Genetic diversity and vegetative compatibility among Trichoderma harzianum isolates

Trichoderma harzianum is the collective name of a set of asexual fungal strains which exhibit heterogeneity in genome structure, DNA sequence and behavior. Contour-clamped homogeneous field (CHEF) electrophoresis of the chromosomes of ten isolates of T. harzianum revealed six clearly distinct electrophoretic karyotypes. Of the ten isolates analyzed, four (GH12, G109, Y and YF) could be classified in a single group with identical karyotypes, while the strains T35 and 315 formed a second group. The genome size characteristic of the different isolates fell into a broad range varying from 29.6 to 56.1?Mb. Gene assignments to the resolved chromosomes showed that all genes analyzed were localized on equivalent chromosomes in the isolates belonging to the same group. Analysis of randomly amplified polymorphic DNAs from the ten isolates confirmed the classification into groups and allowed us to distinguish between isolates T35 and 315, as well as between isolates GH12, G109, Y and YF. Direct confrontation assays using isolates of the same group showed compatible interactions, whereas the same experiment carried out with isolates of different groups showed an incompatible interaction characterized by an area of cell damage. Microscopic observation of the compatible interactions showed hyphal fusions between the isolates, similar to those described for vegetative compatible groups in other fungi. The molecular karyotypes correlated well with the compatibility of the isolates. In addition, we have evaluated both electrophoretic karyotype and randomly amplified polymorphic DNAs analysis as criteria for grouping isolates within the genus according to their capacity for biocontrol of plant pathogens.     

Genomic organization of the yeast Yarrowia lipolytica

We produced electrophoretic karyotypes of the reference strain E150 and of seven other isolates from different geographical origins to study the genomic organization of the dimorphic yeast Yarrowia lipolytica. These karyotypes differed in the number and size of the chromosomal bands. The karyotype of the reference stain E150 consisted of five bands of between 2.6 and 4.9 Mb in size. This strain contained at least five rDNA clusters, from 190 to 620 kb in size, which were scattered over most of the chromosomes. The assignment of 43 markers, including rRNA genes and three centromeres, to the E150 bands defined five linkage groups. Hybridization to the karyotypes of other isolates with pools of markers of each linkage group showed that linkage groups I, II, IV and V were conserved in the strains tested whereas group III was not and was split between at least two chromosomes in most strains. Use of a meganuclease I-SceI site targeted to one locus of E150 linkage group III showed that two chromosomes actually comigrated in band III of this strain. Our results are compatible with six chromosomes defining the haploid complement of strains of Y. lipolytica and that, despite an unprecedented chromosome length polymorphism, the overall structure of the genome is conserved in different isolates. Received: 27 March 1997; in revised form: 8 July 1997 / Accepted: 9 July 1997     

Meiotic Instability of Pythium Sylvaticum as Demonstrated by Inheritance of Nuclear Markers and Karyotype Analysis

Progeny from a sexual outcross between opposite mating types of Pythium sylvaticum were analyzed for inheritance of RFLP and random amplified polymorphic DNA (RAPD) markers. Although most were inherited in expected Mendelian frequencies, several were not. Pulsed field gel electrophoresis was employed to examine these unexpected patterns of marker inheritance at a karyotypic level. Parental oogonial and antheridial isolates had different electrophoretic karyotypes and minimum number of chromosome-sized DNAs (13 and 12, respectively), however, summation of the sizes of all chromosomal bands for each isolate was similar at ~37 Mb. Progeny karyotypes differed significantly from each other and the parental isolates, ranging in estimated minimum number of chromosome-sized DNAs from 9 to 13 and the summation of band sizes within each isolate from 28.1 to 39.0 Mb. For the eight isolates most extensively analyzed, 80% of the progeny chromosome-sized DNAs were nonparental in size or hybridization grouping of cDNA clones and isolated RAPD markers. Based on the results of Southern analysis it appears that length mutations and perhaps aneuploidy and translocations have contributed to generation of karyotypic polymorphisms. Nineteen field isolates of P. sylvaticum collected from the same location also exhibited significantly different karyotypes, suggesting that the meiotic instability observed in the laboratory also is occurring in field populations.     

