌活性研究(英文)

[Objective] The study was aimed to provide theoretical foundation for developing a new fungicide with high activity,low toxicity and low residua.[Method] Based on the plant growth rate and inhibition zone methods,the antifungal activity of extracts of every organ of Xanthium sibiricum,the fermentation liquid of endophytic fungi MC4-23,and the extract of mycelium were tested with six kinds of pathogenic fungi and three kinds of pathogenic bacteria respectively.[Result] The extract of every organ of Xanthium sibiricum,the fermentation liquid of endophyic fungi MC4-23,and the extract of mycelium all had antifungal activities.The acetone extracts of Xanthium sibiricum had stronger antifungal activities than the ethanol extracts of Xanthium sibiricum.The extracts of leaf have the strongest antifungal activities against fungi and bacteria.The inhibition rates of the six pathogenic fungi were all above 47.8%,and the inhibition diameters of the three pathogenic bacteria were all above 0.5 centimeter.The fermentation liquid of endophytic fungi also had strong activities against the fungi and bacteria.The inhibition rates of the six pathogenic fungi were all above 68.1%.The extracts of root of Xanthium sibiricum and the mycelium of endophytic fungi were all had antifungal activities.[Conclusion] Xanthium sibiricum and endophyic fungi MC4-23 had great value of development and utilization.     

臭牡丹叶提取液对2种植物病菌的抑制作用研究（英文）

The test was undertaken to reveal the antifungal activity of extracts from Clerodendrum bungei leaves against Pestalotia funereal and Rhizoctonia solani,the results showed that optimal condition for best antifungal activity of extracts against Pestalotia funereal and Rhizoctonia solani are as follows:material-liquid ratio of 1∶6,75% ethanol as extracting solvent,reflux at 90 ℃ for 1.5 h.The substances with good dissolubility in ethanol and water solution such as organic acid,bioflavonoid and alkaloid are main antifungal bioactive substances in Clerodendrum bungei.     

Solvent Effects on the Ultrastructure and Chemical Composition of Cuticular Wax and Its Potential Bioactive Role Against Alternaria alternata in Pingguoli Pear

Effects of different polarity solvents on the ultrastructure and chemical composition of cuticular wax in Pingguoli pear as well as their bioactive role against Alternaria alternate were studied and the results showed that the highest wax content was extracted with chloroform, and its wax content was up to 322.2 gg cm2. Long-chain fatty acids predominated in menthol extracts and n-alkanes were predominant in wax extracted with ether, chloroform and n-hexane. Pingguoli pear fruit surface was covered by a smooth and amorphous wax layer with small, scattered crystal. The morphology of recrystallized wax in vitro after removal with different solvents was not similar to that of the intact fruit surface. Removal of cuticular wax with various solvents significantly enhanced A. alternata infection, except for wax removed by methanol. The solvent extracts of methanol and chloroform stimulated the spore germination and mycelium growth ofA. alternata, but the ether and n-hexane extracts showed antifungal activity.     

Acaricidal Activity of Boenninghausenia sessilicarpa Against Panonychus citri

Acaricidal activity of Boenninghausenia sessilicarpa against Panonychus cirri was tested in the laboratory. Four solvents were used to prepare crude extracts, petroleum ether, chloroform, ethyl acetate, and ethanol, among which ethanol was the most effective one. The results suggested that ethanol extracts of B. sessilicarpa had eminent acaricidal and ovicidal activities. Concentrated extracts were prepared using petroleum ether, chloroform, ethyl acetate, or distilled water as solvent. Mite mortality rates in the concentrated extracts by petroleum ether, chloroform, or distilled water were significantly lower than those by ethyl acetate. The LC50 values of eggs and female mites were 0.7639 and 1.1033 mg mL^-1, respectively. After liquid chromatography and thin layer chromatography, the concentrated extracts were separated into 14 groups of fractions and further tests for their acaricidal and ovicidal activities were conducted. Fraction 2 was found to possess higher acaricidal and ovicidal activities. The mortality of eggs and adult mites were 85.83 and 63.07%, respectively. Moreover, fraction 2 showed moderate oviposition inhibition effect （0.8795） against P. citri when the used dose was higher than 2.5 mg mL^-1     

