Viability and sporulating capability of Coelomycetes preserved under a range of different storage regimes

The viability and sporulating capability of 45 Coelomycetes strains were evaluated. Strain subcultures were maintained under mineral oil, in soil and on agar slant for different periods of time lasting as long as 50 years, 39 years and 2 years, respectively. Of the 34 strains preserved under mineral oil, 20 maintained their viability but lost the sporulating capability with exception of one strain of Pestalotiopsis guepinii. Of the 16 strains also preserved in soil only one was viable and it was not able to sporulate. All 12 endophytic strains, 11 preserved on agar slant and one under mineral oil remained viable; however, the strain preserved under mineral oil lost its sporulating capability, while the strains on agar slant were only able to sporulate after culturing on sterilized alfalfa twigs. The results demonstrate that routine monitoring, and the use of different preservation methods, specially with the addition of sterilized plant tissue on the culture media for promoting conidiomata formation, is necessary for the success of the Coelomycetes long-term preservation.     

Long term storage of callus cultures at low temperatures or under mineral oil layer

Plant tissue cultures from various species were stored at low temperatures or under mineral oil overlay for 4 to 6 months without subcultures. After transfer to normal culture conditions, it was checked, with 3 strains, that growth characteristics and secondary metabolite production were preserved. The storage with a mineral oil overlay (easy to run and economical method) could be a possible alternative to cryogenic or low temperature storage for a large number of strains.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - TLC Thin layer chromatography - HPLC High performance liquid chromatography - CAS Ceric ammonium sulfate     

Change of Mating Type in an EU1 Lineage Isolate of Phytophthora ramorum

All Phytophthora ramorum EU1 lineage isolates tested are of A1 mating type, except for three rare isolates from 2002 to 2003 from Belgium, which were originally assigned the A2 mating type. In one of these isolates (2338), a switch from A2 to A1 mating type was observed in 2006. This observation initiated a larger study in which all cultures and subcultures of the original three EU1 A2 isolates, maintained in three laboratories under different storage conditions, were checked for mating type change. The A2 to A1 mating type switch was observed in four of seven independently maintained isolates that were derived from isolate 2338 in two laboratories, using different transfer regimes and storage conditions. Following the mating type switch to A1 in these four derived isolates, no reversion back to A2 mating was observed, even after up to 5 years of additional isolate maintenance and several more subculturing events. The three other isolates that were derived from isolate 2338 as well as the other EU1 A2 isolates collected in 2002 and 2003 and stored in the same conditions did not display such mating type change. The potential causes of the mating type conversions as well as their epidemiological implications are discussed.     

Storage procedures for yeast preservation: phenotypic and genotypic evaluation

The aim of this study was to evaluate the morphological and biochemical characteristics of yeasts during storage. SixCandida spp. standard strains were stored in agarized medium with mineral oil in distilled water, frozen at ?70°C and freeze dried. Strains were phenotypically characterised before being stored and then periodically for up to 18 months. Randomly Amplified Polymorphic DNA (RAPD) was carried out in time zero, 6, 12 and 18 months of storage. The viability of all samples was preserved except for the strain ofCandida dubliniensis after 12 months of storage with mineral oil. No phenotypic alterations were observed in any of the methods employed. However, variations were observed in some phospholipase or proteinase activities. Changes in the RAPD patterns were not detected. These results seem to indicate that the maintenance methods tested were able to preserve the stability of the yeast phenotypic and genotypic characteristics.     

