Isolierung und Zählung von Bodenactinomyceten auf Erdplatten mit Membranfiltern

Summary A technique is described to separate actinomycetes from other micro-organisms with membrane filters on soil plates. A measured quantity of soil dilution was passed through a membrane filter and the filter with the adhering organisms was placed upside down on a medium provided with soil. After an incubation period of two weeks all actinomycetes had penetrated the filter developing colonies on the sterile surface, whereas the other organisms were retained by the filter. By this procedure the counting and isolation of actinomycetes has been facilitated. The selection of bacteriostatic active forms is practicable by stamping the crowded filter on a medium that contains test bacteria.

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(With a summary)     

Oligotrophy is Helpful for the Isolation of Bioactive Actinomycetes

It is necessary to develop new methods for the isolation of unknown actinomycetes from soils. To evaluate the effects of oligotrophic medium on the isolation of soil actinomycetes and develop a new isolation method, the Gause’s synthetic medium was diluted to one tenth the recommended concentration in the present study. Soil dilution plate technique was used to isolate actinomycetes from the soil samples. Oligotrophy decreased actinomycete and streptomycete counts, as well as the number of antagonistic actinomycete species. Oligotrophy also decreased the number of actinomycete species in five samples. Some actinomycete species were cultured only on the oligotrophic medium, whereas other species could not be cultured. Oligotrophy decreased actinomycete counts more significantly for soils with organic matter content >40 g/kg. We used 16S rRNA sequence analysis to identify 22 actinomycete species that were only cultured on the oligotrophic medium. Oligotrophic medium was helpful for the isolation of Streptomyces spp., Micromonospora spp. and Streptosporangium spp. Slightly more than 80 % of the identified actinomycete species were biologically active. Therefore, we could draw a conclusion that oligotrophic medium could be helpful for the discovery of new antibiotic producers and the exploitation and utilization of new, biologically active compounds.     

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.     

食细菌线虫对土壤微生物量和微生物群落结构的影响

线虫与微生物的相互作用研究往往是在悉生培养体系 (gnotobiotic microcosm)中进行 ,为了研究在自然或开放土壤条件下土壤线虫与微生物的相互作用 ,作者在开放盆栽体系中接种土壤食细菌线虫 (原小杆线虫 ,Protorhabditis sp) ,研究在小麦不同生育期、在有和无根系作用下食细菌线虫对土壤微生物量和微生物群落结构的影响。结果表明 :接种线虫分别使 SMBC、SMBN、SMBP提高了 2 6 .4 %、32 .9%、2 1.8% ,这种促进作用除个别无根系土和非根际土处理外 ,均达到显著性差异。根际土中的 SMBC、SMBN、SMBP>非根际土 >无根系土。从方差解释比例 v来看 ,SMBN受线虫的影响最大 (v=2 4 % )、其次是 SMBC(v=16 % )、然后是 SMBP(v=12 % ) ,线虫对 SMBC的促进作用在根际土中最突出。接种线虫对土壤细菌、真菌和放线菌的数量有明显的影响。在苗期的无根系土和根际土中 ,接种线虫显著降低了细菌的数量、特别在根际土中尤为突出 ,但在其它处理中却增加了细菌的数量。接种线虫对真菌和放线菌数量的促进作用比对细菌更为明显 ,接种线虫后真菌和放线菌数量的总平均值分别比未接种提高了 4 8.5 %和 6 8.2 % ,而细菌数量的总平均值没有变化。细菌数量与微生物量 C相关散点图表明二者相关性在根际土、非根际土和无根系土中均未     

