仙客来软腐病拮抗细菌鉴定及其生物防治效果

目的为筛选出仙客来软腐病拮抗细菌及评价其生防效果。方法通过分离和筛选,从温州泽雅高山基地采集健康的仙客来植株分离到13株内生细菌对仙客来软腐病病原菌有较强的抑制作用,其中菌株Y1活性最强且遗传稳定。通过形态特征、生理生化特性分析、16SrDNA序列测定及其系统发育分析研究。结果菌株Y1鉴定为芽胞杆菌,将菌株Y1以发酵液灌根方式回接正常仙客来植株,菌株Y1的发酵液处理仙客来软腐病的防效均大于60％以上。结论菌株Y1具有生防的特点,在花卉产业上具有应用潜力。     

Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities

To study the effect of plant growth on potato-associated bacteria, the composition and properties of bacteria colonizing the endosphere of field-grown potato were analyzed by a multiphasic approach. The occurrence and diversity of potato-associated bacteria were monitored by a cultivation-independent approach, using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of plant-specific communities. However, endophytic populations correlated to a certain extent with plant growth performance. Endophytes were also isolated from plants that grew well or grew poorly and were identified by partial sequencing of the 16S rRNA genes. A broad phylogenetic spectrum was found among isolates and differently growing plants hosted different bacterial populations. In an approach to investigate the plant-growth-promoting potential of potato-associated bacteria, a total of 35 bacteria were screened by dual testing for in vitro antagonism towards (i) the fungal pathogens Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum, and Phytophthora cactorum and (ii) the bacterial pathogens Erwinia carotovora, Streptomyces scabies, and Xanthomonas campestris. The proportion of isolates with antagonistic activity was highest against Streptomyces sp. (43%) followed by those against Xanthomonas sp. (29%). As all plants showed more or less severe disease symptoms of scab disease caused by Streptomyces scabies, we assume that the presence of the pathogen induced the colonization of antagonists. The antifungal activity of the isolates was generally low. The biotechnological potential of endophytic isolates assessed by their antagonistic activity and by in vitro production of enzymes, antibiotics, siderophores, and the plant growth hormone indole-1,3-acetic acid was generally high. Overall, seven endophytes were found to antagonize fungal as well as bacterial pathogens and showed a high production of active compounds and were therefore considered promising biological control agents.     

具有拮抗空肠弯曲杆菌能力鸡源乳酸菌的筛选及特性

【目的】从鸡粪中筛选具有拮抗空肠弯曲杆菌能力的乳酸菌,研究其肠道益生特性,探讨其对空肠弯曲杆菌鞭毛毒力因子的影响。【方法】利用牛津杯法测定40株鸡粪源乳酸菌菌株的抑菌活性以确定抑菌性能好的菌株,利用16S r RNA基因分析进行菌株鉴定,采用HT-29细胞测定菌株的细胞粘附能力,通过模拟胃肠液实验分析菌株对胃肠道环境的耐受性,利用扫描电镜分析乳酸菌无细胞提取物对空肠弯曲杆菌鞭毛毒力因子的影响。【结果】从鸡粪中分离得到40株菌株,进一步筛选得到X13、X14和G20等3株拮抗空肠弯曲杆菌能力较强的菌株,经16S r RNA基因序列分析分别鉴定为罗伊氏乳杆菌、唾液乳杆菌和鸡乳杆菌;HT-29细胞粘附实验表明X13、X14及G20的粘附指数分别为11.5、20.3和14.3个/细胞,均具有良好的粘附能力;3株乳酸菌对人工胃肠液均具有良好的耐受性;扫描电镜观察表明,与对照组相比,3株纯培养乳酸菌无细胞提取物均能抑制空肠弯曲杆菌鞭毛毒力因子的合成。【结论】从鸡粪中筛选得到了3株能有效抑制空肠弯曲杆菌生长并能抑制其鞭毛合成的乳酸菌,有望作为拮抗性饲用益生菌用于控制禽畜的空肠弯曲杆菌感染。     

Studies of the antagonistic activities of bacteria endemic to cereal seeds.

