Oligonucleotide fingerprinting detects genetic diversity among Ascochyta rabiei isolates from a single chickpea field in Tunisia

Fifty isolates of Ascochyta rabiei (Pass.) Labr. were hierarchically sampled from four well-separated locations of a single chickpea field in Beja (Tunisia), and single-spored. DNA was isolated from in-vitro-grown mycelia, digested with HinfI or RsaI, and hybridized to a set of synthetic oligonucleotides complementary to simple repetitive sequences. According to the fingerprint patterns derived from the probes (CA)₈, (CAA)₅, (CAT)₅ and (GATA)₄, 12 different fungal haplotypes were found at various frequencies within the investigated field. Seven haplotypes were confined to one location only, four occurred at two, one at three, and none at all four locations. Most of the genetic variability originated from diversity within, rather than between, locations. In some cases, more than one haplotype was isolated from the same lesion of a single host plant. Genetic distances between isolates, as calculated from band-sharing data, varied between 0.05 and 0.22. Relatedness between the different haplotypes was evaluated by cluster analysis using UPGMA.     

Virulence and DNA fingerprinting analysis of Xanthomonas oryzae pv. oryzae identify a new pathotype in Guangxi,South China

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases affecting rice worldwide. However, little is known about the population structure of this organism in Guangxi Zhuang Autonomous Region, South China. Here, pathotypic and DNA fingerprint analyses were conducted to characterize the isolates of Xoo collected from rice leaves in five districts of the region from 2013 to 2016. Their pathogenicity was tested by leaf clipping, and the DNA fingerprints were analyzed by repetitive sequence‐based polymerase chain reaction and endogenous insertion sequence element‐based polymerase chain reaction assays using the repetitive extragenic palindromic sequence and enterobacterial repetitive intergenic consensus primers, respectively. Pathogenicity assays of 70 representative isolates were conducted using a series of near‐isogenic lines and two new pathotypes were identified. All the pathotypes were found to be incompatible with xa5 and Xa7. One pathotype was virulent to Xa14, Xa21, and Xa23, whereas another virulent to Xa21 and Xa23, but incompatible with Xa14. A dendrogram generated for the data sets obtained from DNA fingerprinting suggested the prevalence of high genetic diversity of Xoo throughout Guangxi, and no association between the molecular haplotypes and pathotypes was identified.     

Fingerprinting reveals gamma-ray induced mutations in fungal DNA: implications for identification of patent strains of Trichoderma harzianum

We have analyzed different patent strains and gamma-ray induced mutants of Trichoderma harzianum by DNA fingerprinting and PCR fingerprinting (RAPD). Applying wild-type phage M13 DNA, with the oligonucleotides (CT)₈ and (GTG)₅ as probes for hybridization, as well as the oligonucleotides GGCATCGGCC, (GTG)₅, (CAC)₅ and the M13 sequence GAGGGTGGCGGTTCT as primers in PCR, we were able to obtain different and discriminative fingerprint patterns for all strains and mutants investigated. Irradiation of fungi led to mutations which resulted in new fingerprint patterns. Consequently, irradiation-induced mutants can be clearly distinguished from the original wild-type isolates by genomic fingerprinting which is of importance for the patent protection of fungal strains. Sequencing of the ITS-1 and ITS-2 regions of the rDNA gene complex revealed the same sequence for all mutant strains and the original wild-type strain.     

Physical characteristics of the genome of the phytopathogenic fungus Puccinia graminis

The physical characteristics of the genome of Puccinia graminis f. sp. tritici, the wheat stem rust fungus, were determined by reassociation kinetics. The results indicate that the haploid genome contains 67 Mb and consists of three classes of DNA sequences: (1) 64% unique; (2) 30% repetitive; and (3) 4% foldback. The repetitive sequences have a total complexity of 390 kb and are repeated an average of 52 times. The base composition was 45.3% G+C based on an analysis of the DNA melting temperature. The average amount of DNA per ungerminated urediniospore by diphenylamine assay, corrected for losses during extraction, was 435 fg. This was three times the expected value (147 fg) for dikaryotic spores with nuclei in the G₁ phase of the cell cycle, an indication that the spores were in G₂.     

