Extracellular biosynthesis of silver nanoparticles using a novel and non-pathogenic fungus,Neurospora intermedia: controlled synthesis and antibacterial activity

In the present study, the biosynthesis of silver nanoparticles (AgNPs) using Neurospora intermedia, as a new non-pathogenic fungus was investigated. For determination of biomass harvesting time, the effect of fungal incubation period on nanoparticle formation was investigated using UV–visible spectroscopy. Then, AgNPs were synthesized using both culture supernatant and cell-free filtrate of the fungus. Two different volume ratios (1:100 and 1:1) of the culture supernatant to the silver nitrate were employed for AgNP synthesis. In addition, cell-free filtrate and silver nitrate were mixed in presence and absence of light. Smallest average size and highest productivity were obtained when using equal volumes of the culture supernatant and silver nitrate solution as confirmed by UV–visible spectra of colloidal AgNPs. Comparing the UV–visible spectra revealed that using cell-free filtrate for AgNP synthesis resulted in the formation of particles with higher stability and monodispersity than using culture supernatant. The absence of light in cell-free filtrate mediated synthesis led to the formation of nanoparticles with the lowest rate and the highest monodispersity. The presence of elemental silver in all prepared samples was confirmed using EDX, while the crystalline nature of synthesized particles was verified by XRD. FTIR results showed the presence of functional groups which reduce Ag⁺ and stabilize AgNPs. The presence of nitrate reductase was confirmed in the cell-free filtrate of the fungus suggesting the potential role of this enzyme in AgNP synthesis. Synthesized particles showed significant antibacterial activity against E. coli as confirmed by examining the growth curve of bacterial cells exposed to AgNPs.     

库尔勒香梨黑头病拮抗菌的筛选和鉴定

【背景】库尔勒香梨黑头病是近年来发现的一种由芸薹生链格孢菌(Alternaria brassicicola)XL2引起的采后病症,由于其高侵染率和高腐烂率造成了极大的经济损失,目前已成为库尔勒香梨采后储运的主要防治病症之一。【目的】发掘高效的库尔勒香梨黑头病拮抗菌,探索拮抗菌株的抑菌作用,为其生物防治提供潜在资源菌。【方法】从采后健康果蔬表面分离不同微生物,采用平板对峙法,以A.brassicicola XL2为靶标菌筛选具有拮抗作用的菌株,结合形态学观察、生理生化检测和16S rRNA基因序列分析鉴定拮抗菌株分类地位;检测拮抗菌无菌滤液对A. brassicicola XL2的抑制效应,显微观察拮抗菌对A.brassicicola XL2菌丝生长的影响;验证拮抗菌发酵液在库尔勒香梨果实上的抑菌活性。【结果】从新疆油桃表面分离获得90株菌,其中菌株Y2对A. brassicicola XL2有较强拮抗作用,经鉴定其为枯草芽孢杆菌(Bacillus subtilis)。菌株Y2的无菌滤液对A. brassicicola XL2菌落生长具有明显抑制作用,2%的无菌滤液抑菌率达到70.96%;Y2无菌滤液造成A.brassicicola XL2菌丝扭曲变形、分枝增加、尖端出现致密结构等异常现象;Y2发酵液和无菌滤液明显抑制A.brassicicola XL2的孢子萌发;Y2发酵液在库尔勒香梨果实上具有较高抑菌活性,对库尔勒香梨病斑直径抑制率达到37.66%,深度抑制率达到42.74%。【结论】枯草芽孢杆菌(B.subtilis)Y2能有效抑制A. brassicicola XL2的生长,对库尔勒香梨黑头病具有显著的生物防治效果。     