The seeds of two cultivars of rice and wheat were examined for the presence of bacteria antagonistic to the growth of fungal plant pathogens. A yellow-pigmented bacterium was found to predominate on rice seed cv. Sasashigure, and in pure culture strongly inhibited mycelial growth of a wide range of pathogens. A similarly inhibitory, cream-coloured bacterium was predominant on wheat seeds cv. Longbow. This paper describes the isolation and range of antagonism of these organisms, and details their identification as a member of the Erwinia herbicola group and Pseudomonas syringae respectively.     

Changes in gene expression in canola roots induced by ACC-deaminase-containing plant-growth-promoting bacteria

The technique of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) was used to study changes in gene expression over time in canola roots treated with the 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant-growth-promoting bacterium Enterobacter cloacae UW4 and to compare the changes with those in a mutant of E. cloacae UW4 in which the ACC deaminase structural gene acdS was replaced by homologous recombination with acdS with an intentional knockout containing a tetracycline resistance gene. Genes that were either up- or down-regulated over a three-day period in canola plants treated with wild-type or mutant bacteria were isolated, cloned, and sequenced; all appeared to have high homology with Arabidopsis thaliana genes. The upregulated genes included a cell division cycle protein 48 homolog and a eukaryotic translation initiation factor 3 subunit 7 gene homolog. The downregulated genes included one encoding a glycine-rich RNA binding protein with a function in RNA processing or binding during ethylene-induced stress, which is expressed only in roots, and another gene thought to be involved in a defense signaling pathway. All RAP-PCR results were verified using Northern blotting. These data, indicate that roots isolated from canola seeds treated with the ACC deaminase-producing E. cloacae UW4 upregulate genes involved in cell division and proliferation but down-regulate stress genes. This data is in agreement with a model in which ACC deaminase-containing plant-growth-promoting bacteria reduce plant stress and induce root elongation and proliferation in plants, largely by lowering ethylene levels.     

Phosphate-solubilizing bacteria improve the phytoremediation efficiency of Wedelia trilobata for Cu-contaminated soil

In a controlled experiment, we assessed the effect of phosphate-solubilizing bacterium (PSB) on the soil metal (Cu²⁺) phytoremediation by Wedelia trilobata and examined the effect of the interaction of Cu contamination and PSB on the growth of W. trilobata. We also explored the effect of the interaction of Cu contamination and PSB on the soil microflora. The results showed that the removal efficiency of Cu from soil by W. trilobata increased with an increase in the concentration of PSB, and the translocation factors of Cu (i.e., leaf:root and stem:root) were both significantly upregulated by PSB. The PSB significantly promoted the growth of W. trilobata; however, the effect of the Cu–PSB interaction on the leaf net photosynthetic rate (Pn) of W. trilobata was not significant, whereas copper contamination had a significant negative influence on the soil microflora, PSB had a significant positive influence on the soil microflora. Thus, PSB improved the phytoremediation efficiency of W. trilobata in Cu-contaminated soil because of the positive influence on the soil microflora, improving soil quality, which then increased the growth of W. trilobata in Cu-contaminated soil. The vigorous growth of W. trlobata led to higher of Cu absorption and translocation from soil as the ultimate result.     

Long-term survival of the plant-growth-promoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant

Two plant-growth-promoting bacteria, Azospirillum brasilense Cd and Pseudomonas fluorescens 313, immobilized in 1983 in two types of alginate-bead inoculant (with and without skim-milk supplement) and later dried and stored at ambient temperature for 14 years, were recovered in 1996. The population in each type of bead had decreased, yet significant numbers survived (10⁵–10⁶ cfu/g beads). Population numbers depended on the bead type and the three independent bacterial counting methods: the conventional plate-count method, indirect enzyme-linked immunosorbent assay and the limited-enrichment technique. Both bacterial species retained several of their original physiological features. When inoculated onto wheat plants, both species colonized and produced plant-growth effects equal to those of the contemporary strain from a culture collection or to their own 1983 records. This study showed that bacteria can survive in alginate inoculant over long periods. Received: 1 May 1998 / Received revision: 24 August 1998 / Accepted: 3 September 1998     

Characterization of biosurfactant-producing strains of fluorescent pseudomonads in a soilless cultivation system

The use of biosurfactants is a promising alternative in biological control of zoospore-producing plant pathogens. In the present study, biosurfactant production by the indigenous population of fluorescent pseudomonads in a soilless plant cultivation system was studied during the growing season. A total of 600 strains was screened and of these 18.5% were observed to produce biosurfactants. Production of both antibiotics and biosurfactant was uncommon among the isolated strains. A selective effect of the cultivation system filter was observed on the biosurfactant-producing strains and these strains were only occasionally observed after the filter, despite having a significantly higher motility than the nonbiosurfactant-producing strains. The majority of biosurfactant-producing strains were isolated from the filter skin, which suggests that this is a suitable surface for inoculation with biocontrol strains.     

