我国冰核活性细菌的优势种类调查与研究

１９８６￣１９９４年,从国内１７个省、市、自治区的６８种植物上分离到了２５０株冰核活性细菌,经鉴定分别属于３个属的１７个种或致病变种。其中出现最多的是菠萝欧文氏菌,共１３３株,占总数的５３．２％,其次是丁香假单胞菌群,共７０株,占２８％,其它种类的冰核活性细菌共４７株,仅占１８．８％。因此,我国冰核活性细菌的优势菌种是Ｅ．ａｎａｎａｓ,其次是Ｐ．ｓｙｒｉｎｇａｅ ｐｖｓ。在低纬度的南方地区中Ｅ．ａ     

贪婪倔海绵中抗菌活性细菌的筛选及初步鉴定

采用平板涂布法从我国南海三亚周边海域贪婪倔海绵（Dysidea avara）中分离海绵共附生细菌,采用金黄色葡萄球菌、大肠埃希氏菌、荧光假单胞菌、枯草芽孢杆菌、白假丝酵母、宛氏拟青霉、黑曲霉7种指标菌进行抑菌试验筛选抗菌活性菌,同时对于得到的活性菌进行生理生化鉴定。共分离获得个149个细菌菌株,发现20株具有抑制真菌和革兰氏阳性细菌的活性,占细菌总数的13．4％。经过细菌形态观察和生理生化试验,发现此20株活性菌属于革兰氏阳性芽孢杆菌属（Bacillus sp．）。     

生物防治因子

831266用一种限定培养基生产具有冻核形成活性的细菌〔专,英〕/Lynn,5.Y.…/European Patent Appl.EP 0272669.Pub.29,06.55.入ppl.US 944120,f于-肠d咒.12,吕位仁译白七压、.工州;:“‘、4 089] 介绍了具有冰核形成活性的微生物门丁香假单胞菌(尸:。d。二。二:、、脚坛,‘、“夕)、抢光假单胞菌(j,.fl。。r。:二二)、·“大川胜价胞l翁(尸。eoronajaeiens)、/’.了、公:‘反,”一,遭欧文氏菌(Er田艺瓜ah郡bicola)。采用的培养墓包括葡萄糖(作为碳源)以及“一弋汉代)_戈二酸或能形成氧代戊二酸的氨感暇(仁为氮源)气.具体配制时,还可含…     

阿特拉津降解菌株的分离和鉴定*

从农药厂废水中分离到６株能以除草剂阿特拉津为唯一氮源生长的细菌，即假单胞菌（Ｐｓｅｕ－ｄｏｍｏｎａｓ ｓｐｐ．）ＡＤ１、ＡＤ２和 ＡＤ６，土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｓｐ．）ＡＤ４，黄单胞菌（Ｘａｎｔｈｏｍｏｎａｓ ｓｐ．）ＡＤＳ，欧文氏菌（Ｅｒｗｉｎｉａ ｓｐ．）ＡＤ７。ＡＤ１菌株能使无机盐培养基中的 ０．３ｇ／Ｌ阿特拉津在７２ｈ内降解９９，９％。当以ＡＤ１、ＡＤ２、ＡＤ４、ＡＤ５、ＡＤ６和ＡＤ７菌株的总ＤＮＡ为模板进行ＰＣＲ扩增时，除Ａ     

我国家居蜚蠊细菌的研究

从我国不同地区的147头家居蜚蠊中分离获得562株细菌分属于13个属,其优势菌群属于埃希氏菌属、芽抱杆菌属、短杆菌属共422株菌,占总菌数的75．09％;其次为沙雷氏菌属、假单胞菌属共85株,占总数的15．12％;其他8个属仅43株菌,占总数的9．25％。比较了德国小蠊、美洲大蠊和黑胸大蠊及它们的体表、体腔、中肠和粪便所携带的细菌类群和数量,其结果有很大差异。该文讨论了蜚蠊细菌与人类疾病之间的关系。     

细薄星芒海绵中活性菌筛选及混合菌协同效应

通过平板涂布法从我国南海三亚海域细薄星芒海绵中筛选出104株海洋细菌,采用琼脂扩散法、纸片法和细胞浓度记数法进行抗菌活性筛选,发现23株对于大肠埃希氏菌、金黄色葡萄球菌、荧光假单胞菌、枯草芽孢杆菌、黑曲霉、白假丝酵母、宛氏拟青霉具有稳定的抗菌活性,占总细菌的22．2％;根据形态学与生化指标分析初步确认其中4株抗菌活性显著的A05、A08、A72、A75为芽孢杆菌。研究发现,海绵细菌在抗菌活性方面具有正向和负向的协同效应,其中A72-75组合对于白假丝酵母和荧光假单胞菌具有显著的正向协同效应。     

