Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches

Molecular approaches including internal transcribed spacer (ITS) sequences of ribosomal DNA, universal primer polymerase chain reaction (UP-PCR) fingerprinting, and DNA-DNA hybridization were used to study the genetic relatedness of species within Trichoderma sect. Pachybasium. In the analysis of ITS and 5.8S sequences of ribosomal DNA, parsimony analysis demonstrated that forty-one strains were distributed into five main groups supported by high bootstrap values. The species of Trichoderma sect. Pachybasium were clustered into groups I, II, and IV, with the strains of Trichoderma fasculatum and Trichoderma strictipile forming a separate branch, an independent group V. Some species within each group showed nearly identical sequence differences (fewer than 1-3 bp). UP-PCR and DNA-DNA hybridization were further used to clarify the genetic relatedness of these species with highly similar ITS sequences. Highly similar or identical UP-PCR profiles and high values of DNA complementarity (>70%) were observed among some species, Trichoderma hamatum and Trichoderma pubescens; Trichoderma croceum, Trichoderma polysporum and Trichoderma album, Trichoderma crassum and Trichoderma flavofuscum; and Trichoderma strictipile and Trichoderma fasciculatum. Although every species can be differentiated morphologically, the species showed highly similar molecular characteristics in the above cases, indicating that they could be conspecific. However, in some cases (Trithoderma longipile, T. crassum and T. flavofuscum; Trichoderma fertile and Trichoderma minutisporum; Trichoderma tomentosum, Trichoderma inhamatum and Trichoderma harzianum) there were discriminative patterns of UP-PCR and (or) low levels (<50%) of DNA-DNA hybridization; even their ITS sequences were similar, suggesting a closely phylogenetic relationship.     

Characterization of Trichoderma Isolates from Philippine Rice Fields by UP-PCR and rDNA-ITS1 Analysis: Identification of UP-PCR Markers

Forty-two isolates of Trichoderma from rice fields in four provinces in the Philippines were characterized using rDNA-ITS1 analysis and universally primed polymerase chain reaction (UP-PCR). Two groups were clearly distinguishable on the basis of length and restriction pattern of the internal transcribed spacer (ITS) region of the ribosomal DNA and UP-PCR banding profiles using UP primer, L45. The 40 isolates comprising the largest group were very similar with respect to their UP-PCR banding profiles and were assigned to Trichoderma harzianum Rifai following morphological identification of four of the isolates. The two isolates belonging to the second group were identified as Trichodermaviride Pers. ex. Gray on the basis of their morphology, rDNA-ITS1 analysis and distinct UP-PCR banding profiles. One of the T. harzianum isolates with good cellulolytic and competitive saprophytic abilities was analysed using single and pair-wise combinations of UP primers in order to distinguish it from the remaining 41 isolates. A suitable diagnostic marker was identified and this marker will be valuable for monitoring the isolate in field tests.     

Differentiation of the yeasts Williopsis, Zygowilliopsis and Komagataea by karyotypic and PCR analyses

We used polymerase chain reaction with universal and microsatellite primers, and molecular karyotyping to evaluate the extent of divergence between the genomes of the yeasts currently assigned to the heterogeneous genus Williopsis. Pulsed-field gel electrophoresis of chromosomal DNAs indicates that Zygowilliopsis californica, Komagataea pratensis, Williopsis mucosa, Williopsis salicorniae species and Williopsis sensu stricto complex have clearly different karyotypes. In contrast, the latter six species, Williopsis saturnus, W. beijerinckii, W mrakii, W. suaveolens, W. subsufficiens and W. sargentensis, show similar banding patterns and practically cannot be differentiated on the basis of their karyotypes. The data revealed that a PCR method employing the universal primer N21 is appropriate for the distinction of Williopsis, Zygowilliopsis and Komagataea yeasts. Unique fingerprints were generated with this primer for all 10 species studied while strains of the same species showed nearly identical profiles. The data of UP-PCR are in good agreement with genetic classification and provide support for the species status of the yeasts composing the Williopsis sensu stricto complex. Microsatellite primer (GTG)5 allowing molecular typing of individual strains of the same species may be useful for investigating population structure of the saturn-spored yeasts.     

Williopsis saturnus and Williopsis beijerinckii-different taxons from polymerase chain reaction data with nonspecific primers]

Fifteen strains of the yeast Williopsis sensu stricto were analyzed by means of UP-PCR. With the N21 universal primer, this approach showed that the strains could be clearly divided into two groups corresponding to the species W. saturnus (Kl?cker) Zender and W. beijerinckii (van der Walt) Naumov et Vustin. The results obtained are in good agreement with data of genetic and isoenzyme analyses and provide no support for the conspecificity of W. saturnus and W. beijerinckii commonly accepted in modern determination manuals.     

