Divergent Evolution of Symbiotic Bacteria: Rhizobia of the Relic Legume <Emphasis Type="Italic">Vavilovia formosa</Emphasis> Form an Isolated Group within <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.     

Identification and Assessment of Symbiotic Effectiveness of Phage-Typed <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> Strains on Lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik) Cultivars

Symbiotic effectiveness of 19 indigenous and two exotic (USDA 2426 and USDA 2431) strains of lentil Rhizobium belonging to different phage-sensitive and phage-resistant groups was compared under axenic condition. Four strains (USDA 2431, BHULR 104, BHULR 113, and BHULR 115) sensitive to different phages were found significantly superior over others in terms of nodule number, acetylene reduction activity, and total dry weight per plant. Inoculation response of these strains was then evaluated on six lentil cultivars under field condition. A significant symbiotic interaction between rhizobial strains and lentil cultivars was observed. Grain yield enhancement was noticed by the compatible interaction of lentil cultivars HUL-57, L-4147, K-75, and PL-4/DPL-15/DPL-62 with rhizobial strains USDA 2431, BHULR 104, BHULR 113, and BHULR 115, respectively. The authentication of rhizobial strains was accomplished through 16S rDNA sequence analysis. All rhizobial strains had close matching with R. leguminosarum bv. viciae strains. The results have shown that phages can trustfully help selecting out the symbiotically efficient most rhizobial strains for advantageous use with lentil cultivars, in order to strengthen the BNF-based future lentil breeding programs.     

Comparison of the adaptive potential for <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viceae</Emphasis> nodule bacterial populations isolated in natural ecosystems and agrocenoses

Polymorphism analysis was performed in Rhizobium leguminosarum bv. viceae populations isolated from geographically distant regions of Ukraine and Middle Asia. Examination of cultural, biochemical, and symbiotic traits revealed interpopulation differences, which were attributed to the difference in conditions between natural ecosystems and agrocenoses. Vetch has high species diversity and is not cultivated in Middle Asia, and the corresponding rhizobial population displayed higher genetic diversity and higher polymorphism of adaptive traits ensuring saprophytic development in soil and the rhizosphere, including melanin synthesis (35%) and active exopolysaccharide production (90%). Strains of the Ukrainian population had a lower exopolysaccharide production (10%), did not produce melanin, had higher herbicide resistance, and utilized glucose and succinate (main components of plant root exudation) as carbon sources. Strains capable of efficient symbiosis with Vicia villosa Roth. had a higher frequency in the Middle Asian than in the Ukrainian population, especially among strains isolated from soil (80 and 35%, respectively). In addition, strains of the Middle Asian population better competed for nodulation. It was assumed that the formation of rhizobial populations in vetch cultivation regions (Ukraine) is aimed at adaptation to ectosymbiotic (rhizospheric) interactions with plants and anthropogenic stress factors, while strains of the vetch original center (Middle Asia) are mostly adapted to the endosymbiotic interaction and to natural edaphic stress factors.     

The effect of biotic and physical factors on the competitive ability of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis>

Background  

Rhizobium leguminosarum bv. viciae (Rlv) is a soil bacterium which can form nitrogen-fixing symbiotic relationships with leguminous plants. Numerous rhizobial strains found in soils compete with each other. Competition can occur both during the saprophytic growth phase in the rhizosphere and inside plant tissues, during the symbiotic phase. Competition is important as it may affect the composition of rhizobial populations present in the soil and in the root nodules of plants.     

Molecular diversity and phylogeny of indigenous <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> strains associated with <Emphasis Type="Italic">Trifolium repens</Emphasis> plants in Romania

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Structural and functional organization of the plasmid regulons of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> symbiotic genes

