Microsatellite Markers Reveal Genetic Variation within Sclerotinia sclerotiorum Populations in Irrigated Dry Bean Crops in Brazil

Microsatellites are powerful markers to infer population genetic parameters. We used 10 microsatellite loci to characterize the genetic diversity and structure of 79 samples of Sclerotinia sclerotiorum isolated from four Brazilian dry bean populations and observed that eight of them were polymorphic within populations. We identified 102 different haplotypes ranging from 6 to 18 per locus. Analyses based on genetic diversity and fixation indices indicated variability among and within populations of 28.79% (F_ST = 28793) and 71.21%, respectively. To examine genetic relatedness among S. sclerotiorum isolates, we used internal spacer (ITS1‐5.8S‐ITS2) restriction fragment length polymorphism (PCR‐RFLP) and sequencing analysis. PCR‐RFLP analysis of these regions failed to show any genetic differences among isolates. However, we detected variability within the sequence, which does not support the hypothesis of clonal populations within each population. High variability within and among populations may indicate the introduction of new genotypes in the areas analysed, in addition to the occurrence of clonal and sexual reproduction in the populations of S. sclerotiorum in the Brazilian Cerrado.     

Phylogeny,morphology and pathogenicity of Elsinoë ampelina,the causal agent of grapevine anthracnose in Brazil and Australia

Anthracnose caused by Elsinoë ampelina is one of the most important table grape diseases in humid regions in Brazil and Australia. The objective of this study was to characterize E. ampelina isolates from Brazil and Australia by means of phylogenetic analyses, morphological features and pathogenicity tests. Phylogenetic relationships among 35 isolates were determined based on a data set of internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences. In phylogenetic tree analyses, using a combined ITS and TEF sequence alignment, all E. ampelina isolates were clustered together in a single well‐supported clade. In contrast to the absence of genetic variability within ITS and TEF sequences, HIS3 sequences showed 54 polymorphic sites. The haplotype network generated from HIS3 data set showed four distinct haplotypes. EA1 was the predominant haplotype including 29 isolates from both countries. High genetic variability was observed in two Brazilian isolates, haplotype EA4, which may have lost the intron region during species evolution. Colony colours differed between Brazilian and Australian isolates, but showed similar wrinkled colony texture, absence of spores, sparse‐to‐absent white aerial mycelium and slow growth (0.049–0.060 mm/day). Brazilian isolates produced conidia of 5.65 × 2.65 μm, larger than conidia from Australian isolates, which measured 5.14 × 2.30 μm. In pathogenicity tests, all nine Australian isolates inoculated were pathogenic on detached canes and potted vines of table grape.     

Identification and genetic characterization of a new Brazilian genotype of Toxoplasma gondii from sheep intended for human consumption

Recent studies have demonstrated that strains of Toxoplasma gondii in Brazil are frequently different from those detected in other countries, thus making an accurate phylogenetic analysis difficult. The aim of this study was to genetically characterize T. gondii samples from sheep raised in southern Bahia and intended for human consumption, by means of PCR–RFLP and sequencing techniques. Experimental samples were obtained from 200 sheep brains purchased at butcher's shops in Itabuna, Bahia, Brazil. In total, three samples (#54, #124 and #127) were T. gondii-positive. The application of multilocus PCR–RFLP using ten molecular markers (SAG1, SAG2, SAG3, BTUB, c22-8, PK1, GRA6, L358, c-29-2 and Apico) revealed a single genotype common to all samples of this study, which differed from any other published T. gondii genotypes. An atypical allele was detected in the L358 genetic marker; this has not previously been shown in any other South American T. gondii isolates. Phylogenetic analysis on the sequences from multilocus PCR sequencing revealed that these three samples were classified into the same lineage. Extensive indel regions were detected in the Apico genetic marker. Together, our findings revealed a new Brazilian T. gondii genotype. Further research should be conducted to enrich the database of Brazilian T. gondii genotypes from different regions. This will make it possible to understand the phylogenetic relationship between isolates.     

