rDNA RFLP Research Articles

First report of ‘Candidatus Phytoplasma australasia’‐related strains associated with disease of Jasminum multiflorum in India

Jasminum multiflorum (cv. Star Jasmine; family Oleaceae) is an important ornamental shrub native to South-East Asia. It is cultivated across the tropics for its ornamental value, in particular its fragrant, beautiful white flowers. During February 2020, symptoms of leaf yellowing and the premature decline of inflorescences were observed on 6.5% of plants grown in home gardens (Figs. 1, 2) in the Uttara Kannada district of Karnataka state, India. Four symptomatic and two symptomless plants were subjected to genomic DNA extraction using the CTAB method. Nested PCR was performed with universal phytoplasma 16S rRNA primers P1/P7 (Deng & Hiruki, 2; Schneider et al., 6) and R16F2n/R16R2 (Gundersen & Lee, 4). A DNA extract from the periwinkle virescence phytoplasma of group 16SrI, ‘Candidatus Phytoplasma asteris’ (MN223677) was used as the positive control. Amplification of the 16S rRNA gene was obtained only from symptom-bearing jasmine and the positive control, but not from the symptomless plants. The nested PCR products (~1.25kb) were purified and sequenced directly. Sequences were compared to those of reference phytoplasmas and subjected to in silico RFLP with iPhyclassifier (Zhao et al., 2009) and phylogenetic analysis using the neighbour-joining method of MEGA X (Kumar et al., 2018). The R16F2n/R16R2 gene sequences of the J. multiflorum phytoplasmas (GenBank Accession Nos. MW049328-MW049329) showed 99.9-100% sequence identity with those of the reference phytoplasmas of the former Peanut witches’ broom (16SrII) group, ‘Candidatus Phytoplasma aurantifolia’, including ‘Ca. P. australasia’ (Y10097). The virtual RFLP profiles of both J. multiflorum phytoplasmas were identical to each other and to those of the ‘Ca. P. australasia’ related strain (Y10097) of the 16SrII-D subgroup with a similarity coefficient of 1.00. The phylogenetic tree confirmed that the J. multiflorum phytoplasmas were closely related to the 16SrII-D, ‘Ca. P. australasia’ strain cluster (Fig. 3). Based on the results of the 16S rDNA nucleotide sequence comparison, phylogenetic and RFLP analyses, the J. multiflorum phytoplasmas associated with inflorescence yellowing and premature decline were identified as strains of the ‘Ca. P. australasia’, 16SrII-D subgroup. Phytoplasmas of 16SrII, 16SrXI (‘Ca. P. oryzae’) and 16SrI (‘Ca. P. asteris’) groups have been reported previously affecting J. sambac in Oman and India (Al-Zadjali et al., 1; Madhupriya et al., 5; Gopala & Rao, 3). However, this is the first report of the association of ‘Ca. P. australasia’ affecting J. multiflorum in India. Considering the economic importance of jasmine for the ornamental industry, this report provides significant information to support informed phytosanitary measures to prevent the spread of ‘Ca. P. australasia’ to nearby jasmine and other important members of the Oleaceae. The authors thank Department of Science and Technology, Government of India for providing financial assistance through DST-INSPIRE Fellowship scheme (IF.160687).

Phylogenetic diversity and cross-inoculation of indigenous isolated Bradyrhizobium from nodules of peanut in Liaoning province of China.

Arachis hypogaea. L is a legume of economic importance, which is nodulated by Bradyrhizobium, a slow-growing bacteria. However there is no well characterization of this rhizobia in many areas of China. In the present study, cross-inoculation experiments were performed in cowpea and soybean. The isolated bacteria strains were characterized physiologyically, biochemically and identified through 16S rDNA sequence analysis showing that it belongs to Bradyrhizobium japonicum. The genetic diversity of the seventeen isolated strains were assessed through PCR-RFLP of 16S rDNA and 16S-23S rDNA IGS region. Cross inoculation test indicated that isolates could nodulate cowpea but not soybean. The cluster analysis based on physiological and biochemical characteristics showed the lower correlation between isolates and sites. The isolates were grouped into four clusters based on 16S rDNA gene sequence analysis. Thirteen polymorphisms were variable across all observations in 16S rDNA RFLP and six different IGS types from isolates. The results implies that there was some association between geographical factor and phylogenetic diversity of indigenous Bradyrhizobium isolates.