一株马铃薯干腐病拮抗菌的筛选、鉴定及其生物防效

【背景】马铃薯干腐病是一种由镰刀菌引起的田间和储藏期都普遍发生的病害,主要引起块茎腐烂,致使马铃薯品质和产量降低,严重影响其食用价值和经济价值。【目的】发掘有效的生防菌株以控制马铃薯干腐病,并探究其抑菌作用。【方法】从甘肃定西地区马铃薯根际土壤中分离到109株细菌,以硫色镰刀菌(Fusarium sulphureum)为靶标菌,采用平板对峙法筛选拮抗菌,并通过形态学、生理生化特征及16S r RNA基因序列分析对拮抗菌株进行鉴定。检测拮抗菌无菌发酵液对F.sulphureum菌丝生长、孢子萌发、马铃薯块茎损伤接种病斑直径、干腐病发病率及对绿豆种子发芽的影响。【结果】筛选到一株对马铃薯干腐病有较强抑制作用的菌株YL11,经鉴定其为假单胞菌属(Pseudomonas sp.)菌株。YL11菌株无菌发酵液对F.sulphureum菌丝生长、孢子萌发、马铃薯块茎病斑扩展、干腐病发病率、毒素活性均有显著抑制作用。20%无菌发酵液对F.sulphureum菌落生长的抑制率达到87.3%;75%无菌发酵液可完全抑制孢子萌发;无菌发酵液浸泡能有效抑制马铃薯干腐病病斑的扩展,14 d时对病斑扩展的抑制率达到67.1%;90 d后干腐病的发生率降低了68.4%;同时降低了F.sulphureum毒素的活性。【结论】拮抗菌株YL11能显著抑制F.sulphureum的生长,对马铃薯干腐病有较强的生物防治效果,具有潜在的应用价值。     

溶藻细菌BS03(Microbulbifer sp.)对塔玛亚历山大藻生长及抗氧化系统的影响

【目的】研究溶藻细菌BS03(Microbulbifer sp.)胁迫下塔玛亚历山大藻细胞光合作用、抗氧化酶系统和半胱氨酸蛋白酶3(Caspase-3)变化,探讨溶藻细菌BS03对塔玛亚历山大藻的溶藻机制。【方法】通过0.5%、1.0%、1.5%、2.0%不同终浓度BS03上清液处理藻细胞后12、24、36、48h取样,测定溶藻过程藻细胞光合色素、叶绿素荧光效率、抗氧化酶系统、Caspase酶活性变化。【结果】(1)BS03上清液处理藻细胞后,藻细胞叶绿素a含量和叶绿素荧光Fv/Fm比值随BS03上清液处理时间延长和浓度的增加呈逐渐下降趋势;低浓度处理组藻细胞类胡萝卜素含量上升到一峰值,高于对照组后逐渐回落,而高浓度处理组类胡萝素含量呈下降趋势,低于对照组;(2)藻细胞抗氧化酶保护系统(SOD和CAT)活性随着BS03上清液处理浓度增加而升高,但随着处理时间的延长呈现先上升后下降趋势。藻细胞膜脂过氧化产物MDA积累量随着BS03上清液处理时间延长和处理浓度的增加而显著提高;(3)处理组藻细胞Caspase-3活性显著高于对照组,呈现出类似程序性死亡特征。【结论】BS03的抑藻机理可能是通过抑制藻细胞光合作用,降低抗氧化酶活性、加大膜脂过氧化起到对塔玛亚历山大藻的溶解作用,并呈现出类程序性死亡特征。     

Mechanism of Isolated Hemicellulose and Xylan Degradation by Cellulolytic Rumen Bacteria

Although certain strains of cellulolytic rumen bacteria cannot utilize isolated hemicelluloses or xylan as a source of energy, all strains examined can degrade or solubilize these materials from an 80% ethyl alcohol insoluble to a soluble form. Centrifugation and washing of the cellobiose-grown bacterial cells did not affect the rate or extent of utilization or degradation or both. When the level of a nonutilizing culture inoculum (either normal or washed) was doubled, a corresponding increase in the initial rate of degradation was observed. With a nitrogen-free medium, utilization of xylan was almost completely inhibited for a utilizing strain, whereas degradation by either type of organism was not markedly affected. Cellobiose medium cell-free culture filtrates from a nonutilizing strain were able to degrade or solubilize xylan. The percentage of degradation increased with the volume of cell-free filtrate, and all activity was lost when the filtrate was boiled. No utilization (loss in total pentose) was observed with cell-free filtrates from utilizing or nonutilizing strains. The release of free hexose from insoluble cellulose by culture filtrates from a nonutilizing strain was very limited. On the other hand, carboxymethylcellulose (CMC-70L) and cellulodextrins were degraded to an 80% ethyl alcohol soluble form by filtrates from both types of organisms. Similar enzyme activity was obtained in cell-free culture filtrates from four additional strains of cellulolytic rumen bacteria (one xylan utilizer and three nonutilizers). When the assays were carried out aerobically, CMC-70L solubilization was reduced to a much greater extent than xylan or cellulodextrin solubilization. The enzyme or enzymes responsible for the degradation of hemicellulose by cellololytic rumen bacteria unable to utilize the hemicellulose as an energy source appear to be constitutive in nature, and this activity may be a nonspecific action of a beta-1, 4-glucosidase or -cellulase.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle.

