Biovar Research Articles

Virulence and characterization of isolates of potato bacterial wilt caused by Ralstonia solanacearum (Smith) in Rwanda

Bacterial wilt (Ralstonia solanacearum) is one of the major potato diseases in Rwanda. An in vitro study was carried out to identify and characterize the pathogen isolated from three potato cultivars (Kinigi, Kirundo and Gikungu) in Rwanda. This was achieved by cultural and morphological tests on triphenyl tetrazolium chloride (TTC) and casamino peptone glucose (CPG) agar as well as biochemical tests through Gram staining and biovar test. An in vivo experiment was also performed to assess the pathogenicity of those isolates on potatoes. All isolates showed typical morphological traits of virulent R. solanacearum on TTC and CPG media. The test isolates were Gram-negative bacteria. Biovar test confirmed that all the isolates belonged to race 1 biovar 3 of the pathogen. Furthermore, the highest disease severity (DS=100%) and disease incidence (DI=100%) were observed in Gikungu isolate followed by Kinigi (DS=97.33% and DI=98.25) and Kirundo (DS=94.67% and DI=92.61%). From this study, all three isolates were typical R. solanaceraum belonging to race 1 biovar 3 and were all pathogenic to potato plants. Gikungu and Kinigi isolates were highly virulent than Kirundo isolate. Therefore, Gikungu or Kinigi isolates can be used for further studies in plant protection in management of the disease. Key words: Biovar test, Gram-negative, Gram-positive, pathogenicity test.

Genotyping of human Brucella melitensis strains isolated in Shanxi province by MLVA-2B typing and rpoB typing

Objective To analyze the genotypes of Brucella melitensis strains isolated in Shanxi province by using multiple-locus variable-number tandem-repeat 2B typing (MLVA-2B) and rpoB typing. Methods Brucella strains were isolated by culturing the blood samples collected from patients with brucellosis in Shanxi province from 2014 to 2015 and then were identified with conventional methods. MLVA panel 2 (eight loci)was used to identify the genotypes of the Brucella melitensis strains, and the results were further analyzed by cluster analysis. The rpoB gene was molecularly characterized by PCR amplification and DNA sequencing in those strains. The rpoB nucleotide sequences of all Brucella strains were compared with the published rpoB gene sequence of the Brucella melitensis strain 16M to reveal specific nucleotide variations. Results A total of 41 Brucella strains were isolated in 2014 and 2015 and all of them belonged to Brucella melitensis biovar 3. Those strains were divided into five groups and thirty-one MLVA types based on MLVA panel 2B with a genetic similarity ranging from 64.78% to 100%. Four mutation sites in rpoB gene were investigated in the 41 Brucella strains and based on that analysis, those strains were grouped as rpoB-2 (629-GTG/985-GTC/1309-CTA, 95.12%) and rpoB-2 variant (629-GTG/1309-CTA, 4.88%). Conclusion MLVA genotyping was significant for analyzing the sources, epidemics and outbreaks of brucellosis. Analyzing the panel 2B loci was a simple and efficient way for genotyping the Brucella melitensis strains isolated in Shanxi province. The 41 strains belonged to two genotypes on the basis of rpoB typing, including 39 strains of rpoB-2 genotype and 2 strains of a new genotype rpoB-2 variant. There might be a correlation between MLVA and rpoB genotyping. Key words: Brucella; Multiple-locus variable-number tandem-repeat analysis; rpoB gene

The effects of two biovars of ureaplasma urealyticum on sperm motion parameters

Objective To investigate the effects of two biovars of ureaplasma urealyticum on leukocyte concentration and sperm motion parameters. Methods Sperm motion parameters were analyzed with computer-aided sperm analysis (CASA). Leukocytic number of semen was measured with benziding-perxidase staining. Culturing and real-time polymerase chain reaction (RT-PCR) was used to identify biovars of U. urealyticum in the semen. Results Sperm motion parameters including mean curvilinear velocity (VCL), mean straight-line velocity (VSL), mean average path (VAP), and mean beat Cross frequency (BCF) showed significant difference between infertile and normal group (P<0.05). Biovar Ⅱ infected men displayed significant increase in leukocyte counts (P<0.001). When compared to uninfected group, the sperm motion parameters showed significant decreases of VCL, VSL, VAP, mean percentage lineality (LIN), and swing(WOB) in biovar Ⅱ(P<0.05). Conclusions Biovar Ⅱhas closer relationship with leukocyte elevation and sperm motion impairment. Key words: Ureaplasma urealyticum; Spermatozoa; Semen/CY

First report of Brucella suis biovar 2 in a semi free-range pig farm, Italy.

