Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin / 中国中药杂志

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

Induction of defense-related enzymes in patchouli inoculated with virulent Ralstonia solanacearum

Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.

Evaluación del gen que codifica la enzima βHPMEH para la inhibición de la marchitez bacteriana causada por Ralstonia solanacearum

La marchitez bacteriana causada por Ralstonia solanacearum E.F. Smith es una de las enfermedades bacterianas más importantes que ataca a cultivos agrícolas como papa, tomate, banana, entre otros, causando grandes pérdidas en la producción. Desafortunadamente, su control ha sido difícil por su amplio rango de hospederos alternativos, su supervivencia en el suelo y su variación biológica y genética; así como porque no hay variedades con altos niveles de resistencia y porque no existe un control químico efectivo. Quorum sensing (percepción de quorum) es el fenómeno mediante el cual la acumulación de unas moléculas permite a una bacteria saber el número de bacterias que se encuentran en el medio es decir la densidad poblacional. La bacteria R. solanacearum posee un sistema quorum sensing para la regulación de la expresión de genes de virulencia, y en la cual la molécula 3-OH-PAME es el autoregulador de esta señal. Se conoce que la molécula ΒHPMEH hidroliza a 3-OH-PAME, anulando así la señal de autorregulación y por tanto la comunicación quorum sensing en R. solanacearum. Con el objetivo de evaluar el gen βhpmeh, se diseñaron dos vectores que expresen este gen bajo el control de dos diferentes promotores, los cuales fueron verificados por análisis de restricción, secuenciamiento y posteriormente mediante técnicas de agroinfiltración, se observó su expresión y su efecto frente a R. solanacearum en hojas de papa de la variedad Desiree. Los resultados de la expresión transitoria, muestran que el gen βhpmeh retrasó la aparición de síntomas de la marchitez bacteriana y sería un candidato potencial para transformación genética de la planta entera.

Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease that attacks important agricultural crops such as potato, tomato, banana, among others, causing serious yield losses. Control of R. solanacearum is difficult because of its wide range of alternate hosts, its long survival in soil, its biological and genetic variation, the lack of natural resistance sources and the insufficiency of the appropriate chemical control measures. Quorum sensing is the term that describes the phenomenon whereby the accumulation of molecules allows bacteria to know the number of bacteria found in the environment (population density). R. solanacearum has a quorum sensing system for the regulation of the expression of virulence genes; the molecule 3-OH-PAME is the self-regulatory signal. The molecule ΒHPMEH hydrolyzes 3-OH-PAME nullifying the signal of virulence, and thus, the quorum sensing communication in R. solanacearum. In order to evaluate the βhpmeh gene we designed two vectors that express this gene under the control of two different promoters. Both vectors were verified by restriction analysis and sequencing. Agroinfiltration assays were used to analyze gene expression and the effect against R. solanacearum in potato (Solanum tuberosum) leaves. The results of the transient expression experiments showed that the expression of gene βhpmeh caused a delay in the appearance of symptoms of bacterial wilt and thus is a good candidate for whole genetic plant transformation.

Effect of Solvent Extracts of Some Plants on Ralstonia solanacearum.

Aims: To study the efficacy of different solvent extracts (chloroform, ethanol, methanol and hexane) of ten plants on Ralstonia solanacearum the causal organism of bacterial wilt of tomato. Place and Duration of Study: Departments of Crop Production, Soil and Environmental Management and Biological Sciences, Bowen University, Iwo, Nigeria from August 2011 to April 2012. Methodology: Ten plants namely Ocimum gratissimum, Vernonia amygdalina, Allium sativum, Zingiber officinale, Cymbopogon citratus, Azadirachta indica, Jatropha curcas, Senna obtusifolia, Senna occidentalis and Senna alata were collected from Iwo, air dried and pulverized. Chloroform, ethanol, methanol and hexane were used to extract active ingredients from the ten plants. The solvent extracts were tested against R. solanacearum the causal organism of bacterial wilt of tomato and other plants using the disc diffusion method on Mueller Hinton agar. The minimum inhibitory concentration (MIC) of the effective extracts was determined. Results: The plant extracts from chloroform were the most active and this was followed by methanol and ethanol, the lowest activity was recorded from the hexane extracts. The chloroform extracts of J. curcas had the widest zone of inhibition of 15mm followed by O. gratissimum (13mm). All the solvent extracts of A. sativum were active except the hexane extract. The means and standard error of triplicate tests were recorded. The MIC of the active extracts were studied, the MIC of the A. sativum ethanolic extract was 0.25 mg/ml while it was 0.5mg/ml for the V. amygdalina ethanol extract. The MIC of the A. sativum chloroform extract was 0.25mg/ml; J. curcas chloroform extract MIC was 0.125mg/ml, and the MIC for methanolic extract of both extracts were 0.5mg/ml and 0.25mg/ml respectively. Conclusion: The activities of the different solvent extracts are remarkable when compared with the water extracts. Hence, solvent extracts will enhance the efficacy of these phytochemicals in the management of R. solanacearum infections as opposed to water extracts.