268例化脓性中耳炎分泌物细菌培养及对三种滴耳液的筛选

对２６８例（２８２耳）急性化脓性中耳炎分泌物进行需氧菌培养鉴定。分离出致病菌２３８株,阳性率８４．３％。其中革兰氏阳性菌１４８株,占６２．２％,以金黄色葡萄球菌为主,革兰氏阴性菌９０株,占３２．８％,以假单胞菌属为主。单一菌生长２１２例,复合菌生长２６例。２３８株细菌共分离出１９个种类细菌。我们对其中１２０例分离菌株进行体外药敏测定。对氧氟沙星敏感者Ⅲ株（占９２．９％）,对环丙沙星敏感者１０７株（     

植物青枯病菌细菌素的产生,性质及其利用

细菌素（Ｂａｃｔｅｒｉｏｃｉｎ）是细菌代谢过程中合成的、对同种或近缘种有特异性抑制作用的杀菌蛋白或多肽物质［１］。大多数植物病原细菌如放射农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒａｄｉｏｂａｃｔｅｒ）、密执安棒形杆菌（Ｃｌａｖｉｂａｃｔｅｒ ｍｉｃｈｉｇａｎｅｓｔｓ）、软腐欧文氏菌（Ｅｒｗｉｎｉａｃａｒｏｔｏｖｏｒａ、 Ｅ． ｃｈｒｙｓａｎｔｈｅｍｉ）、丁香假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓｓｙｒｉｎｇａｅ）、甘蓝黑腐黄单胞菌（Ｘａｎｔｈｏｍｏｎａｓ ｃａｍｐｅｓｔｒｉｓ）等都具有产生细菌素的能力［２］…     

丁香假单胞菌冰核基因inaQ变速箱启动子结构与活性分析

序列比对与结构预测显示丁香假单胞菌(Pseudomonas syringae)野生型菌株MB03的冰核基因inaQ启动子为一种在细菌中罕见的变速箱型启动子。通过克隆长度为522bp的inaQ基因启动子区(P522)并与绿色荧光蛋白基因gfp构建融合基因P522gfp后,在恶臭假单胞菌AB92019菌株中进行表达分析。结果表明,包含结构模块A-Box和B-Box的P522在该菌株中具有启动子活性,且在寡营养条件和较低温度下具有更高的活性,是一种可调控启动子。     

铜绿假单胞菌体外抑菌活性研究

目的 观察铜绿假单胞菌抗菌物质对鲍曼不动杆菌等细菌的体外抑菌效果.方法 用交叉条带实验方法检测了铜绿假单胞菌对鲍曼不动杆菌、耐甲氧西林表皮葡萄球菌和粪肠球菌的体外抑制活性.结果 铜绿假单胞菌对鲍曼不动杆菌、耐甲氧西林表皮葡萄球菌和粪肠球菌体外抑菌活性良好,10株铜绿假单胞菌中,有8株对鲍曼不动杆菌的抑制率均达到了100％.另外有8株对耐甲氧西林表皮葡萄球菌的抑菌率均为100％;有6株对粪肠球菌的抑菌率为100％.结论 铜绿假单胞菌对上述3种致病菌具有较强的抗菌活性,具有开发前景.     

Transgenic ice nucleation-active Enterobacter cloacae reduces cold hardiness of corn borer and cotton bollworm larvae

The ice nucleation (IN) gene iceA of Erwinia ananas 110 was integrated into the chromosomes of two Enterobacter cloacae strains (Enc1.2022 and Enc1.181). These two newly derived transgenic strains, designated Enc2022-I and Enc181-I, respectively, possessed ice nucleation activity at -2.5 degrees C, significantly higher than their parent strains (active at approx -10 degrees C or lower). After ingesting these transgenic bacteria, the mean supercooling points (SCPs) of corn borer and cotton bollworm larvae were -3 to -4 degrees C, significantly higher than those of untreated controls. The SCPs remained significantly elevated over the 9-day period after ingestion, which matched well with the efficient gut colonization of the bacteria during this period. All treated larvae froze and eventually died after exposure for 6 h to a temperature of -7 degrees C, and more than 95% died after 12 h at -5 degrees C. In contrast, few or none of the untreated control larvae froze and died under the same conditions. Furthermore, the growth ability of these transgenic ice nucleation-active (INA) En. cloacae strains on corn leaves was reduced, compared to that of wild-type epiphytic E. ananas, as revealed by pot tests conducted in both greenhouse and outdoor conditions. The stable colonization in insect guts and their lower affinity to plants would make these transgenic INA bacteria useful as a novel tool for biological control of insect pests in agricultural fields.     