Using mitochondrial and nuclear markers to evaluate the degree of genetic cohesion among Echinococcus populations

Based on the distinctiveness of their mitochondrial haplotypes and other biological features, several recent publications have proposed that some Echinococcus granulosus strains should be regarded as separate species. However, the genetic cohesion of these species has not been extensively evaluated using nuclear markers. We assess the degree of polymorphism of the partial mitochondrial cox1 (366 bp), the nuclear mdh (214 bp) and EgAgB4 (281-283 bp) genes of E. granulosus sensu lato isolates collected from areas where different strains occur sympatrically. Five distinct mitochondrial haplotypes were determined by direct sequencing (G1, G2, G5, G6 and G7). The mdh genotypes were first screened by SSCP: three alleles were identified (Md1-Md3), which were further confirmed by nucleotide sequencing. For EgAgB4, which was analysed by direct sequencing the PCR products, two groups of sequences were found: EgAgB4-1 and EgAgB4-2. No haplotype-specific mdh or EgAgB4 sequences occur. Nevertheless, alleles Md1 and Md2 and type 1 sequences of EgAgB4 showed a higher frequency within the group of haplotypes G1-G2, while allele Md3 and EgAgB4-2 are most frequent in the G5-G7 cluster. By AMOVA it is shown that 79% of the total genetic variability is found among haplotype groups. These findings are compatible with two not mutually exclusive evolutionary hypotheses: (a) that haplotypes share an ancestral polymorphism, or (b) that the reproductive isolation between parasites with distinct haplotypes is not complete, leading to gene introgression. The biologic and epidemiologic consequences of our findings are discussed.     

A host specialized form of Ceratocystis fimbriata causes seed and seedling blight on native Carapa guianensis (andiroba) in Amazonian rainforests

Ceratocystis fimbriata Ellis & Halsted recently was recorded causing seed and seedling blight on Carapa guianensis Aubl. (andiroba), a tree species native to the Amazon Rainforest and prized for its valuable timber and medicinal seed oil. C. fimbriata more commonly causes wilt type diseases in woody hosts, especially on non-native host trees. However, on andiroba the disease occurs on seedlings and seeds, affecting the species regeneration. We studied 73 isolates of C. fimbriata on andiroba from three regions of the Amazon Basin to see if they represented natural or introduced populations. Analysis of ITS rDNA sequences and phylogenetic analysis of mating type genes revealed new haplotypes of C. fimbriata from the Latin American Clade that were closely related to other Brazilian populations of the fungus. In mating experiments, andiroba isolates were inter-fertile with tester strains of C. fimbriata from Brazil and elsewhere, confirming that they belong to a single biological species. Using microsatellite markers, 14 genotypes and populations with intermediate levels of genetic variability were found, suggesting that the fungus is indigenous to the Amazon Basin. Inoculation tests indicated that the andiroba isolates are host-specialized on andiroba, supporting the proposition of the special form C. fimbriata f. sp. carapa.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.     

Genetic polymorphism of Baylisascaris procyonis in host infrapopulations and component populations in the Central USA

Baylisascaris procyonis is a nematode of significant concern to public and domestic animal health as well as wildlife management. The population genetics of B. procyonis is poorly understood. To gain insights into patterns of genetic diversity within (infrapopulation level) and among (component population level) raccoon (Procyon lotor) hosts, and specifically to assess the relative importance of indirect and direct transmission of the parasite for explaining observed population structure, we collected 69 B. procyonis from 17 wild raccoons inhabiting five counties in Missouri and Arkansas, USA. Informative regions of mitochondrial (CO1, CO2) and nuclear (28S, ITS2) genes were amplified and the distribution and genetic variability of these genes were assessed within and across raccoons. Concatenation of the CO1 and CO2 mtDNA sequences resulted in 5 unique haplotypes, with haplotype diversity 0.456?±?0.068. The most common haplotype occurred in 94% of raccoons and 72.5% of B. procyonis. Sequences for 28S rDNA revealed four unique nuclear genotypes, the most common found in 100% of raccoons and 82.6% of B. procyonis. ITS2 genotypes were assessed using fragment analysis, and there was a 1:1 correspondence between 28S and ITS-2 genotypes. Infrapopulation variation in haplotypes and genotypes was high and virtually all hosts infected with multiple sequenced nematodes also harbored multiple haplotypes and genotypes. There was a positive relationship between the size of the analyzed infrapopulation (i.e., the number of nematodes analyzed) and the number of haplotypes identified in an individual. Collectively this work emphasizes the importance of indirect transmission in the lifecycle to this parasite.     

Genetic Diversity of Giardia duodenalis: Multilocus Genotyping Reveals Zoonotic Potential between Clinical and Environmental Sources in a Metropolitan Region of Brazil

Background

Giardia duodenalis is a flagellate protozoan that parasitizes humans and several other mammals. Protozoan contamination has been regularly documented at important environmental sites, although most of these studies were performed at the species level. There is a lack of studies that correlate environmental contamination and clinical infections in the same region. The aim of this study is to evaluate the genetic diversity of a set of clinical and environmental samples and to use the obtained data to characterize the genetic profile of the distribution of G. duodenalis and the potential for zoonotic transmission in a metropolitan region of Brazil.

