Detection and eradication of endophytic bacteria from micropropagated mint plants

Summary Liquid medium and an enriched agar were used to detect endophytic bacteria in micropropagated mints (Mentha spp.) within 2 to 6 d of inoculation. Bacteria isolated from the cultures were screened on several antibiotic concentrations to determine bactericidal doses. Antibiotics were also tested for phytotoxic effects. Shoot tips from infected plants were treated by immersion in liquid MS medium containing antibiotics either singly or in combination. Streptomycin applied at 1000µg/ml for a period of 10 d was effective and less phytotoxic in a larger number of cases than gentamicin (50µg/ml), neomycin (500µg/ml), or rifampicin (30µg/ml). Mint cultures that tested negative for bacteria after antibiotic treatment were multiplied, retested, and cold-stored for 1 yr or longer. Upon regrowth after storage, 25 of 30 treated cultures (83%) tested negative for bacteria. Of the 25, 8 were successfully treated with streptomycin, 1 with gentamicin, 2 with neomycin, 1 with rifampicin, and 1 with streptomycin and gentamicin; 12 required more than one treatment. An early detection system, initial trial treatment with streptomycin for infected plants, and monitoring of treated cultures successfully reduced the spread of bacterial contamination. Antibiotic treatment in liquid MS medium at pH 6.9 resulted in enhanced bactericidal activity over that seen at pH 5.5.     

Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue

Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were inoculated with 1 ml of diluted (OD_600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin^?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin^?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic tissue confirmed T-DNA integration. Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997     

Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures

Endophytic bacterial contamination is a major constraint to the establishment and maintenance of aseptic Aglaonema cultures. The objectives of the present study included the identification of endophytic bacterial contaminants from micropropagated Aglaonema cultures and the investigation of effective antibiotic treatment for their control. Bacterial contaminants isolated from 181 infected stem nodal explants of six Aglaonema cultivars were identified following the amplification of 16S ribosomal RNA gene and partial sequence analysis. A total of thirteen different bacterial species were identified and these were found to be mostly associated with soil and water. The bacteria were subsequently subjected to antimicrobial susceptibility and minimum inhibitory concentration tests. Three antibiotics, including gentamicin, tetracycline and chloramphenicol, were selected for their effectiveness at low concentrations of 4?C32?mg?l^?1 to inhibit bacterial growth in most of the bacterial species found in the present study. The incorporation of these antibiotics into the culture medium was found to effectively reduce the incidence of bacterial contamination in three of the four Aglaonema cultivars tested. Therefore, sanitation of the irrigation water and growth substrate while raising the stock plants, as well as the appropriate use of antibiotics during the in vitro culture stage will be important factors governing the success of Aglaonema micropropagation in the future.     

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

Alterations in growth of tissue-cultured tansy (Tanacetum vulgare L.) treated with antibiotics

Effects of three antibiotics (cefotaxime, rifampicin and gentamicin) were tested on in vitro shoots cultures of tansy (Tanacetum vulgare L.)- These antibiotics were selected because endophytic bacteria isolated from the in vitro shoot cultures of tansy showed that all bacteria were Gram-negative and their growth was reduced by these three antibiotics. Five isolates were Enterobacteriaceae, three were fluorescent Pseudomonas, and two were aerobic bacteria. Increased concentrations of antibiotics caused usually linear or quadratic changes on the initiation of shoot growth, shoot number, growth rate, and shoot height. These changes and changes in pH of the culture media were tansy genotype-dependent following treatments with gentamicin. Also, the treatment with rifampicin or cefotaxime showed a genotype-dependent effect, because they resulted in significantly higher percentage of rooted plants in one of the three tansy genotypes tested. The growth rate and length of shoots were reduced in the media containing both gentamicin and rifampicin, but less so than in media containing both gentamicin and cefotaxime.     

