Molecular characterization of Rhizoctonia solani isolates the incitant of soybean root rot

Rhizoctonia solani isolates used in this investigation were identified as anastomosis-4 (AG-40), collected from different localities from Assiut governorate in Egypt. Pathogenicity test of seven isolates of R. solani was evaluated on soybean Giza 111 cultivar under greenhouse conditions. All tested isolates were able to infect soybean plants causing root rot with different degrees of severities, isolate No. 1, 2 and 3 showed significantly highest root rot severity, while isolate No. 5 gave the lowest percentage of root rot rating. The sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns were used to compare three isolates of R. solani. There are no variations among R. solani isolates except a few exceptions according to their protein patterns. DNA markers obtained from all isolates showed genetic similarity among different isolates obtained from different geographical regions barring few exceptions. Correlation between DNA patterns of R. solani isolates and their virulence was detected, but no correlation with anastomosis groups (AG).     

Molecular Epidemiology of Vibrio cholerae O1 Isolated from Sporadic Cholera Cases in Okinawa,Japan

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V. cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme-labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed-field gel electrophoresis of the chromosomal DNA digested with NotI restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V. cholerae O1 isolated during this period.     

Factors influencing changes in ploidy and nuclear DNA levels in cells from normal,crown-gall and habituated cultures ofHelianthus annuus L.

Summary Normal and crown-gall tissue cultures ofHelianthus annuus were initiated from diploid cells of the stem and diploid tumour cells respectively. For the first 16 months both normal and tumour isolates remained predominantly diploid, but subsequently many isolates showed varying degrees of polyploidy. It was concluded that cultures fromH. annuus were initially stable, but that this stability was lost during prolonged culture. There was no evidence that tumour or habituated isolates had any greater tendency to become polyploid than normal isolates grown in similar conditions. Furthermore normal isolates initiated and maintained on media supplemented with different auxins (2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, 3-indoleacetic acid and 4-chloro-2-oxobenzothiazolin-3-yl acetic acid) remained chiefly diploid during the first 13 months in culture, but subsequently became polyploid. In addition, increasing the concentration of kinetin in the medium had no obvious effects on the occurrence of polyploid nuclei.     

Phytophthora infestans in Poland from 1987–1989; Nuclear DNA Content, Mating Type Distribution and Response to Metalaxyl

Isolates of Phytophthora infestans were collected from all potato growing regions of Poland during the blight seasons of 1987—1989. All 1987 isolates were of Al mating type and were sensitive to metalaxyl. In 1988 and 1989, 46.5 % and 55.3 % of the isolates were sensitive to metalaxyl, respectively. The percentage of highly resistant (R) isolates increased from 25.6 % in 1988 to 39.5 % in 1989; however the percentage of intermediately resistant (I) isolates decreased during that period from 27.9 % to 5.3 %. A significant association was observed between the A1 compatibility type and metalaxyl resistance. The A2 mating type first appeared in 1988, and its frequency increased from 4.7 % of the population in 1988 to 47.6 % in 1989. Coincident with this change in mating type frequency, changes in ploidy levels of isolates were observed. Whereas 3 % of the 1988 isolates were diploid, 90 % of the 1989 A2 isolates and 28.6 % of the 1989 Al isolates were diploid. The approximate 1:1 ratio of the two mating types encountered in 1989, and the predominance of diploidy, indicates that the Polish population of P. infestans has the potential to become sexual.     

NUCLEAR DNA CONTENT OF MYXAMOEBAE AND PLASMODIA IN SIX NON-HETEROTHALLIC ISOLATES OF A MYXOMYCETE,DIDYMIUM IRIDIS

The nuclear DNA content of six non-heterothallic isolates of the myxomycete Didymium iridis was measured by combining the Feulgen reaction with absorption microspectrophotometry. This allowed us to distinguish between homothallic (sexual) and apogamic (non-sexual) isolates. Four of the isolates studied, Panamanian 4 and 5, California 1, and Missouri 1 are homothallic. Moreover, the average DNA content of the myxamoebal and plasmodial nuclei (0.32 and 0.61 respectively) does not differ significantly from the calculated haploid and diploid values for heterothallic isolates of D. iridis (0.34 and 0.63). Hence, it is concluded that in each of these isolates the myxamoebae are haploid and the plasmodia diploid. In two of the isolates investigated, Georgia 1 and Hawaii 1, the DNA content of the myxamoebal and plasmodial nuclei did not differ significantly. Therefore, in both of these isolates the plasmodia appear to develop apogamically. In addition the mean DNA values recorded for the Ha-1 isolate suggest that it is aneuploid.     

