Chemical Studies on Ninhydrin-Positive Compounds in Cured Tobacco Leaves

A new amino acid, 2-pyrrolidine acetic acid and 1-pipecolic acid were isolated from cured tobacco leaves. The former structure was confirmed by comparison with synthetic compound.     

Occurrence of parasporin-producing Bacillus thuringiensis in Vietnam

A total of 63 Bacillus thuringiensis isolates were recovered from urban soils of Hanoi, Vietnam. Of these, 34 were identified to 12 H serogroups. None of the isolates showed larvicidal activities against three lepidopterous insects. Three isolates belonging to the two serovars, colmeri (H21) and konkukian (H34), were highly toxic to larvae of the mosquito Aedes aegypti. Parasporal inclusion proteins of four isolates exhibited cytocidal activities against HeLa cells. Immunologically, proteins of four isolates were closely related to parasporin-1 (Cry31Aa), a parasporal protein that preferentially kills human cancer cells. Haemolytic activities were associated with parasporal proteins of the three mosquitocidal isolates but not with those of the four cancer-cell-killing isolates. PCR experiments and nucleotide sequence analysis revealed that the genes of four anti-cancer isolates are closely related to the gene parasporin-1 (cry31Aa) but are dissimilar to those of the three other existing parasporins. Our results suggest that the soil of northern Vietnam is a good reservoir of parasporin-producing B. thuringiensis.     

Occurrence of Bacillus thuringiensis in Fresh Waters of Japan

Bacillus thuringiensis was recovered at a relatively high frequency from both running and still fresh waters in natural environments of Kyushu, Japan. Of 107 water samples examined, 53 (49.5%) contained this organism. The frequency of B. thuringiensis colonies was 4.4% among 4414 colonies of the Bacillus cereus/B. thuringiensis group. The density of this bacterium in fresh waters averaged 0.45 cfu/ml. Serologically, B. thuringiensis isolates were assigned to 26 H serotypes. Of these, H14/36 (H serovar israelensis/malaysiensis) was the predominant, followed by the serotypes H3abc (kurstaki), H27 (mexicanensis), H3ad (sumiyoshiensis), and H35 (seoulensis). Of 195 isolates, 52 (26.7%) exhibited larvicidal activity against aquatic Diptera; 21 killed Culex pipiens molestus (Culicidae) only, and 31 were active on both the culicine mosquito and the moth-fly, Clogmia albipunctata (Psychodidae). The Diptera-toxic isolates produced spherical or irregularly pointed parasporal inclusions. Received: 4 September 1999 / Accepted: 8 October 1999     

Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food

Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain. A new observation was that 31 out of the 40 randomly selected B. cereus-like strains could be classified as Bacillus thuringiensis due to crystal production and/or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis.     

Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensis-based insecticides on fresh fruits and vegetables

A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic.     

云南烟叶中苏云金杆菌的分布及杀虫特性

采用4%NaCl培养基选择分离法,从云南7个生产烟区采集的450个复烤烟样中,分离出苏云金芽孢杆菌475株。分离出的Bt菌进行PCR毒素蛋白基因鉴定,其中含cryI毒素蛋白基因的Bt菌有7株,出菌率为1.47%;含cryV毒素蛋白基因的Bt菌11株,出菌率为2.32%;没有含cryⅢ毒素蛋白基因的Bt菌。对含毒蛋白基因cryⅠ及cryⅤ的18个Bt菌株用二龄烟草甲虫进行生物毒力测定,试验后9d有9个Bt菌件生物毒力测定校正死亡率均超过80%;试验后12d有5个Bt菌株生物毒力测定校正死亡率均超过95%。可见,用苏云金芽孢杆菌防治烟叶仓贮害虫是有潜力的。     

Isolation of Bacillus thuringiensis from Stored Tobacco and Lasioderma serricorne (F.)

Bacillus thuringiensis was isolated from dried tobacco residues and dead tobacco beetles (Lasioderma serricorne (F.); Coleoptera: Anobiidae) collected in a large number of locations worldwide. Eighty-eight samples of stored tobacco were analyzed and yielded 78 B. thuringiensis strains which were characterized on the basis of parasporal crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles, and the results of an immunoblot analysis of the insecticidal crystal proteins. Flagellar antigen identification was used to differentiate selected isolates. Strains that produced rhomboidal crystals associated with the Coleoptera-specific pathotype (Cry III group) were the most abundant strains (59% of the isolates). Preliminary toxicity assays were performed with L. serricorne larvae, and the results suggested that activity is not restricted to isolates related to the Coleoptera-specific group. The results of our survey indicate that B. thuringiensis is part of the natural microflora in the stored-tobacco environment and that this special habitat represents a source of B. thuringiensis isolates that may be used to control stored-product pests.     

Isolation of Sugar Compounds of Asparagine,Phenylalanine, Tyrosine and Valine from Cured Tobacco Leaves

Scanning electron microscopy and rigidity characterization were used in this study of myosin gelation in the presence of actin. The heat-induced gel formation of myosin was aided by the addition of actin to myosin at a molar ratio of 1: 2.7. However, this actin effect was not observed when the actin-binding site(s) of myosin was blocked by p-chloromercuri-benzoate treatment or denaturation, or when the myosin-binding site(s) of actin was blocked by trinitrophenylation.

The effect also disappeared with an aging myosin-actin mixture, with ATP or pyrophosphate as dissociating agent prior to thermal treatment of the system.

