Indigenous bacteria with antagonistic and plant-growth-promoting activities improve slow-filtration efficiency in soilless cultivation

In tomato soilless culture, slow filtration allows one to control the development of diseases caused by pathogenic microorganisms. During the disinfecting process, microbial elimination is ensured by mechanical and biological factors. In this study, system efficacy was enhanced further to a biological activation of filter by inoculating the pozzolana grains contained in the filtering unit with 5 selected bacteria. Three strains identified as Pseudomonas putida and 2 as Bacillus cereus came from a filter whose high efficiency to eliminate pathogens has been proven over years. These 5 bacteria displayed either a plant growth promoting activity (P. putida strains) or antagonistic properties (B. cereus strains). Over the first months following their introduction in the filter, the bacterial colonisation of pozzolana grains was particularly high as compared to the one observed in the control filter. Conversely to Bacillus spp. populations, Pseudomonas spp. ones remained abundant throughout the whole cultural season. The biological activation of filter unit very significantly enhanced fungal elimination with respect to the one displayed by the control filter. Indeed, the 6-month period needed by the control filter to reach its best efficacy against Fusarium oxysporum was shortened for the bacteria-amended filter; in addition, a high efficacy filtration was got as soon as the first month. Fast colonization of pozzolana grains by selected bacteria and their subsequent interaction with F. oxysporum are likely responsible for filter efficiency. Our results suggest that Pseudomonas spp. act by competition for nutrients, and Bacillus spp. by antibiosis and (or) direct parasitism. Elimination of other fungal pathogens, i.e., Pythium spp., seems to differ from that of Fusarium since both filters demonstrated a high efficacy at the experiment start. Pythium spp. elimination appears to mainly rely on physical factors. It is worth noting that a certain percentage of the 5 pozzolana-inoculated bacteria failed to colonise the filter unit and were, thus, driven to the plants by the nutrient solution. Their contribution to the establishment of a beneficial microbial community in the rhizosphere is discussed.     

Diversity of Bacterial Communities that Colonize the Filter Units Used for Controlling Plant Pathogens in Soilless Cultures

In recent years, increasing the level of suppressiveness by the addition of antagonistic bacteria in slow filters has become a promising strategy to control plant pathogens in the recycled solutions used in soilless cultures. However, knowledge about the microflora that colonize the filtering columns is still limited. In order to get information on this issue, the present study was carried out over a 4-year period and includes filters inoculated or not with suppressive bacteria at the start of the filtering process (two or three filters were used each year). After 9 months of filtration, polymerase chain reaction (PCR)–single strand conformation polymorphism analyses point out that, for the same year of experiment, the bacterial communities from control filters were relatively similar but that they were significantly different between the bacteria-amended and control filters. To characterize the changes in bacterial communities within the filters, this microflora was studied by quantitative PCR, community-level physiological profiles, and sequencing 16SrRNA clone libraries (filters used in year 1). Quantitative PCR evidenced a denser bacterial colonization of the P-filter (amended with Pseudomonas putida strains) than control and B-filter (amended with Bacillus cereus strains). Functional analysis focused on the cultivable bacterial communities pointed out that bacteria from the control filter metabolized more carbohydrates than those from the amended filters whose trophic behaviors were more targeted towards carboxylic acids and amino acids. The bacterial communities in P- and B-filters both exhibited significantly more phylotype diversity and markedly distinct phylogenetic compositions than those in the C-filter. Although there were far fewer Proteobacteria in B- and P-filters than in the C-filter (22% and 22% rather than 69% of sequences, respectively), the percentages of Firmicutes was much higher (44% and 55% against 9%, respectively). Many Pseudomonas species were also found in the bacterial communities of the control filter. The persistence of the amended suppressive-bacteria in the filters is discussed with regards to the management of suppressive microflora in soilless culture.     

Application of terminal restriction fragment length polymorphism (T-RFLP) analysis to monitor effect of biocontrol agents on rhizosphere microbial community of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Microbial communities in hot pepper (Capsicum annuum L.) cultivation fields under different cultivation methods were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis. Rhizosphere soil and leaf samples were collected from control, conventional and nature-friendly cultivation fields between May and July, 2009. Two Bacillus subtilis strains were applied to nature-friendly cultivation fields as biocontrol agents during the sampling period. Relative abundances of bacteria and plant pathogenic fungi related T-RFs were also measured to monitor the effect of biocontrol agents on potential plant pathogenic fungi. In the principal component analysis (PCA) based on T-RFLP profiles, the microbial communities from rhizosphere soil samples in July, including bacteria and fungi, showed distinct difference between nature-friendly cultivation fields and other cultivation fields. However, there was no correlation between cultivation methods and leaf microbial communities at any sampling period. Changes in the abundance of bacteria related T-RF in the rhizosphere of nature-friendly cultivation fields were observed clearly two months after application of biocontrol agent, while the abundance of plant pathogenic fungi related T-RFs significantly decreased.     