Methodology/Principal Findings

The genetic assemblages and subtypes of G. duodenalis isolates obtained from hospitals, a veterinary clinic, a day-care center and important environmental sites were determined via multilocus sequence-based genotyping using three unlinked gene loci. Cysts of Giardia were detected at all of the environmental sites. Mixed assemblages were detected in 25% of the total samples, and an elevated number of haplotypes was identified. The main haplotypes were shared among the groups, and new subtypes were identified at all loci. Ten multilocus genotypes were identified: 7 for assemblage A and 3 for assemblage B.

Conclusions/Significance

There is persistent G. duodenalis contamination at important environmental sites in the city. The identified mixed assemblages likely represent mixed infections, suggesting high endemicity of Giardia in these hosts. Most Giardia isolates obtained in this study displayed zoonotic potential. The high degree of genetic diversity in the isolates obtained from both clinical and environmental samples suggests that multiple sources of infection are likely responsible for the detected contamination events. The finding that many multilocus genotypes (MLGs) and haplotypes are shared by different groups suggests that these sources of infection may be related and indicates that there is a notable risk of human infection caused by Giardia in this region.     

基于ITS2和16S rRNA的西施舌群体遗传差异分析

目前,我国西施舌群体分子遗传差异研究结果存在争议。分析我国南北沿海（9个群体）、与广西北海毗邻的越南（1个群体）西施舌核DNA的内转录间隔区2（ITS2）和线粒体DNA的16S rRNA基因（16S）片段核苷酸序列及其二级结构,为解决争议问题提供分子生物学资料。扩增获得西施舌ITS2片段和16S序列,其长度分别为389-402 bp和306 bp,加之下载序列共147条;序列分析显示,74个ITS2序列共有17种基因型,73个16S序列有15种单倍型,其中,长乐（CL）群体独享9种ITS2基因型和5种16S单倍型,非长乐群体（nCL）多数为群体间交叉共享1种或几种基因（单倍）型;基因（单倍）型核苷酸变异位点占5.7%（ITS2）和11.8%（16S）;基于ITS2和16S的CL群体和nCL群体间的遗传距离与群体内遗传距离之比分别为2.42和11.08,nCL群体间的平均遗传距离均为0.007;二级结构显示CL群体ITS2的9种基因型和16S的5种单倍型均区别于nCL群体,nCL的ITS2和16S二级结构分别相似;ITS2和16S基因的系统发育分析显示,CL西施舌形成支持率很高（98,96）的单系支,而nCL群体则交叉聚为另一支（98,96）。研究结果揭示,福建西施舌是腔蛤蜊属（Coelomactra）的一个新种。     

Characterizations of Enterocytozoon bieneusi at new genetic loci reveal a lack of strict host specificity among common genotypes and the existence of a canine-adapted Enterocytozoon species

Molecular characterizations of the microsporidian pathogen Enterocytozoon bieneusi at the ribosomal internal transcribed spacer (ITS) locus have identified nearly 500 genotypes in 11 phylogenetic groups with different host ranges. Among those, one unique group of genotypes, Group 11, is commonly found in dogs. Genetic characterizations of those and many divergent E. bieneusi genotypes at other genetic loci are thus far impossible. In this study, we sequenced 151 E. bieneusi isolates from several ITS genotype groups at the 16S rRNA locus and two new semi-conservative genetic markers (casein kinase 1 (ck1) and spore wall protein 1 (swp1)). Comparison of the near full (~1,200 bp) 16S rRNA sequences showed mostly two to three nucleotide substitutions between Group 1 and Group 2 genotypes, while Group 11 isolates differed from those by 26 (2.2%) nucleotides. Sequence analyses of the ck1 and swp1 loci confirmed the genetic uniqueness of Group 11 genotypes, which produced sequences very divergent from other groups. In contrast, genotypes in Groups 1 and 2 produced similar nucleotide sequences at these genetic loci, and there was discordant placement of ITS genotypes among loci in phylogenetic analyses of sequences. These results suggest that the canine-adapted Group 11 genotypes are genetically divergent from other genotype groups of E. bieneusi, possibly representing a different Enterocytozoon sp. They also indicate that there is no clear genetic differentiation of ITS Groups 1 and 2 at other genetic loci, supporting the conclusion on the lack of strict host specificity in both groups. Data and genetic markers from the study should facilitate population genetic characterizations of E. bieneusi isolates and improve our understanding of the zoonotic potential of E. bieneusi in domestic animals.     

Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from different hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random amplified polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS fragments. There was considerable variability among isolates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identified among 23 isolates. Seven isolates were only self‐compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD profiles, 11 haplotypes (A to K) were identified. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) amplification with primers 4R and 5F amplified two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as ‘ITS types’. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non‐soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most‐parsimonious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two different ‘ITS types’, showed three well‐supported groupings. The neighbour‐joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major difference was that in the neighbour‐joining tree the ‘ITS type’ 11 was resolved and grouped in one clade. These results show that the ‘ITS types’ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS‐based phylogeny and isolate origin.     

Mitochondrial and nuclear markers reveal a lack of genetic structure in the entocommensal nemertean Malacobdella arrokeana in the Patagonian gulfs

Malacobdella arrokeana is an entocommensal nemertean exclusively found in the bivalve geoduck Panopea abbreviata, and it is the only representative of the genus in the southern hemisphere. To characterize its genetic diversity, population structure and recent demographic history, we conducted the first genetic survey on this species, using sequence data for the cytochrome oxidase I gene (COI), 16S rRNA (16S) and the internal transcribed spacer (ITS2). Only four different ITS2 genotypes were found in the whole sample, and the two main haplotypes identified in the mitochondrial dataset were present among all localities with a diversity ranging from 0.583 to 0.939. Nucleotide diversity was low (π = 0.001–0.002). No significant genetic structure was detected between populations, and mismatch distribution patterns and neutrality tests results are consistent with a population in expansion or under selection. Analysis of molecular variance (AMOVA) revealed that the largest level of variance observed was due to intrapopulation variation (100, 100 and 94.39 % for 16S, COI and ITS2, respectively). F _st values were also non-significant. The observed lack of population structure is likely due to high levels of genetic connectivity in combination with the lack or permeability of biogeographic barriers and episodes of habitat modification.     

Molecular Epidemiology of Human Oral Chagas Disease Outbreaks in Colombia

Background

Trypanosoma cruzi, the causative agent of Chagas disease, displays significant genetic variability revealed by six Discrete Typing Units (TcI-TcVI). In this pathology, oral transmission represents an emerging epidemiological scenario where different outbreaks associated to food/beverages consumption have been reported in Argentina, Bolivia, Brazil, Ecuador and Venezuela. In Colombia, six human oral outbreaks have been reported corroborating the importance of this transmission route. Molecular epidemiology of oral outbreaks is barely known observing the incrimination of TcI, TcII, TcIV and TcV genotypes.

Methodology and Principal Findings

High-throughput molecular characterization was conducted performing MLMT (Multilocus Microsatellite Typing) and mtMLST (mitochondrial Multilocus Sequence Typing) strategies on 50 clones from ten isolates. Results allowed observing the occurrence of TcI, TcIV and mixed infection of distinct TcI genotypes. Thus, a majority of specific mitochondrial haplotypes and allelic multilocus genotypes associated to the sylvatic cycle of transmission were detected in the dataset with the foreseen presence of mitochondrial haplotypes and allelic multilocus genotypes associated to the domestic cycle of transmission.

Conclusions

These findings suggest the incrimination of sylvatic genotypes in the oral outbreaks occurred in Colombia. We observed patterns of super-infection and/or co-infection with a tailored association with the severe forms of myocarditis in the acute phase of the disease. The transmission dynamics of this infection route based on molecular epidemiology evidence was unraveled and the clinical and biological implications are discussed.     

Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex

The internal transcribed spacer (ITS) of the rRNA gene cluster has been used as a model for the study of the action of concerted evolution and molecular drive on repeated sequence families. In contrast to this general finding, preliminary DNA sequence analysis of cloned representatives of the ITS from the West African black fly species complex Simulium damnosum s.1. demonstrated extensive intra-individual and intra-specific polymorphisms. Variability in the ITS was primarily confined to the ITS1 domain. The degree and type of intra-individual and intra-specific variability within the ITS was further characterized using gel electrophoresis, DNA hybridization, and heteroduplex analysis of the PCR products generated from the ITS1 domain. ITS1 copies from individual S. damnosum s.1. differed in length and sequence composition. These results, when taken together, demonstrate that a large degree of intra-individual and intra-specific heterogeneity exists in the ITS of S. damnosum s.1. The intra-individual heterogeneity was greater in the savanna-dwelling than forest-dwelling sibling species of S. damnosum s.1. This heterogeneity may be due in part to inter-breeding among sympatric sibling species, coupled with disturbance of S. damnosum s.1. populations resulting from intensive vector control efforts.      