Antifeedant Activity and Active Ingredients Against Plutella xylostella from Momordica charantia Leaves

With the bioguided fractionation of the ethanol extracts from the leaves of Momordica charantia, we obtained two most active compounds against the feeding of the diamondback moth, Plutella xylostella larvae. The antifeedant activity of momordicine Ⅰ and momordicine Ⅱ against the second and the third instar larvae of Plutella xylostella were tested using leaf discs of cabbage in the laboratory. The results showed that momordicin Ⅰ and momordicin Ⅱ had significant antifeedant activity on the larvae of P. xylostella, and momordicin Ⅱ was more active than momordicin Ⅰ. The concentrations for 50% antifeedant effects （AFC50） of momordicin Ⅱ against the second and the third instar larvae of P. xylostella were 76.69 and 116.24μg mL^-1, whereas that of momordicin I was 144.08 and 168.42μg mL^-1, respectively. In addition, momordicin Ⅰ and momordicin Ⅱ had significant inhibitive effect on the rate of weight gain and survival of P. xylostella larvae.     

河朔荛花农药生物活性初探(英文)

[Objective] The insecticidal and antibacterial bioactivity of Wikstroemia chamaedaphne Meissn were screened and bioactive substances in it were separated and purified. [Method]The Wikstroemia chamaedaphne Meissn was conducted ultrasonic extraction in petroleum ether, ethyl acetate and methanol. The insecticidal activity of Wikstroemia chamaedaphne Meissn to Mythimna separata walker and aphid were determined. The antibacterial activity of Wikstroemia chamaedaphne Meissn to Fusarium graminearu, Glomerella cingulata, F.oxysporium f.sp niveum, Alternaria solani and Fusarium oxysporium were also determined. The bioactivity-guided methods such as opencolumn chromatography and Pre-HPLC method were used to separate active components in petroleum ether extract from Wikstroemia chamaedaphne Meissn. [Result] When the concentration was 500 mg/L, 3 kinds of extracts from Wikstroemia chamaedaphne Meissn didn’t show obvious antibacterial bioactivity to 5 kinds of test samples. When the concentration was 5%, petroleum ether extract show certain topical toxicity to aphids. The ethyl acetate extract showed certain antifeedant activity to 3rd instar Larvae of Mythimna separata Walker. The fraction F4 of petroleum ether extract possessed highest topical toxicity to aphids and the lethality was 60.00%.[Conclusion] Wikstroemia chamaedaphne Meissn contained many insecticidal constituents whose active parts and mechanism were needed further researches.     

Antibacterial Activity of Crude Extracts of Prinsepia utilis Royle in vitro

The purpose of this study is to isolate the active fraction from the crude extract of Prinsepia utilis Royle and to determine its antibacterial activity against the standard and drug-resistant bacteria strains in vitro. The Prinsepia utilis Royle was extracted in three portions by using petroleum ether, ethanol and water by the continuation recirculate extraction. The antibacterial activity of the extracts were evaluated by the determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values. The results showed that the ethanol extract and aqueous extract of petroleum ether showed the weakest inhibition. MIC values of the aqueous Prinsepia utilis Royle were found that they had significant effects against bacteria, while ethanol extracts against three standard bacteria (S. aureus ATCC25923, E. coli 44102, Salmonella 50041) were 25, 25, 50 mg · mL-1 and 50, 100, 100 mg · mL-1 , MIC values of three drug-resistant bacteria (S. aureus GL17, E. coli EYAC08-56, Salmonella STQD2G.) were 50, 50, 50 mg · mL-1 and 100, 100, 200 mg·mL-1 , respectively. MBC values of two extracts against three standard strains were 25, 50, 100 mg·mL-1 and 50, 100, 200 mg·mL-1 , MBC values of three drug-resistant bacteria were 100, 100, 100 mg·mL-1 and 200, 200, 200 mg·mL-1 , respectively.     

