Single-basidiospore Isolates Research Articles

Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II

Gymnopus dichrous exists in the southern Appalachians (USA) as two distinct entities with essentially identical nuclear ribosomal ITS1 sequences but differing ITS2 and LSU sequences (for convenience, called G. dichrous I and II). F1 ITS heterozygotes between the two are routinely collected from nature. Cloning of ITS PCR products from F1 heterozygotes produced sequences of both parental haplotypes but also numerous chimeric sequences (21.9%). The location of template switching was non-random leading to recovery of the same chimera several times and the chimeric region varied from 45bp to 300bp. By comparison, single-basidiospore isolates from heterozygote F1 fruitbodies showed no recombinant haplotypes within the ITS + LSU span and clones derived from P1 homozygotes were identical to the P1 parent. Thus, chimeric sequences are likely an artifact of the PCR-cloning process and not a consequence of natural recombination events found in nature, nor are they due to hidden existing variation within the ribosomal repeat. Chimeras and PCR-induced mutations are common in cloned PCR products and may result in incorrect sequence information in public databases.

Progeny derived from Thanatephorus cucumeris (Rhizoctonia solani) AG-2-2 IV isolates on sugar beet foliage show more clonal diversity in somatic compatibility grouping than those from roots and petioles

Twenty-nine isolates of Thanatephorus cucumeris (Rhizoctonia solani) AG-2-2 IV were collected from the roots, petioles, and leaves of diseased sugar beets in Hokkaido, Japan. We examined the genetic variation of the field isolates using somatic compatibility grouping of progeny (cultured isolates derived from induced basidiospores) based on analysis of hyphal anastomosis reactions (i.e., hyphal perfect fusion) and inter-simple sequence repeats (ISSRs). The number of somatic compatibility groups (SCGs) of single basidiospore isolates was strongly correlated with the host plant tissue from which the parental isolates were obtained. Parental isolates from roots and petioles tended to be genetically heterogeneous and generated plural SCGs, whereas isolates (except for two non-self-anastomosing isolates) from leaves tended to be homogeneous and generated a single SCG. Heterogeneous sibling basidiospore isolates yielded homogeneous progeny within a few generations. These findings were supported by the results of ISSR analysis, which showed that the homogeneous isolates generated progeny with the same genotype, whereas heterogeneous isolates generated progeny with different genotypes. However, like SCGs, ISSR genotypes of heterogeneous progeny tended to be homogeneous within two or three generations. Additionally, we examined clonal diversity during the basidiospore infection process over a 2-year period. A heterogeneous isolate generated a large number of progeny SCGs, thereby increasing clonal diversity. In contrast, progeny SCGs were almost all the same in a homogeneous isolate plot with a few exceptions. These results indicate that infection of T. cucumeris basidiospores play a role in the clonal diversity of AG-2-2 IV isolates, as indicated by progeny SCGs in the field.

Mechanism of the Generation of New Somatic Compatibility Groups within <i>Thanatephorus cucumeris</i> (<i>Rhizoctonia solani</i>)

Single-basidiospore isolates (SBIs) were obtained from field isolates of Thanatephorus cucumeris (Rhizoctonia solani) AG-1 IC and AG-2-2 IV. Formation of distinctive tufts, a recognized feature of heterokaryon synthesis, was observed, and isolates derived from hyphal-tipped tuft hyphae were obtained following pairings between various strains. Three distinctive types of tufts were formed: the fibrous type of mating-compatible homokaryon-homokaryon (Hom-Hom) pairings, the sparse type between heterokaryon-homokaryon (Het-Hom) pairings originating from one parent, and the compact type between Het-Hom pairings originating from different parents. Amplified Fragment Length Polymorphism (AFLP) profile of fingerprints of these tuft isolates verified that they were all heterokaryotic. Because of heterokaryotic vigor, the growth and pathogenicity of the majority of tuft isolates increased compared with their contributing SBIs. New somatic compatibility groups (SCGs) that were different from parental field isolates occurred following heterokaryon formation within T. cucumeris. Tuft isolates produced by Hom-Hom and Het-Hom pairings among isolates of different parents yielded no somatic compatibility with the original parent isolates and a high frequency of new SCGs (62–100%). This was in contrast to those produced by Hom-Hom and Het-Hom pairings among isolates with a common parent that yielded only 12–37% new SCGs. The SCG diversity of R. solani in the field may be attributed to new fitter heterokaryons formed between a heterokaryon of one pair of parents and a homokaryon of another parent pair. This mechanism greatly contributes to genetic diversity in the field and accounts for the failure to recover the expected distribution of SCGs from a field population.