Monitoring of Biocontrol Agents Based on Trichoderma Strains Following Their Application to Glasshouse Crops by Combining Dilution Plating with UP-PCR Fingerprinting

Universally primed (UP) PCR analysis was used to characterize strains of Trichoderma spp., which constitute the active ingredients of commercial products for biocontrol of phytopathogens. Several UP primers were able to generate distinct and reproducible fingerprints for each strain, allowing them to be differentiated from a collection of other Trichoderma spp. strains. In order to test whether the UP-PCR method in combination with dilution plating could be useful to detect and enumerate propagules of biocontrol strains of Trichoderma spp. when applied on a commercial scale, sampling was carried out in three commercial glasshouse operations. Dilution plating was carried out using a semi-selective medium and a number of the Trichoderma spp. recovered were subjected to UP-PCR analysis with one selected UP primer. The results showed that the method could be used to identify the isolates from the biocontrol products applied in the different glasshouses. Furthermore, it was found that a biocontrol strain also colonized an untreated bench in one of the glasshouses. The combined methods enable verification of the active ingredient concentration, facilitating monitoring establishment and spread of the Trichoderma strains.     

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of Clonostachys rosea and Development of a Strain-Specific PCR Detection Assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

UP-PCR Genotyping and rDNA Analysis of Ascochyta pisi Lib

Isolates of Ascochyta pisi, previously identified as separate pathotypes, were genotyped by rDNA-RFLP and UP-PCR using 8 UP-primers and 2 arbitrary primers individually or in pair-wise combinations. The appearance of polymorphic UP-PCR products for all studied isolates facilitated their differentiation. The markers were suitable in identifying isolates, and may be useful for developing isolate- or pathotype-specific PCR-based diagnostic assays. In studying genetic relatedness by UP-PCR among A. pisi isolates, two distinct clusters were revealed. rDNA analysis of the isolates based on endo-ntjclease digestion of amplified ITSl and 1TS2-25S nuclear rDNA regions did not separate the isolates, whereas the variabihty of the isolates using UP-PCR was more than 40%.     

Assessment of genetic variation in timothy (Phleum pratense L.) using RAPD and UP-PCR

DNA-based fingerprinting technologies including random amplified polymorphic DNA (RAPD) and universally primed PCR (UP-PCR), a novel method for studying genetic variation, were employed as genetic markers for assessing genetic diversity and relationships in timothy (Phleum pratense L.). This study sought to identify the genetic background of the genotypes used in timothy breeding. Thirty eight genotypes from fifteen countries were used as test materials. RAPD and UP-PCR dendrograms based on 132 (from 3 primers) and 44 highly reproducible bands, respectively, were analyzed. The electrophoretic gels showed that the PCR products were informative and polymorphic. Different geographic genotype groups were distinguished according to the combined RADP and UP-PCR results. The results demonstrate that methods based on molecular fingerprinting can be used for timothy identification.     

Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization

To study the role of Trichoderma in sick building syndrome, it is essential to be able to accurately identify species. Forty-four strains of Trichoderma spp. isolated from Danish buildings damaged by water leaks were identified using ITS1 ribotyping and universally primed PCR, UP-PCR. Ribotyping allowed the assignment of the strains into three distinct groups. High similarity of UP-PCR banding profiles of the strains allowed species designation for almost all strains (43 out of 44) when compared with the UP-PCR banding profiles obtained from reference strains of T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum and T. viride. However, cross hybridization of UP-PCR products showed that the latter strain had high DNA homology to the ex-type strain of T. hamatum. The combined approach is a convenient way for reliable identification of Trichoderma strains.     

Williopsis saturnusand Williopsis beijerinckiiAre Recognized as Distinct Taxa by Means of the Polymerase Chain Reaction with Nonspecific Primers

Fifteen strains of the yeast Williopsissensu stricto were analyzed by means of UP-PCR. With the N21 universal primer, this approach showed that the strains could be clearly divided into two groups corresponding to the species W. saturnus(Klöcker) Zender and W. beijerinckii(van der Walt) Naumov et Vustin. The results obtained are in good agreement with data of genetic and isoenzyme analyses and provide no support for the conspecificity of W. saturnusand W. beijerinckiicommonly accepted in modern yeast taxonomic manuals.     

Characterization of Xanthomonas oryzae pv. oryzae recX, a gene that is required for high-level expression of recA

A universally primed (UP)-PCR cross hybridization assay was developed for rapid identification of isolates of Rhizoctonia solani into the correct anastomosis group (AG). Twenty-one AG tester isolates belonging to 11 AGs of R. solani were amplified with a single UP primer which generated multiple PCR fragments for each isolate. The amplified products were spotted onto a filter, immobilized and used for cross hybridization against amplification products from the different isolates. Isolates within AG subgroups cross hybridize strongly, whereas between different AGs little or no cross hybridization occurs. Sixteen Rhizoctonia isolates from diseased sugar beets and potatoes were identified using the assay. The results were supported by restriction fragment length polymorphism analysis of the ITS1-5.8S-ITS2 region of the nuclear encoded ribosomal DNA. Through standardization and use of quick non-radioactive labeling techniques, the UP-PCR cross hybridization assay has potential for routine use by modern DNA chip technology.     

Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards

Genetic diversity of 50 isolates of Neofusicoccum parvum, the predominant species of the Botryosphaeriaceae recovered from grapevines displaying symptoms of dieback and decline in New Zealand, was compared to that of isolates from Australia, South Africa, and California. The eight universally primed polymerase chain reaction (UP-PCR) primers distinguished 56 genotypes, with only four clonal pairs found. Seven main groups were identified in a neighbour-joining (NJ) tree with isolates from different regions and vineyards of New Zealand, Australia, and California distributed in different groups, indicating a high level of intra and intervineyard genetic variation. All of the South African isolates were positioned in a separate UP-PCR group, indicating that these isolates were less related to the other N. parvum isolates. When compared to fungi that reproduce sexually the genetic diversity and Shannon diversity indices were low (0.076-0.249; 0.109-0.367, respectively), genetic identity levels were high (0.76-0.95), and genetic distance levels were low (0.04-0.27). The large number of genotypes and the low number of clones in the New Zealand N. parvum populations may be explained by parasexual recombination as anastomosis was observed between nonself pairings. Pathogenicity tests using isolates from different UP-PCR groups inoculated onto either green shoots or 1-y-old grapevines detected virulence diversity, indicating intra and intervineyard variation between isolates, however, no correlation was detected between UP-PCR group and virulence.     

Intraspecific variability and exopolysaccharide production inAureobasidium pullulans

Forty seven strains of the black yeasts,Aureobasidium pullulans andHormonema dematioides, and the type strain ofHormonema macrosporum were examined using PCR-ribotyping and universally primed PCR with subsequent hybridization. Four groups (populations) were distinguished withinA. pullulans with PCR-ribotyping, which largely coincided with UP-PCR/hybridization groups. The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied. Five strains identified asHormonema dematioides on the basis of physiological and morphological data formed a group recognizable with PCR-ribotyping and UP-PCR/hybridization, which also includedH. macrosporum. Aureobasidium pullulans is characterized by the absence of RsaI restriction sites in rDNA amplified with primers 5.8S-R and LR7, whileHormonema species possessed several bands after RsaI digestion. For analysis of distance between populations, PCR-ribotyping with AluI and MspI is sufficient. Strains ofA. pullulans produce exopolysaccharides in liquid media with different nitrogen sources, while the strains ofHormonema synthesize minor amounts of polysaccharides in media with peptone. Populations ofA. pullulans differ slightly from each other in their optimal, medium-dependent production of polysaccharides.     

Identification of Zygowillopsis californica strains of various origin by polymerase chain reaction with universal primers]

After reevaluation of the taxonomic position of 27 yeast collection strains of different origin by UP-PCR followed by dot-hybridization, only 22 strains were assigned to the biological species Zygowilliopsis californica (Lodder) Kudriavzev. Four strains were identified as Williopsis suaveolens (Kl?cker) Naumov et al. Universal primers L45 and N21 are recommended for identification of the Z. californica yeasts.     

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.     

瓜类保护地土壤镰孢菌种群及UP-PCR多样性分析

对采自辽宁省部分地区瓜类保护地的36份土壤样品进行镰孢菌（Fusarium）分离培养,共获得112株镰孢菌,采用传统形态学分类和现代分子生物学方法,确定属于11个种.对其中25株镰孢菌及3株对照镰孢菌进行了通用引物PCR（UP-PCR）多样性分析.结果表明：6条引物扩增出73条带,其中多态性条带66条,占总条带数的90.4%.对供试菌株进行UP-PCR聚类图谱分析,当相似系数为0.736时,可将其划分为8个类群,其中14株尖孢镰孢菌聚为一类.UP-PCR分析体现了镰孢菌菌株间的亲缘关系及差异性,可以作为镰孢菌分类的辅助方法.     

Comparison of Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Aquatic Bacterial Communities: an Ecological Perspective

Two primer sets for automated ribosomal intergenic spacer analysis (ARISA) were used to assess the bacterial community composition (BCC) in Lake Mendota, Wisconsin, over 3 years. Correspondence analysis revealed differences in community profiles generated by different primer sets, but overall ecological patterns were conserved in each case. ARISA is a powerful tool for evaluating BCC change through space and time, regardless of the specific primer set used.     

A specific primer pair for the diagnosis and identification of Acanthamoeba astronyxis by random amplified polymorphic DNA-polymerase chain reaction

Random amplified polymorphic DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that is useful for species identification. In this study, a distinctive 485-bp band in Acanthamoeba astronyxis band patterns was found, using the OPC20 primer (ACTTCGCCAC). The band specificity was confirmed by hybridization, using it as a probe, against all OPC20 amplifications from different Acanthamoeba species. Once the fragment was sequenced, we used it to design a specific primer pair that was useful for the identification of different isolates as A. astronyxis species.