Rapid identification and differentiation of yeasts by DNA and PCR fingerprinting

We have used the techniques of DNA fingerprinting and polymerase chain reaction (PCR) with probes specific for hypervariable repetitive DNA sequences (mini- and microsatellite DNAs) to analyze 36 yeast strains belonging to 10 species and 2 genera. Using (GTG)₅, (GACA)₄, phage M13 DNA and the M13 sequence GAGGGTGGCGGTTCT as probes and primers, respectively, we obtained DNA polymorphisms which allowed us to discriminate 23 biotechnologically important strains of the yeast Saccaromyces cerevisiae and to distinguish them from strains of S. pastorianus, S. bayanus and S. willianus. Our results demonstrate that both DNA and PCR fingerprinting are suitable tools for an easy, fast and reliable molecular typing of yeasts. The DNA fingerprinting method seems to be more sensitive than PCR fingerprinting with respect to the individualization of strains. Nevertheless, using the PCR fingerprinting technique we were able to unambigously dicriminate between yeast genotypes of different species. Therefore, PCR fingerprinting might become a useful tool in the classification of yeasts on the basis of phylogenetic relatedness.     

Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus <Emphasis Type="Italic">Ascochyta rabiei</Emphasis>

Ascochyta blight caused by the ascomycete fungus Ascochyta rabiei, is a major biotic constraint of chickpea (Cicer arietinum L.), resulting in disastrous crop losses worldwide. To study early stages of development and pathogenic mechanisms of the fungus, two binary vectors for the constitutive expression of the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constructed. Furthermore, we have developed an improved and highly reproducible Agrobacterium tumefaciens-mediated transformation protocol for A. rabiei. Transformation events were confirmed through Southern hybridizations that suggest single-copy integration of reporter genes in majority of the transformants. High level expression of both DsRed and EGFP proteins was obtained both in spores and in mycelia as detected by fluorescence microscopy. Intense fluorescence was used as a highly efficient vital marker to visualize early developmental changes of the fungus. The formation of infection structures like appressoria and germ tubes were observed both in vitro and in planta. This work will be useful to develop methodologies for understanding the mechanisms of Ascochyta–chickpea interaction and functional genomics of A. rabiei towards the isolation of virulence genes.     

Identification and function of a polyketide synthase gene responsible for 1,8-dihydroxynaphthalene-melanin pigment biosynthesis in Ascochyta rabiei

Ascochyta rabiei produces and accumulates one of the well-known fungal polyketides, 1,8-dihydroxynaphthalene-melanin pigment (DHN-melanin), in asexual and sexual fruiting bodies. Degenerate PCR primers were used to isolate an ArPKS1 of A. rabiei encoding a polypeptide with high similarity to polyketide synthase (PKS) involved in biosynthesis of DHN-melanin in other ascomycetous fungi. Site-directed mutagenesis of ArPKS1 in A. rabiei generated melanin-deficient pycnidial mutants but caused no significant reduction of pathogenicity to chickpea. Pycnidiospores in ArPKS1-mutant pycnidia showed higher sensitivity to UV light exposure compared to pycnidiospores in melanized pycnidia of the wild-type progenitor isolate. Integration of an orthologous PKS1 gene from Bipolaris oryzae into the genome of the mutants complemented the dysfunctional ArPKS1 gene. This study demonstrated that A. rabiei uses a DHN-melanin pathway for pigmentation of pycnidia and this molecule may protect pycnidiospores from UV irradiation.     

Heterobasidion annosum 5.8s ribosomal DNA and internal transcribed spacer sequence: rapid identification of European intersterility groups by ribosomal DNA restriction polymorphism

Using conserved fungal ribosomal gene sequences the internal transcribed spacer (ITS) regions one and two (ITS1, ITS2) and the 5.8s ribosomal RNA gene (rRNA) of Heterobasidion annosum were amplified by the polymerase chain reaction (PCR). The nucleotide sequence was determined in three European intersterility groups (ISG-S,-F and-P). Three sequence variants of the ITS were found in ISG-S isolates. The sequence of the ITS of ISG-F differed by two residues from the major ISG-S sequence variant. The ISG-P sequence differed from ISG-S and ISG-F at 15–16 and 16 residues, respectively. Amplified intergenic spacer elements were informative for ISG fingerprinting following digestion with various 4-cutter restriction endonucleases. All differences in the restriction fragments between the ISGs were because of sequence differences in the ITS regions. The fingerprint patterns of isolates from the same intersterility group but from different European localities were identical. These results show that ribosomal DNA finger-printing is a rapid technique to identify ISGs in Heterobasidion annosum.     