Distribution,Disease Level and Virulence Variation of Bremia lactucae on Lactuca sativa in the Czech Republic in the Period 1999–2011

We report the distribution and disease level of downy mildew on lettuce (Lactuca sativa) and virulence variation in populations of its causal agent (Bremia lactucae) in the Czech Republic during the period 1999–2011. Disease incidence was not high; among a total of 92 different localities surveyed, 43.4% of them were infected by lettuce downy mildew at least once during the whole period. However, among individual years, differences were found in disease incidence that ranged from 4.8% (2009) to 66.7% (2004). A total of 128 isolates of B. lactucae collected from infected leaf samples in 35 different localities during the surveying period were included in the virulence analysis. Virulence was examined on a set of 19 differential genotypes of Lactuca sativa and Lactuca serriola (EU‐A test set). Isolates exhibited quite a broad variation in virulence to individual Lactuca differential genotypes. Eighteen of 19 virulence factors (v‐factors) tested were present in the samples. The most frequently detected factors were v1–4, v5/8, v6, v7, v10–14, v16, v36 and v38; factor v17 was not found. The most pronounced temporal shift was recorded for factors v36 and v38 whose frequency increased during the studied period. V‐factors 15, 17, 18 and 37 were present in low frequencies in a pathogen population, and their corresponding gene (Dm15) or resistance factors (R17, R18 and R37) may have the best potential for resistance breeding in the Czech Republic. Broad diversity of v‐phenotypes (63 different ones) was identified during the study period. The numbers of v‐factors per v‐phenotype (resp. isolate) varied within a range of 5–15. Within the 128 analysed isolates, only 9 v‐phenotypes were recorded repeatedly (three or more times). Possible reasons of recorded virulence variation are discussed.     

The structure and interrelationship of fungal populations in native and cultivated soils

Mitochondrial DNA haplotypes have been characterized for 120 isolates of the asexual fungus Fusarium oxysporum. Sixty of these isolates were obtained from soil in a native grassland in the San Joaquin Valley of California, including 20 isolates from each of six different sampling locations. The same sampling strategy was used to obtain 60 additional isolates from an agricultural field of the same soil type directly adjacent to the native soil. Twenty-three different mitochondrial DNA haplotypes were identified among the 120 isolates, including 11 haplotypes represented by two or more isolates and 12 that were unique. The five most common mitochondrial DNA haplotypes accounted for 93 (78%) of the 120 isolates. Isolates representing each of these five mitochondrial DNA haplotypes were found both in the cultivated and in the native soil. Seventy-two per cent of the isolates found in the cultivated soil were associated with the same mitochondrial DNA haplotype as one or more isolates in the native soil. The remaining isolates in the cultivated soil were associated with comparatively rare mitochondrial DNA haplotypes, most of which showed a close relationship to one of the haplotypes found in the native soil. Hierarchial gene diversity analysis indicated that a significant proportion of the mitochondrial DNA haplotype diversity was attributable to differences between sampling sites in the native soil but not in the cultivated soil. This may reflect significant spatial structuring of genetic diversity in populations of F. oxysporum in a native soil. The proportion of mtDNA haplotype diversity attributable to differences between populations in the native and cultivated soils was not significant. This suggests that our entire collection, encompassing strains from both native and cultivated soils, is representative of a single population of F. oxysporum.     

Simple Method to Accurately Differentiate <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates Concurrently Using Polymorphic Patterns of PCR-Amplified,Species-Specific Nuclear and Mitochondrial Targets

We describe a simple method to accurately differentiate Candida albicans isolates by concurrent use of the restriction enzyme digestion patterns for PCR products, targeting two species-specific DNA regions originating from genetically different sources, the nuclear and mitochondrial genomes. The target sequence we used as the nuclear gene was derived from the PHO85 gene, a negative regulator of the PHO system, in which we found a restriction size polymorphism within the two alleles of PHO85 in the diploid genome of this fungus. The mitochondrial target was derived from EO3, a species-specific DNA fragment possessing a small size polymorphism among various clinical isolates. Our results should provide a new tool for molecular epidemiological surveys of patients suffering from candidiasis caused by C. albicans.     

