Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Abstract Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases Eco RI or Hae III. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus . Digestion with Eco RI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae . Digestion of genomic and mitochondrial DNA with Hae III showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium .     

Presence of a 20 bp insertion/deletion in the ITS1 region of Verticillium lecanii

Polymerase chain reaction (PCR) amplification of the complete ribosomal RNA internally transcribed spacer (ITS) region of 36 isolates of Verticillium lecanii and related species gave a single 620 bp product in 31 isolates. Five isolates received as V. lecanii, however, gave a single product of 600 bp. Restriction fragment analysis of the PCR products from all isolates gave consistent patterns for the 31 isolates with a 620 bp product. The five isolates with the 600 bp product showed only minor discrepancies to these, generally related to the size of only one restriction fragment. The total ITS region was sequenced from 10 typical 620 bp isolates and one 600 bp isolate. Sequence variation between the isolates varied from 0 to 14.5%, and the 20 bp size discrepancy was found to relate to an insertion or deletion in the centre of the ITS1 region.     

Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility

A diverse collection of isolates identified as Colletotrichum acutatum, including a range of fruit-rot and foliar pathogens, was examined for mtDNA RFLPs and RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase (GS) gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene. RFLPs of mtDNA, RFLPs of the 900-bp GS intron and sequence analysis of each intron identified the same seven distinct molecular groups, or clades, within C. acutatum sensu lato. Sequence analysis produced highly concordant tree topologies with definitive phylogenetic relationships within and between the clades. The clades might represent phylogenetically distinct species within C. acutatum sensu lato. Mating tests also were conducted to assess sexual compatibility with tester isolates known to outcross to form the teleomorph Glomerella acutata. Mating compatibility was identified within one clade, C, and between two phylogenetically distinct clades, C and J4. The C clade represented isolates from a wide range of hosts and geographic origins. J4 clade contained isolates from Australia or New Zealand recovered from fruit rot and pine seedlings with terminal crook disease. That isolates in two phylogenetically distinct clades were capable of mating suggests that genetic isolation occurred before reproductive isolation. No other isolates were sexually compatible with the mating testers, which also were in groups C and J4. Certain clades identified by mtDNA and intron analysis (D1, J3 and J6) appeared to represent relatively host-limited populations. Other clades (C1, F1 and J4) contained isolates from a wide range of hosts. Isolates described as C. acutatum f. sp. pineum were clearly polyphyletic.     

Selection of Verticillium lecanii Isolates with High Potential for Biocontrol of Cucumber Powdery Mildew by Means of Components Analysis at Different Humidity Regimes

To identify characteristics for the selection of Verticillium lecanii isolates with high potential for biocontrol of Sphaerotheca fuliginea under glasshouse conditions, an exploratory study was performed on the effect of water limitation on the development of 14 isolates. The conidial germination, growth and sporulation of isolates of V. lecanii were studied in a tritrophic system on cucumber leaves and in a ditrophic system in Petri dishes. Their mycoparasitic ability was studied in S. fuliginea and Cladosporium cladosporioides . All characteristics were clearly affected by humidity. Four isolates showed good biocontrol potential. The performance of isolates on agar had less predictive value than on powdery mildew. The germination of isolates of V. lecanii was lower and the mycelial growth faster on agar than on mildewed leaves under corresponding humidity conditions. The results suggest that conditions in the phyllosphere differed from the set humidity in the surrounding air. A correlation was found between the lysis of C. cladosporioides growing in dual culture on agar with isolates of V. lecanii and the parasitism of powdery mildew on detached, rooted leaves. C. cladosporioides might offer a suitable substrate for testing isolates of V. lecanii for mycoparasitic potential under various environmental conditions. Conidial germination, growth and sporulation had limited predictive value.     

Rapid mitochondrial probes for analysis of polymorphisms in Fusarium oxysporum special forms

A method is described for the production of simple mitochondrial DNA probes from filamentous fungi for the partial characterization of mitochondrial DNA without the need for cloning, gradient centrifugation or PCR amplification. A probe (P449) consisting of a 3·38 kb mitochondrial fragment from an isolate of Fusarium oxysporum special form cubense was used to determine RFLPs in restriction digests of total DNA from 28 isolates of F. oxysporum from a variety of hosts and locations. The probe showed mtDNA polymorphisms within and between different special forms.     

Genetic,morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.     

