Genetic analysis has long been restricted to a few fungi, especially those that could be easily grown on simple media in the laboratory. The characteristics of the genome in terms of the number of chromosomes and the microscopic morphology of each of these are called the karyotype. In conventional gel electrophoresis DNA molecules smaller than 50 kb can be separated by size. Larger DNA molecules have a minimal velocity that is proportional to their length and therefore separated by size. The patterns produced are highly polymorphic, allowing discrimination between isolates of one species if sufficient primers are screened. The internal transcribed spacer region as well as the intergenic spacer of the nuclear rDNA repeat evolve much faster, and sequence differences in these regions occur between species within one genus or even between different populations of one species.

Genetic analysis of the asexual fungus Aspergillus niger is based on genetic recombination in somatic cells and requires proper genetic markers. Transformation in Aspergillus results from integration of the transforming DNA sequences in the genome of the recipient. Recombination of homologous transforming DNA may be at the homologous site either by single crossing over, by double crossing over, or by gene conversion. In special cases it may be useful to start transformation with a diploid strain constructed from two haploid strains together carrying markers for each chromosome. In this way time-consuming isolation of heterozygous diploids from each individual transform ant strain with a tester is avoided. Vectors with a heterologous marker may integrate at different sites in the genome, and multicopy transformants usually carry the plasmids as a single insert. An insert with a heterologous marker can be used as marker in genetic mapping and for the localization of genes lacking a detectable phenotype.

Bacterial-fungal interactions are widespread in nature and there is a growing number of studies reporting distinct fungus-associated bacteria. However, little is known so far about how shifts in the fungus-associated bacteriome will affect the fungal host's lifestyle. In the present study, we describe for the first time the bacterial community associated with the saprotrophic fungus Mucor hiemalis, commonly found in soil and rhizosphere. Two broad-spectrum antibiotics that strongly altered the bacterial community associated with the fungus were applied. Our results revealed that the antibiotic treatment did not significantly reduce the amount of bacteria associated to the fungus but rather changed the community composition by shifting from initially dominating Alpha-Proteobacteria to dominance of Gamma-Proteobacteria. A novel approach was applied for the isolation of fungal-associated bacteria which also revealed differences between bacterial isolates obtained from the original and the antibiotic-treated M. hiemalis. The shift in the composition of the fungal-associated bacterial community led to significantly reduced fungal growth, changes in fungal morphology, behavior and secondary-metabolites production. Furthermore, our results showed that the antibiotic-treated isolate was more attractive and susceptible to mycophagous bacteria as compared to the original isolate. Overall, our study highlights the importance of the fungus-associated bacteriome for the host's lifestyle and interactions and indicate that isolation with antibacterials is not sufficient to eradicate the associated bacteria.

Filamentous fungi are of great industrial value due to their ability to excrete large amounts of enzymes. In this study we investigate the possibility of improving starch degrading glucoamylase by experimental evolution. By allowing 80 parallel lines of A. niger to acquire mutations over 240 generations we were able to select for mutants with higher fitness on starch medium. In addition, we screened 30 natural isolates from a worldwide collection on starch use efficiency, providing insights into new starting material for strain improvement. The black Aspergilli, with model organism Aspergillus niger in particular (Kwon et al., 2012; Meyer et al., 2011), are filamentous fungi with a long history of use as industrial enzyme producers. The capacity to excrete extracellular enzymes in large amounts, ranging up to 20 grams per liter of medium (Finkelstein, 1987), combined with the ability to express a vast diversity in primary and secondary metabolites such as proteins and organic acids makes this fungus a useful cell factory in many branches of industry. Its tolerance to diverse growing conditions raises possibilities for culturing in broad ranges of pH, salinity and temperature. In addition to this, A. niger is able to degrade low-cost and abundant substrates such as starch, cellulose and pectin. The most abundant of the extracellular enzymes secreted by A. niger is glucoamylase (E.C. 3.2.1.3, glucan 1,4α-glucosidase, γamylase) which can make up more than 50% of excretome in liquid culture (Lu et al., 2010). Glucoamylase is one of the most commonly used enzymes in the food industry (Reilly, 1999) as it is useful in conjunction with αamylase by its ability to almost fully convert starch into free glucose by hydrolyzing both α1,4 and α-1,6 linkages (Kumar and Satyanarayana, 2009). Interest in strain improvement for glucoamylase production has historically been high and many methods have been successfully used to increase glucoamylase production. In the past strategies such as random mutagenesis (Suresh et al., 1999) and directed evolution (Wang et al., 2006) have been used to improve glucoamylase overall yield or specific desirable properties such as increased thermostability. More recently A. niger has become a model species for many industrially relevant fungi (Kwon et al., 2012) as the public availability of ten genomes of the most important Aspergilli since 2010 have opened up new possibilities for research. Information is a crucial bottleneck in directed strain improvement. Despite the recent advances that were made, the most commonly used methods share a common dependence on detailed information about the processes that need to be improved: recombination requires genetic markers, whereas the effect of inserting multiple gene copies via genetic engineering depends on the specific location of insertion and gene copy number. Directed evolution, although very effective in selecting single beneficial mutations in a gene is restricted by the effects of epistasis (Suresh et al., 1999; Wang et al., 2006) and its application is usually limited to the protein coding part of a single gene (Dalby, 2003). Recent transcriptomic and metabolomic research (Lu et al., 2010) has provided novel insights into glucoamylase metabolism and can reveal potential targets for directed strain improvement. Furthermore, selection systems based on markers such as hph, ble, oliC3 (antibiotic resistance); pyrG, pyrE, argB, adeA, adeB, niaD, trpC, sC (auxotrophic); and amdS, ptrA (nutritional) have been developed (Jin et al., 2004; Meyer et al., 2011), which in combination with recent versatile vectors offer good and flexible control for genetic manipulation of A. niger strains. These novel genetic engineering strategies for expression of heterologous proteins by A. niger have been recently reviewed (Fleißner and Dersch,