Viability, morphological characteristics and dimorphic ability of fungi preserved by different methods

The viability, morphological characteristics and dimorphic ability of fungi were evaluated. Strain subcultures were maintained under mineral oil and in soil for different periods of time, ranging from 49 to eight years. Of the 16 Blastomyces dermatitidis strains, four maintained viability and were able to complete the dimorphic process to the M phase producing a large amount of conidia, but were unable to form Y cells at 36 degrees C. Of the 15 Histoplasma capsulatum var. capsulatum strains, only one was viable but it was impossible to check its identity because it lost sporulating and dimorphic ability. Of the 53 Sporothrix schenckii strains, 37 were viable, 28 able to sporulate and 12 of them completed the whole M <=> Y dimorphic process. All subcultures in soil became inviable. The results demonstrate that the preservation methods used here affected the morphology and sporulating and dimorphic ability of the strains. B. dermatitidis and S. schenckii were considered to be species that survive better than H. capsulatum var. capsulatum under mineral oil. Thus, it is necessary to establish routine monitoring and appropriate environmental and culture conditions, using less widely spaced transplants and choosing the exact time of intervention to induce growth and development restriction in each strain.     

Differences between subcultures of the Mesorhizobium loti type strain from different culture collections

Because of differences in the reported 16S rRNA gene sequence of the Mesorhizobium loti type strain available from different culture collections, we collected different subcultures of this strain and compared them by 16S rDNA sequencing, SDS-PAGE of whole-cell protein extracts and RAPD-PCR. Our results indicate that the 16S rDNA sequence differences can be explained by the presence of two different organisms in one of the subcultures. In addition, even for subcultures of the type strain that had identical 16S rDNA sequences, small differences could be observed in the protein profiles and in the RAPD-PCR patterns. These latter observations indicate that maintenance procedures necessary for long-term preservation by freeze-drying can cause subcultures of the same original strain to undergo changes, effectively leading to different fingerprints even though 16S rDNA sequences remain identical.     

Fruit-body production of test cultures ofFlammulina velutipes preserved for seven years by freezing at three different temperatures

Two strains ofFlammulina velutipes were cultured on PDA plates, and mycelial disks punched out using a cork borer were used for preservation. Five disks of a strain were put into a vial containing one of three cryoprotectants, 10% glycerol, 5% DMSO or 10% polyethylene glycol. Vials were then stored for 7 yr at −20°C, −85°C or liquid nitrogen temperature. The mycelial growth on PDA plates of the cryopreserved mycelial disks, as well as the usual subcultures, were tested two times. After the second test, spawns were prepared for fruit-body production tests by bottle cultivation from selected plates of the second growth tests. The yields of fruit-bodies varied among the cultures derived from the mycelial disks of the same strain preserved under different conditions. Variation in yields was observed even among the mycelial disks preserved at liquid nitrogen temperature, although the range of yield variation was narrower. The yield variation was obvious for the cultures which showed large retardation in the growth test. Four mycelial disks out of the six preserved at −20°C showed higher yields than those preserved at other temperatures. Among the cultures derived from strain FMC224, the control cultures preserved by subculture showed the lowest yield.     

Induced azygospore formation in Mucor (Rhizomucor) pusillus by Absidia corymbifera

Attempts to determine the mating reaction type of heterothallic strains of Mucor pusillus in interspecific contrasts with Mucor strains of known mating reaction type were unsuccessful. Contrasts with Absidia corymbifera strains resulted in the production of azygospores in Mucor pusillus.     

Morphological, Biochemical and Molecular Approaches for Comparing Typical and Atypical Paracoccidioides brasiliensis Strains

We evaluated the morphology of typical and atypical Paracoccidioides brasiliensis strains and the expression of its 43 kDa glycoprotein (GP43). Strains of P. brasiliensis preserved under mineral oil for long periods of time presented different morphological patterns on peptone, yeast-extract and glucose (PYG) agar. The intravenous inoculation in BALB/c mice confirmed that a strain bearing morphological alterations was non-virulent. In contrast, another strain also maintained under mineral oil but which did not exhibit such morphological dysfunction was as virulent as the well characterized Pb 339 and Pb 18 strains. The expression of the main antigen expressed by P. brasiliensis, GP43, was assessed in culture filtrates by western immunoblots. Typical and atypical strains were capable of secreting the glycoprotein, except strain Pb IOC 1059. The identity of the atypical strains was confirmed by PCR using specific primers for gp43, though the single PCR-fragment varied in size for the atypical strains. The PCR fragments from an atypical strain, Pb IOC 1210, and the typical Pb 339 and Pb IOC 3698 strains were sequenced and blasted to the gp43 gene from the Pb 18 strain (GenBank AY005429). These results ensured the identity of the atypical strains as P. brasiliensis, and suggested a relationship between the alteration of morphological differentiation and the virulence factor following storage under mineral oil.     