Improvement of the efficacy of Verticillium lecanii used in biocontrol of Sphaerotheca fuliginea by addition of oil formulations

Biocontrol of cucumber powdery mildew, Sphaerotheca fuliginea, by Verticillium lecanii is seriously hampered at low humidities. The effect is especially marked at low humidity (60% RH) during the three hours following the application of V. lecanii spores suspended in water. Formulations of V. lecanii spores in oil might improve the situation. Arachid oil (peanut oil) and two invert emulsions using either Sunspray 6N or paraffin oil were tested in formulations of V. lecanii spores. Arachid oil gave the best development of V. lecanii on mildewed cucumber leaves. V. lecanii formulated with arachid oil showed significantly better control of mildew than without. A concentration of 0.5% arachid oil was somewhat toxic to mildew but 0.05% was not. Arachid oil did not show toxicity to V. lecanii. The humidity requirements of V. lecanii formulated with and without 0.05% arachid oil were compared at 95, 90 and 85% RH. Arachid oil significantly reduced the humidity dependence of V. lecanii. Since arachid oil is safe for human consumption and not phytotoxic to cucumber leaves, low concentrations of arachid oil are recommended as an additive to increase the effectiveness of V. lecanii as a biocontrol agent of S. fuliginea.     

轮枝菌形态的低温扫描电镜观察

本文报道寄生在昆虫、锈菌、蜘蛛及植物上的7株轮枝菌(Verticillium)和虫草菌(Cordyceps)的轮枝菌无性型的低温扫描电镜观察结果。试验表明该方法可以清晰地观察到保持完好而未变形的轮枝菌头状分生孢子球及分生孢子链,蜡蚧轮枝菌寄生在昆虫与锈菌号的形态具有较大差异。     

Imputing missing yield trial data

Summary Intraspecific mitochondrial DNA (mtDNA) diversity was determined in 23 Phaseolus vulgaris genotypes, and compared to previously observed variability of morphoagronomic characters and isozyme loci. Twenty of the lines were collected from Malawian landraces; the other three were pure-bred cultivars. The mtDNAs were digested with eight restriction endonucleases, revealing complex banding patterns. Southern hybridization using cosmid clones covering about 200-kb of the genome showed a considerable amount of uniformity of the mtDNA banding patterns. However, five restriction fragment length polymorphisms (RFLPs) were detected, dividing the bean lines into two groups corresponding to the previously known Mesoamerican and Andean gene pools of P. vulgaris. The cultivar Mecosta was separated from the rest of the lines by an additional RFLP. At least two out of the six RFLPs are believed to be due to base-pair mutation events. Our results provide the first evidence that the cytoplasms of the two major germ plasm pools of beans are distinct.     

Restriction Fragment Length Polymorphisms of Mitochondrial and Nuclear DNAs among Korean Races of Magnaporthe grisea

Three KJ-races of Magnaporthe grisea (virulent to only japonica type rice cultivars) and seven KI-races (virulent to either indica or japonica type cultivars) isolated from various rice-growing areas of Korea were assayed for restriction fragment length polymorphisms (RFLPs) in mitochondrial and nuclear DNAs of the fungus. The size of mitochondrial DNA of M. grisea was estimated to be approximately 39. 8 kb. No RFLP in the mt DNA was detected among the 10 Korean races, indicating an extensive conservation in the gene composition of mt DNA without sequence variation. The identical restriction patterns of mt DNA also suggest that mt DNA of M. grisea may not be responsible for pathotypic diversity and variability. Southern blot analysis with five single-copy DNA probes showed considerable polymorphisms. Much diversity was detected in the three KI-races predominated during 1978–1985 in Korea. In contrast, no genetic variation was detected between other four KI-races and three KJ-races. RFLPs in nuclear DNA were correlated to some extent with the prevailing races in Korea. However, relationship between RFLPs in nuclear DNA and virulence of M. grisea races was inconclusive.     