A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences.

Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.

Fitodefensivos em plantas medicinais: macromoléculas hidrofílicas de folhas de mil folhas (Achillea millefolium L. ) inibem o crescimento in vitro de bactérias fitopatogênicas / Agrochemicals in medicinal plants: hydrophilic macromolecules from leaves of "mil folhas" (Achillea millefolium L. ) inhibit in vitro growth of phytopathogenic bacteria

Extratos aquosos da planta medicinal Achillea millefolium contêm macromoléculas de interesse para desenvolver fitodefensivos para a agricultura. Duas frações de mil folhas foram obtidas por ultrafiltração, E1 (contendo moléculas maiores que 30 kDa), e E3 (peptídeos entre 1 e 10 kDa) que inibiram o crescimento das bactérias fitopatogênicas Ralstonia solanacearum, gram-negativa, e Clavibacter michiganensis subsp. michiganensis, gram-positiva, com dependência de concentração. Os valores de concentração inibitória mínima (CIM) para ambos os extratos e bactérias foram baixos, entre 20 e 80µM. A CIM relativa à proteína total evidenciou a presença de macromoléculas muito ativas em E3, embora com baixa concentração proteica. E3 se aplica à prospecção de peptídeos antimicrobianos. Estimar a CIM relativa à quantidade de amostra vegetal valorizou o potencial antimicrobiano natural de E1, que contém alta concentração proteica. E1e E3 se aplicam ao desenvolvimento de fitodefensivos para uso biotecnológico. A ultrafiltração fracionou as amostras de forma nativa, rápida, e com baixo custo; além de dessalinizar, clarificar, purificar, e concentrar E1 e E3. Esse estudo inédito sobre a separômica e a ação antimicrobiana de extratos macromoleculares aquosos de mil folhas sugere que plantas cicatrizantes podem apresentar grande potencial para desenvolver fitodefensivos agrícolas naturais não danosos, à semelhança de medicamentos fitoterápicos.

Aqueous extracts from the medicinal plant Achillea millefolium contain macromolecules of interest to develop agrochemicals for agriculture. Two fractions of "mil folhas" were obtained by ultrafiltration, E1 (containing molecules larger than 30 kDa) and E3 (peptides between 1 and 10 kDa), which inhibited the growth of phytopathogenic bacteria Ralstonia solanacearum, gram-negative, and Clavibacter michiganensis subsp. michiganensis, gram-positive, concentration-dependent. The values of minimum inhibitory concentration (MIC) for both extracts and both bacteria were low, ranging from 20 to 80µM. The MIC relative to total protein evidenced the presence of very active macromolecules in E3, although showing low protein concentration. E3 applies to the prospection of antimicrobial peptides. The estimated MIC relative to the amount of plant sample valued the natural antimicrobial potential of E1, which contains high protein concentration. E1 and E3 can be used in the development of agrochemicals for biotechnological purposes. The ultrafiltration procedure fractionated the samples in a rapid and native way and at a low cost; it also desalted, clarified, concentrated and purified both E1 and E3. This pioneering study on the separomics and on the antimicrobial activity of macromolecular aqueous extracts from "mil folhas" suggests that healing plants have great potential to develop non-harmful agricultural natural agrochemicals, similarly to the available phytotherapic drugs.