Phenotypic expression of primary lesions of genetic material in Saccharomyces yeasts

"Illegitimate" mating of yeasts (alpha x alpha), either spontaneous or induced by uv light or ethyl methanesulfanate, in a selective system for "cytoduction" revealed that about 95% of cytoductants expressed their original (alpha) mating type. Inducing the mating by treating the recipient of cytoplasm with uv light reached two orders of magnitude. An additional copy of MAT alpha in the alpha recipient almost completely eliminated the effect, which means that nonheritable mating type changes observed are formally recessive and are localized within MAT alpha complex. About 1% of cytoductants obtained were nonmating types and some of them were identified as mat alpha l mutants. Radl8 mutant as a recipient showed a considerably elevated spontaneous frequency of illegitimate hybridization and cytoduction. The cytoductants also preserved the original mating type. These facts suggest that nonheritable changes of mating type are due to repairable primary (premutational) lesions in MAT alpha genetic material. The significance of these results for understanding the mechanism of nonheritable variability is discussed.     

Stability of the immunogenic properties of plague vaccine strain EV, Research Institute of Epidemiology and Hygiene line, during long-term storage]

The experiments in guinea pigs showed that the immunogenic properties of plague vaccine strain EV, line NIIEG, freeze-dried in 1947 and stored under vacuum without animalization, remained unchanged for 30 years. The subcultures prepared from this train showed, after three passages in guinea pigs, good immunogenic properties which preserved for 6--10 years (the term of observation). After 30-years storage the stock culture of strain EV, line NIIEG, can be used for the preparation of NIIS live plague vaccine.     

Mating type idiomorphs from a French population of the wheat pathogen Mycosphaerella graminicola: widespread equal distribution and low but distinct levels of molecular polymorphism

Septoria tritici blotch caused by the heterothallic ascomycete Mycosphaerella graminicola is currently the most frequent and the most economically damaging disease on wheat worldwide. Five hundred and ten strains of this fungus were sampled from 16 geographical locations representing the major wheat producing areas in France. Multiplex PCR amplification, PCR-RFLP-SSCP screening and sequencing of parts of mating type encoding sequences were performed in order to assess the distribution and molecular polymorphism of the mating type idiomorphs. The two idiomorphs were scored at similar frequencies within all sampled locations. Both mating types were also identified at the leaf spatial scale, on 42% of leaves from which two or three strains were isolated. No correlation was found between distribution of mating types and either host cultivars from which the sampling was carried out or in vitro colony phenotypes observed during the culture of strains on potato dextrose agar (PDA) medium. PCR-RFLP-SSCP assay highlighted only one MAT1-1 strain exhibiting a profile distinct from all other MAT1-1 strains, whereas ten MAT1-2 strains (among which two and four with same profiles, respectively) showed profiles differing from the other MAT1-2 strains. Sequencing revealed that all polymorphisms corresponded to single nucleotide variations and all strains displaying the same single strand conformation polymorphism (SSCP) profiles showed identical nucleotide sequences, thereby confirming the high sensitivity of SSCP. Only two out of the disclosed nucleotide variations were nonsynonymous. This study strongly suggests a large potential for sexual reproduction in the French population of M. graminicola and reports a high conservation of mating type sequences in the fungus at both nucleotide and population levels, with a great difference in molecular variability between the two idiomorphs.     