喀斯特石漠化治理区钙果种植年限对根内细菌群落的影响

存在于健康植物根中的内生细菌不但能够与宿主植物建立共生关系,而且还具有促进植物生长、提升植物对营养元素摄取能力等功能,从而对维持陆地生态平衡、提升喀斯特石漠化综合治理成效具有重要的意义。【目的】探究宿主植物根中的内生细菌群落结构,为深入认识宿主植物-内生细菌的互作机制提供理论依据。【方法】以被引种到喀斯特断陷盆地石漠化综合治理区不同种植年限钙果根中的内生细菌和根际土为研究对象,分析根内生细菌群落特征和根际土理化性质。【结果】钙果种植年限对研究区土壤质量整体有着直接、显著的影响,并间接影响根内生细菌群落。根内生细菌群落以共生互作为主,通过共网络结构识别出种植第一年和第三年的前三位优势属为链霉菌属(Streptomyces)、卡氏伯克霍尔德菌属(Burkholderia-Caballeronia- Paraburkholderia)和噬几丁质菌属(Chitinophaga),种植第五年的前三位优势属为Streptomyces、Chitinophaga和海无柄孢囊黏细菌属(Haliangium)。根内生细菌群落的形成主要由随机性过程中的生态漂变所主导。【结论】不同生长阶段钙果根内生细菌群落结构的差异是由于随机性过程赋予了微生物物种多样化。内生细菌群落的共生互作关系以及优势菌具有生防功能,可增强钙果定殖能力和生长,进而提升钙果在喀斯特断陷盆地石漠化综合治理区的生态效益和经济效益。     

Molecular identification and control of endophytic contamination during in vitro plantlet development of <Emphasis Type="Italic">Fagonia indica</Emphasis>

Microbial contamination is the major cause of economic losses in commercial and scientific plant tissue culture laboratories. For successful micropropagation, it is important to control contamination during in vitro cultures. The present study was designed to isolate, identify and eradicate endophytic contaminants from in vitro cultures of medicinally important plant Fagonia indica. A total of eight distinct bacterial isolates from in vitro grown plantlets of F. indica were selected based on analysis of colony morphology. The endophytic bacterial contaminants identified at the species level through 16S rRNA sequence analysis were Enterobacter xiangfangensis, Bacillus vallismortis, Bacillus tequilensis, Terribacillus halophilus, Pantoea dispersa, Serratia marcescens subsp. Sakuensis, Staphylococcus epidermidis and Bacillus atrophaeus. It was observed that almost 60% of seedlings were contaminated with Bacillus sp. and out of those, Bacillus tequelensis contributed to most infections (70% out of the Bacillus infections). The other most frequently occurring bacteria were Bacillus vallismortis, Terribacillus halophilus and Serratia marcescens subsp. sakuensis. Furthermore, the addition of antimicrobials to the media either completely inhibited or drastically decreased the growth of endophytic bacteria as compared to the control in which 92% of the plantlets were contaminated with these endophytes. Nine different antibiotics (rifampicin, teicoplanin, gentamicin, vancomycin, ciprofloxacin, tobramycin, tetracycline, doxycycline and ampicillin) were tested for their activity against the identified endophytes. Antibiotics such as ciprofloxacin and tobramycin showed a good response and inhibited the growth of all the bacterial isolates at low doses compared to the other antibiotics. Tobramycin was the most effective as it inhibited the growth of five of the bacterial isolates at a dosage as low as 4 mg/L. In case of tetracycline (16 mg/L) and doxycycline (64 mg/L), the contamination frequency in plantlets was 25.6 and 45%, respectively. It is, therefore, important to search for more endophytes, causing adverse effects during in vitro cultures and should devise a feasible anti-microbial strategy for controlling such contamination.     

Sanitizing long-term micropropagated grapes from covert and endophytic bacteria and preliminary field testing of plants after 8 years <Emphasis Type="Italic">in vitro</Emphasis>

Summary Micropropagated grape (Vitis vinifera L.) cv. Arka Neelamani cultures showed a decline in root and shoot growth performance after 6–7 yr of continuous in vitro culture. Indexing the culture medium using nutrient agar or 523 bacteriological medium (Viss et al., 1991) revealed covert bacteria in 75–100% cultures. Testing the tissue from different parts of in vitro plantlets on nutrient agar showed bacteria comprising of six or more morphotypes in 100% of root and collar tissue samples but less frequently in stem segments. The shoot tips had the lowest incidence of bacterial association. The whole shoots treated with NaOCl (4% chlorine) or HgCl₂ (0.1%) showed endophytic bacterial survival. Culturing the HgCl₂-treated (5 min) shoot tips on antibiotic overlaid medium (1 ml of 50 mg l⁻¹ gentamycin and/or cefazolin) in culture tubes (150×25 mm) for 1 mo. facilitated the cleansing of cultures with 75% recovery of contaminant-free shoots as monitored through indexing for the next 2 yr. Repeated indexing of medium and tissue from various plant parts during the first two to four subculture cycles following antibiotic treatment was instrumental in reliably identifying clean cultures and preventing bacterial re-emergence. Antibiotic incorporation in the medium was detrimental to grape microcuttings. Bacteria-freed cultures showed 80–100% rooting and a high number of plantlets that could be acclimatized. The plants put in the field after 8 yr of active micropropagation showed some juvenile characteristics initially, which disappeared in 6–8 mo., and the pruned shoots showed flowering and bunch development within 1 yr of field planting. This indicated the feasibility of keeping grape plants in vitro for long periods if covert microbes were eliminated.     