Gut colonization by an ice nucleation active bacterium, Erwinia (Pantoea) ananas reduces the cold hardiness of mulberry pyralid larvae.

To evaluate the suitability of using ice nucleation active (INA) bacteria for the biological control of insect pests, the supercooling point (SCP) of larvae of mulberry pyralid, Glyphodes duplicalis, and silkworm, Bombyx mori, ingesting INA strains of Erwinia (Pantoea) ananas and Pseudomonas syringae was determined. Mean SCP of the guts of silkworm larvae ingesting INA strains of E. ananas ranged from -2.5 to -2.8 degrees C, being 5 degrees C higher than that in control treatments. Similarly, mean SCP of mulberry pyralid larvae ingesting INA strain of E. ananas, which can grow well in the gut, was -4.7 degrees C at 3 days after treatment, being 6.5 degrees C higher than that in control treatments. On the other hand, mean SCP of the larvae-ingesting INA strain of P. syringae, which cannot grow in the gut, was -9.0 degrees C at 3 days after treatment, rising by only 2.5 degrees C higher than that in the control treatments. In addition, more than 80% of the larvae of mulberry pyralid ingesting the INA strain of E. ananas froze and eventually died when exposed to -6 degrees C for 18 h, while only 36% of the larvae ingesting the INA strain of P. syringae, or approximately 20% of the control larvae, froze and died. Thus, the gut colonization by INA strains of E. ananas reduced remarkably the cold hardiness of the insects. These findings suggest that INA strains of E. ananas could be effective as a potential biological control agent of insect pests.     

冰核细菌在我国北方玉米上的消长动态规律

研究证明,菠萝欧文氏菌(Erwinia ananas)为我国北方玉米上优势冰核细菌种类,占总体INA细菌95 %以上。采用定量定性和定期取样分离方法,首次研究INA细菌在玉米上的消长动态规律。结果表明:玉米不同生长发育阶段是影响INA细菌在玉米上数量分布和消长动态变化的重要因素,以抽雄至成熟期间分布INA细菌数量最多,高达10 7～10 8CFU/ g,比拔节至抽雄期高出2～3个数量级,比苗期至拔节期高出4～5个数量级;同时还指出,玉米不同播期,对INA细菌数量分布影响显著,差异很大,其中INA细菌分布数量消长变化,以正常播种(1.9×10 7CFU / g) >中期播种(7.9×10 5CFU/ g) >晚期播种(5 .0×10 4 CFU/ g) ;研究指出,处于抽雄至成熟期间的玉米上分布的INA细菌数量最多,因此期间(8月上旬至9月下旬) ,气温逐渐降低,昼夜温差大,田间结露多,加上玉米处于成熟阶段,抗INA细菌能力弱,这些因素有利于低温(5～2 0℃范围内生长)型INA细菌生长繁殖,故使INA细菌分布数量最多     

A method for estimation of population densities of ice nucleating active Pseudomonas syringae in buds and leaves of mango

Active ice nucleation strains of Pseudomonas syringae pv. syringae have been associated with a necrotic disease in mango trees growing in Málaga (southern Spain). In this paper a simple multiple-tube test is described to estimate the number of active ice nucleation bacteria associated with plant tissues and, also from suspensions of isolated bacterial strains. This method is based on the most probable number technique developed for microbiological analysis of water. The tube test presented a higher detection sensitivity of active ice nuclei than the traditional drop-freezing test, because a larger amount of plant material could be analysed routinely. Both methods demonstrated a similar accuracy. A high correlation was obtained between the tube test-estimated number of ice nuclei and populations of Ps. syringae -like organisms enumerated on King's agar B.     

Phosphatidylinositol, a phospholipid of ice-nucleating bacteria.

The nature of the phospholipids of the various bacteria that have ice nucleation activity in supercooled water has been determined. The seven bacteria studied included Pseudomonas syringae, Erwinia herbicola, three Escherichia coli K-12 strains that are phenotypically Ice+ because they contain plasmids with different amounts of either P. syringae or E. herbicola cloned DNA, and two E. coli K-12 strains without cloned ice gene DNA. All five Ice+ bacterial strains contained small amounts (0.1 to 1.0% of the total phospholipids) of phosphatidylinositol (PI), a phospholipid not previously detected in E. coli, Pseudomonas, or Erwinia species. The Ice- E. coli strains also contained trace level of PI that amounted to 2 to 30% of the level found in the Ice+ E. coli strains. Extracts of Ice+ strains contained low but measurable activities of PI synthase, while the activities in Ice- strains amounted to only 8 to 12% or less of that found in extracts of Ice+ bacteria. The functioning of the ice gene apparently increased both the PI synthase activity and the PI content of Ice+ strains from low endogenous levels. The relative ice nucleation activity at -4 degrees C or above (class A nucleation activity) of all Ice+ strains was found to be proportional to their PI content. The addition of myo-inositol (5 x 10(-4) M) to synthetic culture media increased the class A nucleation activity of both Ice+ E. coli strains and P. syringae up to sevenfold but had no stimulating effect on ice nucleation at lower temperatures (class B and class C nucleation activities). If these cells after fusion with PI vesicles were incubated with an energy source, the class A nucleation activity increased 70-fold over that present before fusion. These results indicate that PI plays an important role in ice nucleation at warm temperatures and is a likely precursor or component of the class A structure.     