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Induction of trap formation in nematode-trapping fungi by a bacterium

Three soil bacterial strains were identified as Chryseobacterium sp. TFB on the basis of their 16S rRNA gene sequences. Conidia of Arthrobotrys oligospora produced a few mycelial traps (MT) and conidial traps (CT) when cultured with bacterial cells that they did not produce when cultured with a bacterial cell-free culture filtrate. However, co-culture of A. oligospora with bacterial cells and bacteria-free filtrate simultaneously induced MT and CT in large amounts. With the increased concentration of bacteria-free filtrate, the number of typical CT increased, but conidial germination was progressively inhibited. Scanning electron microscopy of A. oligospora co-cultured with bacteria revealed that bacterial attachment to hyphae was a prerequisite to trap formation and that bacteria-free filtrate facilitated bacterial attachments to hyphae. The results that the addition of nutrients in co-culture medium decreased the number of traps suggest that this type of trap formation may be favoured at a low nutrient status. Eight fungi tested were able to form MT and CT when co-cultured with bacterial cells and bacteria-free culture filtrate, but the abilities varied among species. This study provides novel evidence that under laboratory conditions, soil bacteria attaching to hyphae could induce traps in nematode-trapping fungi.     

Umbilical Cord Leucocytes transformed by Lymphoid Cell Filtrates from Healthy People

SEVERAL investigators have shown that a cell-free filtrate, or lethally irradiated whole cells, from Burkitt's lymphoma or cells from acute leukaemic blood can transform human leucocytes in vitro^1–6. Transformed leucocytes can multiply rapidly and, perhaps indefinitely, in vitro. Recently, my associates and I reported that a cell-free filtrate prepared from the Kaplan cell—an established line derived from the blood of an infectious mononucleosis patient—can also transform leucocytes⁷. Furthermore, we described a simple procedure for the investigation of leucocyte transformation⁸. Using this technique, it has been possible to show that a cell-free filtrate prepared from lymphoid cell lines of healthy individuals can transform umbilical cord blood leucocytes.     

Cysteine peptidases, secreted by Trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture

Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.     

Inhibitory efficacy of endophytic Bacillus subtilis EDR4 against Sclerotinia sclerotiorum on rapeseed

Stem rot, caused by Sclerotinia sclerotiorum, is a serious disease of rapeseed worldwide. This paper tested the inhibitory effect of an endophytic bacterial Bacillus subtilis strain, EDR4, on the sclerotial germination and hyphal growth of S. sclerotiorum. The cell-free filtrate solution and cell suspension of strain EDR4 were sprayed on rapeseed leaves and stems one day before, at the same time and one day after inoculation in the greenhouse experiments. There was no significant difference in inhibitory efficacy between the cell-free filtrate solution and cell suspension. The best biocontrol efficacy was achieved by spraying either the cell-free filtrate solution or cell suspension at the same time as inoculation. In the field trials, the efficacy of two applications of EDR4 cell suspension at the initial flowering stage and full bloom stage was the best, but there was no significant difference in efficacy between the one-application and two-application treatments during the initial flowering stage. The efficacy decreased gradually with the culture suspension dilutions. Scanning electron microscopy revealed that EDR4 cells significantly suppressed the hyphal growth. The bacterial treatment caused shrink, cytoplasm leakage and irregular tip swelling of fungal hyphae. The hyphal cells in the treated groups had higher numbers of vacuoles in the cytoplasm than the non-treated hyphal cells. The hyphal cytoplasm was disintegrated; the hyphal biomass was reduced; the formation of infection cushions was delayed; and the infection was suppressed after spraying the bacterial culture on rapeseed leaves. The results showed that the EDR4 bacterial strain could be used to control stem rot of rapeseed.     