A ‘Candidatus Phytoplasma omanense’-related strain detected in yellowing grapevine, stunted bindweed and Cixiidae planthoppers in Lebanon

Grapevine yellows (GY) affecting Lebanese vineyards are reported to be associated with ‘Candidatus Phytoplasma solani’, the bois noir (BN) etiological agent. However, during a field survey in June 2014 for BN in Mansoura municipality of West Bekaa, Lebanon, ‘Candidatus Phytoplasma omanense’ was detected in a grapevine sample, cultivar Syrah, exhibiting leaf scorch and discoloration, using a phytoplasma universal nested-PCR and sequencing of the 1.2 kbp 16SrDNA amplicon. The same 1.2 kbp 16SrDNA sequence could be amplified from Hyalesthes obsoletus and Reptalus sp. Cixiidae planthoppers collected on Convolvulus arvensis (bindweed) plants in June 2014 in Aammiq municipality of West Bekaa. Yellowing and stunted bindweed plants collected in 2015 in Kefraya and Aamiq municipalities of West Bekaa were also found infected with the same phytoplasma strain. A 16S rDNA RFLP assay was designed to differentiate this phytoplasma from ‘Ca. P. solani’ and ‘Ca. P. phoenicium’, which are endemic to the Bekaa region. ‘Ca. P. omanense’ is reported for the first time in grapevine and in Cixiidae planthoppers already known to respectively host and vector ‘Ca. P. solani’ strains associated with BN disease in grapevine. This result highlights the need for a precise survey of phytoplasmas associated to grapevine yellows in the Eastern Mediterranean basin, and for investigating the possible role of ‘Ca. P. omanense’ as a new threat to Euro-Mediterranean viticulture.

FIRST REPORT OF ‘CANDIDATUS PHYTOPLASMA SOLANI’ IN PEPPER AND CELERY IN BOSNIA AND HERZEGOVINA

In August 2015, in Bijeljina (Semberija province of Bosnia and Herzegovina) pepper and celery plants were surveyed for phytoplasma infections. During the survey pepper plants (Capsicum annuum ) of cvs Fortesa, Niska Sipka and Amanda showing stunting and leaf yellowing were observed, while celery plants (Apium graveolens L.) variety ‘Giant Prague’, expressed leaf whitening and stunting; the percentages of symptomatic plants ranged from 20 to 30%, respectively. Leaf samples from symptomatic and asymptomatic plants were tested to verify phytoplasma presence using nested-PCR/RFLP analyses. Results of 16S rDNA RFLP analyses with Tru1I and tuf typing using HpaII (Langer and Maixner, 2004) indicated the presence of a ‘Candidatus Phytoplasma solani’-related strain in all the symptomatic samples tested. There was no variability in the tuf gene and only tuf-type b was detected. Four symptomatic pepper and two celery samples were selected for further characterization amplifying vmp1 and stamp genes (Fialova et al., 2009; Fabre et al., 2011). The nucleotide sequences of the obtained amplicons were submitted to GenBank (accession Nos. KU340846-51 and KU295501-06 for vmp1 and stamp sequences, respectively). Homology with ‘Ca. P. solani’ sequences from database was 96- 100% for the vmp1 and 99-100% for the stamp gene. Combined RFLP analyses using Tru1I and Hpy188I on stamp and RsaI on vmp1 genes distinguished four ‘Ca. P. solani’ lineages indicating the presence of genetic variability among the phytoplasmas studied. This is the first report of ‘Ca. P. solani’ presence in symptomatic pepper and celery in Bosnia and Herzegovina.

First Report of a 16SrII-C Phytoplasma Associated with Asymptomatic Acid Lime (Citrus aurantifolia) in Brazil.