Biological control of sheath-blight of rice in india with antagonistic bacteria

Strains of fluorescent and nonfluorescent bacteria that were isolated from rice rhizospheres of Southern India and showed antagonism towardsRhizoctonia solani were evaluated for biological control of rice sheath-blight (ShB). Efficient strains of bacteria inhibited mycelial growth ofR. solani, affected sclerotial viabilityin vitro and protected IR 20 and TKM 9 rice seedlings from infection byR. solani in greenhouse tests. Pretreatment of sclerotia in bacterial suspensions resulted in reductions in ShB lesion sizes up to 31 to 44% in IR20 and 58 to 74% in TKM 9 rice. In field plots, IR 50 and TKM 9 rice plants raised from bacterized seeds had 65 to 72% less ShB than those plants from untreated seeds.     

Novel diphenylalkyl piperazine derivatives with dual calcium antagonistic and antioxidative activities

Two types of novel diphenylalkyl piperazine derivatives containing the thio or aminopropanol moiety substituted by phenyl or benzyl group were synthesized, and evaluated for their calcium antagonistic and antioxidative activities. These compounds showed apparent inhibitions against KCl-induced contractions in isolated rat aorta. Among them, phenylamino compound 9 and benzylamino compound 13 also possessed potent inhibitory activities against auto-oxidative lipid peroxidations in canine brain homogenates. Two representative compounds 3a and 9 were evaluated for their inhibitory activities against KCl-induced contractions in isolated canine arteries (basilar, coronary, mesenteric, and renal). Both compounds showed the most potent inhibitions to basilar artery.     

Intestinal bacteria antagonistic to Clostridium difficile in mice

Overgrowth by Clostridium difficile has been reported in conventional mice injected intraperitoneally with ampicillin. In this study, we aimed to determine which types of indigenous intestinal bacteria were eliminated by ampicillin to allow overgrowth by C. difficile. C. difficile overgrowth was associated with a decrease in the numbers of lactobacilli, an increase in bacteroidaceae and a slight decrease in the frequency of isolation of fusiform-shaped bacteria (clostridia). C. difficile cytotoxin was detected in caeca from mice in which the numbers of C. difficile were greater than 10(5) per gram of faeces. Gnotobiotic mice were inoculated with various groups of intestinal anaerobes to determine which members of the indigenous flora would antagonize C. difficile. Gnotobiotic mice inoculated with three strains of lactobacilli, 37 strains of bacteroides or 46 strains of clostridia isolated from limited-flora mice were unable to eliminate C. difficile. C. difficile was eliminated, however, from the gastrointestinal tracts of gnotobiotic mice inoculated with whole faeces or chloroform-treated faeces from conventional mice or whole faeces from limited-flora mice containing only clostridia.     

The antagonistic action of Trichoderma sp. hyphae to Lentinula edodes hyphae changes lignocellulotytic activities during cultivation in wheat straw

Lentinula edodes (Berk.) Pegler was cultivated in sterilized or pasteurized wheat straw both with and without inoculation with Trichoderma sp. Enhancements of -mannosidase and laccase activities and lowering of Mn-dependent peroxidase activity were observed seven days after inoculation in substrates inoculated with Trichoderma sp. These enzymes were not produced by Trichoderma sp. Most of the polysaccharidase activities were higher in substrates with Trichoderma sp. than in absence of Trichoderma sp. The area of the substrate contaminated with T. harzianum significantly correlated with cellulase, laccase and Mn-dependent peroxidase activities measured in the substrate. The increase of cellulase activity was due to enzymes produced by Trichoderma sp. and the decrease of Mn-dependent peroxidase activity was due to diminished growth of L. edodes. The stimulation of laccase activity was linked with the formation of brown lines (oxidation of polyphenols) at the contact between the mycelia of the two antagonists.     