For the genetic dissection of sexual sporulation in Aspergillus nidulans, we started a collection of ascosporeless mutants. After mutagenization of conidiospores with high doses of UV, we isolated 20 mutants with defects in ascospore formation. We crossed these mutants in two successive rounds with the wild-type strain. Eighteen of the 20 isolated mutants produced progeny with the original mutant phenotype in these crosses, and these mutants were further analyzed. All 18 analyzed mutations were recessive to wild type. We assigned them to 15 complementation groups, based on crosses between mutants. The mutants could be classified as follows according to their cytological phenotype: (1) no croziers, (2) arrest at prekaryogamy, (3) arrest in early meiotic prophase, (4) arrest in late meiotic prophase, (5) arrest in meiotic metaphase I, (6) defective postmeiotic mitosis and/or deliniation of ascospores, and (7) slow progression through the postmeiotic stages of ascospore formation. A large proportion of the mutants, namely 11 of 18, arrested in meiotic prophase or metaphase I. We discuss a possible approach for isolating the wild-type alleles of the genes that carry the sexual sporulation mutations.

The intraspecific variability of theAspergillus viridinutans species was examined using various techniques including morphological examinations, carbon-source-utilization tests, restriction-enzyme analysis of the mitochondrial and nuclear DNA, and sequence analysis of part of the β-tubulin gene. Although the genetic distances between the isolates were higher than between,e.g., A. fumigatus andNeosartorya fischeri, mostA. viridinutans isolates, together withN. aureola andN. udagawae strains, were found to belong to a single cluster based on sequence data. Strain FRR 1266, which was earlier classified as a highly divergentA. fumigatus isolate, was found to belong to theA. viridinutans species. The ochratoxin A producingA. viridinutans strain IMI 306 135 was most closely related to an asexual isolate. These two latter strains were more closely related toA. fumigatus andN. fischeri than to anyA. viridinutans strains, and possibly represent a new species in theAspergillus sectionFumigati. The dendrogram based on carbon source utilization data and results of restriction analysis of the mitochondrial and nuclear DNA of the strains supported most of the evolutionary relationships observed on the basis of sequence data. The results indicate that the presence or absence of nodding conidial heads is not an unequivocal morphological character for the identification of species within theAspergillus sectionFumigati.

Isolates representing newly described Neosartorya species, and isolates with abnormal morphologies from Aspergillus section Fumigati were examined by phylogenetic analysis of sequences of part of their β-tubulin gene. Phylogenetic analyses supported the earlier suggestions that heterothallism is a derived character, and that sexuality was lost several times during the evolution of Aspergillus section Fumigati. The heterothallic N. fennelliae and N. udagawae strains were found to be closely related to the homothallic Neosartorya sp. NRRL 4179 and N. aureola, respectively. Aspergillus sp. FRR 1266, which was earlier described as a variant of A. fumigatus, was found to be closely related to A. viridinutans. Another abnormal asexual isolate was found to be closely related to A. fumigatus and N. fischeri. Phylogenetic relationships among newly described Neosartorya species and other taxa were successfully established based on phylogenetic analysis of β-tubulin sequences.

Fungi normally do not senesce, but in some species mitochondrial plasmids are known to occur that induce senescence. In this paper models for the dynamics of a senescence plasmid in a fungal population are developed and analysed. In the first model it is assumed that total fungal biomass density is constant, while in the second model the resource dynamics and its effect on fungal growth is modelled explicitly. An additional death rate describes the effect of the plasmid on the senescent subpopulation. Plasmids can be transferred to non-senescent fungus. Criteria for the coexistence of the non-senescent and senescent fungal strains are derived, all of which have a clear biological interpretation. It is shown that coexistence is not possible in the first model, but is possible in the second model for a large range of parameter values. We show that the interplay between resource dynamics, fungal growth and plasmid transmission is crucial for coexistence. We develop a biological interpretation of how these mechanisms have to interact to promote coexistence. A numerical study of the second model further clarifies the relations between the numerical value of several parameters and coexistence of non-senescent and senescent fungal strains.