This communication describes the isolation of Brucella suis (B. suis) biovar 2 in semi‑free‑range pigs located in the province of Rome, Italy. Sera of 28 pigs from a herd with reproductive problems were tested for brucellosis. Twenty-five sera (89%) were found positive to Rose Bengal Test (RBT), while 22 (79%) were positive to Complement Fixation Test (CFT). Two positive pigs were slaughtered, organs were collected and tested for the presence of bacteria. Brucella spp. was isolated from the spleens and the abdominal lymph nodes of the 2 subjects. The isolates were identified as B. suis biovar 2 by biochemical and Polymerase Chain Reaction (PCR) tests. The frequent infringement in the fences of the premises and the birth of striped piglets provided evidence that sows mated with wild boar, the major reservoir of B. suis biovar 2. Conversely, the isolation of B. suis biovar 2 from spleens and lymphnodes of seropositive slaughtered animals only, as well as the constant negative results from all vaginal swabs and the abortion materials tested, raise doubts on the implication of B. suis biovar 2 in the infertility of the holding.

Virulence to tomato of Iranian isolates of Ralstonia solanacearum.

Four strains belonging to biovars 2A and 2T (N2) of Ralstonia solanacearum and two mutants impaired in extracellular polysaccharide (EPS) production were studied for their virulence to tomato, colonization of host tissue and survival in the soil. Mutants impaired in EPS biosynthesis were generated using a plasmid containing Tn5. Results showed that whereas biovar 2A is more virulent than 2T, they did not differ in the ability to survive in the soil. Susceptibility of various tomato cultivars to R. solanacearum strains was evaluated. Results showed that besides the bacterial biovar, the host cultivar is effective in determining disease severity. However, no significant differences were observed in bacterial populations from tomato cultivars with different susceptibility. Mutants defective for EPS production were less virulent than the wilde-type strain, highlighting the role of EPS in pathogenicity. No difference was observed in the colonization of tomato stems by either mutant impaired in EPS production and the wild-type.

CHARACTERIZATION OF RALSTONIA SOLANACEARUM CAUSING BACTERIAL WILT IN BELL PEPPER IN THE STATE OF PERNAMBUCO, BRAZIL

Seventy-seven bacterial strains obtained from wilted bell pepper plants from the Agreste and Mata mesoregions of Pernambuco State (northeastern Brazil), were identified as Ralstonia solanacearum using multiplex PCR. The strains were further analyzed to characterize their biochemical, physiological, and molecular diversities. The biovars and biotypes were determined biochemically, and a molecular characterization of the strains was made using multiplex PCR, rep-PCR, and ISSR. The genotypic diversity was assessed, considering the number of genotypes observed and how they were distributed throughout the populations, for their differences in terms of richness, evenness and diversity. Strains of biovar 3, biotype 8 and phylotype I were predominant (97.40%), but biovar 1, biotypes 3 and 6 and phylotype II were also present. Rep-PCR analysis using REP and BOX primers, and ISSR showed similarities among the majority of the strains; however, these primers did not allow strain separation by biovars, biotypes, phylotypes or areas. Analysis of the genotypic diversity revealed a moderate diversity in the overall population, with a high variability in the strains from the same municipality.

Identification and genetic characteristics on the bacteria isolate from a case of human Brucellosis in Guizhou province

A suspected Brucella (B.) strain(GZZA), isolated from a case of anti-Brucella antibody positive patient was identified and its' genetic characteristics was analyzed, to provide etiologic basis for the confirmation of patient in Guizhou province. Conventional methods and polymerase chain reaction(PCR)were used to identify the bacteria strain, with genetic characteristics analyzed by MLVA-16. The bacteria strain was identified as B. melitensis biovar 3 under the conventional and PCR methods. Results from the MLVA-16 analysis indicated that the bacteria strain was closely clustered with B. melitensis biovar 3, and differences of repeated numbers at VNTR loci bruce42, bruce04, bruce09 and bruce16 were also displayed. Both traditional and molecular methods to identify one bacteria strain isolated from the human patient as B. melitensis biovar 3 and the genetic characteristics of the strain was closely related to that of B. melitensis biovar 3. Differences of repeated numbers at part of VNTR loci were also showed. The results of this study provided etiologic evidences for the confirmation of Brucella infection of the patient, also providing scientific basis for the control and prevention of Brucellosis in Guizhou province.