The structure of the plasmid locus containing the sym-genes (nod-, nif-, and fix-operons) was investigated in eight Rhizobium leguminosarum strains differing in their origin and host specificity, including five strains of the viciae biovar—symbionts of pea (3), forage beans (1), and Vavilovia (1)—as well as three strains of the biovar trifolii (clover symbionts). Strains of R. leguminosarum bv. viciae, which possess the nodX gene (controlling acetylation of the Nod factor, which is responsible for the ability of rhizobia to form symbioses with a broad spectrum of hosts, including the “Afghan” pea lines, homozygous by the allele sym^2A), are characterized by a less compact location of the sym-genes than the strains lacking the nodX gene. The size of the symbiotic cluster in the strains possessing nodX was 94.5 ± 3.5 kb, with the share of the sym-genes of 36.5 ± 1.5%, while for the strains lacking nodX these values were 61.7 ± 3.7 kb and 56.3 ± 1.4%, respectively (significant difference at P ₀ < 0.01). Syntenic structures were revealed in the symbiotic regions of strains Vaf12, UPM1131, and TOM, as well as syntenic structures of non-symbiotic regions in strains Vaf12, TOM, and WSM1689. The correlation coefficients between the matrices of genetic distances in the analyzed strains for the nodABC, nifHDK, and fixABC operons were on average 0.993 ± 0.002, while their values for the plasmid sites located between the sym-genes were considerably less (0.706 ± 0.010). In these regions, 21 to 27% of the genes were involved in amino acid transport and metabolism, which was substantially higher than the average for the genome of R. leguminosarum bv. viciae (11–12%). These data suggest that the evolution of R. leguminosarum bv. viciae, defined by narrowing of the host specificity (associated with a loss of the nodX gene), was accompanied by reduction of the regions of plasmids located between the sym-genes, as well as by specialization of these areas to perform the functions related to symbiotic nitrogen fixation. The observed increase of density in the cluster of sym-genes may be associated with intensification of their horizontal transfer in the populations of rhizobia, which determines the speed of evolution of the symbiotic system.     

Genetic diversity of root-nodulating bacteria isolated from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Effect of the synthetic polysaccharide MOD-19 on the formation and function of symbiosis between <Emphasis Type="Italic">Pisum sativum</Emphasis> L. and <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

The physiological action of the MOD-19 polysaccharide (PS), synthesized similarly to bacterial glucans, on the nodule bacteria Rhizobium leguminosarum bv. viciae and pea seeds was studied. It was found that MOD-19 stimulated nodule bacterium growth and bacterial biomass accumulation. It also altered metabolism in rhizobia grown in solid and liquid media containing this polymer. Treatment of pea seeds with MOD-19 before sowing increased the intensity of root formation, plant tissue peroxidase activity, and general symbiosis efficiency owing to secondary nodule formation on lateral roots and prolongation of their intense nitrogen fixation.     

<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii rosR</Emphasis> is required for interaction with clover,biofilm formation and adaptation to the environment

Background  

Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.     

Diversity and symbiotic divergence of endophytic and non-endophytic rhizobia of <Emphasis Type="Italic">Medicago sativa</Emphasis>

Knowledge of rhizobium diversity is helping to enable the utilization of rhizobial resources. To analyze the phenotypic and genetic diversity and the symbiotic divergence of rhizobia of Medicago sativa, 30 endophytic and non-endophytic isolates were collected from different parts of five alfalfa varieties in three geographic locations in Gansu, China. Numerical analyses based on 72 phenotypic properties and restriction fragment length polymorphism (RFLP) fingerprinting indicated the abundant phenotypic and genetic diversity of the tested strains. According to the phylogenetic analysis of 16S RNA, atpD, glnII, and recA gene sequences, Rhizobium and Ensifer were further classified into four different genotypes: Rhizobium radiobacter, Rhizobium sp., Rhizobium rosettiformans, and Ensifer meliloti. The differences in architecture and functioning of the rhizobial genomes and, to a lesser extent, environment diversification helped explain the diversity of tested strains. The tested strains exhibited similar symbiotic feature when inoculated onto M. sativa cvs. Gannong Nos. 3 and 9 and Qingshui plants for the clustering feature of their parameter values. An obvious symbiotic divergence of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants because of the scattered parameter values. Their symbiotic divergence differed according to alfalfa varieties, which indicated that the sensitivity of different alfalfa varieties to rhizobial strains may differ. Most of the tested strains exhibited plant growth-promoting traits including phosphate solubilization and production of indole-3-acetic acid (IAA) when colonizing plant tissues and soil.     

Genetic variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> strains nodulating soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill]

Brazil has succeeded in sustaining production of soybean [Glycine max (L.) Merrill] by relying mainly on symbiotic N₂ fixation, thanks to the selection and use in inoculants of very effective strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. It is desirable that rhizobial strains used in inoculants have stable genetic and physiological traits, but experience confirms that rhizobial strains nodulating soybean often lose competitiveness in the field. In this study, soybean cultivar BR 16 was single-inoculated with four B. japonicum strains (CIAT 88, CIAT 89, CIAT 104 and CIAT 105) under aseptic conditions. Forty colonies were isolated from nodules produced by each strain. The progenitor strains, the isolates and four other commercially recommended strains were applied separately to the same cultivar under controlled greenhouse conditions. We observed significant variability in nodulation, shoot dry weight, shoot total N, nodule efficiency (total N mass over nodule mass) and BOX-PCR fingerprinting profiles between variant and progenitor strains. Some variant strains resulted in significantly larger responses in terms of shoot total N, dry weight and nodule efficiency, when compared to their progenitor strain. These results highlight the need for intermittent evaluation of stock bacterial cultures to guarantee effective symbiosis after inoculation. Most importantly, it indicates that it is possible to improve symbiotic effectiveness by screening rhizobial strains for higher N₂ fixation capacity within the natural variability that can be found within each progenitor strain.     

Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

Genetic diversity of rhizobia associated with <Emphasis Type="Italic">Vicia faba</Emphasis> in three ecological regions of China

Great genetic diversity was revealed among 75 rhizobal isolates associated with Vicia faba grown in Chinese fields with AFLP, ARDRA, 16S rDNA sequencing, DNA–DNA hybridization, BOX-PCR and RFLP of PCR-amplified nodD and nodC. Most of the isolates were Rhizobium leguminosarum, and six isolates belonged to an unnamed Rhizobium species. In the homogeneity analysis, the isolates were grouped into three clusters corresponding to (1) autumn sowing (subtropical) region where the winter ecotype of V. faba was cultivated, (2) spring sowing (temperate) region where the spring ecotype was grown, and (3) Yunnan province where the intermediate ecotype was sown either in spring or in autumn. Nonrandom associations were found among the nod genotypes, genomic types and ecological regions, indicating an epidemic symbiotic gene transfer pattern among different genomic backgrounds within an ecological region and a relatively limited transfer pattern between different regions. Conclusively, the present results suggested an endemic population structure of V. faba rhizobia in Chinese fields and demonstrated a novel rhizobium associated with faba bean. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Core Genome Haplotype Diversity and <Emphasis Type="Italic">vacA</Emphasis> Allelic Heterogeneity of Chinese <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains

The human gastric pathogen, Helicobacter pylori, has co-evolved with its host and established itself in the human stomach possibly millions of years ago. Therefore, the diversity of this bacterium is important in its clinical manifestations. Our aim has been to evaluate the genetic diversity of 40 H. pylori clinical isolates from four different parts of China. The methods of multi-locus sequence typing and vacA allele genotyping were used to assess their genetic diversity. To discriminate MLST, the vacA genotype method was used to identify strains. Patients from the northern, eastern, southern, and southwestern parts of China were recruited randomly from the cities of Beijing, Shanghai, Guangzhou, and Chongqing, respectively. Most of the sequence types are new and have never been reported in the database of the H. pylori multi-locus sequence typing system. The most prevalent vacA genotype in patients was s1a/m2 (80.0%), followed by s1b/m2 (17.5%). In contrast, the s1a/m1 genotype was scarcely represented (2.5%). The vacA genotype varied for each ST. These results showed that the MLST method offers high resolution of the H. pylori isolates in China when compared to vacA genotyping. The vacA allelic s1a has been correlated with the peptic ulcer. Because of the paucity of data on human isolates due to the absence of systematic investigations of H. pylori in China, the data provide useful information for understanding the epidemiology of H. pylori in China from the viewpoint of nucleotide sequence databases.     

The <Emphasis Type="Italic">pssA</Emphasis> gene encodes UDP-glucose: Polyprenyl phosphate-glucosyl phosphotransferase initiating biosynthesis of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> exopolysaccharide

Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum bv. viciae VF39 secrete an acidic heteropolysaccharide, the biosynthesis of which involves the stage of polyprenyl diphosphate octasaccharide formation with its carbohydrate fragment corresponding to the repeating polymer unit. The amino acid analysis of the product of the pssA gene, we have earlier identified, showed its homology to bacterial polyisoprenyl phosphate hexose 1-phosphate transferases catalyzing the formation of phosphodiester bonds between polyprenyl phosphates and hexose 1-phosphates, whose donors are nucleotide sugars. The immunoblotting demonstrated that Rhizobium cells synthesize a protein with a molecular mass of 25 kDa, which implies the translation of the open reading frame occurring from the second initiating codon followed by the protein processing. It was shown that PssA is an integral membrane-bound protein involved in glucose 1-phosphate transfer from UDP-glucose to polyprenyl phosphate to form polyprenyl diphosphate glucose. These results suggest that the pssA gene encodes UDP-glucose:polyprenyl phosphate-glucosyl phosphotransferase.     