At present, the principal bacterial disease of citrus in Brazil is Huanglongbing, caused by the alpha-proteobacterium 'Candidatus Liberibacter spp.' (although a phytoplasma of the 16SrIX group is also associated with this disease [4]). While there is a wide diversity of phytoplasmas in crop species in Brazil (3), there have been no reports of symptoms associated with phytoplasma in Brazilian citrus. Asymptomatic infections of citrus cannot be excluded as a possibility and such plants could serve as a reservoir of phytoplasma inoculum. The aim of this study was to assess the presence of phytoplasma in asymptomatic Citrus aurantifolia (acid lime) in Brazil. Thirty-three leaf samples (young leaves from the upper canopies) were randomly collected from different plants in the states of Minas Gerais (n = 23), Santa Catarina (n = 2), and São Paulo (n = 8). Two additional samples of C. limonia ('Rangpur' lime) and one of C. latifolia ('Persian' or 'Tahiti' lime) were collected in Minas Gerais. Total DNA extraction was performed using NucleoSpin Plant II Kit (Macherey-Nagel) according to the manufacturer's recommendations. PCR was carried out with a universal P1/P7 primer set followed by nested primers R16F2n/R16R2 (2). Additionally, direct PCR was performed using primers specific for phytoplasma immune-dominant membrane protein IMP3F/IMP3R (1). 'Rangpur' and 'Tahiti' lime were not infected with phytoplasma. Of the C. aurantifolia samples, 52% were positive for phytoplasma in the direct and nested PCR assays. The numbers of positive samples in Minas Gerais, Santa Catarina, and São Paulo states were 12, 1, and 4, respectively. Of these, five were selected for DNA purification and 1,246-bp fragments were ligated to the pGEM T-easy vector (Promega) and partial 16Sr DNA was sequenced. Nucleotide sequences of Brazilian phytoplasma strains BR:MG:FNS10:2011, BR:MG:FNS53:2011, BR:SP:FNS73:2011, BR:SC:FNS86:2011, and BR:MG:FNS126:2012 (GenBank Accession Nos. KJ158173, KJ158174, KJ158175, KJ158176, and KJ158177, respectively) were subjected to RFLP analyses. The 16S rDNA RFLP in silico patterns for the five strains were identical to each other and to Cactus witches'-broom phytoplasma (16SrII-C subgroup, AJ293216). In addition, the highest similarity coefficient (5) and nucleotide sequence identity of Brazilian phytoplasma strains were 0.99 and 99%, respectively, with Cactus witches'-broom phytoplasma. PCR-RFLP analyses using the enzymes Bstu I, EcoR I, and Hpa II were consistent with RFLP in silico results, showing the same pattern as the 16SrII-C subgroup. Phylogenetic analyses based on 16S rDNA sequences (1,246 bp) demonstrated that all the Brazilian strains grouped in the same clade with other representative sequences from the 16S rDNAII group. To confirm the absence of any macroscopic symptoms, morphological characteristics of 10 uninfected and 10 phytoplasma-infected plants randomly selected from a single field in Minas Gerais were analyzed. There were no significant differences in leaf area, stalk diameter, or numbers of leaves, flowers, or fruits per branch. To our knowledge, this is the first report of the 16SrII-C subgroup phytoplasma associated with C. aurantifolia in Brazil, and the first report of asymptomatic citrus plants infected with phytoplasma.

Farklı Su ve Süt Kaynaklarından İzole Edilen Bacillus sp.'ların 16S-ITS rDNA RFLP Profillerinin Karşılaştırılması

The aim of this research was the isolation of thermophilic Bacillus sp. from different sources and grouping by 16S-ITS rDNA RFLP. Seventy-four thermophilic strains were isolated from various environmental samples. Since fifteen isolated strains were Gram (+), endospore forming rods, they were identified as Bacillus sp. These strains were screened for the existence of five extracellular enzyme activities. These were, lipases, amylases, proteases, xylanases, cellulases. 16S-ITS rDNA RFLP profiles were produced by using two restriction endonucleases Taq I and Hae III. The isolated strains were clustered into eigth groups by Taq I restriction profiles of 16S-ITS rDNA while seven groups were obtained by Hae III digestion profiles.

Clavibacter michiganensis subsp. phaseoli subsp. nov., pathogenic in bean

A yellow Gram-reaction-positive bacterium isolated from bean seeds (Phaseolus vulgaris L.) was identified as Clavibacter michiganensis by 16S rRNA gene sequencing. Molecular methods were employed in order to identify the subspecies. Such methods included the amplification of specific sequences by PCR, 16S amplified rDNA restriction analysis (ARDRA), RFLP and multilocus sequence analysis as well as the analysis of biochemical and phenotypic traits including API 50CH and API ZYM results. The results showed that strain LPPA 982T did not represent any known subspecies of C. michiganensis. Pathogenicity tests revealed that the strain is a bean pathogen causing a newly identified bacterial disease that we name bacterial bean leaf yellowing. On the basis of these results, strain LPPA 982T is regarded as representing a novel subspecies for which the name Clavibacter michiganensis subsp. phaseoli subsp. nov. is proposed. The type strain is LPPA 982T (=CECT 8144T=LMG 27667T).

