Differentiation of strains and isolates of chicken reovirus using PCR and heteroduplex mobility assay in amplified cDNA

The method of chicken reovirus strain differentiation was worked out on the basis of RT-PCR and heteroduplex mobility assay (HMA). The S3 gene cDNA (633-896 b.p.) of some Russian and Italian chicken reovirus isolates was amplified by RT-PCR. The analysis of these cDNA samples was carried out by HMA. The relation between nucleotide differences and relative mobility of compared cDNA heteroduplex was reflected by the regression curve. The equation of linear regression was derived (y = 91.726-0.89x; where y is the level of nucleotide difference of compared cDNA (%), x is the relative mobility of compared cDNA heteroduplex (%)). This method made it possible to take correct results within 5-35% of nucleotide difference in heteroduplex sequences.     

Position and degree of mismatches and the mobility of DNA heteroduplexes

Heteroduplex mobility assay (HMA) is a fast and inexpensive method for determining relatedness between DNA sequences. Rapidly evolving viruses such as HIV-1 develop marked sequence differences in their genomes over the course of the epidemic and infection in a single individual. HMA can be used to monitor both processes. Here, we systematically evaluated the influence of single base mismatches on heteroduplex mobility. The impact of mismatches at nine different positions in 559 bp double-stranded DNA molecules, within a background of overall sequence divergence ranging from 1.97 to 9.65%, was evaluated in both non-denaturing and partially-denaturing acrylamide gels. We found that the electrophoretic mobility of heteroduplexes was proportional to the level of mismatch when that level exceeded 4.5%. Overall, mismatches near the center of the fragment and clustered mismatches tended to have an exaggerated influence on the mobility of heteroduplexes. Thus, the use of HMA for quantitative inference of genetic distances under the conditions we describe is of greatest utility at levels of mismatch >5%.     

Molecular and phenotypic analyses reveal association of diverse Colletotrichum acutatum groups and a low level of C. gloeosporioides with olive anthracnose

Anthracnose (Colletotrichum spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of Colletotrichum spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128 Colletotrichum spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and beta-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed Colletotrichum acutatum to be dominant (>97%) with limited occurrence of Colletotrichum gloeosporioides (<3%). Among C. acutatum populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other C. acutatum groups and C. gloeosporioides were sporadic in their occurrence, mostly related to marginal areas of olive cultivation. C. gloeosporioides, isolated from olive fruits with symptoms indistinguishable from those of C. acutatum, showed same virulence rating as the most virulent C. acutatum isolate from group A2. C. acutatum and C. gloeosporioides isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from beta-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.     

Electrophoretic analysis of ITS from Piscirickettsia salmonis Chilean isolates

Piscirickettsia salmonis is the most important pathogen in salmonid mariculture in Chile. Since it was reported numerous piscirickettsiosis outbreaks have occurred differing in virulence and mortality. Genetic variability of P. salmonis isolates has been suggested as one factor to explain this. However until now isolates obtained from outbreaks have not been analyzed. Knowledge of genetic variability of P. salmonis is very limited and also a useful screening method for genetic variations in isolates without sequencing is not available. Here we report an electrophoretic analysis of internal transcribed spacer region (ITS) of eleven P. salmonis isolates obtained from different salmon species and places in southern Chile. When PCR products were submitted to polyacrylamide gel electrophoresis (PAGE) a characteristic electrophoretic pattern was observed, distinguishable from ITS of other bacteria, including fish pathogens. Even though this pattern is conserved in all isolates, a difference in ITS electrophoretic mobility was observed, determining clearly two groups: ITS with higher or with lower electrophoretic mobility, including LF-89 and EM-90 isolates, respectively. A higher ITS sequence homology inside each group was shown by heteroduplex mobility assay (HMA). Our results show that genetic variability between Chilean P. salmonis isolates allows the differentiation of two groups with similar behavior observed previously when six P. salmonis isolates from three geographic origins were analyzed by 16S, 23S and ITS sequencing. PAGE analysis of ITS and HMA could be a basis to develop an assay for screening genetic variability between P. salmonis isolates.     

Development and optimization of sequence-tagged microsatellite site markers to detect genetic diversity within Colletotrichum capsici, a causal agent of chilli pepper anthracnose disease

Genomic libraries enriched for microsatellites from Colletotrichum capsici, one of the major causal agents of anthracnose disease in chilli pepper (Capsicum spp.), were developed using a modified hybridization procedure. Twenty-seven robust primer pairs were designed from microsatellite flanking sequences and were characterized using 52 isolates from three countries India, Sri Lanka and Thailand. Highest gene diversity of 0.857 was observed at the CCSSR1 with up to 18 alleles among all the isolates whereas the differentiation ranged from 0.05 to 0.45. The sequence-tagged microsatellite site markers developed in this study will be useful for genetic analyses of C. capsici populations.     