The soilborne plant pathogen Thanatephorus cucumeris (anamorph Rhizoctonia solani) is a basidiomycete that occurs worldwide and causes damage to a large variety of agricultural crops. The lack of knowledge of the genetic basis of incompatibility in T. cucumeris hampers the development of environmentally friendly control measures for this plant pathogen. To clarify incompatibility mechanisms in T. cucumeris, sexual and vegetative compatibility were investigated simultaneously in anastomosis group (AG)-I. Sporulation was induced in vitro for a field isolate belonging to AG-I, and single spores were isolated, giving rise to homokaryotic colonies. The homokaryons were paired, and the contact area between isolates was studied macro- and microscopically. Mating processes (formation of heterokaryotic tufts between paired homokaryons) occurred independently from vegetative incompatibility processes (lysis of anastomosed cells), showing that in T. cucumeris AG-1 sexual and vegetative incompatibility are two mechanisms that operate independently. Vegetative incompatibility was variable and irreproducible, indicating vegetative compatibility in T. cucumeris AG-1 is a complex mechanism. Furthermore, heterokaryotization of homokaryotic mycelium (Buller phenomenon) was observed. A novel phenomenon is described, consisting of the spontaneous lysis of the cells of some of the homokaryotic progeny of the field isolate.

Mitochondrial chloramphenicol and oligomycin resistance mutations were used to investigate mitochondrial inheritance in A. nidulans. Mitochondrial RFLPs could not be used to distinguish between paternal and maternal mitochondria because none were detected in the 54 isolates investigated. Several thousand ascospores from each of 111 hybrid cleistothecia from 21 different crosses between 7 heterokaryon incompatible isolates were tested for biparental inheritance. All mitochondrial inheritance was strictly uniparental. Not one instance of paternal inheritance of mitochondria was observed. The implications of our results for the theory that uniparental inheritance evolved to avoid cytoplasmic conflict are discussed. Possible explanations for the maintenance of strict uniparental inheritance of mitochondria in an inbreeding homothallic organism are suggested. The chloramphenicol resistance marker was inherited preferentially to the oligomycin resistance marker probably due to the inhibited energy production of mitochondria with the oligomycin resistance mutation. The maternal parent was determined for 93 hybrid cleistothecia from 17 crosses between 7 different strains. Contrary to previous reports A. nidulans strains functioned as both maternal and paternal parent in most crosses.

We analysed the distribution of mitochondrial plasmids among 82 Neurospora intermedia isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on Neurospora plasmids.

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5′ ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5′ ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.

We analysed the distribution of mitochondrial plasmids among 82 isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on plasmids.

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.

The mitochondrial DNAs (mtDNAs) and the ribosomal repeat unit (ribosomal DNA, rDNA) of black Aspergillus isolates collected in various parts of the world were examined. Wide-ranging mtDNA variation was observed in natural populations of the Aspergillus niger aggregate. Most isolates were classifiable as A. niger or Aspergillus tubingensis according to their rDNA and mtDNA patterns. The mtDNA variation was distributed unevenly in the populations studied. The mtDNAs of most of the isolates collected in Australia were of the A. tubingensis type, with an unexpectedly high degree of variation, while the rDNA of these isolates exhibited the same A. tubingensis pattern as that of isolates from other locations. Some other local populations displayed very little polymorphism in their mtDNA and rDNA. Hybridization experiments in which cloned A. niger and Aspergillus nidulans mtDNA fragments were used revealed that the two main mtDNA groups corresponding to A. niger and A. tubingensis are more distantly related than concluded earlier. Six of the 13 Brazilian isolates examined exhibited mtDNA and rDNA types different from those of all the other strains and could not be classified into the above species. Classical taxonomic examination of these strains is in progress.

We have investigated the horizontal transfer of two mitochondrial plasmids and the Kalilo senescence phenotype in the fungus Neurospora without the use of heterokaryon-forcing markers. The Kalilo senescent state was only transferred between fully-compatible N. crassa strains, but not between strains differing at any of the loci het-c, het-d, het-e or mating-type. However, the linear plasmid kalDNA and the circular plasmid Han-2 were transferred following incompatible vegetative interactions. Our data suggest that vegetative incompatibility due to allelic differences at het-c is more effective in preventing transfer than that due to het-d, het-e or mating-type. Based on these observations we have developed a novel test for assessing vegetative incompatibility between Kalilo and non-Kalilo field isolates of N. intermedia. In this procedure combinations of Kalilo and non-Kalilo field isolates of N. intermedia were grown together and tested for senescence. Compatibility is inferred if the young non-Kalilo strain dies along with the senescent Kalilo strain, whereas incompatibility is inferred when the Kalilo strain dies without imposing its senescent state onto the non-Kalilo strain. Our results suggest that each of the nine Kalilo strains tested is incompatible with each of 20 non-Kalilo isolates from the same N. intermedia population of the Hawaiian island of Kauai. However, the observed incompatibility did not completely prevent cytoplasmic exchange, and in several cases plasmid transfer could be detected.