16S rDNA analysis of bacterial communities associated with the hyper accumulator Arabidopsis halleri grown on a Zn and Cd polluted soil

Having a glimpse of the relationships between the zinc and cadmium hyper accumulator Arabidopsis halleri and its rhizospheric bacteria may represent an interesting step towards the implementation of a rhizoremediation strategy based on this plant. Until now, these relationships remained unraveled. This work aimed to give some insights into the composition of the bacterial communities surrounding A. halleri when it grows on a soil harboring elevated amounts of zinc and cadmium, as well as into the influence that plant roots may exert on them. Industrial highly polluted soils containing different amounts of zinc and cadmium and surrounding A. halleri plants were sampled and their bacterial contents were harvested at various distances from A. halleri root system. Genetic diversity was assessed through DNA extraction, PCR amplification, 16S rDNA libraries construction, RFLP analysis and partial sequencing. Nine mini-libraries of hundred clones each were established and analyzed. Up to 481 independent clones were sequenced and identified at genus level. Twelve genera among 48 were found particularly well represented in the different sub-fractions analyzed. At class level, ten different classes were identified. Alpha-proteobacteria, Gamma-proteobacteria, Flavobacteria and Sphingobacteria were the main contributors to flora diversity. In the less polluted soil, roots proximity seems to affect negatively Alpha-proteobacteria and Flavobacteria and positively Gamma-proteobacteria. A rhizospheric selection limited to some specific genera was also observed when pollution raised.

Polyphasic characterization of rhizobia isolated from Leucaena leucocephala from Panxi, China

Leucaena leucocephala was introduced into Panxi, Sichuan, China, in the 1980s and 1990s for afforestation and preventing water loss and soil erosion in this area. The co-introduction of rhizobial symbionts of introduced plants has drawn attention since they may influence local soil communities. We studied the phylogenetic position of the L. leucocephala isolates and assessed if the rhizobia were introduced together with the host to Panxi, Sichuan, China. The glnII and atpD genes of fifteen representative isolates were sequenced and analyzed, and applied multilocus sequence analyses in which the housekeeping genes recA, glnII and atpD were included. Furthermore, we estimated the within species diversity directly with 23S rDNA and IGS RFLP and indirectly through phenotypic analysis of forty L. leucocephala isolates. The isolates represented seven species and 38 diversified strains in the genera Ensifer, Mesorhizobium, Bradyrhizobium and Rhizobium. The within species diversity of the Ensifer isolates was large, proposing a potential to occupy novel niches. There was not conclusive evidence to show that any of the strains would have been co-introduced with L. leucocephala. On the contrary, we came to a conclusion that the possible introduction should not be inferred from sequence data alone.

A novel phytoplasma associated with witches’ broom disease of Ligustrum ovalifolium in Turkey

California privet (Ligustrum ovalifolium Hassk.) plants exhibiting leaf yellowing, witches’ broom, dieback and decline symptoms were observed for two years (2010–2011) in three gardens at Adana region (Turkey). DNA isolated from symptomatic and healthy plants was used to amplify 16S rDNA fragments by direct and nested-PCR. Phytoplasmas were detected in 21 symptomatic plants, out of 30 samples collected, whilst no PCR amplifications were obtained from asymptomatic plants. BLAST analysis of the 16S rDNA showed that the phytoplasma found in L. ovalifolium from Turkey, denoted as Turkish Ligustrum witches’ broom phytoplasma (TuLiWB), most closely resembled members of group 16SrII (peanut witches’ broom group) and shared up to 92 % sequence identity. Based on in silico 16S rDNA RFLP analysis and automated calculation of the pattern similarity coefficient, TuLiWB showed molecular characteristics different from all previously described phytoplasma species to represent a new taxon. Similar indication also emerged from the phylogenetic tree which allocated it in a novel discrete subclade within the phytoplasma clade. This is the first report on the presence of a phytoplasma affecting L. ovalifolium and whether this novel phytoplasma is the same agent reported as a mycoplasma-like organism (MLO) and associated with witches’ broom disease of Ligustrum in Korea (1989) is yet to be determined.