Mitotic recombination in the absence of synaptonemal complexes in Saccharomyces cerevisiae.

Summary Mitotic cells of a diploid strain of Saccharomyces cerevisiae with appropriate markers for the detection of mitotic crossing-over and mitotic gene conversion were irradiated with X-rays. Induction of these recombinational events was strong. After irradiation, cells were incubated in a rich growth medium and samples were removed for studying the possible formation of synaptonemal complexes up to a time when most cells had completed the first post-irradiation cell division. No complexes were found during the entire period of sampling, during which mitotic recombination in G₁ (mitotic gene conversion), DNA replication and G₂ (mitotic crossing-over) had occurred. These results are interpreted to mean that synaptonemal complexes are not required for mitotic recombination.     

Variation of Fungicide Resistance in Czech Populations of Pseudoperonospora cubensis

During the growing seasons between the years 2001 and 2004, 98 isolates of Pseudoperonospora cubensis from nine regions of Czech Republic were collected and screened for tolerance/resistance to the three frequently used fungicides (propamocarb, fosetyl‐Al, metalaxyl). Fungicides were tested in five different concentrations, using a floating disc bioassay. Fungicide effectiveness varied considerably. Propamocarb appeared most effective and all the isolates collected in the years 2001–2003 were found sensitive to all tested concentrations [607–9712 μg active ingredient (a.i.)/ml]. In 2004, some strains with increased resistance to propamocarb were detected. These strains were characterized by tolerance at the lowest concentrations (607 μg a.i./ml, eventually on 1214 μg a.i./ml); however, they were controlled by 2428 μg a.i./ml. Fosetyl‐Al was effective at the recommended concentration of 1600 μg a.i./ml against all isolates. However, the occurrence of isolates (collected in 2001) which sporulated at low concentrations (400 and 800 μg a.i./ml) indicated that the selection for tolerance occurs in the pathogen population. Nevertheless, this phenomenon was not confirmed with the P. cubensis isolates collected between the years 2002 and 2004. Metalaxyl was found ineffective, because 97% of the isolates showed the resistance to the recommended concentration (200 μg a.i./ml), and the other 3% of isolates expressed tolerant response. The majority of the isolates showed profuse and/or limited sporulation at higher concentrations (400 and 800 μg a.i./ml). A substantial shift to highly metalaxyl resistant strains was evident in the Czech P. cubensis populations during 2001–2004.     

COMPARATIVE MEASUREMENTS OF NUCLEAR DNA IN A HETEROTHALLIC AND A SELF-FERTILE ISOLATE OF THE MYXOMYCETE,DIDYMIUM IRIDIS

Comparative measurements were made of the nuclear Feulgen-DNA content of a heterothallic and a self-fertile isolate of the myxomycete Didymium iridis. Plasmodial nuclei of both isolates contain the diploid amount of DNA. The replicated diploid (4C) values for the heterothallic and the self-fertile isolates are 5.66 and 5.95, respectively. Myxamoebae, however, are quite dissimilar in their nuclear DNA content. Those of the heterothallic isolates, Honduran 1–2 (A¹) and Panamanian 2–4 (A⁷), have mean values of 3.81 and 3.69, whereas myxamoebae of the self-fertile Philippine-1 isolate were found to have a mean value of 6.07. Myxamoebae of the Ph-1 isolate are, therefore, at the same ploidy level as the Ph-1 Plasmodium. Mean DNA values for Ph-1 sporangial nuclei were in category 4C. Measurement of the DNA content of mitotic metaphases in sporangia at T = 6 hr confirmed that the mean DNA content of both Ph-1 myxamoebae and plasmodial nuclei is equivalent to 4C. It is concluded that nuclear phase alternance is lacking in the Ph-1 isolate and that the Plasmodium of this isolate develops by apogamy.     

Parasexuality in Race 65 Colletotrichum lindemuthianum Isolates

ABSTRACT. Heterokaryosis is the initial step of the parasexual cycle, a process that provides genetic variability in filamentous fungi through the production of heterozygous diploid nuclei. To characterize the parasexual cycle in Colletotrichum lindemuthianum, we evaluated the presence of heterokaryosis, vegetative compatibility reactions, and diploid formation among isolates of Race 65 collected from different Brazilian states. Vegetative compatibility groups were identified among the isolates according to their ability to form heterokaryons. Two heterozygous diploids were selected from compatible heterokaryons, which were characterized by the segregation of the parental auxotrophic markers and by RAPD profiles.     