Cloning of the mating type locus from Ascochyta lentis (teleomorph: Didymella lentis) and development of a multiplex PCR mating assay for Ascochyta species

The mating type (MAT) locus of the lentil pathogen, Ascochyta lentis, was cloned and characterized using thermal asymmetric interlaced and inverse PCR with primers designed to the HMG-box of Ascochyta rabiei. A multiplex PCR assay for mating type was developed based on MAT idiomorph and flanking sequences. Primers were designed to specifically amplify MAT from several Ascochyta spp. including A. pisi, A. fabae and A. viciae-villosae in addition to A. lentis. Four hundred and fifty and 700 bp fragments were amplified from MAT1-1 and MAT1-2 isolates, respectively, and fragment size correlated perfectly with laboratory crosses using mating type tester strains. MAT-specific PCR allowed rapid scoring of mating type in crude DNA extracts from geographically diverse population samples of A. viciae-villosae from California and Washington State, USA. This co-dominant MAT-specific PCR assay will be a valuable tool for studying the population structure, biology and epidemiology of these fungi.     

Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.)

Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular and cytogenetic characterization of site-specific repetitive DNA sequences in the Chinese soft-shelled turtle (<Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>, Trionychidae)

A novel family of repetitive DNA sequences that are components of constitutive heterochromatin were cloned from BglI-digested genomic DNA of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae), and characterized by filter hybridization and chromosome in-situ hybridization. The BglI-family of repetitive sequences were classified into four types by their genome organization and chromosomal distribution as follows: the repeated sequences located on (1) two pairs of microchromosomes, (2) four pairs of microchromosomes,(3) about half the number of microchromosomes and (4) the interstitial region of the short arm of chromosome 2. The presence of microchromosome-specific repetitive sequences has also been reported in the Struthioniformes and Galliformes, suggesting that turtle chromosomes retain some similarity to the chromosome structure as well as the karyotypes of avian species     

Differentiation of species and strains among filamentous fungi by DNA fingerprinting

Summary We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)₈, (GTG)₅ and (GACA)₄, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)₄ produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.     

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the A and B chromosomes of the Korean field mouse (<Emphasis Type="Italic">Apodemus peninsulae</Emphasis>, Muridae,Rodentia)

Three novel families of repetitive DNA sequences were molecularly cloned from the Korean field mouse (Apodemus peninsulae) and characterized by chromosome in-situ hybridization and filter hybridization. They were all localized to the centromeric regions of all autosomes and categorized into major satellite DNA, type I minor, and type II minor repetitive sequences. The type II minor repetitive sequence also hybridized interspersedly in the non-centromeric regions. The major satellite DNA sequence, which consisted of 30 bp elements, was organized in tandem arrays and constituted the majority of centromeric heterochromatin. Three families of repetitive sequences hybridized with B chromosomes in different patterns, suggesting that the B chromosomes of A. peninsulae were derived from A chromosomes and that the three repetitive sequences were amplified independently on each B chromosome. The minor repetitive sequences are present in the genomes of the other seven Apodemus species. In contrast, the major satellite DNA sequences that had a low sequence homology are present only in a few species. These results suggest that the major satellite DNA was amplified with base substitution in A. peninsulae after the divergence of the genus Apodemus from the common ancestor and that the B chromosomes of A. peninsulae might have a species-specific origin.     

Morphological characteristics and pathogenicity of fungi associated with Roselle (Hibiscus Sabdariffa) diseases in Penang, Malaysia