Morphological, anatomical and molecular investigation into witches’ broom disease of mamejvo (Enicostemma axillare)

Mamejvo (Enicostemma axillare Raynal) is a perennial herb with diversified health benefits. It was found to be affected by a witches’ broom disease under the field conditions at Anand, Gujarat, India. Affected plants were 53.33% reduced in height due to shortened internodes, leading to typical witches’ broom symptoms. Flower size was reduced significantly in the affected plants compared with the healthy ones and their petal color gradually turned green (virescence). Affected plants produced 2.5 times more dry herbage than the normal plants but with less active ingredient (swertiamarin). Symptomatic plants died early while root suckers from such plants failed to establish after transplanting. DAPI stained transverse sections of affected shoot tips showed the presence of discrete greenish fluorescence in the phloem cells under UV light. Amplification of 1.2 kb phytoplasma specific rDNA fragment from diseased tissue confirmed the presence of the pathogen. Further characterization of the pathogen through virtual rDNA–RFLP pattern and rDNA sequence based phylogeny suggested that the pathogen, Enicostemma witches’ broom phytoplasma, belonged to 16SrII–C group of ‘Ca Phytoplasma aurantifolia’.

Geographical variation of foxtail millet, Setaria italica (L.) P. Beauv. based on rDNA PCR–RFLP

The rDNA PCR–RFLP of foxtail millet (Setaria italica) germ-plasm collected throughout Eurasia and from a part of Africa was investigated with five restriction enzymes according to our previous study. Foxtail millet germ-plasms were classified by length of the rDNA IGS and RFLP; clear geographical differentiation was observed between East Asia, the Nansei Islands of Japan-Taiwan-the Philippines area, South Asia and Afghanistan-Pakistan. We also found evidence of migration of foxtail millet landraces between the areas. We calculated diversity index (D) for each region and found that center of diversity of this millet is East Asia such as China, Korea and Japan.

Phenotyping and genotyping of streptococci in bovine milk in Argentinean dairy herds

Most veterinary and milk hygiene laboratories identify streptococci and enterococci based on serological and biochemical tests. The analysis of 16S rDNA was suggested to be used for more exact identification; however, its use has not been considered so far in monitoring studies. The objective of the present study was to compare a conventional phenotypic method with restriction fragment length polymorphism analysis of 16S rDNA (16S rDNA RFLP) for identification of streptococci isolated from composite milk samples collected in connection with intramammary infection (IMI) in six Argentinean dairy farms. Composite milk samples (n = 1223) from cows belonging to six herds were collected for bacteriological analysis. Twelve reference strains and fifty streptococci or streptococcuslike isolates were identified to species level by the API 20 Strep system, conventional biochemical tests and 16S rDNA RFLP in a blind assay. The remaining streptococci or streptococcus-like isolates (n = 40) were identified to the species level both by 16S rDNA RFLP and conventional biochemical tests. As indicated by Kappa values, agreement between the 16S rDNA RFLP and the conventional scheme for identification of Streptococcus agalactiae, S. dysgalactiae, S. uberis, S. equinus and Enterococcus faecalis was 0.91, 0.73, 0.92, 0.81 and 0.85, respectively. Together with the less frequently isolated streptococcal species, the conventional scheme correctly identified 77 out of 90 isolates (85.5%). Thus, the use of 16S rDNA RFLP is considered valuable for monitoring studies due to its affordable cost for standard laboratories.

Validation of a partialrpoBgene sequence as a tool for phylogenetic identification of aeromonads isolated from environmental sources

A collection of 50 aeromonads isolated from environmental sources were studied, together with all known Aeromonas nomenspecies, by phenotypic, amplified 16S rDNA restriction analysis (16S rDNA RFLP) and by partial sequence alignment of both 16S rDNA and rpoB genes. Although most of the type strain showed a unique phenotypic pattern, a database constructed on type strain phenotype allowed the identification of only 24% of the isolates. Analysis of 16S rDNA RFLP and the rpoB sequence were almost concordant in identifying environmental isolates at the species level, except for strains belonging to Aeromonas caviae spp., which were not differentiated from Aeromonas aquariorum, nor Aeromonas hydrophila susbsp. dhakensis by 16S rDNA RFLP. In addition, rpoB gene analysis clustered separately a group of isolates found in snails within the A. hydrophila species. In contrast to 16S rDNA RFLP and rpoB, the partial 16S rDNA sequence analysis was weak in resolving species identity. Part of these results, phenotypic and phylogenetic data, showed that Aeromonas molluscorum and Aeromonas sharmana are distant from all other Aeromonas species and that the type species of A. hydrophila subsp. anaerogenes is similar to A. caviae and should be considered synonymous.