Spatial and temporal analysis of the microbial community in slow sand filters used for treating horticultural irrigation water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

番茄茎内生细菌的分离鉴定及青枯病拮抗菌的筛选

采用化学法进行表面灭菌处理,运用平板涂布法及平板划线法从番茄茎内得到17株内生细菌.通过形态观察和生理生化指标鉴定,17株内生细菌分属于6个属,即葡萄球菌属(Staphylococcus)、短芽孢杆菌属(Brevi-bacillus)、芽孢杆菌属(Bacillus)、棍状杆菌属(Clavibacter)、欧文氏菌属(Erwinia)和乳杆菌属(Lactobacillus).采用滤纸片法从17株内生菌中筛选出1株对青枯菌(Pseudomonas solanacearum)有拮抗作用的菌株,编号为TS-06,属于芽孢杆菌属(Bacillus).其抑菌圈半径为2.5 mm.     

Bacteriocin diversity inBacillus sphaericus

Nondenaturing polyacrylamide gel electrophoresis revealed the presence of diversity among bacteriocins produced by strains of Bacillus sphaericus. Bacteriocin bands of six strains (pathogenic and non pathogenic) were found to be located just below the stacking gel. However, in two other strains (1 pathogenic and 1 collection strain) more than one protein band with bacteriocin activity were seen in the middle of resolving gel. In bacteriocin-treated cultures, electron-microscopy studies revealed the growth of lysedswollen ghost cells, and loss of viability among sensitive strains.     

毛泡桐内生真菌的分离、拮抗细菌菌株的筛选和鉴定

为寻找新的能产生抗生素的植物内生真菌,采用3种培养基对毛泡桐进行内生真菌的分离,以大肠埃希菌、枯草芽胞杆菌、金黄色葡萄球菌、青枯假单胞菌为指示菌,利用琼脂块法和滤纸片扩散法筛选能抑制细菌的菌株;通过形态特征和ITS序列分析鉴定高活性菌株。结果从毛泡桐的根、茎、叶中共分离得到46株内生真菌,至少能抑制一种指示菌的有9株,其中菌株KLBMP-Pt630、KLBMP-Pt675和KLBMP-Pt686活性较强,鉴定结果显示:KLBMP-Pt630为三线镰刀菌、KLBMP-Pt675为棒曲霉、KLBMP-Pt686属于肉座菌目真菌。     

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．）。     

Survival and colonization of rhizobacteria in a tomato transplant system

Plant-growth-promoting rhizobacteria (PGPR) are used on crops most often as seed treatments; however, an alternative application method for transplanted vegetables is mixing PGPR into the soilless medium in which the transplants are grown. Studies were undertaken to compare root colonization and persistence of rifampicin-resistant mutants of PGPR strains Bacillus pumilus SE34 and Pseudomonas fluorescens 89B61, SE34r and 89B61r, on tomato as a function of application method. When the bacteria were incorporated into Promix soilless medium at log 6, 7, and 8 colony- forming units/g, populations of strain SE34r per gram of medium maintained the initial inoculum densities, while populations of 89B61r decreased approximately one to two orders of magnitude by 4 weeks after planting. The populations of each PGPR strain colonizing roots after application into the soilless medium showed a similar pattern at 6 weeks as that at 4 weeks after planting, with higher populations on the whole roots and lateral roots than on the taproots. Strain SE34r but not 89B61r moved upwards and colonized the phyllosphere when incorporated into the soilless medium. Following application as seed treatment, populations of SE34r were significantly higher on upper roots and on the taproot than were populations following application through the soilless medium. Conversely, populations were higher on lower roots and lateral roots following application through the soilless medium than were populations following application as seed treatment. While strain SE34 enhanced plant growth with application both to the medium and as seed treatment, the level of growth promotion was significantly greater with application in the soilless medium. The results indicate that PGPR can be successfully incorporated into soilless media in vegetable transplant production systems.     