新疆北部主要盐生植物根部内生细菌群落结构的高通量分析

【目的】揭示同一盐渍环境中不同种盐生植物根部内生细菌群落多样性特征和分布规律,结合根际土壤理化因子探讨其对内生细菌群落结构的影响。【方法】通过罗氏454高通量测序获得内生细菌16S r RNA片段,然后进行生物信息分析。【结果】研究的16种盐生植物其内生细菌群落主要由Proteobacteria、Tenericutes、Actinobacteria和Firmicutes 4个门的细菌组成。从植物"种"的水平来看,不同种盐生植物内生细菌群落存在差异;从植物"属"的水平来看,同一属的盐生植物内生细菌相似;从植物"科"的水平来看,藜科盐生植物内生细菌以Actinobacteria和Proteobacteria门为主;蒺藜科盐生植物内生细菌以Proteobacteria门为主;柽柳科盐生植物内生细菌以Tenericutes门为主;白花丹科盐生植物内生细菌以Proteobacteria、Fimicutes和Actinobacteria门为主。根际土壤中Cl~–含量对盐生植物内生细菌群落变化具有显著影响;而Cl~–、Mg~(2+)和总氮组成的集合与内生细菌群落结构相关性最高。【结论】盐生植物内生细菌多样性丰富。在同一盐渍生境中,盐生植物内生细菌群落分布呈现宿主的种属特异性,根际土壤中Cl~–是影响其内生细菌群落变化的主要驱动因素之一。     

Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments

Aims: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. Methods and Results: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin/penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. Conclusions: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l^?1), streptomycin/penicillin (25 mg l^?1) and nystatin (10 mg l^?1) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. Significance and Impact of the Study: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures.     

The dual role of carbenicillin in shoot regeneration and somatic embryogenesis of horseradish (<Emphasis Type="Italic">Armoracia rusticana</Emphasis>) in vitro

Carbenicillin, a well-known antibiotic, has been reported to have growth regulator-like activity in vitro for some plant species. In the present paper we add horseradish (Armoracia rusticana) to the list of plants exhibiting such responses. This project began as an effort to eliminate latent bacterial contamination among established in vitro horseradish plants. Carbenicillin (100 mg L⁻¹) added to regeneration medium eliminated all visible bacterial contaminants. Unexpectedly, carbenicillin-grown explants regenerated adventitious shoots faster (14 days) than those on control medium (21 days). In addition eight of 11 horseradish cultivars grown on carbenicillin produced more adventitious shoots per explant than control. At much higher levels (2,000 mg L⁻¹) carbenicillin was found to retard somatic embryogenic callus induction. Based on these observations we suggest that carbenicillin at moderate levels enhances shoot development in horseradish. The mode of action of carbenicillin’s growth regulator-like activity needs to be investigated.     

Targeting of Antibiotics in Bacterial Infections Using Pegylated Long-Circulating Liposomes

Abstract

PEGylated long-circulating liposomes were used as a delivery system of antibiotics providing enhancements in antibiotic pharmacokinetics and penetration to infected sites. Pharmacokinetic and therapeutic efficacy studies were performed in the model of unilateral pneumonia/septicemia caused by Klebsiella pneumoniae in rats with intact host defense or leukopenic rats. Gentamicin was encapsulated in PEGylated liposomes designed to achieve delivery of antibiotic to the infected left lung tissue. Our data show that the efficacy of liposomal gentamicin was superior to free gentamicin particularly in difficult to treat infection due to impaired host defense (leukopenia) or low antibiotic susceptibility of the infectious organism. In leukopenic rats infected with a high gentamicin-susceptible bacterial strain, free gentamicin must be administered at the maximum tolerated dose to be therapeutically effective. The addition of a single dose of liposome-encapsulated gentamicin on the first day of treatment with free gentamicin leads to full therapeutic efficacy while keeping the antibiotic doses low. In even more difficult to treat infection due to both an impaired host defense (leukopenia) and low gentamicin-susceptibility of the bacterial strain, free gentamicin is not effective, and the addition of the liposome-encapsulated form of gentamicin is needed to achieve full therapeutic efficacy. In this respect, the lipid composition of the liposomes is an important determinant in establishing both sufficient antibiotic levels in blood and sufficient release of antibiotic from the liposomes at the infectious focus.

