基于荧光增强现象用于炭疽芽孢杆菌生物标志物DPA的检测

以柠檬酸和六水合硝酸铕为原料,采用水热法合成了微米级的铕配位聚合物(Europium coordination polymer,Eu-CPs),在0.2 mol/L HCI酸性环境伴随着280 nm紫外光连续照射,且存在炭疽芽孢杆菌生物标志物2,6-吡啶二羧酸(Dipicolinic acid, DPA)的情况下,在390 nm 处产生了新的发射峰,并且该发射峰强度随着光照时间的延长而增加。根据280 nm紫外光照前后390nm处发射峰增强的荧光强度差与DPA浓度之间的线性关系,测得DPA检测范围为1-60 μmol/L,最低检测限为28.16 nmol/L。最后我们将其用来实时监测枯草芽孢杆菌孢子中DPA的释放,得到DPA在孢子内的释放几乎在60分钟内完成。

基于铜掺杂碳纳米点荧光测定炭疽生物标志物

以柠檬酸、乙二胺为前体,硫酸铜为金属掺杂剂,采用一步水热法制备了一种高荧光量子产率的水溶性铜掺杂碳纳米点(Cu-CDs)。根据2,6-吡啶二甲酸(DPA)与碳纳米点的强螯合作用建立了铜掺杂碳纳米点荧光猝灭测定炭疽生物标志物DPA的新方法。在最佳实验条件下,DPA在5~100nmol/L(r2=0.9941)和150~400nmol/L(r2=0.9976)浓度范围内与Cu-CDs的荧光猝灭率呈良好的线性关系,检出限为2.3nmol/L。该分析方法成本低、专属性强、灵敏度高、操作简便,在炭疽生物标志物检测方面具有良好的应用前景。

Curcuminoids Inhibit Botrytis cinerea and Colletotrichum gloeosporioides

The growth rate method was adopted to measure the inhibitory effect of curcumin,tetrahydrocurcumin,demethoxycurcumin,and bisdemethoxycurcumin on the mycelial growth of Botrytis cinerea and Colletotrichum gloeosporioides.The results showed that the four curcuminoids inhibited the mycelial growth of the two pathogens in a concentration-dependent manner.Bisdemethoxycurcumin at 600 mg/L exerted the strongest inhibitory effect on the mycelial growth of B.cinerea and C.gloeosporioides,with the relative inhibition rates of 98.19%and 100%,respectively;followed by demethoxycurcumin;curcumin exerted the worst inhibitory effect.Toxicity test results also showed that four curcuminoids all had a certain toxicity to B.cinerea and C.gloeosporioides,among which,bisdemethoxycurcumin exhibited the strongest toxicity,with the EC_(50)of 131.125 and 122.235 mg/L,respectively;while curcumin had the lowest toxicity,with the EC_(50)of 273.143 and 194.943 mg/L,respectively.

Study on Pathogeny Identification and Biological Characteristics of Anthracnose in Cassava

[Objective] The research aimed to isolate and identify the anthracnose pathogen of Cassava and study its biological charcteristics.[Method] Two isolates of anthracnose （CCGHN01 and CCGHN03） in Cassava were isolated from the diseased leaves collected from Hainan of China.They were identified through the morphological observation of conidia and ITS sequence analysis.And the morphological characteristics were studied.[Result] The morphological observation of conidia and ITS sequence analysis indicated that the two isolates were Colletotrichum gloeosporioides.The morphological characteristics study results showed that optimum medium for the growth of two strains was PSA,the optimum temperatures were 26 ℃ and 30 ℃ respectively,the optimum pH was 8.0,the optimum light conditions were alternative light and dark and complete darkness respectively.For the conidia germination of two strains,the optimum temperatures were 28 ℃ and 30 ℃ respectively and the lethal temperature was 55 ℃ remaining 10 min.[Conclusion] The research laid the foundation for further control of anthracnose in Cassava.