Phytophthora infestans Field Isolates from Gansu Province,China are Genetically Highly Diverse and Show a High Frequency of Self Fertility

The genetic diversity of 85 isolates of Phytophthora infestans collected in 2007 from Gansu province in China was determined and compared with 21 isolates collected before 2004. Among them, 70 belonged to the A1 mating type and 15 were self‐fertile (SF). The mitochondrial DNA haplotypes revealed both Ia (25%) and IIa (75%) haplotypes. Metalaxyl resistance occurred with high frequency (54%) in Gansu. Simple sequence repeat (SSR) genotyping revealed 26 genotypes (13 from the Tianshui region) among the 85 isolates, and 18 genotypes among the 21 isolates collected before 2004, without overlap in genotypes detected in the two groups. Cluster analysis showed clear subdivisions within the different mating type isolates. Among Gansu's isolates, Nei's and Shannon's diversity indices were highest in isolates collected in Tianshui where both A1 and SF isolates were found. Analysis of molecular variance of isolates from Gansu indicated that 51% and 49% of the variance was explained by within‐area and among‐area variance, respectively. The results suggest that the occurrence of SF isolates increases the risk of sexual reproduction, the formation of oospore as initial inocula in the field, and affects the genotypic diversity in the population.     

Sequence Analysis of cytb Gene in Echinococcus granulosus from Western China

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.     

Characterization of a Chromobacterium haemolyticum population from a natural tropical lake

Aim: To study genetic diversity of Chromobacterium haemolyticum isolates recovered from a natural tropical lake. Methods and Results: A set of 31 isolates were recovered from a bacterial freshwater community by conventional plating methods and subjected to genetic and phenotypic characterization. The 16S ribosomal RNA (rRNA) gene phylogeny revealed that the isolates were related most closely with C. haemolyticum. In addition to the molecular data, our isolates exhibited strong β‐haemolytic activity, were nonviolacein producers and utilized i‐inositol, d ‐mannitol and d ‐sorbitol in contrast with the other known chromobacteria. Evaluation of the genetic diversity in the 16S rRNA gene, tRNA intergenic spacers (tDNA) and 16S‐23S internal transcribed spacers (ITS) unveiled different levels of genetic heterogeneity in the population, which were also observed with repetitive extragenic palindromic (rep)‐PCR genomic fingerprinting using the BOX‐AR1 primer. tDNA‐ and ITS‐PCR analyses were partially congruent with the 16S rRNA gene phylogeny. The isolates exhibited high resistance to β‐lactamic antibiotics. Conclusion: The population genetic heterogeneity was revealed by 16S rRNA gene sequence, ITS and BOX‐PCR analysis. Significance and Impact of the Study: This study provides for the first time an insight into the genetic diversity of phylogenetically close isolates to C. haemolyticum species.     

Diversity of Trichoderma spp. causing Pleurotus green mould diseases in Central Europe

The present study includes the molecular characteristics of Trichoderma pleurotum and Trichoderma pleuroticola isolates collected from green moulded cereal straw substrates at 47 oyster mushroom farms in Poland. The screening of the 80 Trichoderma isolates was performed by morphological observation and by using the multiplex PCR assay. This approach enabled specific detection of 47 strains of T. pleurotum and 2 strains of T. pleuroticola. Initial identifications were confirmed by sequencing the fragment of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the rRNA gene cluster and the fragment including the fourth and fifth introns and the last long exon of the translation–elongation factor 1-alpha (tef1) gene. ITS and tef1 sequence information was also used to establish the intra- and interspecies relationship of T. pleurotum and T. pleuroticola originating from the oyster mushroom farms in Poland and from other countries. Comparative analysis of the ITS sequences showed that all T. pleurotum isolates from Poland represent one haplotype, identical to that of T. pleurotum strains from Hungary and Romania. Sequence analysis of the tef1 locus revealed two haplotypes (“T” and “N”) of Polish T. pleurotum isolates. The “T” type isolates of T. pleurotum were identical to those of strains from Hungary and Romania. The “N” type isolates possessed a unique tef1 allele. Detailed analysis of the ITS and tef1 sequences of two T. pleuroticola isolates showed their identicalness to Italian strain C.P.K. 1540.     

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.