Variants of Sporothrix schenckii with attenuated virulence for mice

Strains of Sporothrix schenckii preserved under mineral oil were examined for virulence in BALB/c mice. The mice were inoculated with S. schenckii conidia and development of cutaneous lesions, signs of inactivity, weight loss, survival rates, number of viable yeast cells in lung and spleen, splenomegaly and organ lesions were evaluated. After intravenous injection of 7.5 x 10(6) conidia, two of five S. schenckii strains were unable to induce systemic disease and to kill the mice, only producing cord-like lesions on the tail that regressed with mouse maturation. Very small numbers of viable cells isolated from the spleen confirmed the lower invasive ability of these strains when compared with other strains studied here. These results suggest a relationship between the attenuation of virulence and the storage method under mineral oil after long periods of time.     

Mating compatibility and competitiveness of transgenic and wild type Aedes aegypti (L.) under contained semi-field conditions

We conducted the world??s first experiments under semi-field conditions (ACL-2 field house) to assess the mating competitiveness of genetically sterile RIDL male mosquitoes (513A strain). The field house is a state-of-the-art, fully-contained trial facility, simulating the living space for a household of 2?C4 people in Peninsular Malaysia. Ten genetically sterile RIDL male A. aegypti mosquitoes competed with ten wild type males inside this field house to mate with ten wild type females. Hatched larvae from mated females were screened under a fluorescent microscope for genetic markers to determine if they were fathered by RIDL male or wild type male, and all results were cross-checked by PCR. Two such experiments were conducted, each repeated sufficient number of times. All strains were on a Malaysian lab strain background for the first experiment, while the RIDL males alone were on a recently-colonised Mexican strain background for the second experiment. A total of 52 % of the matings were with RIDL males in the first experiment, while 45 % of the matings were with RIDL (Mexican) males in the second experiment. Statistically, this is not significantly different from 50 % of the matings expected to take place with RIDL males if the latter were as competitive as that of the wild type males. This shows that A. aegypti RIDL-513A has excellent mating competitiveness under semi-field conditions, verifying earlier trends obtained in small lab cages. We also observed high mating compatibility between recently-colonised Mexican RIDL males and lab-reared Malaysian wild type females.     

Directed Replacement of Mt a by Mt a-1 Effects a Mating Type Switch in Neurospora Crassa

To test the functions of a mating type genes, we developed an efficient strategy to select transformants of Neurospora crassa in which resident A mating type DNA was replaced by cloned DNA from the mt a idiomorph. Cloned a idiomorphic DNA could specify all functions, including fertility, of a mating type, but only when it replaced A DNA at the mating type locus. Only the mt a-1 region of the a idiomorph was necessary in order to specify a mating type. Gene replacement events involved the homologous sequences flanking the unique mating type idiomorphic DNA, resulting in apparently isogenic a and A strains. These isogenic strains were fertile when crossed with one another, indicating that no determinants outside the transforming DNA are necessary for fertility as a and that no host sequences of A strains interfere with fertility as a. One a replacement strain bore a duplication of the transforming mt a-1 and hph DNA. The duplication strain had unexpected properties. Although mating type segregated 1:1 in crosses of this strain to A, the duplicated regions were efficiently altered during the sexual process to generate a single copy in the progeny. No progeny were recovered that had undergone RIP (repeat induced point mutation) sufficient to inactivate the mt a-1 gene. We infer that the mt a-1 gene is necessary and sufficient to specify a mating type identity in all vegetative and sexual activities. Mt a-1 may also play an essential role in ascosporogenesis after fertilization.     