Isolation and Identification of Colletotrichum gloeosporioides in Pears and Its Biological Characteristics

[Objective] This study aimed to explore the biological characteristics of Col etotrichum gloeosporioides in pears. [Method] Twenty-five C. gloeosporioides strains were isolated and identified from the diseased samples. Their pathogenicity was identified by inoculating the surface of punctured pears with fungal discs. The effects of different temperatures, pH values, carbon sources and nitrogen sources on the growth of C. gloeosporioides mycelia were explored by incubating fungal discs on the center of plates. [Result] Among the twenty-five C. gloeosporioides strains, three had strong pathogenicity, and eighteen had intermediate pathogenicity, and four strains had weak pathogenicity. Those highly-pathogenic strains had darker colonies, with dense mycelia, whereas those lowly-pathogenic ones had white colonies, with sparse mycelia. Those with fast-growing colonies showed strong pathogenicity, while those with slowly-growing colonies displayed weak pathogenicity. There was no relationship between conidia yield and pathogenicity. The optimum temperature for the growth of C. gloeosporioides mycelia was 25-30 ℃, and the optimum pH was 5.0-7.0. C. gloeosporioides could make use of various carbon sources （monosaccharide and disaccharide）, inorganic and organic nitrogen sources, and the optimal carbon source and nitrogen source were sucrose and beef extract, respectively. [Conclusion] Our study benefits further understanding of C. gloeospori-oides and helps to control pear anthracnose more effectively.

Control Effect of Four Kinds of Agents for Anthracnose in Autumn Greenhouse Cowpea

In order to mitigate the occurrence and damage of cowpea anthracnose and reduce the chemical pesticide consumption, residue and environmental pollution, the methods of forecasting, randomized block design and statistical analysis were conducted to study the control effect of biopesticide chitosan, biochemical compound Prochloraz-Chitosan and chemical pesticides difenoconazole and thifluzamide by field efficacy trials. The results showed that under serious occurrence of anthracnose in autumn greenhouse cowpea, when the 2% chJtosan AS （2 250 g/hm^2）, 46% Prochloraz-Chitosan EW （450 g/hm^2）, 10% difenoconazole WG （900 g/hm^2, CK） or 24% thifluzamide SC （360 ml/hm^2） were applied three times with an interval of 5-8 d, the control efficiency on day 7, 14 and 20 reached 83%, 78% and 73%, respectively. No significant difference was found in control efficiency among the four kinds of agents. These four kinds of agents, especially chitosan and Prochloraz-Chitosan, could be used as ideal agents for controlling anthracnose, as well as for modern agricultural demonstration zones, pollution-free agricultural products, green agricultural products and organic agricultural products,thereby meeting the develop- ment needs of ＇modern agriculture, green agriculture, organic agriculture and ecological agriculture.

Biocontrol of Strawberry Anthracnose Caused by Colletotrichum fragariae

[Objective] The aim was to explore biocontrol approaches of strawberry an-thracnose. [Method] With hyphal growth inhibition method, bacteriostatic activities of Bacil us subtilis and Osthole on strawberry anthracnose were measured and field test was carried out. [Result] The results show that both of Bacil us subtilis and Osthole were of higher bacteriostatic activity on strawberry anthracnose, and the values of EC50 were 0.007 5 mg/L and 1.063 0 ml/L, respectively. The result of field test show that the prevention effects of Bacil us subtilis （600-750 g/hm2） and 25% prochloraz （600 ml/hm2） both achieved higher than 76%, 7 and 14 d after triple medical applica-tions with rains sheltered or in open field. But the effects were of extremely signifi-cant differences with that of Osthole （1 800-2 700 ml/hm2） （P〈0.01）. [Conclusion] Bacil us subtilis can be made use of for control ing strawberry anthracnose.