Antifungal Potential of Chenopodium album L. against Chickpea Blight

Chenopodium album L. （bathu） leaves and roots were selected to evaluate their antifungal potential against the Ascochyta rabiei responsible for chickpea blight that causes destructive yield losses. Methanolic extract of C. album leaves and roots was prepared and their various applied concentrations, viz., 1%, 2%, 3%, 4% and 5% were tested against A. rabiei. Leaf extracts were found more effective and showed significant antifungal activity over the root extracts. C. album leaf was found effective in screening bioassays, so this was subjected for fractional guided bioassays. Different various organic fractions of leaf extract were isolated, viz., n-hexane, chloroform, ethyl acetate and n-butanol. These isolated fractions were serially diluted to check their minimum inhibitory concentration （MIC） along with a synthetic fungicide （72% WP, Puslan）. The MIC of various concentrations from 500 mg/mL to 1.95 mg/mL was recorded after 24 h and 48 h intervals. Ethyl acetate and synthetic fungicides were found most effectual in retarding conidial germination with MIC of 1.95 mg/mL after 48 h incubation periods. Chloroform fraction also inhibited the fungal growth whereas n-hexane and n-butanol fraction were found to be ineffective.     

Antifeedant Activity and Active Ingredients Against Plutella xylostella from Momordica charantia Leaves

With the bioguided fractionation of the ethanol extracts from the leaves of Momordica charantia, we obtained two most active compounds against the feeding of the diamondback moth, Plutella xylostella larvae. The antifeedant activity of momordicine Ⅰ and momordicine Ⅱ against the second and the third instar larvae of Plutella xylostella were tested using leaf discs of cabbage in the laboratory. The results showed that momordicin Ⅰ and momordiein Ⅱ had significant antifeedant activity on the larvae of P. Xylostella, and momordicin Ⅱ was more active than momordicin Ⅰ. The concentrations for 50% antifeedant effects (AFC50) of momordicin Ⅱ against the second and the third instar larvae of P. Xylostella were 76.69 and 116.24 μg mL-1, whereas that of momordicin Ⅰ was 144.08 and 168.42 μg mL-1, respectively. In addition, momordicin Ⅰ and momordicin Ⅱ had significant inhibitive effect on the rate of weight gain and survival of P. Xylostella larvae.     

Studies on Acaricidal Bioactivities of Artemisia annua L. Extracts Against Tetranychus cinnabarinus Bois. （Acari： Tetranychidae）

The aim of this study was to determine the best extraction technique, the most suitable solvent, the optimal plant parts, and the acaricidal activities of Artemisia annua L. The petroleum ether （30-60℃）, petroleum ether （60-90℃）, ethanol, acetone, and water parallel and sequenced extracts were obtained from the leaves, stems and roots of different period of A. annua L. in April, May, June, July and September respectively. And then the acaricidal bioactivities against Tetranychus cinnabarinus of all extracts were determined by the slide-capillary method in the laboratory. The results indicated that the acaricidal bioactivities elevated as the development of A. annua plant at the concentration of 5 mg mL-L The general tendency exhibited the sequence of July 〉 June 〉 May 〉 April, but September decreased comparing to July. However, the most effective extracts in five months were all acetone parallel extract of A. annua leaf, and the corrected mortalities treated after 48 h ranged from 74 to 100%. The median lethal concentrations （LC50） against T. cinnabarinus of acetone parallel extracts ofA. annua leaves in September, July, June, May and April were 0.5986, 0.4341, 0.8376, 0.9443 and 1.3817 mg mL^-1, respectively, treated after 48 h. The 13 groups were isolated from acetone extracts ofA. annua leaves in July by column chromatography, both the 1 lth and 12th groups exhibited strong bioactivities. The median lethal concentrations of the 1 lth and 12th groups against T. cinnabarinus were 0.3683 and 0.1586 mg mL^-1, respectively. The acetone parallel extract ofA. annua leaf in July was the most toxic to T. cinnabarinus and the corrected mortality was 100% after 48 h. The acetone parallel extract of the 1 lth and 12th groupswere the most active components, acted as the emphases in further study.     