Isolation and analysis of differentially expressed genes from peanut in response to challenge with Ralstonia solanacearum

Background: Bacterial wilt caused by Ralstonia solanacearum is the most devastating disease in peanut. Planting resistant peanut cultivars is deemed as the sole economically viable means for effective control of the disease. To understand the molecular mechanism underlying resistance and facilitate breeding process, differences in gene expression between seeds of Rihua 1 (a Virginia type peanut variety resistant to bacterial wilt) inoculated with the bacterial pathogen suspension (10(9) cfu ml-1) and seeds of the same cultivar treated with water (control), were studied using the GenefishingTM technology. Results: A total of 25 differentially expressed genes were isolated. Expression of genes encoding cyclophilin and ADP-ribosylation factor, respectively, were further studied by real time RT-PCR, and full length cDNAs of both genes were obtained by rapid amplification of cDNA ends. Conclusions: The study provided candidate genes potentially useful for breeding peanut cultivars with both high yield and bacterial wilt resistance, although confirmation of their functions through transgenic studies is still needed.

Quick adaptation of Ralstonia Solanacearum to copper stress to recover culturability and growth in water and soil

Cells of Ralstonia solanacearum were exposed to Cu in distilled water, and the resulting Cu-stressed non-culturable cells were inoculated to natural (non-pasteurized) and pasteurized soils in order to examine their culturability and recovery. Exposing the cells to 20 µM CuSO4 produced transitory non-culturable cells, which exhibited a remarkable recovery in culturability after incubation in the solution for 36 h, reaching a density near the initial level by 108 h. To determine whether such non-culturable cells actually "resuscitated" or multiplied after adapting to Cu toxicity, growth curves were constructed in order to contrast the rates of increase in culturable cell numbers between Cu-stressed or non-stressed inocula. Additionally, fresh non-stressed cells were exposed to CuSO4 in the presence of nalidixic acid by adding the antibiotic at different times after the onset of Cu stress to verify any cell multiplication during the population increase. The results revealed that the non-culturable cells surviving Cu toxicity adapted very quickly to Cu and began multiplying within 12 h, because only the Cu-stressed cells that were increasing in the exponential growth phase, but not those in the stationary phase, were killed by the antibiotic. Such cells exhibited an apparent tolerance to this metal when inoculated to a freshly prepared solution of CuSO4, and also detoxified the ion in the solution in which they grew. The presence of nutrients greatly counteracted the effect of Cu in water microcosms, since culturable cells were detected and increased in number even when exposed to 40 µM CuSO4. In contrast, when inoculated to non-pasteurized soil, Cu-stressed cells showed no such recoveries. However, when the soil was pasteurized before inoculation or added with nutrients, culturable cells were recovered and increased in number. This indicates that increased nutrient availability in soil allows Cu-stressed cells to quickly overcome the stress and increase in culturable populations.

Isolation of pathogenic Ralstonia solanacearum from Pogostemon cablin and determination of its pathogenicity / 中草药

Objective: To isolate the pathogenic Ralstonia solanacearum from the infected Pogostemon cablin plants and to study the bacteria characteristics, so as to lay a foundation for the control of bacterial wilt. Methods: Pathogenic strains of R. solanacearum were isolated from the infected P. cablin plants by being streaked on a tetrazolium chloride (TZC) medium. These strains were used for the classification based on oxidative utilization of three kinds of disaccharide (lactose, maltose, and cellobiose) and three kinds of hexy I alcohol (mannitol, dulcitol, and sorbitol). The pathogenicity was tested by the methods of cutting and soaking roots. Results: Seven strains of pathogenic R. solanacearum were isolated from the infected P. cablin plants which contain four biotypes. Strains of HX5 and HX7 belong to biotype I, HX1 and HX6 belong to biotype II, HX2 and HX4 belong to biotype III, and HX3 belongs to biotype V. The result of pathogenicity test indicated that the most strains had strong pathogenicity. HX4 and HX5 have more serious virulence and can cause higher plant death rate. Conclusion: Strains of R. solanacearum with different biotypes from the P. cablin plants have different pathogenicity.

Fontes de resistência à murcha bacteriana em germoplasma de Capsicum spp. do estado do Amazonas / Sources of resistance against bacterial wilt in Capsicum spp. germoplasm of the Amazonas state

A murcha bacteriana, causada por Ralstonia solanacearum, é uma das doenças mais importantes do gênero Capsicum no Brasil. No Amazonas, as condições de elevada temperatura e umidade favorecem o desenvolvimento da doença. O objetivo deste trabalho foi avaliar a resistência à murcha bacteriana de germoplasma, selvagem e comercial, de Capsicum spp. Foram avaliados 22 acessos de Capsicum em casa de vegetação. A inoculação foi feita mediante ferimento das raízes, seguido de adição no solo, ao redor das plantas, de suspensão bacteriana na concentração de 10(8) ufc mL-1. A avaliação foi feita diariamente a partir do quarto dia após a inoculação, em função desenvolvimento dos sintomas. A partir das médias de progresso dos sintomas foi construída a área abaixo da curva de progresso da doença (AACPD), e os dados submetidos ao teste de Scott-Knott ao nível de 5 por cento de probabilidade, utilizando o programa estatístico SAEG 9.1. Foram selecionados os acessos 30, 20 e 17, da espécie C. chinense, como resistentes à murcha bacteriana para ensaios futuros em programas de melhoramento genético.