A polymerase chain reaction-based test for the identification of Trichoderma harzianum biotypes 2 and 4, responsible for the worldwide green mold epidemic in cultivated Agaricus bisporus

We describe a polymerase chain reaction (PCR)-based test that is specific for the pathogenic European biotype 2 (Th2) and North American biotype 4 (Th4) of Trichoderma harzianum, responsible for the green mold epidemic in the cultivated mushroom, Agaricus bisporus. A PCR primer pair was designed that targets a 444-bp arbitrary sequence in the genome of Th4. The primers also amplified the same product with Th2, but showed no reactivity with other biotypes of T. harzianum, several biocontrol Trichoderma, or with 31 other genera and species of fungi. The PCR-based test should have application in disease management programs, and in the evaluation of biocontrol Trichoderma for potential pathogenicity on mushrooms. Received: 23 November 1998 / Received revision: 19 February 1999 / Accepted: 5 March 1999     

Colonisation of phase II compost by biotypes of Trichoderma harzianum and their effect on mushroom yield and quality

Colonisation assessments confirmed that Trichoderma harzianum biotypes Th1, Th2a, Th2b and Th3 inoculated into two distinct compost types at spawning became established by first flush assessment; the extension rate of two Th2 isolates was over 1000 times that of Th1 and Th3. Results subsequently confirmed that while Th1 and Th3 did not significantly affect yield, Th2 could reduce mushroom quality and productivity by as much as 80%. Analysis of compost type also indicated that the speed and magnitude of T. harzianum colonisation was influenced by key compost characteristics, most notably, moisture, ash content and degree of fermentation. This study has shown that compost parameters which have a positive influence on Agaricus growth and productivity also resulted in increased compost colonisation by T. harzianum. Commercially acceptable yields obtained from uninoculated compost confirmed that production of a high quality, productive substrate does not confer inherent immunity to colonisation by T. harzianum. Received: 25 September 1998 / Received revision: 30 November 1998 / Accepted: 5 December 1998     

Nucleolar Organizing Regions, 18S and 5S rDNA in Astyanax Scabripinnis (Pisces, Characidae): Populations Distribution and Functional Diversity

Astyanax scabripinnis specimens from four distinct populations in Brazil were studied with respect to their karyotype macrostructure, nucleolar organizer regions, and 18S and 5S rRNA genes. The four populations showed a 2n = 50 chromosomes (3 M + 11 SM + 5 ST + 6 A pairs) and 1–2 B chromosomes. No chromosomal differentiations were observed between sexes. Although a karyotypic diversity has been characterized in this fish group, the populations now analyzed presented the same macrokaryotypic pattern. Chromosome mapping of 5S rDNA showed a total of eight sites located in four distinct chromosomal pairs, with no apparent differences among populations. A comparative study on 18S rDNA locations and Ag-NORs showed some secondary NOR sites that are not usually expressed in karyotypes and a probable differential NOR activity among populations. Correlations between these data, environmental conditions and B chromosomes are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Comparative Analysis of ISSR and RAPD Markers for Study of Genetic Diversity in Shisham (<Emphasis Type="Italic">Dalbergia sissoo</Emphasis>)

Shisham (Dalbergia sissoo) is one of the most preferred timber tree species of South Asia. Two DNA-based molecular marker techniques, intersimple sequence repeat (ISSR) and random amplified polymorphism DNA (RAPD), were compared to study the genetic diversity in this species. A total of 30 polymorphic primers (15 ISSR and 15 random) were used. Amplification of genomic DNA of 22 genotypes, using ISSR analysis, yielded 117 fragments, of which 64 were polymorphic. Number of amplified fragments with ISSR primers ranged from five to ten and varied in size from 180 to 1,900 bp. Percentage polymorphism ranged from 0 to 87.5. The 15 RAPD primers produced 144 bands across 22 genotypes, of which 84 were polymorphic. The number of amplified bands varied from five to 13, with size range from 180 to 2,400 bp. Percentage polymorphism ranged from 0 to 100, with an average of 58.3 across. RAPD markers were relatively more efficient than the ISSR assay. The mental test between two Jaccard’s similarity matrices gave r ≥ 0.90, showing very good fit correlation in between ISSR- and RAPD-based similarities. Clustering of isolates remained more or less the same in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.734 to 0.939, 0.563 to 0.946, and 0.648 to 0.920 with ISSR, RAPD, and combined dendrogram, respectively.     