Mitochondrial DNA variation in Russian populations of Stavropol krai,Orel and Saratov oblasts

Mitochondrial DNA (mtDNA) polymorphism was examined in three Russian populations from the European part of Russia (Stavropol krai, Orel oblast, and Saratov oblast). This analysis showed that mitochondrial gene pool of Russians was represented by the mtDNA types belonging to haplogroups H, V, HV*, J, T, U, K, I, W, and X. A mongoloid admixture (1.5%) was revealed in the form of mtDNA types of macrohaplogroup M. Comparative analysis of the mtDNA haplogroup frequency distribution patterns in six Russian populations from the European part of Russia indicated the absence of substantial genetic differences between them. However, in Russian populations from the southern and central regions the frequency of haplogroup V (average frequency 8%) was higher than in the populations from more northern regions. Based on the data on mtDNA HVS1 sequence variation, it was shown that the diversity of haplogroup V in Russians (h = 0.72) corresponded to the highest h values observed in Europe. The reasons for genetic differentiation of the Russian population (historical, ecological, and adaptive) are discussed.     

Mating behavior of Phytophthora parasitica: Evidence for sexual recombination in oospores using DNA restriction fragment length polymorphisms as genetic markers

Cloned nuclear DNA fragments that detected restriction fragment length polymorphisms (RFLPs) in homozygous loci of isolates of Phytophthora parasitica were used as genetic markers to investigate sexual recombination during oospore formation. It was found that the majority of the 23 oospore progeny studied in each of the two crosses carried both of the parental markers. However, aberrant recombination patterns were observed; some of the progeny were homozygous at one RFLP locus, whereas at another locus both of the parental markers were present. Only two of the progeny of each cross did not show sexual recombination with any of the four or five RFLP markers used. Mitochondrial DNA (mtDNA) was uniparentally inherited. In both crosses the majority of the progeny carried the mtDNA type of one of the common parental strains.     

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Genetic variants of ribosomal DNA and mitochondrial DNA between swamp and river buffaloes

To clarify the genetic relationship between Swamp and River buffaloes, the restriction fragment length polymorphisms (RFLPs) of nuclear genomic ribosomal DNA (rDNA) and cytoplasmic mitochondrial DNA (mtDNA) were analysed. Blood or liver samples from 73 Swamp and three River buffaloes were collected in East and South-east Asian countries. DNA samples from cattle, goats and sheep were used for comparisons. The analysis of rDNA allowed water buffaloes, cattle, goats and sheep to be characterized by four distinct repeat-types. However, swamp and river buffaloes showed the same repeat-type. Divergence of water buffalo and cattle is considered to have occurred approximately four to six million years ago. The RFLPs for mtDNA divided water buffaloes into three haplotypes, swamp-1, swamp-2 and river types. Swamp-1 accounted for 91% of all swamp buffaloes while swamp-2 was observed only in water buffaloes from Thailand (9%). All river buffaloes were of the same haplotype. No differences were observed between swamp and river buffaloes at the rDNA level. In contrast, a few distinct differences between them were found at the mtDNA level. Therefore, mtDNA polymorphisms provide an adequate means for classifying water buffaloes into either swamp or river buffaloes.     

Mitochondrial DNA Variation in Russian Populations of Stavropol Krai, Orel and Saratov Oblasts

Mitochondrial DNA (mtDNA) polymorphism was examined in three Russian populations from the European part of Russia (Stavropol krai, Orel oblast, and Saratov oblast). This analysis showed that mitochondrial gene pool of Russians was represented by the mtDNA types belonging to haplogroups H, V, HV*, J, T, U, K, I, W, and X. A mongoloid admixture (1.5%) was revealed in the form of mtDNA types of macrohaplogroup M. Comparative analysis of the mtDNA haplogroup frequency distribution patterns in six Russian populations from the European part of Russia indicated the absence of substantial genetic differences between them. However, in Russian populations from the southern and central regions the frequency of haplogroup V (average frequency 8%) was higher than in the populations from more northern regions. Based on the data on mtDNA HVS1 sequence variation, it was shown that the diversity of haplogroup V in Russians (h= 0.742) corresponded to the highest h values observed in Europe. The reasons for genetic differentiation of the Russian population (historical, ecological, and adaptive) are discussed.     