Roselle, or Jamaica sorrel (Hibiscus sabdariffa) is a popular vegetable in many tropical regions, cultivated for its leaves, seeds, stems and calyces which, the dried calyces are used to prepare tea, syrup, jams and jellies and as beverages. The main objectives of this study were to identify and characterise fungal pathogens associated with Roselle diseases based on their morphological and cultural characteristics and to determine the pathogenicity of four fungi infecting Roselle seedlings, namely Phoma exigua, Fusarium nygamai, Fusarium tgcq and Rhizoctonia solani in Penang. A total of 200 fungal isolates were obtained from 90 samples of symptomatic Roselle tissues. The isolates were identified based on cultural and morphological characteristics, as well as their pathogenicity. The fungal pathogen most frequently isolated was P. exigua (present in 45% of the samples), followed by F. nygamai (25%), Rhizoctonia solani (19%) and F. camptoceras (11%). Pathogenicity tests showed that P. exigua, F. nygamai, F. camptoceras and R. solani were able to infect both wounded and unwounded seedlings with different degrees of severity as indicated by the Disease severity (DS). R. solani was the most pathogenic fungus affecting both wounded and unwounded Roselle seedlings, followed by P. exigua that was highly pathogenic on wounded seedlings. F. nygamai was less pathogenic while the least pathogenic fungus was F. camptoceras, infecting only the unwounded seedlings but, surprisingly, not the wounded plants.     

Characterization and genetic diversity of sugarcane streak mosaic virus causing mosaic in sugarcane

Sixty-three sugarcane leaf samples were collected from fifty-eight sugarcane varieties, evolved from eleven major sugarcane growing states in India, Australia, South Africa and USA. In RT-PCR, using gene specific primers for sugarcane streak mosaic virus (SCSMV)-CP, 58 of 63 sugarcane samples were found positive to the virus infection and rest of the five samples were negative. Partial CP gene sequences of 42 SCSMV isolates including an isolate from aphid colony (Melanaphis indosacchari) infested on sugarcane variety from this study were characterized after cloning and sequencing for selective isolates represented by at least one isolate from each location. The new sequences identified in the study were named as SCSMV-CB isolates. Fifty two sequences including the 10 database sequences (complete CP cds) deposited earlier from this institute were compared with each other as well as GenBank database sequences of Potyviridae members viz., Rymovirus, Potyvirus, Ipomovirus, Tritimovirus and eight sequences of SCSMV reported from elsewhere. Among the SCSMV-CB isolates sequenced in the study, 85.7–100% (nucleotide) and 89.9–100% (amino acid) sequence identities were observed and with the other data base sequences of SCSMV, the respective identities were 82.2–97.5 and 89.7–98.6%. Grouping of the isolates by the maximum likelihood with molecular clock model, distributed 60 SCSMV sequences including the eight database sequences deposited by other SCSMV working groups from India and USA in 16 different phylogenetic groups. Although the isolates of SCSMV were relatively close to Ipomovirus and Tritimovirus, they were sandwiched between Rymovirus and Ipomovirus. The sequence comparison and phylogenetic studies revealed that the relatedness of SCSMV with the potyviral related genera was comparatively low to consider it as a member of earlier described potyviral genera, hence the genus “Susmovirus” (sugarcane streak mosaic virus) has been proposed, with SCSMV as the sole species to be included. The 52 SCSMV-CB isolates from this institute were distributed in 14 phylogenetic groups and the grouping pattern revealed that the virus isolates could not be grouped based on geographical origin of the host varieties or longevity of the host variety.     

Characterization and evolutionary dynamics of a complex family of satellite DNA in the leaf beetle <Emphasis Type="Italic">Chrysolina carnifex</Emphasis> (Coleoptera,Chrysomelidae)

The present study characterizes the complex satellite DNA from the specialized phytophagous beetle species Chrysolina carnifex. The satellite DNA is formed by six monomer types, partially homologous but having diverged enough to be separate on the phylogenetic trees, since each monomer type is located on a different branch, having statistically significant bootstrap values. Its analysis suggests a common evolutionary origin of all monomers from the same 211-bp sequence mainly by means of base-substitution mutations evolutionarily fixed to each monomer type and duplications and/or deletions of pre-existing segments in the 211-bp sequence. The analysis of the sequences and Southern hybridizations suggest that the monomers are organized in three types of repeats: monomers (211-bp) and higher-order repeats in the form of dimers (477-bp) or even trimers (633-bp). These repetitive units are not isolated from others, and do not present the pattern characteristic for the regular tandem arrangement of satellite DNA. In-situ hybridization with biotinylated probes corresponding to the three types of repeats showed the pericentromeric location of these sequences in all meiotic bivalents, coinciding with the heterochromatic blocks revealed by C-banding, indicating in addition that each type of repeat is neither isolated from others nor located in specific chromosomes but rather that they are intermixed in the heterochromatic regions. The presence of this repetitive DNA in C. haemoptera, C. bankii and C. americana was also tested by Southern analysis. The results show that this satellite DNA sequence is specific to the C. carnifex genome but has not been found in three other species of Chrysolina occupying similar or different host plants.     