Intra-specific variation in female remating in Callosobruchus chinensis and C. maculatus

The effects of mating duration on female remating (exp. 1) and under different male densities (exp. 2) were examined in two strains of the adzuki bean beetle, Callosobruchus chinensis and in one strain of the bruchid beetle, C. maculatus. In experiment 1, the frequency of female remating was markedly different between the two strains of C. chinensis. Females of the jC strain, reared long-term in the laboratory, did not remate after being allowed to mate freely (=monogamy), whereas females of the isC strain, recently established from the field, showed high remating frequencies (=polyandry). In both strains, the frequency of female remating increased after the duration of the first mating was deliberately shortened. The relation between mating duration and remating frequency was significantly different, however, between the two strains. In a closely related species, C. maculatus, which manifests polyandry, this relation was more similar to that of the field-derived (=isC) than to that of the laboratory-derived (=jC) strain of C. chinensis. The reasons for the inter-strain variation observed in the remating frequencies of C. chinensis are also discussed. In experiment 2, the mating duration of the three strains was compared under different male densities. Only the lab-derived strain demonstrated a significantly shorter mating duration when one female was placed together with five males than when paired with one male. The shorter mating duration (approximately 26 s) was similar to that of females allowed to remate in the monogamous strain in experiment 1.     

Strain-dependent variation in ribosomal DNA arrangement in Absidia glauca

Restriction analysis of total DNA from the zygomycete Absidia glauca reveals a pattern of prominent bands on a homogeneous background. By Southern blot analysis with 32P-end-labelled ribosomal RNA most of these bands could unequivocally be identified as repetitive copies of ribosomal DNA. There are marked differences in restriction patterns of rDNA between all seven strains tested, even of strains belonging to mating type pairs, presumably isolated from the same location. By using purified rRNAs as probes in hybridization experiments, evidence is presented that 5S rRNA is part of the ribosomal repetitive unit. A more detailed analysis of one strain pair [A. glauca CBS 100.48 (+)/101.48 (-)] provided evidence that the (+) strain, in addition to one rDNA repeat unit common to both strains, contains a second one, derived from the common form by a small deletion.     

表型与亲本不易区分的茯苓同核体菌株新类型

茯苓Wolfiporia hoelen是我国传统中药材之一,也是一种食药兼用的大型真菌,目前已规模化栽培,但由于其交配系统一直不明确,影响了种质改良。前期我们发现了茯苓的同核体,明确了茯苓的交配系统和生活史,并建立了以培养特性和分子标记区分同核体的方法,但未明确是否适用于茯苓种群的不同个体。在对多个菌株的研究中,发现了同核体表型与亲本不易区分的茯苓菌株。本研究主要以来自日本的菌株775 (NBRC 30628)为亲本,对其同核体菌株进行收集鉴定,并对同核体菌株的培养特性、交配现象和杂交等进行了研究。此类菌株的同核体菌株可通过与亲本对峙培养进行鉴定,但菌丝生长、菌落形态和吃料速度等与亲本没有显著差异,不同交配型的同核体之间交配时没有明显的交配现象。rpb2杂合位点标记可以用于鉴定该类型同核体菌株,且能验证是否交配。该类型同核体与之前发现的同核体类型之间可以进行杂交,杂交菌株可与两亲本都产生拮抗现象。该发现补充了之前建立的茯苓同核体鉴定方法,加深了对茯苓物种群体的了解,同时丰富了茯苓的育种资源。     

Inoculation experimental animals withParacoccidioides brasiliensis strains: An attempt to reestablish the dimorphic process and variation in pathogenicity as a function of time of preservation under mineral oil

In an attempt to reestablish the dimorphic process in strains ofParacoccidioides brasiliensis in the transition phase (Y ⇄ M) and to reisolate them, five strains in the transitional phase due to the long time of preservation under mineral oil and two strains in the yeast-like phase were inoculated into male albino rats. The animals were then studied for the presence of paracoccidioidomycotic granulomata. Of the seven strains inoculated, five caused granulomatous nodules in several organs of the animals and only two of these five strains, which had been preserved for the shortest period of time (9 years) were reisolated in culture. Two strains were unable to provoke infection, with no lesions detected in any organ. It is assumed that the long period of time during which the strains were left under oil favored the alteration of celt wall contents, leading to differences in pathogenicity.