Intraspecific Differentiation of Colletotrichum gloeosporioides sensu lato Based on In Silico Multilocus PCR-RFLP Fingerprinting

Colletotrichum gloeosporioides sensu lato is one of the most common and widely distributed plant pathogens in the world. Understanding fungal biodiversity is hinged on accurate identification and delimitation at the inter- and intraspecific levels. Sequences of the ITS1-5.8S-ITS2 region (ITS), β-tubulin (TUB), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes of 30 C. gloeosporioides sensu lato isolates, collected from anthracnose infected papaya fruits grown in the main production areas in Trinidad, were analyzed by in silico PCR-RFLP analysis with the aim of identifying which gene region(s) had the highest level of intraspecific polymorphism. Restriction site polymorphisms generated from 13 restriction enzymes enabled the identification of specific enzymes that were successful at intraspecific discrimination of the C. gloeosporioides isolates. Genetic distance values were reflective of the level of polymorphisms obtained for the four different gene regions. In both cases (calculated genetic distance and percentage of polymorphic loci from RFLP profiles), ACT and ITS gene regions had the highest level of restriction site polymorphisms and genetic diversity, GPDH and TUB had the lowest. Cluster analysis based on PCR-RFLP genetic distance data revealed sub-specific placement of the isolates which appeared to be gene-dependent. The implications of these findings are discussed relative to biodiversity monitoring and the need for multilocus, polyphasic investigations which must take into account the possibility of exaggerated estimates of genetic diversity.     

NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER REGIONS (ITS1 AND ITS2) DEFINE DISCRETE BIOGEOGRAPHIC GROUPS IN CLADOPHORA ALBIDA (CHLOROPHYTA)1

Nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2), the 5.8S, and short stretches of the adjacent 18S and 26S coding regions were determined in isolates from four disjunct Cladophora albida (Huds.) Kütz. populations (NE-America, W-Europe, Japan, and W-Australia). The two Pacific isolates share nearly identical ITS sequences as do the two Atlantic isolates. In contrast, interoceanic comparisons exhibit a 21% sequence difference. Variation within ITS regions is useful for identification of population groups on a regional or oceanic scale. However, both spacers are characterized by numerous repeat motifs as well as point mutations, which result in alignment problems at the interspecific level within Cladophora.     

Genetic analysis of human immunodeficiency virus type 1 strains from patients in Cyprus: identification of a new subtype designated subtype I.

DNA sequences encoding the C2 to V3 region of envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) were amplified by PCR from uncultured peripheral blood mononuclear cells obtained from 24 of 25 HIV-1-seropositive patients from Cyprus. By using a heteroduplex mobility assay (HMA), all amplified products were studied genetically and compared with 16 previously characterized HIV-1 strains belonging to subtypes A through F. HMA results revealed that HIV-1 gp120 sequences from 15 of our patients were of subtype B of HIV-1, whereas one isolate was of subtype C. However, gp120 sequences from eight patients had no obvious similarities to the known subtypes as defined by HMA. DNA sequencing and phylogenetic analyses of molecular clones confirmed the HMA results and placed the eight undefined HIV-1 isolates into three distinct genetic clusters. On the basis of branch topology and lengths of the phylogenetic tree, we conclude that one group consisting of three clones from two patients represents a new HIV-1 env subtype, which we have termed subtype I. The remaining two sequence clusters, consisting of five sequences from four patients and two sequences from two other patients, are distally related to subtypes A and F. These data demonstrate the extensive heterogeneity of HIV-1 in Cyprus, including the presence of new subtype.     