Transformation of the plant pathogenic fungus,Rhynchosporium secalis

  The barley leaf scald fungus, Rhynchosporium secalis, was transformed to hygromycin-B and phleomycin resistance using the hph gene from E. coli and the ble gene from Streptoalloteichus hindustanus under the control of Aspergillus nidulans promoter and terminator sequences. Plasmid DNA was introduced into fungal protoplasts by PEG/CaCl₂ treatment. Transformation frequencies varied from 59 to 493 transformants per 10 μg of DNA and 5 × 10⁷ protoplasts. The antibiotic-resistant phenotype appeared to be stable under selective, as well as under non-selective, conditions for several generations. Co-transformation using the E. coli uidA gene under the control of A. nidulans promoter and terminator sequences on a non-selectable plasmid occurred at frequencies of up to 66%. Received: 20 September 1995 / 26 January 1996     

Genetic transformation of the fungal plant wilt pathogen,Fusarium oxysporum

Summary A system for transformation of the fungal plant pathogen Fusarium oxysporum has been developed. The system employs plasmids which contain a bacterial hygromycin B phosphotransferase gene (hph) linked to Aspergillus regulatory sequences and which confer hygromycin B resistance in Fusarium. Transformation resulted from integration of the vectors into heterologous regions of the Fusarium genome and occurred at a frequency of approximately one transformant per µg DNA. No evidence was found for autonomous replication of the vector in the fungus. The transformed, drug resistant phenotype was mitotically stable with or without selection. However, modification of integrated DNA could occur during vegetative growth.     

Comparative genomic in situ hybridization (cGISH) analysis on plant chromosomes revealed by labelled Arabidopsis DNA

A new approach for comparative cytogenetic banding analysis of plant chromosomes has been established. The comparative GISH (cGISH) technique is universally applicable to various complex genomes of Monocotyledonae (Triticum aestivum, Agropyron elongatum, Secale cereale, Hordeum vulgare, Allium cepa, Muscari armenaticum and Lilium longiflorum) and Dicotyledonae (Vicia faba, Beta vulgaris, Arabidopsis thaliana). Labelled total genomic DNA of A. thaliana generates signals at conserved chromosome regions. The nucleolus organizing regions (NORs) containing the majority of tandemly repeated rDNA sequences, N-band regions containing satellite DNA, conserved homologous sequences at telomeres and additional chromosome-characteristic markers were detected in heterologous FISH experiments. Multicolour FISH analysis with repetitive DNA probes simultaneously revealed the chromosome assignment of 56 cGISH signals in rye and 61 cGISH signals in barley. Further advantages of this technique are: (1) the fast and straightforward preparation of the probe; (2) the generation of signals with high intensity and reproducibility even without signal amplification; and (3) no requirement of species-specific sequences suitable for molecular karyotype analysis. Hybridization can be performed without competitive DNA. Signal detection without significant background is possible under low stringency conditions. The universal application of this fast and simple one-step fluorescence banding technique for plant cytogenetic and plant genome evolution is discussed.     

Comparison of the chicken and zebra finch Z chromosomes shows evolutionary rearrangements

Using fluorescent in-situ hybridization (FISH) of zebra finch (Taeniopygia guttata) bacterial artificial chromosome (BAC) clones, we determined the chromosomal localizations of 14 zebra finch genes that are Z-linked in chickens: ATP5A1, CHD1, NR2F1, DMRT1, PAM, GHR, HSD17B4, NIPBL, ACO1, HINT1, SMAD2, SPIN, NTRK2 and UBE2R2. All 14 genes also map to the zebra finch Z chromosome, indicating substantial conservation of gene content on the Z chromosome in the two avian lineages. However, the physical order of these genes on the zebra finch Z chromosome differed from that of the chicken, in a pattern that would have required several inversions since the two lineages diverged. Eight of 14 zebra finch BAC DNA showed cross-hybridization to the W chromosome, usually to the entire W chromosome, suggesting that repetitive sequences are shared by the W and Z chromosomes. These repetitive sequences likely evolved in the finch lineage after it diverged from the Galliform lineage.