铜绿微囊藻(Microcystis aeruginosa)对惠氏微囊藻(Microcystis wesenbergii)的化感作用

通过混合培养和添加过滤液两种方式观察铜绿微囊藻和惠氏微囊藻的生长曲线,探讨两种微囊藻之间的化感作用。结果表明:在混合培养条件下,两者能够形成相互抑制作用;当两者起始藻密度高于0.5×106cells.mL-1、混合比为1:1时,惠氏微囊藻的生长因化感作用而受到显著抑制(P<0.05),同时惠氏微囊藻也会对铜绿微囊藻产生一定的胁迫作用;处于对数生长期的铜绿微囊藻过滤液能抑制惠氏微囊藻的生长,且惠氏微囊藻起始藻密度低于0.5×106cells.mL-1,连续滴加该过滤液后,其生长受到极显著抑制(P<0.01)。     

枣缩果病拮抗菌的鉴定及其拮抗效应

【目的】发掘有效生防菌株以控制枣缩果病,并探究生防菌株的拮抗特性。【方法】采用梯度稀释法和平板对峙法筛选出2株对枣缩果病菌细极链格孢(Alternaria tenuissima)具有显著拮抗作用的菌株STO-12和STO-45,通过PCR扩增得到其16S rRNA基因序列,进行同源比对分析和分子系统树的构建,并结合形态学观察和生理生化实验对菌株进行鉴定。采用皿内对峙、显微观察及蛋白质抑菌试验等测定了菌株STO-45的拮抗效应。【结果】两拮抗菌株STO-12和STO-45均鉴定为枯草芽孢杆菌,但菌株STO-12能产生橘红色色素,且两菌株在大小、生理生化特征及系统发育关系上具有生理差异。这两株拮抗细菌的发酵液、发酵上清液及发酵滤液均对枣缩果病菌具有显著抑菌活性。STO-45发酵滤液在0.8%的低体积浓度下即达到抑制中浓度,可使病原菌A. tenuissima MY5的菌丝及芽管部分畸形膨大,发酵液中的蛋白类物质可能是其拮抗物质之一。【结论】拮抗菌株STO-12和STO-45对枣缩果病的生物防治均具有潜在的应用价值,其抑菌机制及生物制剂的开发利用值得进一步研究。     

A convenient synthesis of 3-keto bile acids by selective oxidation of bile acids with silver carbonate-Celite.

A number of 3-keto bile acids were synthesized by the selective oxidation of bile acid methyl esters with silver carbonate-Celite in refluxing toluene. The pure 3-keto bile acids were isolated simply by filtering the reaction mixture and concentrating the filtrate. The relation of the bile acid structure to the oxidation rate is also discussed.     

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.     

Herbivore dung as food for dung beetles: elementary coprology for entomologists

1. How do dung beetles and their larvae manage to subsist on herbivore dung consisting of plant remains that are at least partly indigestible, mixed with various metabolic waste products? To clarify what is known and not known about this basic aspect of dung beetle biology, the present review summarises information on dung composition and discusses the feeding of beetles (food: fresh dung) and larvae (food: older dung) in relation to this information. 2. There is 70–85% water in typical fresh dung, and undigested lignocellulose or ‘fibre’ constitutes about 70% of the organic matter which also contains 1.5–3% N. About 75% of this is ‘metabolic faecal nitrogen’, mostly associated with dead and alive microbial biomass. As all essential amino acids and cholesterol are probably present, additional synthesis by microbial symbionts may not be needed by the beetles. 3. Beetles minimise the intake of lignocellulose by filtering fibre particles out of their food which is probably microbial biomass/debris with much smaller particle size. Excess fluid may be squeezed out of this material by the mandibles before ingestion. 4. All larvae are bulk feeders and unable to filtrate, but little is known about the composition of their food, i.e. older dung in pats or underground brood masses. Larvae in dung pats may depend on easily digestible dung components, probably microbial biomass, whereas the nutritional ecology of larvae in brood masses is still not understood. Unravelling the composition of their food might answer some of the so far unanswered questions.     

Relationship between macrotetrolide production and specific activity of some hydrolases in a high and a low producing strain of Streptomyces griseus

The activities of five hydrolytic enzymes in the culture filtrate and in cell-free extracts from strains of Streptomyces griseus, differing in macrotetrolide production, have been determined over a fermentation period of 200 h. The specific activities of phosphatase, phosphodiesterase, and adenosine triphosphatase in the medium, and phosphatase and phosphodiesterase in the cell-free extract were lower in the low than in the high producing strain. No significant difference was found between the strains, for adenosine triphosphatase and protease activity in the cell-free extract or protease activity in the medium. The specific activity of esterase was higher in the low than in the high producing strain.     