The amounts of nuclear DNA and the duration of DNA synthetic period (S) in related diploid and autotetraploid species of oats

The relative amounts of nuclear DNA of root meristematic cells of two related diploid Avena species, A. strigosa 2x and A. pilosa, which have different karyotypes, and an autotetraploid of one, A. strigosa 4x, were measured by Feulgen microspectrophotometry. The durations of various periods of their mitotic cycles were studied by autoradiography of cells pulse-labeled with tritiated thymidine. The results show that the autotetraploid, with twice the amount of nuclear DNA of its diploid, has the same duration of S period as the diploid, while A. pilosa, with intermediate nuclear DNA content, has a longer S period. These results support the hypothesis that homologous chromosomes or genomes require similar duration for their DNA synthesis and suggest that the structures of chromosomes are involved in temporal control of the DNA synthesis in cells.     

Genetic diversity of the ectomycorrhizal fungus Pisolithus tinctorius based on RAPD-PCR analysis

 Twenty Pisolithus tinctorius isolates from different geographic locations and different hosts were characterized by the random amplified polymorphic DNA technique. Thirteen arbitrary primers generated 87 DNA fragments, all of them polymorphic. These data were used to calculate genetic distances among the isolates. The pairwise genetic distances ranged from 1 to 100%, with an average of 58.7%. Cluster analysis based on the amplified fragments grouped the isolates according to their host and geographical origins. Group I contained isolates collected in Brazil and group II those collected in the Northern Hemisphere. In addition to the diversity seen at the molecular level, the isolates also showed host specificity. Greenhouse experiments demonstrated that isolates from the Northern Hemisphere colonized mainly Pinus whereas isolates from Brazil colonized only Eucalyptus. The molecular data suggest that the Pisolithus tinctorius isolates analyzed belong to two distinct groups. The data also suggest new guidelines for future investigations on the taxonomy and systematic of this important fungus species. Furthermore, these results support future experiments aimed at the selection and development of improved isolates of P. tinctorius. Accepted: 3 October 1997     

Notes on the life histories of Boergesenia and Valonia (Siphonocladales,Chlorophyta)

Life history studies were carried out with isolates of Boergesenia forbesii (from Western Australia), Valonia fastigiata (Hawaii) and V. utricularis (Canary Islands, Mediterranean Sea) cultivated under laboratory conditions. Ploidy levels of nuclei were identified by micro spectrophotometric DNA content measurements after Feulgen staining. Fundamentally, the life history in both genera is diplohaplontic. Autodiploidization phenomena have been observed favoring the diploid generation. In contrast to other genera of the Siphonocladales s. str. producing biflagellate diploid zoospores (e.g. Ernodesmis, Boodlea), Valonia develops haploid and diploid zoospores with four flagella. No diploid zoospores were observed in Boergesenia.     

Population structure of Magnaporthe oryzae isolates from green foxtail in Japan examined by DNA fingerprint analysis

The population structure of Magnaporthe oryzae from green foxtail (Setaria viridis) in Japan was examined by DNA fingerprint analyses using the transposable elements MGR586 and MAGGY as probes. Fifteen M. oryzae isolates from green foxtail were collected from 11 Japanese prefectures so that a macrogeographic population of this pathogen is represented. All the 15 isolates were sorted into distinct haplotypes by DNA fingerprint analyses with both probes. Furthermore, similarities between the DNA fingerprint profiles of the 15 isolates were exclusively low; i.e., if lineages are arbitrarily established based on greater than 70% similarities in isolates, the 15 isolates could be categorized into 13 distinct lineages by DNA fingerprinting with both probes. We also examined the MGR586 DNA fingerprint variations of this pathogen in 9 microgeographic populations each of which contained 20 to 24 isolates collected from a 1 m² or 50 m² area. In all the 9 populations, more than 2 haplotypes, which shared less than 70% similarities, were identified in the DNA fingerprint profiles. These results suggested that M. oryzae isolates from the green foxtail in Japan possessed a complex lineage structure, even at the microgeographic scale.     