Inheritance of chromosome length polymorphisms inCoprinus cinereus

The sexually compatible strains ofCoprinus cinereus 5302 and Dd 13 revealed chromosome length polymorphisms in their electrophoretic karyotypes. The dikaryon derived from two monokaryons contained a mixture of the two electrophoretic patterns. F₁ progenies were isolated by crossingC. cinereus 5302 and Dd 13 strains and it showed unique karyotypes. Chromosome length polymorphisms of both parental strains were inherited at random in the F₁ progenies. As a result, several novel electrophoretic karyotypes which had not been observed in either parental strains were found in the F₁ progeny. The rDNA probe hybridized with one chromosome in both parental strains, with two chromosomes in the hybridization pattern of both parental strains in the dikaryon, and with one to two chromosomes in the F₁ progenies. The relation between mating type and hybridization pattern has thus not been made clear in the case of F₁ progeny.     

A taxon-specific oligonucleotide probe for temperate zone soil isolates of Glomus mosseae

 The 5.8 S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus mosseae FL156 and UK118 were amplified by polymerase chain reaction (PCR) using ITS1 and ITS4 as primers. The amplification product from template DNA of UK118 was cloned and sequenced (569 bp); the amplified DNA from FL156 was sequenced directly (582 bp). There was a 95% sequence similarity between DNAs amplified from the two isolates; in contrast, major dissimilarities with partial sequences of seven other glomalean taxa were observed. Four oligonucleotide sequences unique to Glomus mosseae were identified as potential primers. Their specificity to Glomus mosseae was assessed by PCR amplification of genomic DNA from spores from 36 glomalean fungi: 13 isolates of Glomus mosseae, two Glomus monosporum, 10 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The Glomus mosseae isolates were from a broad range of temperate zone agricultural soils. Oligonucleotide pair GMOS1 : GMOS2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all Glomus mosseae isolates and two isolates of the closely related Glomus monosporum. This primer pair did not prime PCR when the template consisted of DNA from any of the other glomalean fungi or any of the nonmycorrhizal controls. In addition, a 24-mer oligonucleotide, designated GMOS5, hybridized with Glomus mosseae and Glomus monosporum DNA amplified by PCR using primer pairs ITS1 : ITS4 and GMOS1 : GMOS2. Colony-blot assays showed that GMOS5 hybridized to 100% and 97% of E. coli pUC19 clones of amplification products from Glomus mosseae FL156 and UK118 DNA templates, respectively, indicating that nearly all clones contained an homologous sequence. GMOS5 was used successfully to detect specifically Glomus mosseae in DNA extracted from colonized sudan grass (Sorghum sudanense L.) roots and amplified by PCR using the primer pair GMOS1 : GMOS2. The results confirm several previous indications that Glomus mosseae and Glomus monosporum are indistinguishable taxonomic entities. Accepted: 14 February 1998     

Comparative molecular analysis of <Emphasis Type="Italic">Fusarium solani</Emphasis> isolates by RFLP and RAPD

Fusarium solani is an important pathogen causing wilt disease of guava in India. In this work, we analyzed seven representative isolates of F. solani, collected from different places of India, by restriction fragment length polymorphism (RPLP) using HindIII or DraI restriction endonucleases and random amplified polymorphic DNA (RAPD). Pattern of restriction enzyme revealed a similar restriction cut type cluster in the isolate namely, Allahabad (isolate-3), Faizabad (isolate-4), Unnao (isolate-5) and Lucknow (isolate-6) region, while other cluster was consist of isolate from Ranchi (isolate-2) and Ludhiana (isolate-7). Slightly variable results were obtained when 10 randomly amplified polymorphic DNA markers (OPA01–OPA10) tested in the genome of Fusarium solani and grouped on basis of obtained allelic data. RAPD fingerprinting showed a higher variability than RFLP, and each isolate had a unique electrophoretic pattern with five of the ten primers used. Our results show that RAPD much efficient to distinguish between all F. solani isolate tested.     

Specific PCR Assays for the Detection and Quantification of DNA from the Biocontrol Strain Trichoderma harzianum 2413 in Soil

Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68°C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.     