HOST RANGE VARIATION AND GENETIC DIVERSITY OF IRANIAN ISOLATES OF RALSTONIA SOLANACEARUM FROM POTATO AND TOMATO WITH RAPD AND (GTG)5-PCR

Of 170 Iranian isolates of Ralstonia solanacearum from wilting potato and tomato plants, 130 were ana- lyzed for genetic and pathogenicity diversity. Specific PCR assays identified all the isolates as R. solanacearum biovar N2 (2T) and 2A. Based on pathogenicity and PCR tests, the isolates were divided into two pathogenic groups. Group I, corresponding to race 3, was patho- genic to potato and tomato, whereas group II, corre- sponding to race 1, was pathogenic to tomato, potato, eggplant and tobacco. Molecular analysis was done with random amplified polymorphic DNA (RAPD) and rep- PCR using (GTG)5 primer. RAPD and (GTG)5-PCR analysis divided the 130 Iranian isolates of R. solanacearum into eight haplotypes that were grouped in three clusters. Cluster I comprised all biovar 2A isolates with four haplotypes, cluster II and III contained biovar N2 isolates with four haplotypes. Both RAPD and (GTG)5-PCR enabled discrimination of R. solanacearum strains according to biovar and the host of origin.

Development and application of one-step polymerase chain reaction(PCR) for rapid identification of Brucella species and some biovars

Objective To develop a one-step PCR assay for rapid discrimination of six Brucella species and some intraspecific biovars.Methods Using 6 pairs of primers in one-step PCR to differentiate six classical Brucella species and some biovar in ordinary PCR instrument.The tested strains including 27 reference strains of six Brucella species and 239 Brucella strains were estimated by the PCR assay and biological identification methods.Results The six Brucella species could be precisely differentiated by the onestep PCR assay from the tested strains.Five biovars and vaccine strain of B.suis species could be determined,and biovars 1,3,4 and biovars 2,5,6,7,9 of B.abortus species could be identified at the level of their biovar,moreover,biovars 1,2 and 3,and vaccine strain Rev 1 of B.melitensis species were also discriminated at the biovar and strain level.The accurate rates of the biological identification method and the PCR assay were 98.33％ and 100％ respectively.Conclusion One-step PCR assay was a rapid,specific,and low cost method for identification of Brucella species and discriminating biovars in ordinary PCR instrument. Key words: Brucella ; Polymerase chain reaction; Identification

Survival of &lt;i&gt;Pseudomonas syringae&lt;/i&gt; pv &lt;i&gt;actinidiae&lt;/i&gt; in nonkiwifruit green compost

Pseudomonas syringae pv actinidiae (Psa) the causal agent of bacterial canker of kiwifruit can survive in the environment outside its host Determining where Psa can survive in the environment is important to limit the incidence and spread of this disease Composts are sometimes used in kiwifruit orchards and the goal of this study was to determine the risk associated with such a practice Three commercially prepared composts made from nonkiwifruit green waste were analysed for presence of Psa and for the ability of Psa to survive Psa was not found in any of the composts analysed An antibioticresistant derivative of Psa biovar 3 (previously PsaV) used in this experiment could not multiply in any of the composts and its population declined rapidly Live Psa could not be detected 7 days after inoculation In sterile composts Psa could be detected for at least 8 days after inoculation

Identification and analysis of a putative pathogenicity island Uu146-Uu170 in Ureaplasma urealyticum collected from sterile patients