The bacterial wilt caused by Ralstonia solanacearum is one of the most important in the genus Capsicum in Brazil. In the state of Amazonas, high temperatures and humidity favor the development of the disease. The objective of this work was to evaluate resistance in germoplasm of wild and commercial Capsicum spp. to bacterial wilt. Twenty two accesses of Capsicum spp. were evaluated in greenhouse conditions. The inoculation was made by means of wounds in the roots, followed by addition of bacterial suspension in the concentration of 10(8) ufc ml-1 in the soil, around the plants. Plant evaluation was made daily after the fourth day of the inoculation (DAI) considering the symptoms progress. From the average progress of symptoms was constructed the area under the disease progress curve (AUDPC), and the data submitted to the Scott-Knott test at 5 percent of probability, using SAEG statistical program. From the average severity notes, we constructed the area under the disease progress curve (AUDPC). The accesses 30, 20 and 17 were selected from C. chinense as resistant to the bacterial wilt, for future use in genetic breeding programs.

Variabilidad genética de la bacteria Ralstonia solanacearum (Burkholderiales: Burholderiaceae) en la zona bananera de Urabá (Colombia)

Genetic variability of the bacterium Ralstonia solanacearum (Burkholderiales: Burholderiaceae) in the banana-growing region of Uraba (Colombia). The banana moko disease, caused by the bacterium Ralstonia solanacearum, is one of the most important phytopathological problems of the banana agribusiness in tropical countries. In Uraba and Magdalena (Colombia), the main exporting regions of banana in Colombia, this disease causes a destruction estimated in 16.5ha/year. The bacterium presents an extremely high level of genetic variation that affects control measures. This is the first study of its variation in Colombia and was done with AFLP molecular markers on a population of 100 isolates from banana plants, soils and "weeds". The high level of genetic diversity, with Nei and Shannon indexes of h=0.32 and I=0.48, respectively, and the AMOVA, showed that this population is subestructured (Fst=0.66): the host is the main factor of differentiation. Even so, previous tests show that all varieties have pathogenicity on Musa. Rev. Biol. Trop. 58 (1): 31-44. Epub 2010 March 01.

La enfermedad del moko de las musáceas, causada por la bacteria Ralstonia solanacearum, es uno de los problemas fitopatológicos más importantes de la agroindustria del banano en los países tropicales. En Urabá y el Magdalena (Colombia), las principales regiones exportadoras de banano en Colombia, esta enfermedad provoca una destrucción estimada en 16.5ha/año. La bacteria presenta un nivel extremadamente alto de variación genética que afecta las medidas de control. Este es el primer estudio de su variación en Colombia y se hizo con los marcadores moleculares AFLP en una población de 100 aislamientos de plantas de banano, suelos y arvenses. El alto nivel de diversidad genética, con índices de Nei y de Shannon de h=0,32 y I=0,48, respectivamente, y la AMOVA, demostró que esta población es subestructurada (Fst=0,66): el hospedero es el principal factor de diferenciación. Aun así, las pruebas anteriores mostraron que todas las variedades presentan patogenicidad en Musa.

Bactérias endofíticas como agentes promotores do crescimento de plantas de tomateiro e de inibição in vitro de Ralstonia solanacearum / Endophytic bacteria as agents of plant growth promotion in tomato and inhibition in vitro of Ralstonia solanacearum

A partir de 150 isolados de bactérias endofíticas obtidos de folhas, caules e raízes de tomateiros sadios, 53 destacaram-se quanto à habilidade em promover o crescimento de plantas de tomateiro (Solanum lycopersicum L.). Submetidos a uma nova seleção, os isolados UFV-E17, UFV-E22, UFV-E25, UFV-E26, UFV-E27, Bacillus cereus (UFV-E29), UFV-E49, UFLA 06-LS, UFLA 08-LS e UFLA 11-LS apresentaram maior promoção do crescimento. Avaliações semanais de altura e número de folhas e folíolos das plantas aconteceram durante 45 dias. Após a sexta avaliação, mensurou-se a área foliar e o peso da matéria fresca e seca da parte aérea e da raiz das plantas. O isolado UFV-E49 apresentou melhor resultado para altura, área foliar, número de folhas e peso da matéria fresca e seca, tanto da parte aérea quanto da raiz. Dos isolados selecionados, somente dois apresentaram efeito inibitório direto in vitro a Ralstonia solanacearum.