强壮前沟藻化感物质分析

微藻化感作用是一种极其复杂的生理、生态学现象。选取强壮前沟藻指数生长初期Ⅰ和平台生长初期Ⅱ两个阶段的滤液对中肋骨条藻、海洋原甲藻、锥状斯氏藻及球等鞭金藻生长的影响进行了研究,并萃取了阶段Ⅱ的粗提物,抑藻检测表明其具有"杀藻"效应,通过GC/MS分析该粗提物中具有潜在化感作用的物质种类。研究发现强壮前沟藻两个生长阶段的滤液对中肋骨条藻均产生强烈致死效应(phaseⅠ:F=15.18475,P=0.00298<0.05;phaseⅡ:F=6.24559,P=0.03149<0.05);锥状斯氏藻在强壮前沟藻滤液中生长,实验结束时两个阶段中的细胞密度分别是对照组的79.3%和68.9%;海洋原甲藻在强壮前沟藻生长阶段Ⅱ滤液实验的最后3d,其生长受到显著抑制(F=4.84438,P=0.04925<0.05);而等鞭金藻在强壮前沟藻两个生长阶段滤液中被抑制现象不明显(P>0.05)。强壮前沟藻滤液实验表明,强壮前沟藻能够向微环境中分泌代谢产物来抑制中肋骨条藻和海洋原甲藻的生长,并且这种抑制效应具有种类特殊对应性。上述实验结果还表明,强壮前沟藻生长阶段Ⅱ的滤液具有的生长抑制作用较为明显。采用乙酸乙酯萃取强壮前沟藻生长阶段Ⅱ滤液中的代谢产物,检测发现其代谢粗提物具有溶藻效应,GC/MS分析结果表明粗提物中存在4种可能产生化感抑制作用的物质,其中二丁基羟基甲苯(Butylated Hydroxytoluene BHT)被认为具有抗滤过性病原体和抗微生物活性。     

Functions of lipopeptides bacillomycin D and fengycin in antagonism of Bacillus amyloliquefaciens C06 towards Monilinia fructicola

In previous studies, Bacillus amyloliquefaciens C06 has been proven to be effective in controlling brown rot of stone fruit caused by Monilinia fructicola. When tested in vitro, cell-free filtrate of B. amyloliquefaciens C06 significantly inhibited mycelial growth and conidial germination of the fungal pathogen. This study aimed to determine the role of the antifungal compound(s) in the cell-free filtrate of B. amyloliquefaciens C06 by an approach combining a DNA-based suppression subtractive hybridization (SSH) method with MALDI-TOF-MS analysis. It was demonstrated that B. amyloliquefaciens C06 harbored two genes, bmyC and fenD, involved in biosynthesis of bacillomycin D and fengycin, two lipopeptides belonging to the iturin and fengycin family, respectively. To determine the roles of bacillomycin D and fengycin of B. amyloliquefaciens C06 in suppressing M. fructicola, the mutants of B. amyloliquefaciens C06 deficient in producing bacillomy- cin D, fengycin or both were constructed, and evaluated in vitro together with the wild-type B. amyloliquefaciens C06. The results indicated that bacillomycin D and fengycin jointly contributed to the inhibition of conidial germination of M. fructicola, and fengycin played a major role in suppressing mycelial growth of the fungal pathogen.     

Characteristics of a plant deleterious rhizosphere pseudomonad and its inhibitory metabolite(s)

A cell-free culture filtrate of the plant growth inhibitory bacterial isolate Å313, identified as Pseudomonas fluorescens, was tested for its effect on wheat root elongation in vitro, with and without various pretreatments. The filtrate showed a strongly inhibitory effect on root elongation and could be heated to 100°C for 5 min or incubated at a pH within the range of 4–10 without losing its activity. Unlike other root growth-inhibitory bacterial metabolites the effect of the filtrate was not reversed by methionine, nor was any inhibitory activity present in the water phase after extraction with chloroform. The inhibitory agent(s) was formed when the bacterium was grown in an artificial medium as well as in root exudates from wheat. Two wheat cultivars differing in reaction to inoculation with living cells of Å313 showed the expected difference in growth reduction when exposed to culture filtrate, indicating a cultivar difference in sensitivity to the metabolite(s) formed by the bacterium. Isolate Å313 invaded intercellular spaces of the root cortex of gnotobiotically grown wheat plants, but did not produce pectolytic enzymes in vitro, nor induce a hypersensitive response in tobacco plants.