Molecular and Conventional Identification and Pathogenicity of Rhizoctonia solani Isolates from Tobacco (Nicotiana tabacum L.) in Samsun, Turkey

AbstractA total of 101 fungal samples were isolated from tobacco (Nicotiana tabacum L.) plants in Samsun, Turkey and identified as Rhizoctonia solani. Of these samples, three were identified as belonging to Anastomosis Group (AG) ‐1‐IB, 6 to AG‐2‐1, 25 to AG‐4‐HG‐I and 67 to AG‐4‐HG‐II, using ITS1‐5.8S‐ITS2 rDNA RFLP analysis, base sequence phylogenies and conventional characters, such as anastomosis reactions, morphology and thiamine requirement. For five isolates from both the AG‐4‐HG‐I and AG‐4‐HG‐II groups and for all isolates of the AG‐1‐IB and AG‐2‐1 groups, molecular analysis and pathogenicity tests were performed. All selected Rhizoctonia solani isolates were highly virulent to tobacco plants, causing symptoms resembling damping‐off, except for isolate 55TkTB63, which was only moderately virulent.

Polyphasic taxonomic studies of lactic acid bacteria associated with Tunisian fermented meat based on the heterogeneity of the 16S–23S rRNA gene intergenic spacer region

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in traditionally fermented meat collected from different areas of Tunisia. A polyphasic study, which involves phenotypic tests and ribosomal DNA-based techniques, was used to identify Gram-positive and catalase-negative isolates. PCR amplification of the 16S-23S rDNA ISR of 102 isolates and other reference LAB strains gave (1) one type of rrn operon (M-ISR) for lactococci, (2) two types of rrn operon (S-ISR and M-ISR) for enterococci, (3) two types of rrn operon (S-ISR and L-ISR) for Lactobacilli, and (4) three PCR amplicons (S-ISR, M-ISR, and L-ISR) obtained for Pediococcus spp. and Weissella genus. The clustering and comparison of ISR-RFLP profiles given by the isolates with those given by reference LAB strains, allowed their identification as Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Enterococcus sanguinicola, Enterococcus hawaiiensis, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus alimentarius, Pediococcus pentosaceus, and Weissella confusa. Combined 16S-23S rDNA ISR and RFLP patterns can be considered as a good potential target for a rapid and reliable differentiation between isolates of LAB and provided further information on the organization of their rrn operons.

GENETIC VARIABILITY OF STOLBUR PHYTOPLASMA IN ANNUAL CROP AND WILD PLANT SPECIES IN SOUTH MORAVIA

SUMMARY The genetic diversity of stolbur phytoplasma isolates was assessed by different molecular typing methods in a wide range of plant hosts in a limited geographical area of the Czech Republic. Plants of Apium graveolens, Capsicum annuum, Solanum lycopersicum, Solanum tuberosum, Amaranthus retroflexus, Calystegia sepium, Cirsium arvense, Convolvulus arvensis, and Datura stramonium displaying symptoms of proliferation, discoloration, and malformation of flower were collected in vegetable crop plots in the vicinity of the village of Lednice (south Moravia) and examined for the presence of phytoplasmas. Using 16S rDNA amplification and RFLP assays, stolbur phytoplasma was detected in some of the symptomatic plants. The genotype of the phytoplasma isolates detected was investigated by restriction pattern or sequence analysis of two non-ribosomal house-keeping genes (tuf and secY) and a recently characterized polymorphic gene (vmp1). According to tuf genotyping, all isolates were of the tufAY-b genotype sensu Langer and Maixner (2004), a genotype known to be associated with bindweed (C. arvensis) reservoirs. A finer isolate differentiation was obtained with vmp1 genotyping as four genetic variants carrying different vmp1 RFLP patterns were found, especially in C. arvensis. In addition, preliminary sampling in other south Moravian localities and secY sequence analyses diclosed also the presence of stolbur isolates different from those previously reported in binweed (C. arvensis) and grapevine.