Out of one hundred and fifty isolates of endophytic bacteria from leaves, stems and roots of healthy tomatoes (Solanum lycopersicum L.), fifty three showed ability to promote tomato plant growth, among these, ten isolates UFV-E17, UFV-E22, UFV-E25, UFV-E26, UFV-E27, Bacillus cereus (UFV-E29), UFV-E49, UFLA 06-LS, UFLA 08-LS and UFLA 11-LS, provided the largest plant growth promotion. Weekly assessment of plant height and number of leaves and leaflets were carried out during 45 days. After the sixth evaluation, the leaf area and the fresh and dry weight of the aerial part of plants and of the roots were measured. Isolate UFV-E49 provided the largest values of height, leaf area, number of leaves and fresh and dry weight of the aerial part of plants as well as the root. From the selected isolates, only two presented antimicrobial activity against Ralstonia solanacearum.

Effect of Cultural Condition on Fatty Acid Composition of Ralstonia solanacearum / 微生物学通报

Fatty acids of Ralstonia solanacearum cultured under different temperatures, times, pH values and cultural media were detected by using gas chromatography (GC) method. Rs-J.1.4-010704-01v, a viru- lent strain of R. solanacearum isolated from ginger was chosen for the experiment. The results showed that the kind of fatty acid of Rs-J.1.4-010704-01v fluctuated from 14 to 33. The contents of its three plentiful fatty acids, C16:1?7c/C15:0 ISO 2OH, C16:0 and C18:1?7c (with retain times of 10.644, 10.950 and 14.177 min, respectively), also varied in a range of 55.66% to 75.69%. The diversification of the bacterium’s fatty acids at various cultural conditions was clustered into four groups by cluster analysis, according to the kinds and percentage contents of the fatty acids detected. The pathogenicities of Rs-J.1.4-010704-01v under 20?C and 25?C were deduced to be mid-virulent, with C16:0 less than C16:1?7c/C15:0 ISO 2OH. The bac- terium showed as a virulent strain under the other cultural conditions including 30?C~40?C, 24 h~96 h, pH 5~9 and four cultural media (LB、NA、TTC and TSB), with C16:0 more than C16:1?7c/C15:0 ISO 2OH.However, the difference between C16:0 and C16:1?7c/C15:0 ISO 2OH raised significantly from 2.35 to 13.23 under 40?C and 48 h~96 h. Meanwhile, the kind of fatty acid increased more than 30 as the cultural time increased. It was concluded that temperature and cultural time had more significant effects on the fatty acid composition of R. solanacearum than pH value and cultural medium.

Study on Population of Bacterial Physiological Groups in Tomato with Different Resistance to Ralstonia solanacearum / 微生物学通报

The population of bacterial physiological groups in tomato with different resistant to Ralstonia solanacearum was studied. The results suggested that endophytic bacterial communities and population in tomato variety changed with different resistant cultivars, different stages of tomato and seasons. It was con-ducted that the amount of ammoniation bacteria was the highest among the seven physiological bacterial groups. There were more ammoniation bacteria in high resistant tomato cultivars than that in high suscepti-ble cultivars. It may indicate that ammoniation bacteria played a key role in the occurrence of tomato bacte-rial wilt. In addition, the total amount of physiological bacteria in resistant cultivars was more than that in susceptible cultivars in different stages of tomato, and the tendency of changing displayed fluctuation. The average level of quantities of the ammoniation bacteria, nitrifiers bacteria, erobic nitogenfixing bacteria and desulphate reducer bacteria in summer were higher than that in winter, while the population of the sulphate reduced bacteria in winter was higher than that in summer. Furthermore, the amount of anaerobic bacteria was the least among them.

Optimization of Ralstonia Solanacearum Preparation Methods by HPLC Analysis / 中国生物工程杂志

16M?) ). Using this method could not only avoid the interference of medium on the chromatographic behavior of Ralstonia solanacearum, but also keep the cell viability and cell surface properties.