Impact of short-term acidification on nitrification and nitrifying bacterial community dynamics in soilless cultivation media

Soilless medium-based horticulture systems are highly prevalent due to their capacity to optimize growth of high-cash crops. However, these systems are highly dynamic and more sensitive to physiochemical and pH perturbations than traditional soil-based systems, especially during nitrification associated with ammonia-based fertilization. The objective of this study was to assess the impact of nitrification-generated acidification on ammonia oxidation rates and nitrifying bacterial community dynamics in soilless growth media. To achieve this goal, perlite soilless growth medium from a commercial bell pepper greenhouse was incubated with ammonium in bench-scale microcosm experiments. Initial quantitative real-time PCR analysis indicated that betaproteobacterial ammonia oxidizers were significantly more abundant than ammonia-oxidizing archaea, and therefore, research focused on this group. Ammonia oxidation rates were highest between 0 and 9 days, when pH values dropped from 7.4 to 4.9. Pyrosequencing of betaproteobacterial ammonia-oxidizing amoA gene fragments indicated that r-strategist-like Nitrosomonas was the dominant ammonia-oxidizing bacterial genus during this period, seemingly due to the high ammonium concentration and optimal growth conditions in the soilless media. Reduction of pH to levels below 4.8 resulted in a significant decrease in both ammonia oxidation rates and the diversity of ammonia-oxidizing bacteria, with increased relative abundance of the r-strategist-like Nitrosospira. Nitrite oxidizers (Nitrospira and Nitrobacter) were on the whole more abundant and less sensitive to acidification than ammonia oxidizers. This study demonstrates that nitrification and nitrifying bacterial community dynamics in high-N-load intensive soilless growth media may be significantly different from those in in-terra agricultural systems.     

稻株上拮抗细菌的定殖及其对土著细菌的影响

在温室条件下,通过分批播种、接种纹枯病菌Rhizoctonia solani,以及在水稻分孽盛期喷雾拮抗细菌Pseudomonas fluorescens Pf7-14 (天然的抗萘啶酮酸菌株) 和B5423-R(Bacillus subtilis B5423的利福平抗性突变体)的菌悬浮液,并通过定期取样,平板系列稀释法回收,测定了菌株Pf7-14、B5423-R和土著细菌群体在水稻健株和纹枯病株上的种群数量,所获结果如下:①当相同的浓度(约2.0×108cfu/ml)的菌悬浮液喷雾到叶片时,菌株Pf7-14定殖的时间比菌株B5423-R长,且在相同的时间内,菌株Pf7-14的平均群体数量高于菌株B5423-R;②在健康的水稻茎部,菌株Pf7-14的两个高、低不同浓度处理的平均群体数量均表现出随时间降低的趋势;相比,较低浓度(4.0×107cfu/ml)的B5423-R在茎部的平均群体数量随着时间的下降,而较高浓度(2.0×108cfu/ml)的B5423-R的平均群体数量在水稻乳熟至黄熟期保持稳定或略有增长;③当病斑面积占取样茎面积的比率为20%～35%时,在应用1和14d后菌株Pf7-14在健茎的平均群体数量分别是病茎的6倍多和2倍多,差异均达到显著性的水平(P=0.05),而菌株B5423-R 在应用1d后病茎的数量比在健茎显著性地低大约1倍,但在7～14d后,病茎的数量比在健茎显著性地高5～6倍,群体在病茎表现出相对的增长;④土著细菌群体在病斑茎部是健茎的6～7倍.这些结果表明两类拮抗细菌有着明显不同的定殖习性,在病斑上B5423比Pf7-14具有更强的竞争能力,是一类更好的生防制剂;同时表明引入的拮抗细菌同土著细菌群体在营养和空间上竞争激烈,且土著细菌群体更具有竞争优势.     