华西雨屏区天然常绿阔叶林土壤可培养微生物数量对模拟氮沉降的响应

为了解氮沉降对华西雨屏区天然常绿阔叶林土壤微生物数量的影响,从2013年11月至2014年12月,通过野外模拟N(NH_4NO_3)沉降,氮沉降水平分别为对照(CK 0 kg N hm~(-2)a~(-1))、低氮沉降(L 50 kg N hm~(-2)a~(-1))、中氮沉降(M 150 kg N hm~(-2)a~(-1))和高氮沉降(H 300 kg N hm~(-2)a~(-1)),研究了氮沉降对天然常绿阔叶林0—10cm和10—20cm土层土壤可培养微生物数量的影响。结果表明:华西雨屏区天然常绿阔叶林0—10cm土层的细菌、真菌和放线菌数量均显著大于10—20cm土层,氮沉降未改变原有垂直分布格局。L处理对0—10cm和10—20 cm土层土壤微生物总量无显著影响,M和H处理则显著降低了土壤微生物总量。氮沉降降低了0—10cm和10—20cm土层的细菌数量,且抑制作用随氮沉降量的增加而增强。氮沉降降低了0—10cm土层的真菌数量,但下降幅度与氮沉降量之间无明显规律;在10—20cm土层,M和H处理在夏季显著增加了真菌数量,表明适量氮沉降能有效缓解夏季土壤真菌的氮限制状态。氮沉降对0—10cm土层放线菌数量的影响表现为先促进再抑制,L和M处理增加了放线菌数量,H处理降低了放线菌数量;氮沉降增加了10—20cm土层的放线菌数量,其中M处理的促进作用最大。氮沉降对土壤微生物数量的影响随土壤深度的增加而减弱。     

Microwave irradiation is a useful tool for improving isolation of actinomycetes from soil

Actinomycetes are an important source of novel, biologically active compounds. New methods need to be developed for isolating previously unknown actinomycetes from soil. The objective of this experiment was to study microwave irradiation of soil as a means for isolating previously unknown actinomycetes. Soil samples were collected at ten elevations between 800 m and 3670 m on Taibai Mountain, Shaanxi Province, China. Moistened soil samples were irradiated at 120 W heating power (2450 MHz) for 3 min using a household microwave oven. Irradiation increased total actinomycete, streptomycete, and antagonistic actinomycete counts on three types of culture media. Irradiation also increased the number of culturable actinomycete isolates. Some actinomycete isolates were culturable only after the soil was irradiated, whereas other isolates could not be cultured after irradiation. Irradiation of soil from elevations >3000 m increased actinomycete counts significantly but had little effect on the number of culturable actinomycete isolates. In contrast, irradiation of samples from elevations <3000 m had relatively little effect on actinomycete counts, but significantly increased the number of culturable actinomycete isolates. We used 16S rDNA sequence analysis to identity 14 actinomycete isolates that were only culturable after irradiation. Microwave irradiation of soil was helpful for isolating Streptomyces spp., Nocardia spp., Streptosporangium spp., and Lentzea spp. Slightly more than 90% of the identified actinomycete species were biologically active. In conclusion, microwave irradiation is a useful tool for isolating biologically active actinomycetes from soil.     

Application of actinomycetes to soil to ameliorate water repellency

AIMS: The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil. METHODS AND RESULTS: Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency. CONCLUSIONS: Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil.     

放线菌对干旱胁迫下黑麦草生长及抗氧化特性的影响

采用菌剂接种及盆栽生物实验,研究了干旱缺水条件下放线菌对黑麦草生长和抗氧化特性的影响。结果显示:(1)干旱胁迫下,土壤中接种放线菌显著促进黑麦草的生长,其中根分蘖及根部生物量分别显著增加35.00%和37.47%;(2)接种放线菌后,黑麦草叶片的叶绿素总量和类胡萝卜素含量分别显著增加了12.02%和10.38%;(3)加菌后3种主要的抗氧化酶超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)较不加菌对照均增加,其增率分别为12.72%、2.38%和24.83%,其中SOD和CAT的活性增加显著。研究表明,干旱胁迫下放线菌接种土壤后,提高了黑麦草叶绿素含量和类胡萝卜素含量,增强了黑麦草抗干旱胁迫的抗氧化酶活性,可显著促进黑麦草的生长,增加其生物量。     