(T2AG3)n Telomeric Sequence Hybridization Suggestive of Centric Fusion in Karyotype Marsupials Evolution

It has been suggested that the karyotype of the marsupials derived from a low diploid number (2n = 14) which originated, through fissions of biarmed chromosomes, the karyotypes with a higher 2n. The telomeric sequence (T₂AG₃)_nwas in situhybridized to the chromosomes of Gracilinanus microtarsusand G. emiliae, Micoureus demeraraeand Marmosa murina, species with 2n = 14, in Monodelphissp., M. domestica, M. kunsiand M. brevicaudatawith 2n = 18, and in Lutreolina crassicaudata, Didelphis albiventris, Chironectes minimus, Philander opossumand P. frenata, all of them with 2n = 22. The probe hybridization occurred in the telomeric regions of both arms, short and long, of all chromosomes of the complement of all individuals of all species analysed. However, in some pairs of the karyotypes of Gracilinanus microtarsusand Micoureus demerarae(with 2n = 14), and in Monodelphissp., M. domestica, M. kunsiand M. brevicaudata(2n = 18) ectopic signs of hybridization were detected proximal to the centromeres, suggesting the retention of this telomeric sequence in the centromeric regions of some chromosomes of these species. Based on these results, it is proposed that the karyotype of marsupials evolved from a 2n = 22 to a 2n = 14, by means of chromosomal fusions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers

 The potential of PCR-based markers for construction of a genetic linkage map in Einkorn wheat was investigated. From a comparison of polymorphisms between two Einkorn wheats, Triticum monococcum (Mn) and T. boeoticum (Bt), we obtained 49 polymorphic bands produced by 33 primers for inter-simple sequence repeat (ISSR) and 36 polymorphic bands shown by 25 combinations of random amplified polymorphic DNA (RAPD) primers for mapping in 66 individuals in the F₂ population. Although 44 ISSR fragments and 29 RAPD fragments statistically showed a 3 : 1 segregation ratio in the F₂ population, only 9 markers each of the ISSR and RAPD bands were able to be mapped on the RFLP linkage map of Einkorn wheat. ISSR markers were distributed throughout the chromosomes. The mapped positions of the ISSR markers seemed to be similar to those obtained by the RFLP markers. On the other hand, 4 of the 9 RAPD markers could map the RFLP marker-poor region on the short arm of 3A^m, suggesting a potential to map novel regions containing repetitive sequences. Comparisons of the genetic linkage map of Einkorn wheat to the linkage map and cytological map of common wheat revealed that the marker orders between the two maps of Einkorn wheat and common wheat coincided except for 4A, which harbors chromosome rearrangements specific for polyploid wheats, indicating a conservatism between the two genomes. Recombinations in Einkorn wheat chromosomes took place more frequently around the centromere and less at the distal part of chromosomes in comparison to those in common wheat. Nevertheless, recombinations even in Einkorn wheat chromosomes were strongly suppressed around the centromere. In fact, the markers located within 1 cM of the centromere were located almost in the central part of the chromosome arm. Received: 7 June 1997 / Accepted: 17 June 1997     

Electrophoretic karyotypes and chromosome numbers in Candida species

The electrophoretic karyotypes of five Candida albicans isolates and of five other Candida species have been determined, using orthogonal field alternating gel electrophoresis (OFAGE). None of the C. albicans isolates had the same electrophoretic karyotype. By comparing all five strains, we arrived at a chromosome number of nine to ten, but since the organism is diploid, we cannot distinguish genetically different chromosomes from homologues which resolve. We determined minimal chromosome numbers of 9 for Candida stellatoidea, 10 for C. glabrata and 6 for C. guilliermondii.     

A doubled haploid rye linkage map with a QTL affecting α-amylase activity

A rye doubled haploid (DH) mapping population (Amilo × Voima) segregating for pre-harvest sprouting (PHS) was generated through anther culture of F₁ plants. A linkage map was constructed using DHs, to our knowledge, for the first time in rye. The map was composed of 289 loci: amplified fragment length polymorphism (AFLP), microsatellite, random amplified polymorphic DNA (RAPD), retrotransposon-microsatellite amplified polymorphism (REMAP), inter-retrotransposon amplified polymorphism (IRAP), inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers, and extended altogether 732 cM (one locus in every 2.5 cM). All of the seven rye chromosomes and four unplaced groups were formed. Distorted segregation of markers (P ≤ 0.05) was detected on all chromosomes. One major quantitative trait locus (QTL) affecting α-amylase activity was found, which explained 16.1% of phenotypic variation. The QTL was localized on the long arm of chromosome 5R. Microsatellites SCM74, RMS1115, and SCM77, nearest to the QTL, can be used for marker-assisted selection as a part of a rye breeding program to decrease sprouting damage.     

Analysis of Genetic Diversity of Fusarium oxysporum f. sp. phaseoli Isolates, Pathogenic and Non-pathogenic to Common Bean (Phaseolus vulgaris L.)