不同产地美花石斛内生细菌分离及促生潜力比较

对采自广西、云南和广东的美花石斛(Dendrobium loddigesii Rolfe)野生植株根、茎和叶内的内生细菌进行分离并测定其促生特性,采用扩增核糖体DNA限制性酶切分析(ARDRA)与UPGMA聚类分析相结合的方法对内生细菌菌株进行分类并确定优势属;在此基础上,对具有解磷、解钾和产生长素能力的菌株进行促生潜力评价。结果显示:从不同产地美花石斛植株不同部位共分离得到67株内生细菌菌株,其分布呈现出组织与地区的特异性;其中,来源于广西的植株中菌株数量最多(42株),分离自茎的菌株数量最多(34株)。67株内生细菌菌株可分为31个ARDRA簇,经16S rDNA序列比对鉴定为12个属,包括假单胞菌属(Pseudomonas)、微杆菌属(Microbacterium)、肠杆菌属(Enterobacter)、芽孢杆菌属(Bacillus)、鞘氨醇单胞菌属(Sphingomonas)、葡萄球菌属(Staphylococcus)、嗜冷杆菌属(Psychrobacter)、短波单胞菌属(Brevundimonas)、涅斯捷连科氏菌属(Nesterenkonia)、副球菌属(Paracoccus)、泛菌属(Pantoea)和沙雷氏菌属(Serratia),其中芽孢杆菌属、微杆菌属和肠杆菌属为优势属;来源于广西的植株中内生细菌的丰度与多样性均高于其他两地。在67株内生细菌菌株中,有30株菌株具有解无机磷和解有机磷的双重能力、22株具有解钾能力、24株具有产生长素能力,其中仅8株菌株兼具3种促生能力。组培实验结果显示:在培养基中接种1×106CFU·mL-1芽孢杆菌DLB20菌株,对株高2~3 cm和3~4 cm的美花石斛试管苗生长有促进作用,且更有利于株高3~4 cm试管苗的生根,表明具有解磷、解钾和产生长素能力的菌株对美花石斛试管苗有一定的促生潜力。     

Synonymous codon ordering: a subtle but prevalent strategy of bacteria to improve translational efficiency

Background

In yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB) is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes.

Methodology/Principal Findings

In this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes.

Conclusions

Overall, a high level of synonymous codon pairing bias was detected in 73% investigated bacterial species, suggesting the synonymous codon ordering strategy has been prevalently adopted by prokaryotes to improve their translational efficiencies. The findings in this study also provide important clues to better understand the complex dynamics of translational process.     

相似穿孔线虫拮抗细菌的筛选及分子鉴定

【背景】相似穿孔线虫是一种重要的检疫性有害生物,其寄主范围广,危害具有毁灭性,已给我国农业生产造成潜在威胁。由于目前大多化学杀线剂毒性较高,筛选获得对相似穿孔线虫具有拮抗活性的微生物菌株对线虫防控具有重要意义。【方法】广泛从染病香蕉植株根际采集土壤样本,运用等比稀释涂布平板法分离菌株;采用摇瓶发酵法制备拮抗菌株上清液和发酵液。通过室内生物测定和盆栽试验,筛选出对相似穿孔线虫具有拮抗作用的细菌菌株,并通过16S rDNA的PCR扩增和序列分析,对拮抗菌株进行种类鉴定。【结果】获得5株对相似穿孔线虫具有较理想拮抗活性的细菌菌株。其中,菌株HD-86的作用效果最好,其发酵上清液处理24 h后的相似穿孔线虫死亡率达100%。盆栽试验表明,菌株HD-86发酵液处理42和70 d后对相似穿孔线虫的相对防效分别为77.34%和90.51%,均优于对照药剂阿维菌素。【结论与意义】菌株HD-86对相似穿孔线虫具有很强的拮抗作用,将其鉴定为洋葱伯克氏菌Burkholderia cepacia。本研究可为相似穿孔线虫生防制剂的研发和病害的防控提供依据。     

Biological control of bacterial wilt caused by Pseudomonas solanacearum in India with antagonistic bacteria

From among 125 strains of fluorescent and 52 strains of nonfluorescent bacteria initially screened in the laboratory for their antibiosis towards the bacterial wilt pathogen, Pseudomonas solanacearum, strain Pfcp of Pseudomonas fluorescens and strains B33 and B36 of Bacillus spp., were chosen and evaluated further in greenhouse and field tests. Pfcp treated banana (Musa balbisiana), eggplant and tomato plants were protected from wilt upto 50, 61 and 95% in greenhouse and upto 50, 49 and 36% respectively in field. Protection afforded by the Bacillus strains was lower. In bacteria-treated plants which were subsequently inoculated with P. solanacearum plant height and biomass values increased and were close to those of nontreated and noninoculated control plants.