To identify and analyze a putative pathogenicity island (PAI) Uu146-Uu170 of Ureaplasma urealyticum (Uu) and explore the relationship between PAI and infertility. Fifty-one Uu isolates were collected from healthy females, 57 isolates from sterile females with fallopian tube disease, 42 isolates from healthy males and 38 isolates from sterile males. Biovar-typing was performed based on the gene of multiple-banded antigen (MBA). The distribution of PAI was analyzed by PCR. The association of clinical phenotype with biovar type and PAI distribution was calculated by Fisher's exact test. In female healthy group and female disease group, biovar type 1 account for 68.6% (35/51) and 73.7% (42/57), whereas the positive rate of PAI were 15.7% (8/51) and 12.3% (7/57) in the two groups, both difference were not statistically significant. In male healthy group and male disease group, biovar type 1 account for 71.4% (30/42) and 65.8% (25/38), whereas the positive rate of PAI were 21.4% (9/42) and 18.4% (7/38) in the two groups, both difference were not statistically significant. The positive rate of PAI in biovar type 2 was significantly higher than that in biovar type 1 in the fallopian tube disease and male infertility groups (5/15 vs 2/42, 5/13 vs 2/25, both P < 0.05) whereas no significant difference was found between two biovar types in normal groups (both P > 0.05). There is insufficient evidence that Uu with presence of PAI is more pathogenic to cause infertility. The Uu strain of biovar type 2 with PAI Uu146-Uu170 may be associated with infertility.

Molecular epidemiological study of Ureaplasma urealyticum and Mycoplasma hominis in urogenital tract of elementary and high school students in Qingdao area

Objective To detect Ureaplasma urealyticum (Uu) and Mycoplasma hominis (Mh) in urogenital tract of healthy elementary and high school students,and to determine the biovars and serotypes of Uu isolates.Methods Urine specimens were collected from 957 healthy school children,including 404 elementary school students,260 junior high school students and 293 senior high school students,and submitted for Uu and Mh culture in selective liquid medium.Thc specimens positive for Uu and (or) Mh culture were subsequently subjected to PCR.The biovars and serotypes of Uu were determined.Results Uu was detected in 4.81％ (46/957) of the school children.Among the 46 Uu positive specimens,32 (69.57％) harbored Ureaplasma Parvum (biovar 1 ),14 (30.43％) Ureaplasma urealytieum (biovar 2); 12 (2.97％) were from elementary school students,including 9 (75.00％) cases of Ureaplasma Parvum and 3 (25.00％) Ureaplasma urealytieum,8 (3.08％) were from junior high school students,including 5 cases of Ureaplasma Parvum and 3 Ureaplasma urealytieum,26 (8.87％) were from senior high school students,including 18 (69.23％) cases of Ureaplasma Parvum and 8 (30.77％)Ureaplasma urealytieum.Mh was positive in 9 (0.94％) school students,including 1 elementary school student,1 junior school student and 7 senior high school students.The detection rate of Uu was significantly higher in females than in males in all the 3 groups (elementary school,junior and senior high school) of students.Conclusions Mh and Uu are commensals in urogenital tract of elementary and high school students without symptoms.Ureaplasma Parvum is the predominant biovar in this population,though Ureaplasma urealytieum accounts for a significant proportion,which implies that Uu is commensal in healthy elementary and high school students,and females are more liable to be colonized than males. Key words: Ureaplasma urealyticum; Mycoplasma hominis; Urogenital system; Epidemiology; Students

Study on the exocellular polysaccharide of Ureaplasma urealyticum biofilm in vitro

Objective To investigate the extracellular polysaccharide distribution and components of Ureaplasma urealyticum (Uu) after biofilm having been developed in.Methods The standard serotype 3 and serotype 14 belong to biovar Parvo,and the standard serotype 4 and serotype 8 belong to biovar T960 were employed to form biofilrns in vitro.Scanning electron microscope and confocal laser scanning microscope were used to analysis the biofilms and extracellular polysaccharide.We used combination of two different labeled lectins,Canavalia ensiformis(FITC-ConA) and Erythrina cristagalli(ECA) which bind to specific polysaccharide residues to visualize extracellular polysaccharide in biofilms,and average uorescence intensity was evaluated Results All the strains can form the biofilmsin vitro.The biofilm was honeycomb-Like structures mainly,and extracellular polymeric substances accounts for majority of proportions.All the extracellular polysaccharide could be combined with FITC-ConA and ECA,and the total average fluorescence intensity of FITC-ConA was higher than ECA( P＜0.001 ).Conclusion Ureaplasma urealyticum biofilm is honeycomb-like structures mainly.The extracellular polysaccharide contains,galactose,and N-acetyl glucan residual,and the glucose,mannose residual are the main components. Key words: Ureaplasma urealyticum; Biofilm; Exopolysaccharide