Identification of airborne propagules of the Gibberella fujikuroi species complex during maize production

The airborne dispersal of the anamorphs of the Gibberella fujikuroi species complex was studied under pre- and postharvest maize (corn) production conditions using a 3-stage Andersen sampler. The aim of this study was to identify and analyse the size distribution of such species in air samples. Differences were observed between the concentration of large- and small-sized propagules (identified as aggregates and single microconidia, respectively), but the difference was only significant during a high concentration period (October 2007, P = 0.009). No correlation was found between the concentration of fusaria found at different sampling heights (10 and 150 cm above ground level). Fusarium isolates were collected and identified based on morphological characters and using species-specific PCR assays. The PCR analysis confirmed morphological identification of F. verticillioides, F. proliferatum and F. subglutinans. High concentrations were found during the maize harvest, loading and corn shelling. Our results showed that the monitoring of F. verticillioides should be performed at a single sampling height.     

Frequent occurrence of multidrug‐resistant CC17 Enterococcus faecium among clinical isolates in Sweden

Aims: To screen for the globally spread cluster of Enterococcus faecium, clonal complex 17 (CC17) and characterize the genetic profile of Swedish clinical Ent. faecium isolates. Methods: A total of 203 consecutive isolates collected from 2004 to 2007 from patients with bacteraemia in Sweden. All isolates were genotyped using multiple‐locus variable‐number tandem repeat analysis (MLVA) and 20 isolates representing different MLVA types (MT) were chosen for multilocus sequence typing (MLST). Minimal inhibitory concentrations against clinically relevant antibiotics were determined with agar dilution. Presence of the virulence genes esp and hyl was investigated using PCR. Results: A total of 65% (n = 109) of all isolates belonged to MT‐1, and the second most common MLVA type was MT‐159 (13%, n = 21). MLST analysis confirmed the presence of CC17 during the entire study period. The number of isolates resistant to gentamicin and vancomycin, as well as the presence of hyl, increased significantly during the investigation period. Conclusions: The present study demonstrates that nosocomial infections caused by Ent. faecium CC17 are commonly occurring in Sweden. Significance and Impact of the Study: This is the first report of CC17 Ent. faecium in Sweden. The increase of antibiotic resistance and virulence indicates that these strains are further adapting to the hospital environment.     

Variations in body length, weight, fat content and survival inCoccinella septempunctata at different hibernation sites

Adults ofCoccinella septempunctata L. (Coleoptera; Coccinellidae) were sampled in four overwintering sites: one in England and three at different altitudes in the Czech Republic (350–1420 m above sea level) in autumn 1992 and spring 1993. Body length, dry weight and fat weight were measured. There were significant differences in body length between sampling sites in spring 1993, but not in autumn 1992. The average sex ratio (♀) in all samples was 1∶0.60 in 1992 and 1∶0.72 in 1993. Females were significantly larger and heavier, and contained more fat than males in both sampling periods. Dry weight and fat weight were positively correlated to body length at all sampling sites except at Ruzyne, in the Czech Republic. Adults hibernating at higher altitudes and away from their breeding and feeding habitats had significantly more fat than those hibernating at lower altitudes. Fat reserves were reduced by 30% during the hibernation at the top of the Krkonose mountain, in the Czech Republic, where temperatures were much lower, but more than half of the fat reserves were consumed during the overwintering period at the other sampling sites. The significance of body length and fat reserves in relation to adult hibernation sites and overwintering survival is discussed.     

Molecular detection of <Emphasis Type="Italic">Entoloma</Emphasis> spp. associated with roots of rosaceous woody plants

Seventeen isolates of Entoloma clypeatum, a fungus associated with rosaceous woody plants and suspected to act as a root pathogen, were obtained from fruit-bodies collected at 7 localities in the Czech Republic. The fungus grew best on the medium supplied with cellobiose. The growth of E. clypeatum was inhibited by fungicide preparation Ridomil Gold MZ 68WP. A part of the LSU rRNA gene of 11 selected isolates was sequenced and two reverse primers recognizing the sequence motifs in the LSU rRNA gene of E. clypeatum and other related species associated with rosaceous woody plants were designed. One of these primers, NL4EC2, showed high specificity towards DNA of Entoloma spp. of interest and may be used for detection of these organisms in soil, in roots or in the substrate where the fruit trees are cultivated. The results of in vitro inoculation experiment suggest a resistance of roots of young Prunus domestica plants to colonization by Entoloma clypeatum, and the fungus could be detected only in the surrounding substrate. We were able to detect Entoloma spp. in soil and in roots of Prunus avium but only if the fructification of the fungus occurred at the locality. The method is robust towards the false positive detection caused by nonspecific amplification of DNA of other soil microorganisms.