微波处理对土壤放线菌分离效果的影响

采用稀释平皿分离测数及琼脂块法研究了微波预处理对土壤放线菌分离效果的影响.结果表明: 1)微波处理能显著增加可培养放线菌数量.随着微波处理时间的增加,高有机质土壤放线菌总数呈先增后减的趋势.处理3～15 min,GA及HA培养基上放线菌总数分别较对照提高8.3%～92.6%及24.4%～108.5%;处理18～24 min,放线菌总数分别较对照降低了62.1%～78.8%及41.4%～79.8%.微波处理对低有机质土壤放线菌数量无明显影响.2)微波处理对可培养放线菌种类有明显影响.随着微波处理时间的增加,高有机质土壤中新出现的放线菌种类呈先增后减趋势. 处理3～24 min,GA与HA培养基上新出现放线菌种类占放线菌总种类的比例为62.5%～85.7%与66.7%～83.3%,新出现了原小单孢菌属、链轮丝菌属等稀有放线菌属;链霉菌类群也有明显变化.低有机质土壤具有类似趋势.3)微波处理对可培养放线菌中拮抗性放线菌株数占供试放线菌总株数的比例也有明显影响.微波处理6、9和15 min,拮抗性放线菌株数所占比例分别较对照提高66.7%、66.7%和83.3%,其中新出现的拮抗性放线菌株数分别占拮抗性放线菌总株数的70.0%, 90.0%和81.8%.     

青海沙珠玉人工植被系统土壤放线菌生态分布及拮抗性

为探索人工植被重建对土壤放线菌的影响,采用稀释平板法及琼脂块法对青海沙珠玉地区植被重建后沙土中放线菌的生态分布及拮抗性进行了研究.结果表明：随着高寒干旱沙地植被重建和覆盖度的提高：土壤放线菌数量显著增加,其中退耕还草地土壤中放线菌总数较沙地提高145.4%,小单孢菌数量约为沙地的6倍;拮抗性放线菌平均筛出率大幅度增加,退耕还草地土壤中拮抗性放线菌、天然草地土壤中抗细菌放线菌及林地土壤中抗病原真菌放线菌的平均筛出率约分别为沙地的2、3.2及1.5倍;地表植被盖度与土壤养分对放线菌数量以及拮抗性放线菌数量有很大影响,其中有机质、碱解氮含量以及鲜草产量与放线菌总数的相关性达到极显著水平(P<0.01),有机质含量、鲜草产量与拮抗性放线菌株数的相关性达到极显著水平(P<0.01).此外,植被盖度、土壤全氮、全磷、全钾、全盐以及速效钾含量与放线菌总数、链霉菌数、小单孢菌数的相关性也达到显著水平(P<0.05).     

嗜酸丝状放线菌的选择性分离与多样性

摘要:【目的】针对酸性土壤中的嗜酸丝状放线菌,建立有效的选择性分离方法,并了解其多样性。【方法】用不同的样品预处理方式和分离培养基,并添加不同的抑制剂进行分离;根据放线菌的菌落数和出菌率确定最佳分离方法组合。采用最佳分离方法对从江西采集的17份酸性土壤样品进行分离;根据培养特征对分离菌株进行分群,进一步通过对各类群的显微形态观察和pH梯度生长实验确定代表菌株;对代表菌株进行16S rRNA基因序列分析研究其多样性。【结果】嗜酸丝状放线菌的最佳分离方法为：土壤样品经分散差速离心预处理后,涂布添加了放线菌酮、制霉菌素和萘啶酮酸（各50 mg/L）的GTV培养基。用此方法共分离到放线菌369株,归为10个不同的颜色类群,其中6.6%为严格嗜酸放线菌,72.4%为中度嗜酸放线菌,21.0%为耐酸放线菌。52株嗜酸放线菌代表菌株分布于放线菌目中的12个属：链霉菌属(Streptomyces)、小单孢菌属(Micromonospora) 、诺卡氏菌属(Nocardia)、野野村菌属(Nonomuraea) 、韩国生工属(Kribbella) 、小双孢菌属(Microbispora)、马杜拉菌属(Actinomadura)、拟无枝菌酸菌属(Amycolatopsis)、指孢囊菌属(Dactylosporangium)、伦茨氏菌属(Lentzea)、游动四孢菌属(Planotetraspora) 和链嗜酸菌属(Streptacidiphilus),其中链霉菌分离菌株在系统发育树上形成12个不同的进化类群。【结论】所建立的选择性分离方法可用于土壤嗜酸丝状放线菌的高效分离;江西酸性土壤含有丰富多样的嗜酸丝状放线菌种属。     