BIOLOGICAL AND MOLECULAR CHARACTERISTICS OF IRANIAN BIOVARS 2A AND 2T OF RALSTONIA SOLANACEARUM

The biological and molecular characteristics of 8 strains of biovars 2A of Ralstonia solanacearum from the Iranian province of Isfahan and of 7 isolates of biovar 2T from the province of Khuzestan were investigated morphologically, physiologically, biochemically, and using pathogenicity tests, BOX-PCR, ERIC-PCR and RFLP. Biovar 2A strains showed irregular, creamy-white and highly fluid colonies with pink center on TZC medium, slow spreading necrosis in hypersensivite reaction (HR) after 48 h, produced melanin in tyrosin test, were less pectolytic and did not utilize ribose and tryptophan. By contrast, biovar 2T strains had small size, less fluid, round and deep red colonies with a thin white margin, did not produce melanin, had a higher pectolytic activity, rapid HR on tobacco leaves after 24 h and utilized ribose and tryptophan. Comparative analysis of the BOX-PCR and ERIC-PCR fingerprint of the 15 strains at the 45% similarity level, revealed two main clusters. comprising all biovar 2A strains (cluster 1) or all biovar 2T strains (cluster 2). RFLP profiles of both biovers did not show any polymorphism. Typically, biovar 2A isolates were more pathogenic and bacterial density was higher in the xylem of all hosts tested. There was no significant difference in biovar 2A and 2T survival in loamy-clay soil, which was up to 186 days under controlled conditions.

Genotypic Diversity of Iranian strains of Ralstonia solanacearum

Normal 0 14 false false false During 2007–2009, 170 bacterial isolates were obtained from wilting potato and tomato plants from Iran. Specific polymerase chain reaction (PCR) analyses identified all the isolates as Ralstonia solanacearum . Diversity among the Iranian isolates was determined by a study based on biovar determination, pathogenicity, repetitive sequence PCR and phylotype classification. On the basis of genotypic, phenotypic and pathogenic characteristics two distinct clusters obtained. Cluster I comprised all biovar 2 isolates and cluster II comprised all biovar N2 (2T) isolates. Phylotype-specifi c multiplex PCR (pmx-PCR) indicated that all biovar 2 and N2 belong to phylotype II. This is the first report of phylotype determination of biovars of R. solanacearum with pmx-PCR in Iran.

OCCURRENCE AND PATHOGENICITY OF PSEUDOMONAS FLUORESCENS AND P. PUTIDA ON TOMATO PLANTS IN ITALY

SUMMARY During 2006-2008, a survey conducted in soilless and soil-grown tomato crops in eastern and southeastern Sicily (Italy) disclosed the presence of widespread leaf chlorosis and necrosis associated with yellow, pink or brown discolouration of vascular and pith tissues. Bacteria were always isolated from symptomatic tissues on KB and NA media. A total of 158 isolates showed the LOPAT characters of group Va (-+-+-) and group Vb (++-+-) and 21 of these strains were identified as different biovars of Pseudomonas fluorescens and P. putida. Koch’s postulates showed that 42 fluorescent Pseudomonas isolates are able to induce vascular discolouration of tomato plants but not leaf chlorosis and necrosis. The interactions of vascular infections and abiotic stresses were studied in growth chamber experiments. Differences in virulence were found among P. fluorescens biovar I and P. putida bv. A strains on tomato plants grown under saline stress conditions. Both bacteria were able to induce leaf chlorosis, necrosis or death of tomato plants under saline stress conditions.

COLEUS, A NEW HOST FOR RALSTONIA SOLANACEARUM RACE 1 BIOVAR 3 IN INDIA

Ralstonia solanacearum was isolated from wilting coleus plants (Coleus forskohlii) in six commercial nurseries around Bangalore (south India). Colonies of all isolates were irregular with smooth margins, slimy dull white with pink to red centre on SMSA medium, Gramnegative, non-capsulated and non-spore forming. Bacterial identity was confirmed by PCR with species-specific 16S rDNA-based primers OLI1+Y2 and DAS-ELISA. The isolates could infect tomato, potato, and ginger, but failed to infect mulberry and banana. Six isolates were identified as race 1, based on pathogenicity tests, and as biovar 3, based on carbohydrate utilization.