Genetic relationships among Leptographium terebrantis and the mycangial fungi of three western Dendroctonus bark beetles

Morphology, mitochondrial DNA (mtDNA) restriction fragment polymorphisms (RFLPs) and nuclear DNA (nDNA) fingerprinting were used to clarify relationships among the morphologically similar Ophiostoma and Leptographium species associated with mycangia of three Dendroctonus bark beetles (Ophiostoma clavigerum associated with both D. ponderosae and D. jeffreyi, and L. pyrinum associated with D. adjunctus), as well as a closely related nonmycangial bark beetle associate (L. terebrantis). Most isolates of O. clavigerum form long (40-70 μm), septate conidia, while all isolates of L. terebrantis and L. pyrinum form conidia less than 17.0 μm in length. The conidia of L. pyrinum are pyriform, with truncate bases, while the conidia of the other species form only slightly truncate bases. Conidial masses of L. terebrantis are creamy yellow, while the conidial masses of the other species are white. Nuclear DNA fingerprints resulting from probing PstI restrictions with the oligonucleotide probe (CAC)(5) and HaeIII and MspI restrictions of mtDNA, exhibited three major clusters. In the dendrogram developed from mtDNA RFLPs, the L. pyrinum isolates formed one cluster, while the majority of O. clavigerum isolates, including all D. jeffreyi isolates, formed another. A third cluster was composed of all L. terebrantis isolates, as well as several O. clavigerum isolates from D. ponderosae. The inclusion of some O. clavigerum isolates in the L. terebrantis cluster suggests that horizontal transfer of mtDNA has occurred among these fungi. The nDNA dendrogram also exhibited three clusters, and most isolates of L. pyrinum, L. terebrantis and O. clavigerum grouped separately; however, one isolate of O. clavigerum grouped with the L. terebrantis isolates, while one isolate of L. terebrantis grouped with O. clavigerum. No genetic markers were found that distinguished between O. clavigerum associated with D. ponderosae and O. clavigerum associated with D. jeffreyi. Ophiostoma clavigerum might be a recently diverged morphological variant of L. terebrantis, with special adaptations for grazing by young adults of D. jeffreyi and D. ponderosae. The anamorph of O. clavigerum, Graphiocladiella clavigerum, is transferred to Leptographium.     

Analysis of mitochondrial DNA restriction fragment length polymorphisms for examining genetic variability among isolates of Phellinus linteus

 Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNAs (mtDNAs) from nine Japanese wild isolates of Phellinus linteus was carried out to examine their genetic variability. BamHI and EcoRI digests of mtDNAs from these isolates produced four and five distinct RFLP patterns, respectively. By combining the RFLP patterns obtained with the two endonucleases, mtDNAs from the nine isolates could be assigned to five different genotypes, but no mtDNA variation was detected among the isolates collected from a small area. Distance values calculated among all pairs of mtDNA genotypes, based on the presence or absence of comigrating restriction fragments, were clearly smaller than those among the mtDNA genotypes of Lentinula edodes and Pleurotus ostreatus samples collected worldwide, suggesting the necessity of collecting P. linteus wild isolates for genetic resources from geographically wider areas. Received: June 27, 2002 / Accepted: August 19, 2002 Correspondence to:T. Nakamura     

Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice

Summary Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.Florida Agricultural Experiment Station Journal Series No. 8807. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable     

Abundant sequence variation around the mitochondrial origin of replication in the human opportunistic yeast pathogen Candida albicans from a tropical island in China

Due to its haploid nature and its predominantly uniparental mode of inheritance, the mitochondrial genome has been analyzed extensively in population and evolutionary genetic studies of eukaryotes. Among the mitochondrial DNA (mtDNA) fragments, the region surrounding the origin of replication is the most commonly studied. However, most of the studies have focused on animals and little is known about the extent and patterns of sequence variation around the mtDNA origin of replication (mtOri) in fungi. In this study, we found abundant variation in a 597bp fragment surrounding the mtOri for 53 isolates of the pathogenic yeast Candida albicans obtained from a diverse group of hosts in Hainan, a tropical island of China. Within this DNA fragment, a total of 17 haplotypes were found for the 53 isolates. The extent of sequence variation for this group of strains was similar to that for 24 strains that represented the global nuclear genotype diversity. In contrast to those in animals where there were significant biases in favor of transitional mutations (e.g. the transition to transversion ratio is about 20 in human mtDNA), our data showed a transition to transversion ratio of approximately 0.5 around mtOri in C. albicans. Our analysis revealed no apparent geographic pattern of sequence variation based on the birthplaces of the analyzed hosts. However, the sample from patients had a lower genotypic diversity than those from healthy hosts borne either in Hainan or elsewhere in China. Our results suggest that C. albicans mtDNA has a base substitution pattern different from its nuclear genome and that sequences from the mtOri region could enhance our understanding of C. albicans genome evolution and population structuring.