Twenty isolates of Fusarium oxysporum from Brazil, pathogenic and non‐pathogenic to common bean, were analysed using random amplified polymorphic DNA (RAPDs) to study the genetic diversity. RAPD analysis using 23 oligonucleotides resulted in the amplification of 229 polymorphic and 7 monomorphic DNA fragments ranging from 234 to 2590 bp. High genetic variability was observed among the isolates, with the distances varying between 8% and 76% among pathogenic, 2% and 63% among the non‐pathogenic and 45% and 76% between pathogenic and non‐pathogenic isolates. The analysis of genetic distance data showed that the pathogenic isolates tended to group in one group and the non‐pathogenic in another. The genetic distance values of 30% among the pathogenic isolates in cluster A are compatible with the genetic distance values observed within the physiological races, but the distance values among the pathogenic isolates in clusters B and G are not compatible with the distance values observed within the race. Although our results are preliminary, it was not possible to exclude the existence of more than one race of this fungus in Brazil.     

RAPD polymorphism of wild emmer wheat populations, Triticum dicoccoides, in Israel

 Genetic diversity in random amplified polymorphic DNAs (RAPDs) was studied in 110 genotypes of the tetraploid wild progenitor of wheat, Triticum dicoccoides, from 11 populations sampled in Israel and Turkey. Our results show high level of diversity of RAPD markers in wild wheat populations in Israel. The ten primers used in this study amplified 59 scorable RAPD loci of which 48 (81.4%) were polymorphic and 11 monomorphic. RAPD analysis was found to be highly effective in distinguishing genotypes of T. dicoccoides originating from diverse ecogeographical sites in Israel and Turkey, with 95.5% of the 100 genotypes correctly classified into sites of origin by discriminant analysis based on RAPD genotyping. However, interpopulation genetic distances showed no association with geographic distance between the population sites of origin, negating a simple isolation by distance model. Spatial autocorrelation of RAPD frequencies suggests that migration is not influential. Our present RAPD results are non-random and in agreement with the previously obtained allozyme patterns, although the genetic diversity values obtained with RAPDs are much higher than the allozyme values. Significant correlates of RAPD markers with various climatic and soil factors suggest that, as in the case of allozymes, natural selection causes adaptive RAPD ecogeographical differentiation. The results obtained suggest that RAPD markers are useful for the estimation of genetic diversity in wild material of T. dicoccoides and the identification of suitable parents for the development of mapping populations for the tagging of agronomically important traits derived from T. dicoccoides. Received: 13 July 1998 / Accepted: 13 August 1998     

Production of β-Xylosidase Activity by Trichodermaharzianum Strains

Nine Trichoderma harzianum strains were screened for β-xylosidase activity when grown in solid-state cultures on media containing wheat bran as the carbon source. All strains produced β-xylosidase activity, the most active being in extracts of cultures of T. harzianum strain 4. A β-xylosidase was purified by ammonium sulfate precipitation, ultrafiltration, gel filtration, and ion exchange chromatography from solid-state cultures of T. harzianum strain C. Enzyme preparations yielded a single band when stained for protein following eletrophoresis. The molecular weight value, calculated following SDS-PAGE, was determined to be 60 kDa. β-Xylosidase was most active at pH 4.0–4.5 and 70°C. This enzyme had a K _m value of 0.053 mM. The phenol-sulfuric acid method detected the presence of a small amount of carbohydrate in the purified enzyme preparation. β-Xylosidase was active against some p-nitrophenylglycosides. The enzyme was inactive against xylan and PNPG. β-xylosidase activity was inhibited by xylose and SDS. Iodoacetamide, dithiothreitol, gluconolactone, glucose, and mercuric chloride failed to inactivate this enzyme's activity. A synergistic effect was observed when β-xylosidase from T. harzianum strain C and β-xylanase from Aspergillus fumigatus were incubated with pretreated arabinoxylan. Received: 1 December 1995 / Accepted: 11 December 1995     

Electrophoretic karyotyping of the entomogenous fungus Paecilomyces fumosoroseus

Pulsed-field gel electrophoresis was used to compare the electrophoretic karyotypes of isolates of Paecilomyces fumosoroseus. Electrophoretic karyotypes of P. fumosoroseus exhibit a high degree of similarity among the isolates. However, hybridization data indicated that similar sized chromosomes among the isolates did not always bear the same genetic information.