Ralstonia solanacearum biovar 1 associated with a new outbreak of potato brown rot in Portugal

In May 2007, potato plants exhibiting symptoms possibly of brown rot were collected in some potato fields in the Baixo Mondego region (Center), Portugal, as a part of a nationwide programme to monitor Ralstonia solanacearum. All laboratory procedures laid down in Commission Directive 2006/63/EC, including dilution plating on semi-selective medium SMSA, indirect imunofluorescence (IIF), polymerase chain reaction (PCR) using specific primers and bioassays on tomato plants, were strictly followed and the causal agent of the disease was identified as Ralstonia solanacearum. The identity of the pure cultures of the isolated organism was confirmed by PCR, IIF and pathogenicity tests on several other plant species (eggplant, tobacco, pelargonium and eucalyptus). In biovar determination, the failure of the isolates to utilise/oxidise certain carbon sources indicated that the isolates were all biovar 1. This biovar has a broader host range than biovar 2 strains, and affects several crops of economic importance including ornamental plants and forest trees. Comparative analysis of 16S rRNA and endoglucanase (egl) gene sequences of these isolates with sequences that have been deposited at the GenBank revealed a similarity higher than 99% for several Ralstonia solanacearum isolates from biovar 1, including isolate DAR 64836 (Accession number DQ011551). This is the first report of Ralstonia solanacearum biovar 1 in Portugal. All control measures specified in the Commission Directive are being implemented.

INTRODUCTION TO EUROPE OF RALSTONIA SOLANACEARUM BIOVAR 2, RACE 3 IN PELARGONIUM ZONALE CUTTINGS

In December 1999, a sample of imported Pelargonium zonale cuttings was found to be infected by Ralstonia solanacearum in the UK. From September to December 2000, bacterial wilt was diagnosed in several Pelargonium nurseries in Belgium, Germany and the Netherlands. R. solanacearum biovar 2, race 3 was consistently isolated and identified. Surveys traced the origin of infections to cuttings produced in Kenya for the European market. Quarantine measures were imposed to prevent spread and persistence of the organism. Infections of R. solanacearum have not been found on imported cuttings or nurseries in European countries since then. The introduction of the pathogen in Pelargonium raised concern of spread to potato. In Kenya it was found that the contamination most probably occurred through contaminated surface (river) water used for irrigation in the nurseries. Infection was found in different cultivars. The nurseries in Kenya have taken strict hygiene and water disinfection measures and eliminated all infected and probable infected plant material. Since than no further infections have been found. The R. solanacearum isolates from Pelargonium were characterised as R. solanacearum biovar 2, race 3 on the basis of host range, morphology, serology, PCR (also using biovar-specific primers, REP-PCR, AFLP) and fatty acid patterns. Symptoms in Pelargonium, results of strain characterisation and a proposed testing method for latent infections are presented.

Occurrence and distribution of crown gall disease in Jordan

Field inspection indicated that crown gall disease occurs in various fruit-tree-growing areas in Jordan, including Almafraq, Alsalt, Alshobak, Altafila, Alyadoda, Amman, Irbid, Jerash, Jordan Valley and Madaba. Twohundred tumorigenic Agrobacterium isolates were obtained from stone fruit rootstocks (bitter almond, GF677, GF305, Myrobalan, Nemaguard and Mahaleb), pome fruit rootstocks (apple seedling, quince, and clonal rootstock MM106), grapevine, olive, pomegranate, carob, rose and Cichorium pumilum. The pathogenicity of these isolates was proved through artificial inoculation on tomato seedlings and Kalanchoe plants, and through tmr gene detection with PCR. Biochemical and physiological testing of the isolates indicated the occurrence of biovar 1 (60.5%), biovar 2 (23.5%), and biovar 3 (1%), in addition to an intermediate biovar (15%). Seventy-seven percent of isolates were found to be sensitive to agrocin 84; of these, most (66.9%) belonged to biovar 1.