巨桉连栽对土壤微生物生物量和数量的影响

为探讨巨桉连栽对土壤微生物生物量和数量的影响,采用时空互换法,研究了马尾松(Pinus massoniana)林和不同连栽代次的巨桉(Eucalyptus grandis)人工林的微生物生物量、数量与土壤理化性质的关系。结果表明,巨桉一代林的土壤微生物生物量碳、生物量氮和土壤真菌、细菌数量与马尾松林的差异不显著,放线菌数量则显著增加。随巨桉连栽代次增加,土壤微生物生物量碳、生物量氮和土壤细菌、放线菌、真菌数量均递减。回归分析表明,土壤全磷能独立解释微生物生物量氮、细菌和真菌数量71.7%、86.1%和63.0%的变异,与总孔隙度共同解释微生物生物量碳87.9%的变异,与全氮共同解释放线菌数量89.6%的变异。可见,土壤全磷较大程度解释了微生物的变化。     

土壤微生物数量对模拟氮沉降增加的早期响应

通过以南亚热带森林三种主要树种即荷木、锥栗和黄果厚壳桂为主的苗圃试验地的苗圃控制实验,初步探讨土壤微生物数量对模拟氮沉降增加的响应。结果表明,施氮增加对土壤微生物数量的影响根据类群和氮处理水平不同而异。总的来说,施氮增加对土壤微生物数量具有促进作用,这种促进作用对放线菌数量仅在一定(中氮)处理水平以下,超过此水平施氮增加则表现为抑制作用,而施氮对真菌数量则始终表现为抑制作用,尤其以中N处理水平的抑制作用最强。但目前细菌仍占微生物总量的绝对优势,放线菌次之,真菌则占微生物总量的比例最小。分析结果还表明,有效氮与放线菌、有效氮与细菌数量变化呈显著相关关系。     

Selective medium with nalidixic acid for isolating antibiotic-producing Actinomyces

The majority of actinomycetes belonging to various genera proved to be resistant to nalidixic acid concentrations having an inhibitory effect on bacteria with trailing growth i.e. B. subtilis and B. mycoides. The bacteria prevented isolation of actinomycetes as pure cultures. The use of a selective medium with nalidixic acid for isolation of soil actinomycetes resulted in 20 per cent increase in the number of the actinomycetes isolated as pure cultures. Preliminary treatment of the soil samples with calcium carbonate under moist conditions followed by the inoculation to the medium with nalidixic acid made it possible to increase isolation of actinomycetes at most 100-fold. With this complex method 495 actinomycete cultures were isolated, their antibiotic properties were studied and their taxonomic position at the genus level was determined. The complex method including the preliminary treatment of soil samples with calcium carbonate followed by inoculation to the selective medium with nalidixic acid is efficient and may be recommended for screening organisms producing new antibiotics.     

有机物料厌氧发酵液中拮抗苹果再植障碍病原真菌的细菌筛选及其防治效果

有机物料厌氧发酵液(AFOF)能显著改善苹果再植障碍.本研究对AFOF中能拮抗苹果再植障碍主要病原菌(腐皮镰刀菌、层出镰刀菌、尖孢镰刀菌、串珠镰刀菌)的细菌进行了分离筛选,并对其作用效果进行了盆栽验证.结果表明: AFOF能显著抑制病原真菌的生长繁殖;对峙试验共得到4株具有较强拮抗作用的细菌(L11、L12、L13、L14),最高抑菌率达到57.3%,鉴定发现这4株细菌均属于芽孢杆菌属,相互之间没有明显的拮抗作用;在盆栽条件下,与连作土相比,溴甲烷熏蒸处理和拮抗菌菌液处理对平邑甜茶幼苗的生物量均有不同程度的促进作用;在幼苗的长势上,溴甲烷熏蒸处理效果要好于拮抗菌菌液处理;在根系活力上,拮抗菌菌液处理效果要好于溴甲烷熏蒸处理,根系长度、根尖数分别增加了25.1%、70.9%.与连作土处理相比,拮抗菌菌液和溴甲烷熏蒸均能显著降低土壤中的真菌数量,分别降低了71.2%和64.2%,拮抗菌菌液处理能显著增加土壤中的细菌和放线菌数量,分别增加了48.0%和140.2%,使土壤微生物结构向“细菌型”转化;而溴甲烷熏蒸处理显著降低了土壤中的细菌和放线菌数量,说明拮抗菌的确能够抑制土壤中病原真菌的生长.     