Synthesis and Insecticidal Activity of Novel Camptothecin Derivatives Containing Analogs of Chrysanthemic Acid Moieties

Creating high-efifcient and environment-friendly pesticides is very important to produce the pollution free agriculture food and maintain the balance of the survival environmental of the human being. According to reports, camptothecin （CPT） and its derivatives are now being explored as a class of botanical insecticide in agriculture due to its novel mode of action. In order to improve the insecticidal activity of CPT, ten novel camptothecin （1） and 10-hydroxycamptothecin （2） derivatives （1a, 1b, 1c, 1d, 1e;2a, 2b, 2c, 2d, 2e） were designed and synthesized via esteriifcation with analogs of chrysanthemic acid, which have outstanding insecticidal activity. The results showed that compound 2a exhibited potent antifeeding effect and the best contact toxicity among the target compounds against the third-instar larvae of beet armyworm, Spodoptera exigua H＆#252;bner. Compound 2a was also found to be the most effective cytotoxic compound to the tested insect cell lines, IOZCAS-Spex-II, which were established from the fat bodies of S. exigua. It was proposed that the 10-hydroxyl group in the camptothecin derivatives is a key factor for the antifeeding activity of a compound. The nature of the substituents was considered the major factor in determining the insecticidal activity of these compounds.     

Screening of Fungus Antagonists against Six Main Disease Pathogens in Crops

28 soil samples were collected from the rhizosphere of 16 plant species in six different distriers in Hunan. As a result of isolation and purification, 122 fungus strains were obtained of which the antagonistic activity was tested against six fungus pathogens in tmaato, cotton, cucumber, chilli, rice and rape, and 17 strains were found antagonistic to one or more pathogenic fungi.     

马齿苋提取物对枸杞蚜虫毒杀和拒食活性的研究(英文)

[Objective] The experiment studied on insecticidal and antifeedant action of on Portulaca oleracea L.to provide theoretical basis for developing a new botanical insecticide by taking Portulaca oleracea L.as a raw material.[Method] The insecticidal activities of the ethanol extracts and 5 kinds of extracts with petroleum ether,chloroform,ethyl acetate,n-butanol,water from Portulaca oleracea L..against Aphis sp.was studied;Leaves are using drug treatments of 5 extracts anti-feedant activity of Aphis sp.[Result] The results showed that 95% ethanol extract displayed high contact activity to Aphis sp.The corrected mortality after 48 h reached 95.4 % under the concentration of 50.00 mg·ml-1 and the insecticidal activities of 5 extracts against Aphis sp.was in the order that petroleum ether > chloroform > n-butanol > ethyl acetate >water.Five kinds of anti-feeding activity in the extract of the order for the water> ethyl acetate> n-butanol > chloroform >petroleum ether.[Conclusion] The extract from Portulaca oleracea L.has a strong contact toxicity of pesticide active on Aphis sp.substances and for a small polar compounds,the strong anti-feeding activity is a kind of larger polar compounds.     

Study on the Insecticidal and Antifeedant Activity of Extracts from Portulaca oleracea L. on Aphis sp.

[Objective] The experiment studied on insecticidal and antifeedant action of on Portulaca oleracea L.to provide theoretical basis for developing a new botanical insecticide by taking Portulaca oleracea L.as a raw material.[Method] The insecticidal activities of the ethanol extracts and 5 kinds of extracts with petroleum ether,chloroform,ethyl acetate,n-butanol,water from Portulaca oleracea L..against Aphis sp.was studied;Leaves are using drug treatments of 5 extracts anti-feedant activity of Aphis sp.[Result] The results showed that 95% ethanol extract displayed high contact activity to Aphis sp.The corrected mortality after 48 h reached 95.4 % under the concentration of 50.00 mg·ml-1 and the insecticidal activities of 5 extracts against Aphis sp.was in the order that petroleum ether > chloroform > n-butanol > ethyl acetate >water.Five kinds of anti-feeding activity in the extract of the order for the water> ethyl acetate> n-butanol > chloroform >petroleum ether.[Conclusion] The extract from Portulaca oleracea L.has a strong contact toxicity of pesticide active on Aphis sp.substances and for a small polar compounds,the strong anti-feeding activity is a kind of larger polar compounds.     