Heteroduplex mobility assay detects DNA mutations for differentiation of closely related phytoplasma strains

We present the first use of DNA heteroduplex mobility assay (HMA) to detect the point mutations including substitutions and deletions/insertions in 16S rDNA of aster yellows phytoplasma (AY27) and to differentiate phytoplasmas collected from field samples of clover proliferation (CP) and alfalfa witches'-broom (AWB). The phytoplasmal 16S rDNA fragment was amplified from AY27 by polymerase chain reaction (PCR) and cloned into a plasmid vector. The cloned DNA fragment was subjected to in vitro mutation to produce 1- to 4-base substitutions and 1- to 3-base deletions. The mutated 16S rDNA fragments were analyzed by HMA. The results showed that a single two-base substitution or a single-base deletion/insertion in the 529 bp DNA fragment was directly detected and that a DNA divergence at a level of as low as 0.2% was detectable by HMA. Heteroduplex mobilities were affected by the number and composition of the phytoplasma DNA bases in mismatches or gaps and were proportional to the degree of DNA divergences. Gaps caused greater retardation in heteroduplex mobility than mismatches did. HMA was highly sensitive in detecting the mixed infections of phytoplasmas. In analyses of CP and AWB field samples collected in Alberta, two CP and one AWB phytoplasma isolates were differentiated from others by HMA but not by restriction fragment length polymorphism (RFLP). Therefore, HMA provides a simple, rapid, highly sensitive and analytical method to detect and estimate the genetic divergence of phytoplasmas when other methods such as RFLP are not readily applicable.     

A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of <Emphasis Type="Italic">Colletotrichum</Emphasis><Emphasis Type="Italic">capsici</Emphasis>

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.     

广东省柑橘炭疽病病原菌的形态与分子鉴定

炭疽病是柑橘的主要真菌性病害之一。2007年春,广东省德庆县名优柑橘品种贡柑炭疽病暴发流行。为了明确该县及广东省其他地区柑橘炭疽病菌的种类,为防治提供依据,对采集自广东省6个地区柑橘属10个栽培品种上的炭疽病样本进行病原菌分离,共获得柑橘炭疽病菌单孢菌株75株,对其中10株代表性的菌株进行了种类鉴定。通过培养性状和形态学特征观测、核糖体DNA(rDNA)内转录间区(ITS)序列分析、ITS区特异性引物PCR检测和系统发育关系比较等方面的研究,结果表明:10个柑橘炭疽病菌菌株均为盘长孢状刺盘孢Colletotrichum gloeosporioides,未发现国际上其他国家报道的严重危害柑橘花器和幼果部位的柑橘花后落果病病原菌——尖刺盘孢C.acutatum。     

Molecular characterization and pathogenicity of Colletotrichum sp. from guava

Guava (Psidium guajava) fruit is vulnerable to postharvest diseases, such as anthracnose. In the present study, molecular characterisation and pathogenicity of Colletotrichum associated with antharcnose disease of guava fruit were conducted. From anthracnose lesion of guava, 20 isolates were successfully recovered. Based on colony colours, conidia, appressoria and presence or absence of setae, and ITS regions and ß-tubulin gene sequences, the isolates were identified as Colletotrichum gloeosporioides. Phylogenetic analysis based on combined data-sets using neighbour-joining method showed that C. gloeosporioides isolates did not group with C. gloeosporioides epitype strain, and thus the isolates were referred to as C. gloeosporioides species complex or C. gloeosporioides sensu lato. Pathogenicity tests using wounded treatment showed that C. gloeosporioides isolates from guava were pathogenic causing anthracnose on the fruits. The present study showed that C. gloeosporioides sensu lato is the most common species causing antharcnose disease of guava fruit.     

Diversity of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. isolated from chili pepper fruit exhibiting symptoms of anthracnose in Thailand

Colletotrichum spp. are causal agents of anthracnose disease in chili fruits and other tropical crops. The disease is increasing in chili fruits in Thailand and significantly reduces fruit quality and fruit production. Forty-eight isolates of Colletotrichum spp. associated with chili anthracnose were collected from different areas of Thailand during 2010–2015. Based on morphological characteristic identification, 10 isolates were shown to belong to the C. gloeosporioides species complex, 24 isolates belong to the C. acutatum species complex and 14 isolates to C. capsici. For molecular identification, two primer sets, ITS1/ITS4 and ACT528/ACT738, were used for amplification of the internal transcribed spacer of rRNA gene (ITS1–5.8S–ITS2) and partial region actin gene (ACT), respectively. The phylogenetic analysis of individual and combined ITS region and actin nucleotide sequences identified the collected isolates into 4 species: C. gloeosporioides, C. siamense, C. acutatum and C. capsici. The pathogenicity test demonstrated that all four species were pathogenic on intact unwounded and healthy fruits. These results indicated that C. capsici, C. acutatum, C. gloeosporioides and C. siamense were the causal agents of chili anthracnose disease.     

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA

Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B1 and B2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.     