Ciprofloxacin was encapsulated in PEGylated liposomes designed to serve as a microreservoir of antibiotic during circulation in blood. Our data show that the administration of ciprofloxacin in the liposomal form resulted in slow release of ciprofloxacin from the liposomes over time in blood. Delayed ciprofloxacin clearance, as well as increased and prolonged ciprofloxacin concentrations in blood and tissues was observed. The therapeutic efficacy of liposomal ciprofloxacin was superior to that of free ciprofloxacin. PEGylated liposomal ciprofloxacin was well tolerated in relatively high doses (increasing the maximum tolerated dose for free ciprofloxacin), permitting the administration on a once-a-day schedule without loss in therapeutic efficacy.     

Shoot regeneration and free-radical scavenging activity in <Emphasis Type="Italic">Silybum marianum</Emphasis> L.

The morphogenic potential and free-radical scavenging activity of the medicinal plant, Silybum marianum L. (milk thistle) were investigated. Callus development and shoot organogenesis were induced from leaf explants of wild-grown plants incubated on media supplemented with different plant growth regulators (PGRs). The highest frequency of callus induction was observed on explants incubated on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ 6-benzyladenine (BA) after 20 days of culture. Subsequent transfer of callogenic explants onto MS medium supplemented with 2.0 mg l⁻¹ gibberellic acid (GA₃) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) resulted in 25.5 ± 2.0 shoots per culture flask after 30 days following culture. Moreover, when shoots were transferred to an elongation medium, the longest shoots were observed on MS medium supplemented with 0.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, and these shoots were rooted on a PGR-free MS basal medium. Assay of antioxidant activity of in vitro and in vivo grown tissues revealed that significantly higher antioxidant activity was observed in callus than all other regenerated tissues and wild-grown plants.     

Introduction of some new endophytic bacteria from Bacillus and Streptomyces genera as successful biocontrol agents against Sclerotium rolfsii

Endophytic bacteria live inside plant tissues without causing disease and not only promote plant growth but can also protect plants against plant pathogens. During 2010–2011 crop years, some endophytic bacteria were collected and are then biochemically and molecularly identified (16srRNA) from bean farms of East Azarbaijan, Iran. Among these bacteria isolates, four isolates from Bacillus genera and four isolates from Streptomyces genera were selected for evaluation of their ability for biocontrol of Sclerotium rolfsii in laboratory and glasshouse conditions. Except one isolate named Streptomyces parvus, the rest of isolates could significantly inhibit mycelial growth in dual culture on PDA medium. All seven selected isolates showed significant inhibition in disease treatments in glasshouse experiments. Biological traits, such as length, wet and dry weight of roots and stems in endophytic bacterial treatment showed no differences with healthy control.     

Loline alkaloid production by fungal endophytes of Fescue species select for particular epiphytic bacterial microflora

The leaves of fescue grasses are protected from herbivores by the production of loline alkaloids by the mutualist fungal endophytes Neotyphodium sp. or Epichloë sp. Most bacteria that reside on the leaf surface of such grasses can consume these defensive chemicals. Loline-consuming bacteria are rare on the leaves of other plant species. Several bacterial species including Burkholderia ambifaria recovered from tall fescue could use N-formyl loline as a sole carbon and nitrogen source in culture and achieved population sizes that were about eightfold higher when inoculated onto plants harboring loline-producing fungal endophytes than on plants lacking such endophytes or which were colonized by fungal variants incapable of loline production. In contrast, mutants of B. ambifaria and other bacterial species incapable of loline catabolism achieved similarly low population sizes on tall fescue colonized by loline-producing Neotyphodium sp. and on plants lacking this endophytic fungus. Lolines that are released onto the surface of plants benefiting from a fungal mutualism thus appear to be a major resource that can be exploited by epiphytic bacteria, thereby driving the establishment of a characteristic bacterial community on such plants.     

Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide

Endophytic bacteria are ubiquitous in most plant species influencing the host fitness by disease suppression, contaminant degradation, and plant growth promotion. This endophytic bacterial community may be affected by crop management such as the use of chemical compounds. For instance, application of glyphosate herbicide is common mainly due to the use of glyphosate-resistant transgenic plants. In this case, the bacterial equilibrium in plant–endophyte interaction could be shifted because some microbial groups are able to use glyphosate as a source of energy and nutrients, whereas this herbicide may be toxic to other groups. Therefore, the aim of this work was to study cultivable and noncultivable endophytic bacterial populations from soybean (Glycine max) plants cultivated in soil with and without glyphosate application (pre-planting). The cultivable endophytic bacterial community recovered from soybean leaves, stems, and roots included Acinetobacter calcoaceticus, A. junii, Burkholderiasp., B. gladioli, Enterobacter sakazaki, Klebsiella pneumoniae, Pseudomonas oryzihabitans, P. straminea, Ralstonia pickettii,and Sphingomonassp. The DGGE (Denaturing Gradient Gel Electrophoresis) analysis from soybean roots revealed some groups not observed by isolation that were exclusive for plants cultivated in soil with pre-planting glyphosate application, such as Herbaspirillum sp., and other groups in plants that were cultivated in soil without glyphosate, such as Xanthomonas sp. and Stenotrophomonas maltophilia. Furthermore, only two bacterial species were recovered from soybean plants by glyphosate enrichment isolation. They were Pseudomonas oryzihabitans and Burkholderia gladioliwhich showed different sensibility profiles to the glyphosate. These results suggest that the application at pre-planting of the glyphosate herbicide may interfere with the endophytic bacterial communitys equilibrium. This community is composed of different species with the capacity for plant growth promotion and biological control that may be affected. However, the evaluation of this treatment in plant production should be carried out by long-term experiments in field conditions.     

Impact of Agricultural Practices on the Zea mays L. Endophytic Community

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.     

Effect of bacterial inoculation,plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.     

不同水稻组织内生细菌的群落多样性

[目的]为详细了解水稻不同组织内生细菌群落多样性。[方法]对宁粳43号内生细菌的总DNA提取后,采用高通量测序技术对水稻内生细菌的16S rRNA基因进行了序列测定,分析了水稻不同组织部位内生细菌群落结构特征。[结果]叶部共获得内生细菌OTUs 610个,茎部411个,根部174个。物种分类显示,叶部内生细菌种类隶属于22门40纲103目198科399属,其中优势类群是红球菌属（Rhodococcus）和乳酸杆菌属（Lactobacillus）,它们的相对丰度分别为21.00%和9.19%;茎部内生细菌种类隶属于19门31纲85目169科306属,其中优势类群是红球菌属和罗尔斯通菌属（Ralstonia）,它们的相对丰度分别为19.25%和13.52%;根部内生细菌种类隶属于9门19纲44目82科140属,其中优势类群是肠杆菌属（Enterobacter）和埃希氏杆菌属（Escherichia）,它们的相对丰度分别为81.13%和10.89%。根茎叶中相同的OTU有78个,放线菌门（Actinobacteria）与大多数细菌具有相关性。根系内生细菌中具有调控各种代谢网络功能的物种丰度高于茎部和叶部。[结论]不同水稻组织内生细菌具有丰富的群落多样性,其中叶部的内生细菌物种最丰富,根系参与各种代谢调控的细菌丰度最高,各个组织部位的优势菌属各不相同,变形菌门是最重要的水稻内生细菌。     

Masking of antibiotic-resistance upon recovery of endophytic bacteria

During studies on internal plant colonization by rhizosphere bacteria and endophytic bacteria over several years, we frequently observed lack of growth of rifampicin-resistant mutants (rif+) on tryptic soy agar amended with rifampicin (RTSA). Following seed treatment of cucumber with 6 species of rif+ rhizosphere bacteria in one experiment, all strains were recoverable on RTSA when external root colonization was monitored. Following trituration of surface-disinfested roots, only one strain grew directly on RTSA; however colonies isolated on tryptic soy agar (TSA) grew within 18 h after transfer to RTSA. We term this temporary loss of the antibiotic-resistant phenotype ‘antibiotic masking’. Antibiotic masking was also observed with isolation of 7 rif+ endophytic bacterial strains from inside stems of cotton and with isolation of mutants of bacterial endophytes resistant to polymyxin B sulfate from cotton plants. Rifampicin-masking was not accounted for in vitro by inhibitory compounds from cotton plant extracts, by bacterial growth on low nutrient agar, or by competition with other bacteria. Collectively, these results suggest that expression of antibiotic-resistance may be altered in planta, although causes for this antibiotic-masking remain to be elucidated, methods for quantifying internal plant colonization by rif+ bacteria should account for this possibility. ei]Section editor: R O D Dixon