Detection of double stranded RNA in phytopathogenic <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> causing charcoal rot in <Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>

One hundred one isolates of Macrophomina phaseolina from various hosts and eco-geographical locations were employed for elucidating relationships among genetic diversity and virulence. Highly pathogenic, moderately pathogenic, and hypovirulent cluster bean specific isolates were identified. In order to correlate respective phenotypes of plant pathogenic fungus multiple and complex patterns of dsRNA elements were analyzed. Double-stranded ribonucleic acids (dsRNA) are ubiquitous in all major groups and most of them have vast potential as biological control agents for fungi. Rate of virulence and its further association could ascertain by host plant and their fungal genotypes. Variability of the fungal genotypes decides the link between the complexity of dsRNA with different variants and the change in virulence pattern. Double-stranded RNA was identified in approximately 21.7% of M. phaseolina isolates from charcoal rot infected cluster bean varieties. After recurrent laboratory transfer on culture media, the preponderance of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to cluster bean. Macrophomina has successfully showed diversified and reproducible banding profile in dsRNA containing/free isolates. This is the first report of hypovirulence and detection of dsRNA in Macrophomina phaseolina isolates of cluster bean origin.     

Variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">B. elkanii</Emphasis> Seven Years after Introduction of both the Exotic Microsymbiont and the Soybean Host in a Cerrados Soil

The plasticity of rhizobial genomes is far greater than previously thought, with complex genomic recombination events that may be accelerated by the often stressful environmental conditions of the tropics. This study aimed at evaluating changes in soybean rhizobia due to adaptation to inhospitable environmental conditions (high temperatures, drought, and acid soils) in the Brazilian Cerrados. Both the host plant and combinations of four strains of soybean Bradyrhizobium were introduced in an uncropped soil devoid of rhizobia capable of nodulating soybean. After the third year, seeds were not reinoculated. Two hundred and sixty-three isolates were obtained from nodules of field-grown soybean after the seventh year, and their morphological, physiological, serological, and symbiotic properties determined, followed by genetic analysis of conserved and symbiotic genes. B. japonicum strain CPAC 15 (same serogroup as USDA 123) was characterized as having high saprophytic capacity and competitiveness and by the seventh year represented up to 70% of the cultivable population, in contrast to the poor survival and competitiveness of B. japonicum strain CPAC 7 (same serogroup as CB 1809). In general, adapted strains had increased mucoidy, and up to 43% of the isolates showed no serological reaction. High variability, presumably resulting from the adaptation to the harsh environmental conditions, was verified in rep-PCR (polymerase chain reaction) profiles, being lower in strain CPAC 15, intermediate in B. elkanii, and higher in CPAC 7. Restriction fragment length polymorphism (RFLP)-PCR types of the 16S rDNA corresponded to the following: one type for B. elkanii species, two for B. japonicum, associated to CPAC 15 and CPAC 7, and unknown combinations of profiles. However, when nodC sequences and RFLP-PCR of the nifH region data were considered, only two clusters were observed having full congruence with B. japonicum and B. elkanii species. Combining the results, variability was such that even within a genetically more stable group (such as that of CPAC 15), only 6.4% of the isolates showed high similarity to the inoculant strain, whereas none was similar to CPAC 7. The genetic variability in our study seems to result from a variety and combination of events including strain dispersion, genomic recombination, and horizontal gene transfer. Furthermore, the genetic variability appears to be mainly associated with adaptation, saprophytic capacity, and competitiveness, and not with symbiotic effectiveness, as the similarity of symbiotic genes was higher than that of conserved regions of the DNA.     

Molecular Analysis of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates from Clinical Specimens

The aim of this study was to genotype Candida albicans strains isolated from various clinical specimens by using CA-INT-R and CA-INT-L primer pairs designed to span the region that includes the site of the transposable group-1 intron in the 25S rRNA gene. A total of 194 C. albicans isolates (28 invasive and 166 noninvasive) were genotyped. The frequencies of genotypes A, B, C and D were found as 51.0, 29.4, 19.1 and 0.5%, respectively. Statistically significant difference was determined between frequency of genotype distribution between invasive and noninvasive isolates (P < 0.001). Genotype C was more prevalent among invasive isolates while genotype A was in noninvasive ones. Furthermore, six different subtypes were determined among genotype A C. albicans isolates by restriction endonuclease analysis using a previously constructed differentiation scheme consisting of HaeIII and MspI digestions. This study demonstrated the genetic diversity of clinical isolates of C. albicans in our hospital.