Characterization and Pathogenicity of Colletotrichum truncatum Causing Stem Anthracnose of Red‐Fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During August 2010 and January 2011, 10 isolates of Colletotrichum were recovered from stem anthracnose lesions of Hylocereus polyrhizus in the states of Kedah and Penang, Malaysia. Based on the morphological characteristics of colony colour and appearance, and shapes of conidia as well as sequences of internal transcribed spacer regions (ITS), β‐tubulin, actin (ACT) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), the fungus was identified as Colletotrichum truncatum. Pathogenicity test showed that C. truncatum isolates were pathogenic to the artificially inoculated H. polyrhizus stem. This is the first report of C. truncatum causing anthracnose on H. polyrhizus stems in Malaysia.     

A heteroduplex method for detection of targeted sub-populations of bacterial communities

We describe a simple method, based on heteroduplex mobility analysis of 16S rDNA fragments, for targeted detection of sub-populations of bacteria within diverse microbial communities. A small (ca. 200 bp) polymorphic fragment of the bacterial 16S rRNA gene was amplified from sample DNA using universal primers. Sample products were hybridised with a fluorescently labelled fragment amplified from a selected 'reporter' organism representing the target group. The resulting products were resolved and the labelled heteroduplex pairs detected on non-denaturing gels using automated DNA detection technology. A model, based on analysis of samples with known 16S rDNA sequences, demonstrates that heteroduplex mobility is inversely correlated with genetic distance and that beyond 26% genetic difference, heteroduplex products are not detected. The utility of the method was tested by field studies in which stream biofilms could be characterised by heteroduplex profiles generated with heterotrophic and autotrophic reporter organisms representing target groups.     

Molecular diagnosis of Colletotrichum gloeosporioides causing Anthracnose/Dieback disease in Greater Yam (Dioscorea alata L.)

The Greater Yam (Dioscorea alata L.), a significant tropical tuber crop is highly affected by the anthracnose/dieback disease caused by Colletotrichum gloeosporioides, which greatly reduces the yield as well as market acceptability of the tubers. The different methods that could be used for proper identification of the pathogen by PCR were investigated. The studies indicate that species specific polymerase chain reaction assay based on highly conserved regions in ITS in the genome of the pathogen can be the best strategy for detection of this pathogen at species level. The use of genus specific primers was also successful in detection of Colletotrichum spp. The cloning and sequencing of evolutionarily conserved regions such as ITS, PelB and paralleling them with the available sequences in NCBI database is also costly but reliable approach. The various methods are elaborately tested and their use in diagnosis is discussed in detail.     

Identification of Colletotrichum Species Causing Bitter Rot of Apple and Pear in Croatia

Symptoms of bitter rot were observed on apple and pear fruits in the field and in storage in Croatia between 2009 and 2011. Fifteen Colletotrichum isolates from apple and two from pear were collected and identified by sequencing of the ITS1 and ITS2 regions of the ribosomal DNA. Phylogenetic analysis revealed that ten isolates from apple and two isolates from pear could be identified as Colletotrichum fioriniae, five isolates from apple were clustered in Colletotrichum clavatum, while one isolate was in the Colletotrichum acutatum A7 group. All isolates caused typical bitter rot symptoms when inoculated on apple and pear fruits.     

Use of homoduplex ribosomal DNA spacer amplification products and heteroduplex cross-hybridization products in the identification of Salmonella serovars.

When the hypervariable 16S-23S intergenic spacer regions found in prokaryotic ribosomal DNA (rDNA) are amplified from conserved adjacent sequences, homoduplex double-stranded DNA structures and heteroduplex structures containing substantial regions of single-stranded DNA are generated. The electrophoretic separation of these structures results in product profile patterns, which may be organized into highly correlated pattern groups of ribosomal spacer and heteroduplex polymorphism (RS/HP) types. In a test panel of 380 Salmonella strains that were analyzed by this procedure, 36 unique RS/HP types were observed. Of the 28 serovars in the test group, 21 showed single characteristic RS/HP types. The remaining seven serovars each contained multiple RS/HP types, which were also unique to individual serovars. Formation of heteroduplex structures with a substantially reduced electrophoretic mobility was observed in 29 of the 36 RS/HP pattern types. Because the mobility of these heteroduplex structures is sensitive to intergenic spacer sequence composition, the presence of these structures adds an additional diagnostic feature that is extremely useful in the differentiation of Salmonella serovars. The RS/HP types show sufficient diversity to be useful in the identification of many commonly observed Salmonella serovars. This analytical procedure is simple to perform and is well suited to rapid and inexpensive screening of large numbers of Salmonella strains.