马齿苋提取物对枸杞蚜虫毒杀和拒食活性的研究(英文)

[Objective] The experiment studied on insecticidal and antifeedant action of on Portulaca oleracea L.to provide theoretical basis for developing a new botanical insecticide by taking Portulaca oleracea L.as a raw material.[Method] The insecticidal activities of the ethanol extracts and 5 kinds of extracts with petroleum ether,chloroform,ethyl acetate,n-butanol,water from Portulaca oleracea L..against Aphis sp.was studied;Leaves are using drug treatments of 5 extracts anti-feedant activity of Aphis sp.[Result] The results showed that 95% ethanol extract displayed high contact activity to Aphis sp.The corrected mortality after 48 h reached 95.4 % under the concentration of 50.00 mg·ml-1 and the insecticidal activities of 5 extracts against Aphis sp.was in the order that petroleum ether > chloroform > n-butanol > ethyl acetate >water.Five kinds of anti-feeding activity in the extract of the order for the water> ethyl acetate> n-butanol > chloroform >petroleum ether.[Conclusion] The extract from Portulaca oleracea L.has a strong contact toxicity of pesticide active on Aphis sp.substances and for a small polar compounds,the strong anti-feeding activity is a kind of larger polar compounds.     

鸦胆子愈伤组织提取物的杀虫活性研究(英文)

[Objective] The research aimed to lay a foundation for the screening of cell lines producing secondary metabolites of Brucea javanica(L.)Merr.[Method] The insecticidal activities of the extracts from branch and 3 different types of calluses of Brucea javanica(L.)Merr.was detected through methods of leaf disc and potted seedlings against the diamond back moth.[Result] Extracts from four kinds of Brucea javanica(L.)Merr.tissues assumed both the activities of antifeedant and oviposition deterrency against the diamond back moth.Antifeedant effect of extracts was in turn the callus C< callus B< callus A< branches.Oviposition deterrency activity of the extracts was in turn the callus A> branch > callus B>callus C.The insecticidal activities of callus A and B were higher than that of the callus C.[Conclusion] The results show that insecticidal activity of callus and its growth rate is inversely proportional.     

In Vitro Antifungal Activity of the Extract and Compound from Acorus tatarinowii Against Seven Plant Pathogenic Fungi

Acorus tatarinowii Schott is a traditional Chinese medicine plant and has multiple bioactivities in medicine and pesticide field. In this study, the antifungal compound 1,2-dimethoxy-4（2-propenyl） benzene was isolated from A. tatarinowii Schott by activity-directed isolation method, and the inhibitory activity of the extract and 1,2-dimethoxy-4（2-propenyl） benzene against seven plant pathogenic fungi was evaluated. The results showed that the extract and 1,2-dimethoxy-4（2- propenyl） benzene had high inhibitory activity against hyphal growth of Thielaviopsis paradoxa （de Seynes） V. Hohnel, Pestalotia mangiferae P. Henn., Fusarium oxysporum f. sp. niveum （E. F. Smith） Syn. et Hans., Alternaria alternate Tanaka, Colletotrichum musae （Berk et Curt） V. Arx, Sphaceloma fawcettii Jenk., and Mycosphaerella sentina （Fr.） Schroter. The EC50 values of extract were 1.6162, 1.6811, 1.1253, 3.5771, 1.7024, 2.2284, and 2.2221 g L^-1, respectively, and the EC50 values of 1,2-dimethoxy-4（2-propenyl） benzene were 0.1021, 0.0997, 0.0805, 0.1742, 0.1503, 0.1853, and 0.1924 g L^-1, respectively. 1,2-Dimethoxy-4（2-propenyl） benzene also inhibited spores germination of T. paradoxa （de Seynes） V. Hohnel and F. oxysporum f. sp. niveum （E. F. Smith） Syn. et Hans., with the inhibitory rates of 98.81 and 100% at a concentration of 0.4 g L^-1 after 8 h, respectively. 1,2-Dimethoxy-4（2-propenyl） benzene is a potential botanical antifungal agent for controling of plant fungal diseases.