The present findings indicate the indispensability of myosin binding to actin for enhancing the thermal gelation of myosin. The gelation process appears to involve a large electrostatic contribution, as well as such chemical reactions as oxidation of SH groups.     

苏云金芽孢杆菌的一个新亚种(Btsubsp.sinensis)

198 9年自云南昆明市石林的红棕壤中分离到数株苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎ ｓｉｓ,Ｂｔ)菌株[1] ,对其中的一株ＹＫ30 0 4进行了生物学特性、杀虫特性研究及分类鉴定。1　材料与方法1.1　供鉴定的Ｂｔ菌株由云南昆明市石林的红棕壤中分离的苏云金芽孢杆菌ＹＫ30 0 4菌株。1.2　标准Ｂｔ菌株血清型Ｈ1 Ｈ4 1、Ｈ4 4 Ｈ55及Ｈ57 Ｈ69标准Ｂｔ菌株由法国巴斯德研究院ＤｒＬｅｃａｄｅｔ提供 ,其余为本实验室保存。1.3　生物测定用昆虫小菜蛾 (Ｐｌｕｔｅｌｌａｘｙｌｏｓｔｅｌｌａ) 3龄幼虫 ;斜纹夜盗蛾 (Ｐｒ…     

Metalloprotease from Bacillus thuringiensis

Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn2+ or Ca2. The addition of Ca24 was required for enzyme stabilization...     

Occurrence of two serologically distinct groups within Bacillus thuringiensis serotype 3 ab var. kurstaki

Two groups distinguishable on the basis of crystal serology have been identified within Bacillus thuringiensis var kurstaki (serotype 3 ab). The toxicities of these two groups to Trichoplusia ni and Heliothis virescens expressed as $\text{T}\text{H}$ ratio also differed. It is suggested that serotype 3 ab be separated into two subgroups designated as K-1 and K-73.     

苏云金芽孢杆菌(Bt)晶体毒蛋白基因在烟草叶绿体中的表达

将全长３．５ｋｂ的Ｂｔ基因３’端缺失,得到长为２．１ｋｂ、１．８ｋｂ的基因。分别将这３个长度（１．８ｋｂ、２．１ｋｂ、３．５ｋｂ）的基因置于水稻叶绿体ｐｓｂＡ基因的启动子和终止子调控之下,并与选择标记基因ａａｄＡ（编码氨基糖苷－３’－腺苷酸转移酶,具壮观霉素抗性）表达盒相连;以烟草叶绿体基因ｔｒｎＨ－ｐｓｂＡ－ｔｒｎＫ为同源片段,构建成叶绿体转化载体ｐＢＴ３、ｐＢＴ８和ｐＢＴ２２。用基因枪把Ｂｔ基     

Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus

One hundred and thirty-seven strains of Bacillus thuringiensis and 35 strains of Bacillus cereus were tested for the presence or absence of 99 traits. An analysis of these data indicated that strains of B. thuringiensis were indistinguishable from B. cereus, except for their ability to produce parasporal crystals. This conclusion was based on a comparison of the phenotypic properties of B. thuringiensis and B. cereus, as well as on the results of numerical analyses of the data which grouped strains into clusters on the basis of phenotypic similarity. In the resulting dendrograms, strains of B. thuringiensis and B. cereus were interspersed, exhibiting no tendency to segregate. In addition, with the exception of serovar israelensis, strains on B. thuringiensis belonging to the same flagellar serovar showed little or no tendency to group in different clusters. A comparison of the phenotypic differences between serovars indicated that the greater the number of strains in the serovars, the fewer, if any, phenotypic traits separating them. This suggests that the properties reported to differentiate serovars can be attributed to the internal phenotypic diversity of the species. Characterization of 10 mosquitocidal strains of Bacillus sphaericus indicated that the traits employed in this study readily distinguished these highly related organisms from strains of B. thuringiensis and B. cereus.     

The biotechnology of Bacillus thuringiensis

One of the challenges in the application of biotechnology to pest control is the identification of agents found in nature which can be used effectively. Biotechnology offers the potential of developing pesticides based on such agents which will provide environmentally sound and economically feasible insect control alternatives. Such an agent, the insect pathogen Bacillus thuringiensis, is the subject of intense investigations in several laboratories. Insecticides which use the entomocidal properties of B. thuringiensis are currently produced and sold worldwide; new products are currently in the development stage. Herein, the biology and genetics of B. thuringiensis and the problems associated with current products are critically reviewed with respect to biotechnology. Moreover, the economic and regulatory implications of technologically advanced products are evaluated.     

Ultra-violet-resistant mutants of Bacillus thuringiensis

One of the main disadvantages of using Bacillus thuringiensis as an insecticide is that the spore and crystal preparations applied to foliage are readily washed away by rain and are inactivated by sunlight. Spores from some strains of B. thuringiensis have been shown to be highly sensitive to u.v. light. This study has demonstrated how mutants with increased resistance to u.v., isolated by successive rounds of u.v. irradiation, and additionally with increased specific pathogenicity can be isolated. These techniques should be applied to strains that are frequently used in the industrial production of B. thuringiensis toxin.     

Bacteriocinogenic plasmids of Bacillus thuringiensis

When strains of Bacillus thuringiensis v. morrisoni or v. darmstadiensis were plated on solid medium, the appearance of oligosporogenic (Ospo) mutants and bacteriocin non-producing clones (Thc-) was observed. The comparative analysis of the plasmid content of original and mutant strains revealed that the appearance of Ospo and Thc- phenotypes correlated with the loss of certain plasmids.