Isolation and characterization of culturable bacteria from tropical coastal waters

In this study we isolated and characterized some culturable bacteria from tropical coastal waters of Peninsular Malaysia. We obtained between 0.23 and 1.85 × 103 cfu mL–1 in the Zobell 2216E medium, and cultured 0.04% to 0.12% of total bacterial counts. Different bacterial strains were then selected by 16S rDNA RFLP using four restriction enzymes (DdeI, HhaI, RsaI, and Sau3AI), of which HhaI gave the most RFLP patterns. A total of 54 unique strains were obtained and these were identified by their 16S rDNA gene sequence. These bacterial strains could be divided into five classes: 38 strains of γ-Proteobacteria (61.1%); 3 strains of α-Proteobacteria (5.5%); 2 strains of the Cytophaga-Flavobacterium-Bacteroides group (3.7%); 3 strains of high GC, Gram-positive bacteria (5.5%); and 13 strains of low GC, Gram-positive bacteria (24.1%). These isolates have good potential for further biotechnological studies since about 56% of the isolates exhibited amylase activity, whereas 36% and 18% of the isolates had protease and lipase, respectively. Most (>70%) of the isolates also produced poly-ß-hydroxybutyrate.

Utility of Microsatellite Markers and Amplified Fragment Length Polymorphism in the Study of Potentially Ochratoxigenic Black Aspergilli

Microsatellite markers and the results of amplified fragment length polymorphism (AFLP) were compared in the characterization of 68 Aspergillus carbonarius and A. niger aggregate strains of differing ochratoxin-producing ability and from different geographic areas, isolated mainly from grapes and soil. AFLP was applied to both A. carbonarius and A. niger aggregate strains, and it clearly differentiated these species. Microsatellite markers were only applied to A. niger aggregate strains because of the species-specific nature of these markers. Both AFLP and microsatellite marker analyses were able to divide A. niger aggregate strains into the two recognized internal transcribed spacer (ITS)-5.8S rDNA RFLP types, N and T. Clustering of A. niger aggregate strains was similar in both AFLP and microsatellite analyses, yielding an additional separation of N type strains into two groups. Both microsatellite marker and AFLP analyses showed high levels of polymorphism in the A. niger aggregate (index of discriminatory power 0.991 and 1.0, respectively). Of the two techniques, microsatellite marker analysis was quicker and more straightforward to perform. In addition, microsatellite marker analysis is more reproducible, and the results can be expressed as quantitative data, making microsatellite markers a good candidate for use in large-scale studies of genetic diversity in A. niger aggregate species.

Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey

To isolate Bacillus thuringiensis strains from different olive-related habitats (olive groves and olive oil factories) in Turkey and to characterize these strains by molecular methods. A total of 150 samples, consisting of olive grove soil, green olive leaves, olive leaf residues, animal faeces, olive pomace and dust, were examined for the presence of B. thuringiensis. One hundred B. thuringiensis strains were isolated from 54 environmental samples (36%) and characterized in terms of crystal morphology, cry and cyt gene content by polymerase chain reaction, plasmid profiles and 16S-internal transcribed spacer ribosomal DNA restriction fragment length polymorphism (16S-ITS rDNA RFLP). The highest percentage of samples containing B. thuringiensis was found in 38 out of 54 total soil samples (70%). Of the 100 B. thuringiensis isolates, the most frequent crystal shapes were irregularly shaped (24%), spherical-irregular pointed (19%), cuboidal (17%) and spherical (16%). The cry1 plus cry4 genotype was the most abundant genotype in our collection (21%). RFLP analysis of the amplified 16S-ITS rDNA revealed 11 distinct patterns for the isolates and 10 reference strains. Bacillus thuringiensis isolates showed a great genetic diversity and crystal shape heterogeneity. This is the first study on the isolation and characterization of B. thuringiensis from olive-related habitats in Turkey. No correlation was observed between the cry genotypes and insecticidal crystal shapes of the isolates. Restriction profiles of 23% of the isolates were found to be different from those of the 10 reference strains used.