The rhizosphere microflora of wheat grown under controlled conditions I. The effect of soil fertility and urea leaf treatment on the rhizoplane microflora

Summary Microbial colonization of seminal roots of seedlings and of nodal roots of tillering plants was studied on spring wheat ‘Kaspar’ cultivated in growth, chambers. Methods were developed to microbially condition the soil before seeding and to regulate soil humidity. Addition of inorganic nutrients (NPK) to the soil increased the number of rhizoplane bacteria and actinomycetes, but did not effect the number of fungi on seminal and nodal roots. Urea leaf treatments stimulated bacteria and actinomycetes 7 and 9 days after treatment. Fourteen days after urea leaf treatment, however, bacterial numbers were mostly reduced, especially on seminal roots, while numbers of actinomycetes generally equalled the control. Root types and soil fertility did not obviously interact with the effect of urea leaf treatment on rhizoplane bacteria and actinomycetes. The only effect of urea on total numbers of fungi, was a reduction of their numbers on seminal roots 9 days after treatment at both NPK-levels.     

Selective Isolation of Aerobic Actinomycetes

The composition of an arginine-glycerol-salt medium (AGS), suitable for the selective isolation of aerobic actinomycetes, was given. When soil samples were treated with calcium carbonate and plated on the AGS medium, higher total and relative plate counts of actinomycetes were obtained than when other media and methods were used.     

Isolation of Actinomycetes from Soil Using Extremely High Frequency Radiation

A new method employing extremely high frequencies (EHFs) is proposed for the selective isolation of actinomycetes from soil. The pretreatment of soil suspensions with EHF wavelengths of 5.6 and 7.1 mm led to a nonselective isolation of actinomycetes. At the same time, the irradiation of soil suspensions within wavelength bands of 3.8–5.8 and 8–11.5 mm considerably augmented the total number of isolated actinomycetes and increased the fraction of the isolated rare genera by 2 and 7 times, respectively. The rare actinomycete genera were represented by Actinomadura, Microtetraspora, Nonomuraea, Micromonospora, Amycolatopsis, Pseudonocardia, Saccharotrix, and Streptosporangium.     

低剂量混合稀土积累对黄褐土微生物主要类群的生态效应

采用田间小区试验和室内低剂量模拟叠加试验相结合的方法,研究低剂量混合稀土在黄褐土中积累对土壤微生物主要类群的生态效应．结果表明,低剂量稀土的持续积累对土壤细菌、放线菌产生刺激、抑制、再刺激的交替作用;对真菌也产生类似的作用,但抑制作用不显著,而刺激作用持续、明显．混合稀土对3类土壤微生物数量抑制程度顺序为：细菌>放线菌>霉菌．稀土积累至150mg·kg^-1时,土壤各类微生物的种群结构均发生显著的改变,耐稀土微生物数量大幅度增加,细菌中的G^-细菌、链霉菌的白孢类群、真菌中青霉分别成为优势种群．对低浓度稀土积累的田问土壤微生物学参数模拟计算结果表明,稀土对土壤细菌、放线菌和真菌的EC50(半抑制浓度)值分别为24．1、41．6～73．8和55．3～150．1mg·kg^-1,30mg·kg^-1值可以初步确定为稀土在黄褐土中积累的安全临界值．     

Effects of the Biocontrol Agent Aspergillus flavipes on the Soil Microflora and Soil Enzymes in the Rooting Zone of Pepper Plants Infected with Phytophthora capsici

This study examined the effect of ASD strain (Aspergillus flavipes), isolated from continuous cropping soil for pepper and named by the sampling position, on soil microflora and soil enzymes in rooting zone soil of healthy and diseased (Phytophthora capsici) pepper plants. Results showed that the ASD strain could significantly reduce the number of bacteria and actinomycetes, with a significant increase in fungi in the rhizosphere soil of both healthy and diseased plants. With increasing colonization time of the ASD strain, the number of bacteria and actinomycetes decreased initially and then increased gradually, while the number of fungi was first increased significantly and later decreased slowly. The soil enzyme activities of urease, acid phosphatase, invertase and dehydrogenase were significantly increased by the ASD strain, while the activity of catalase was not significantly increased. As time from inoculation with the ASD strain increased, the activities of various enzymes were higher than controls. Maximum enzyme activities were found on the tenth day after ADS inoculation. The response of soil enzyme activities affected by the ASD strain was as follows: urease > dehydrogenase > invertase > acid phosphatase > catalase. These results suggest that the biocontrol of ASD strain could improve the micro ecology of rhizosphere soil.