Spatial and Temporal Analysis of the Microbial Community in Slow Sand Filters Used for Treating Horticultural Irrigation Water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

Detection and validation of volatile metabolic patterns over different strains of two human pathogenic bacteria during their growth in a complex medium using multi-capillary column-ion mobility spectrometry (MCC-IMS)

Headspace analyses over microbial cultures using multi-capillary column-ion mobility spectrometry (MCC-IMS) could lead to a faster, safe and cost-effective method for the identification of pathogens. Recent studies have shown that MCC-IMS allows identification of bacteria and fungi, but no information is available from when on during their growth a differentiation between bacteria is possible. Therefore, we analysed the headspace over human pathogenic reference strains of Escherichia coli and Pseudomonas aeruginosa at four time points during their growth in a complex fluid medium. In order to validate our findings and to answer the question if the results of one bacterial strain can be transferred to other strains of the same species, we also analysed the headspace over cultures from isolates of random clinical origin. We detected 19 different volatile organic compounds (VOCs) that appeared or changed their signal intensity during bacterial growth. These included six VOCs exclusively changing over E. coli cultures and seven exclusively changing over P. aeruginosa cultures. Most changes occurred in the late logarithmic or static growth phases. We did not find differences in timing or trends in signal intensity between VOC patterns of different strains of one species. Our results show that differentiation of human pathogenic bacteria by headspace analyses using MCC-IMS technology is best possible during the late phases of bacterial growth. Our findings also show that VOC patterns of a bacterial strain can be transferred to other strains of the same species.     

Pseudomonas corrugata: plant pathogen and/or biological resource?

Pseudomonas corrugata is the causal agent of tomato pith necrosis yet it is also used in the biological control of plant pathogenic bacteria and fungi. Potentially it could be used in other fields, such as the production of commercial biomolecules with a wide range of application and including bioremediation. This review reports the multiple characteristics of the bacterium, highlights its known molecular features and speculates on the possible underlying mechanisms of action.
Taxonomy:  Bacteria; Proteobacteria ; γ subdivision; order Pseudomonadales ; family Pseudomonadaceae ; genus Pseudomonas .
Microbiological properties:  Gram-negative, oxidase-positive, non-spore forming rods; non-fluorescent on King's B medium; produces wrinkled and rarely smooth colonies on yeast peptone glucose agar or nutrient dextrose agar; yellow to brown diffusible pigments are frequently produced.
Disease symptoms:  The typical symptom on tomato is necrosis and/or hollowing of the pith of the stem; the syndrome determines loss of turgidity of the plant, hydropic/necrotic areas and long conspicuous adventitious roots on the stem.
Biological control agent: In vitro assessed against plant pathogenic fungi and bacteria, as well as the phytotoxin indicator microorganims Rhodotorula pilimanae and Bacillus megaterium ; in vivo used against pre- and post-harvest plant pathogens.     

广西番茄内生细菌的多样性和数量动态

为了探明内生细菌在番茄中的分布和数量变化规律,有目的地筛选防治番茄青枯病的内生细菌,我们对广西可培养的番茄内生细菌的类群和数量动态进行了调查。从广西部分县市采集的303个番茄样本中分离到624株内生细菌菌株,初步确定有芽孢杆菌(Bacillus)、假单胞菌(Pseudomonas)、黄单胞菌(Xanthomonas)、棒杆菌(Corynebacterium)、土壤杆菌(Agrobacterium)、微杆菌(Microbacterium)、肠杆菌(Enterobacter)和欧文氏菌(Erwinia)8个属,其中以芽孢杆菌、假单胞菌和土壤杆菌为芽孢杆菌为优势类群。番茄内生细菌在植株器官中的分布以根部数量最多,其次是茎和叶。内生细菌的总量在番茄生育期的变化趋势是从苗期到花期数量上升,而从结果期到成熟期数量逐渐下降。多数内生细菌种群的数量变化动态符合细菌总量的变化趋势,只有微杆菌在番茄植株整个生育期中始终保持下降的趋势。春季种植的番茄植株的内生细菌类群数量比秋季种植的少。     

A New Filter Based Cultivation Approach for Improving Aspergillus Identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.

     

Culture-based and denaturing gradient gel electrophoresis analysis of the bacterial community structure from the intestinal tracts of earthworms(Eisenia fetida)

The bacterial communities in the intestinal tracts of earthworm were investigated by culture-dependent and - independent approaches. In total, 72 and 55 pure cultures were isolated from the intestinal tracts of earthworms under aerobic and anaerobic conditions, respectively. Aerobic bacteria were classified as Aeromonas (40%), Bacillus (37%), Photobacterium (10%), Pseudomonas (7%), and Shewanella (6%). Anaerobic bacteria were classified as Aeromonas (52%), Bacillus (27%), Shewanella (12%), Paenibacillus (5%), Clostridium (2%), and Cellulosimicrobium (2%). The dominant microorganisms were Aeromonas and Bacillus species under both aerobic and anaerobic conditions. In all, 39 DNA fragments were identified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Aeromonas sp. was the dominant microorganism in feeds, intestinal tracts, and casts of earthworms. The DGGE band intensity of Aeromonas from feeds, intestinal tracts, and casts of earthworms was 12.8%, 14.7%, and 15.1%, respectively. The other strains identified were Bacillus, Clostridium, Enterobacter, Photobacterium, Pseudomonas, Shewanella, Streptomyces, uncultured Chloroflexi bacterium, and uncultured bacterium. These results suggest that PCR-DGGE analysis was more efficient than the culture-dependent approach for the investigation of bacterial diversity and the identification of unculturable microorganisms.     