冰核细菌及冰核基因的应用研究进展

引起水由液态变为固态的物质称为冰核或成核剂。冰核种类繁多,目前已发现4属23种或变种的细菌、4属11种或变种的真菌和1种病毒,它们都具成冰活性。细菌冰核是一类蛋白质,也称冰蛋白,由细菌冰核基因编码。作为生物冰核领域的研究重点,冰核细菌的研究已涉及到促冻杀虫、防霜冻、植物病害等多个领域;同时冰核细菌已成功地应用于人工降雪、制冷和高敏检测等方面,具有广阔的应用前景。主要对冰核细菌的应用研究现状和发展进行综述。     

Molecular cloning of an ice nucleation gene from Erwinia ananas and its expression in Escherichia coli

Abstract An approximately 7 kbp genomic DNA fragment was cloned from an ice nucleation-active (ina) strain of Erwinia ananas and defined as to its restriction enzyme site. When the DNA fragment was introduced into E. coli MM294, a potent ice nucleation activity was expressed. Both 0.7 kbp truncation from the 5'-end and 1.7 kbp truncation from the 3'-end were also effective in expressing the ice nucleation activity in E. coli . Therefore, the resulting DNA fragment of approximately 5 kbp was considered to be an ina gene and named ina A. It existed as a unique gene in this strain of E. ananas . No corresponding ina gene existed in an ice nucleation-inactive strain of E. milletiae .     

冰核细菌(Erwinia ananas 110)冰核基因克隆和序列分析

从作者自行分离的冰核细菌（Erwinia ananas110)中克隆到我国第1个细菌冰核基因,并完成其序列测定和分析,所克隆基因编码区全长3921bp,编码1306aa,氨基酸序列明显分为3个区即N-端（161aa),C-端（41aa)的单一序列区和中部的高度重复序列R区（1104aa)。以16氨基酸为重复单元的R区占整个编码序列的84.5%。序列分析表明我们所克隆的基因为一个新冰核基因,将其命名为iceA。该基因已在GenBank上登录,登录号为：AF387802。     

香梨内生拮抗细菌的筛选及对梨火疫病的生防潜力

该研究以健康香梨树的新鲜花器、当年生新生枝条、叶片和果实为试验材料,采用植物组织内生细菌分离法获得内生细菌菌株,培养菌落,并采用平板对峙法初筛和发酵液复筛对梨火疫病菌(Erwinia amylovora)、梢枯病菌(Pseudomonas syringae pv.syringae)和腐烂病菌(Valsa mali var.pyri)具有抑菌作用的细菌;将筛选获得的拮抗梨火疫病菌的菌株通过喷施香梨离体花序和盆栽杜梨苗测定其防病效果,以发掘内生细菌的生防资源,为探索梨树病害的生物防治途径奠定基础。结果显示:(1)从香梨枝条、叶、花和果实组织中共分离出337个内生细菌菌株,其中有14株具有明显的抑菌作用;进一步筛选出对梨火疫病菌、梢枯病菌和腐烂病菌具有抑菌作用的细菌分别有8株、9株和4株,其中ZN5、SN19和HN9菌株对梨梢枯病菌和腐烂病菌均有拮抗作用,HN89菌株对3种病原菌都具有较强的抑菌作用;共筛选出对梨火疫病菌具有抑菌作用的7个菌株(SN37、HN89、HN98、HN143、HN126、TN68、TN16)和具有竞争作用的TN50菌株。(2)内生菌株TN50预处理(喷施)对预防香梨花腐的效果显著,其防效(52.36%)与农用链霉素(60.67%)相近;HN89的防效(39.66%)其次;内生菌株TN50、HN89和SN37预处理(喷施)能够显著降低盆栽杜梨苗嫩枝的枝枯率和病情指数(P<0.05),7~15 d的平均保护性防效分别为67.20%、54.32%和45.91%,治疗性防效分别为63.88%、52.10%和36.17%。(3)结合形态特征和16S DNA序列分析,TN50、HN89和SN37菌株分别鉴定为克雷伯氏菌属(Klebsiella sp.)、类芽孢杆菌属(Paenibacillus sp.)和假单胞杆菌属(Pseudomonas sp.)。