Effect of Plumeria alba＇s Bioactive Extract against Colletotrichum gleosporoides in Vitro and in Vivo

Plant species in the genus Plumeria of Apocynaceae family are well known for their uses in traditional medicine antimicrobial activity. Plant materials from P. alba were extracted using Soxhlet apparatus and screened for the control of anthracnose disease caused by Colletotrichum gleosporoides on fruit during post harvest storage. For in vitro, the crude extract of P. alba （stem bark and leaves） were completely inhibited conidial germination （sporulation test method） of Colletotrichum gleosporoides at all concentration tested except the lowest concentration （4.4 uL）. In vivo studies show that percentage of infection on fruit after treated with both stem bark （11.11 ± 3.8） and leaves （15.55 ± 3.8） part, at 32,000 mg/L have no significant differences during seven days of storage at ambient temperatures. These results proved that P. alba extracted （stem bark and leaves） were found to be promising as an antifungal agent and to control anthracnose disease in fruits tested.

Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the gene encoding protective antigen receptor-binding domain (the 4 th domain of PA, PAD4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PAD4 by IPTG. The recombinant protein was purified by chromatography and then identified by N-terminal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E.coli . The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

Study of the Action Mode of Wickerhamomyces anomalus against Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is the causal agent of anthracnose disease in fruits and vegetables, representing a global problem. The use of biocontrol agents has proved effective against fungal diseases in a wide variety of products. In this work, the antifungal activity of Wickerhamomyces anomalus against C. gloeosporioides isolated from contaminated avocados was evaluated. The antagonism and volatile compound inhibition were measured on Petri dishes. In the mixed cultures, the mycelia damage was observed by scanning electron microscope （SEM）. Chitinase and glucanase production by the antagonism was quantified by the reducing sugars method, and biofilm formation was evaluated with 1% crystal violet. The yeast W. anomalus could reduce the growth of C. gloeosporioides up to 65% by direct antagonism and 10% by volatile compounds. The antagonist did not allow the conidia germination and mycelia growth in any of the tested formulations. SEM showed mycelial damage caused by W. anomalus. The antagonist showed adhesion to the mycelium by a polysaccharide biofilm. The presence of mycelium stimulated the hydrolytic enzyme production with the maximal activity of 21.4 U/mg for chitinases at 24 h and 10 U/mg for glucanases at 60 h. These results showed that W. anomalus used together different mechanisms to express its antifungal activity against C. gloeosporioides. This study might be the first report for this phytopathogen isolated from avocado fruits, which could represent an opportunity to establish biocontrol of diseases for this agricultural product.

Effects of rhizobacteria Paenibacillus polymyxa APEC136 and Bacillus subtilis APEC170 on biocontrol of postharvest pathogens of apple fruits

In this study, plant growth-promoting rhizobacteria（PGPR） were evaluated as potential biocontrol agents against postharvest pathogens of apple fruits. In vitro bioassays revealed that, out of 30 isolates screened, isolates APEC136 and APEC170 had the most significant inhibitory effects against the mycelial growth of several fungal pathogens. Analysis of 16 S ribosomal RNA（rR NA） sequences identified the two effective isolates as Paenibacillus polymyxa and Bacillus subtilis, respectively. The two strains showed greater growth in brain-heart infusion broth than in other growth media. Treatment of harvested apples with suspensions of either strain reduced the symptoms of anthracnose disease caused by two fungal pathogens, Colletotrichum gloeosporioides and Colletotrichum acutatum, and white rot disease caused by Botryosphaeria dothidea. Increased productions of amylase and protease by APEC136, and increased productions of chitinase, amylase, and protease by APEC170 might have been responsible for inhibiting mycelial growth. The isolates caused a greater reduction in the growth of white rot than of anthracnose. These results indicate that the isolates APEC136 and APEC170 are promising agents for the biocontrol of anthracnose and white rot diseases in apples after harvest, and suggest that these isolates may be useful in controlling these diseases under field conditions.