Bacterial contaminants of micropropagated plant cultures

Bacterial contaminants of micropropagated plant cultures were isolated and characterized with standard bacteriological tests and appropriate API strips. Results obtained were analysed by the API identification software. Of 198 bacterial strains isolated from nine plant species, 90% were identified as Bacillus, Enterobacter, Micrococcus, Staphylococcus, Pseudomonas or Lactobacillus species. Possible sources of contamination are discussed.     

林木病原菌广谱拮抗菌株的筛选及功能研究

以尖孢镰刀菌、腐皮镰孢菌、胶孢炭疽菌、拟盘多毛孢菌等林木常见病原菌为指示菌,用平板对峙法和菌丝球干重法,从土壤固氮细菌群中筛选广谱拮抗菌株,并调查了各菌株的溶磷及解钾能力。结果表明：对4种病原菌拮抗能力较强的菌株有24株,其中11株能溶解无机磷,7株能溶解有机磷,4株有解钾功能。通过拮抗能力复筛得到最优菌株为菌株2024和7014。菌种鉴定结果表明2024菌株为枯草芽孢杆菌亚种（Bacillus subtilis subsp. subtilis）,7014菌株为成团泛菌（Pantoea vagans）。     

大蒜内生细菌的分离及拮抗菌筛选与鉴定

利用常规分离方法对大蒜鳞茎进行内生细菌的分离,采用对峙法和平板涂布法对分离的内生菌进行拮抗试验研究,并对菌株DSP6进行16S rDNA全序列鉴定。结果表明:分离得到19株内生细菌,其中10株菌对2种以上植物病原真菌有不同程度的抑制作用,占分离菌总数的52.6%,DSN7对番茄早疫病的抑菌圈半径最大,为13mm;17株菌对5种病原细菌中至少1种有抑制作用,占分离菌总数的89.5%,其中菌株DSP3对大肠杆菌的抑菌圈半径最大,达到10 mm;菌株DSP6对供试的9种病原菌有较强的抑菌作用,且抑菌圈平均半径最大,为6.88mm;16S rDNA全序列鉴定显示,菌株DSP6与芽孢杆菌属Bacillus axarquiensis相似性为100%,表明菌株DSP6为Bacillus axarquiensis。     

Biofilter performance and characterization of a biocatalyst degrading alkylbenzene gases

A biofilter treating alkylbenzene vapors was characterized for its optimal running conditions and kinetic parame-ters. Kinetics of the continuous biofilter were compared to batch kinetic data obtained with biofilm samples as well as with defined microbial consortia and with pure culture isolates from the biofilter. Both bacteria and fungi were present in the bioreactor. Five strains were isolated. Two bacteria, Bacillus and Pseudomonas, were shown to be dominant, as well as a Trichosporon strain which could, however, hardly grow on alkylbenzenes in pure culture. The remaining two strains were most often overgrown by the other three organisms in liquid phase batch cultures μ _max, K_S, K_I values and biodegradation rates were calculated and compared for the difterent mixed and pure cultures. Since filter bed acidification was observed during biofiltration studies reaching a pH of about 4, experiments were also undertaken to study the influence of pH on performance of the different cultures. Biodegradation and growth were possible in all cases, over the pH range 3.5–7.0 at appreciable rates, both with mixed cultures and with pure bacterial cultures. Under certain conditions, microbial activity was even observed in the presence of alkylbenzenes down to pH 2.5 with mixed cultures, which is quite unusual and explains the ability of the present biocatalyst to remove alkylbenzenes with high efficiency in biofilters under acidic conditions.     

两株生防芽孢细菌筛选、鉴定及拮抗研究

【目的】筛选出广谱、高效的生防芽孢细菌,并对其拮抗作用进行研究。【方法】以8种植物病原真菌为靶标菌,通过皿内拮抗和发酵液拮抗能力的测定筛选出2株广谱性和高效性的芽孢细菌B06和B07。【结果】B06对8种植物病原真菌的R2/R1为0.4-1.8,无菌滤液对8种植物病原真菌的抑制率为66.7%-87.5%。B07对8种植物病原真菌的R2/R1为0.23-1.21,无菌滤液对8种植物病原真菌的抑制率为55.56%-81.25%。经16S rRNA序列鉴定,菌株B06和B07都被鉴定为解淀粉芽孢杆菌。【结论】芽孢细菌能够抑制多种植物病原真菌,具有较好的抑病作用。广谱和高效芽孢细菌的筛选在农业生物防治方面具有很大的开发和应用价值。