Heterokaryon formation in Thanatephorus cucumeris ( Rhizoctonia solani) AG-1 IC

Approximately 50 single-basidiospore isolates (SBIs) obtained from each of 16 field isolates of Thanatephorus cucumeris AG-1 IC were examined for heterokaryon formation. All SBIs obtained from each field isolate were divided into two mating groups (SBIs-M1 and SBIs-M2), and tufts of mycelia were formed in the contact zone between colonies of paired SBIs-M1 and -M2 based on 0.5 % charcoal agar medium. Tufts were produced from all possible pairing between SBIs from non-parental field isolates. Hyphal anastomosis reactions indicated no cell death and random cell death at the contact cell, and was not related to tuft formation. AFLP phenotypes of SBIs from each field isolate were not identical to each other and were different from their parental field isolate. AFLP phenotypes of the tuft isolates formed from SBIs-M1 and SBIs-M2 from each field isolate were heterokaryotic. Moreover, several SBIs also formed tufts with their parental and non-parental field isolates. AFLP phenotypes of these tuft isolates suggested that they were all heterokaryotic. Results of these experiments suggest that T. cucumeris AG-1 IC is heterothallic and bipolar, and that genetic exchange can occur between homokaryotic and heterokaryotic isolates (Buller phenomenon).

Isolation of a homothallic mutant in Lentinula edodes

To obtain a homothallic mutant in Lentinula edodes, basidiospores derived from the common Bmut dikaryon (A1B1mut × A2B1mut) were treated with UV irradiation. Of a total of approximately 5000 monosporous cultures recovered, a single basidiospore isolate was found to produce the hyphae bearing clamp connections without mating. This mutant strain could form fruit bodies, and all its single basidiospore isolates developed into colonies with clamp connections. Such homothallic behaviors were transmitted from the mutant strain to the next generation. During the germination and following hyphal elongation in a single basidiospore of mutant strain, clamp connections were clearly detected in multicellular hyphae, which contained two nuclei in each cell. Their clamp connections were morphologically variable, viz., pseudo, abnormal, and true clamps. Amplified fragment length polymorphism (AFLP) profiles among the basidiospore isolates of mutant strain were identical, indicating that the mutant strain produced isogenic basidiospore progeny.

Isolation of a homothallic mutant in <i>Lentinula edodes</i>

To obtain a homothallic mutant in Lentinula edodes, basidiospores derived from the common Bmut dikaryon (A1B1mut × A2B1mut) were treated with UV irradiation. Of a total of approximately 5000 monosporous cultures recovered, a single basidiospore isolate was found to produce the hyphae bearing clamp connections without mating. This mutant strain could form fruit bodies, and all its single basidiospore isolates developed into colonies with clamp connections. Such homothallic behaviors were transmitted from the mutant strain to the next generation. During the germination and following hyphal elongation in a single basidiospore of mutant strain, clamp connections were clearly detected in multicellular hyphae, which contained two nuclei in each cell. Their clamp connections were morphologically variable, viz., pseudo, abnormal, and true clamps. Amplified fragment length polymorphism (AFLP) profiles among the basidiospore isolates of mutant strain were identical, indicating that the mutant strain produced isogenic basidiospore progeny.

Heterokaryon formation in Thanatephorus cucumeris anastomosis group 2-2 IV

Thirty single basidiospore isolates (SBIs) obtained from four field isolates of the basidiomycete fungus Thanatephorus cucumeris AG 2-2 IV were examined for heterokaryon formation. SBIs of three of four field isolates (Rh509, 92155 and R94) did not produce a tuft of mycelium in the hyphal interaction zone between paired isolates on 2% charcoal agar. Field isolates Rh509, 92155 and R94 indicated no death of interacting mycelium with their progenies on glass slide and microscopic examination. AFLP (amplified fragment length polymorphism) phenotypes of parent and their SBIs were identical. Field isolates Rh509, 92155 and R94 and their SBIs were homothallic. SBIs obtained from field isolate SA-1 were grouped into two mating types (SBI-M1 and SBI-M2), and a tuft of mycelium was formed between paired SBIs-M1 and -M2. SBIs of field isolate SA-1 indicated that no death and death of interacting mycelium were randomly observed. AFLP phenotypes among SBIs of isolate SA-1 were not identical and were also different from their parent isolate. AFLP phenotypes of tuft mycelia produced between heterothallic SBI-M1 and -M2 were heterokaryotic. The mating system of field isolate SA-1 and its SBIs was heterothallic. Both SBIs-M1 and -M2 further produced tuft mycelium with homothallic field isolates and their SBIs. AFLP banding patterns suggested that tuft mycelium was heterokaryotic produced from between heterothallic and homothallic isolates. Results from these experiments clarified that both homothallic and heterothallic isolates exist in population of T. cucumeris AG 2-2 IV, and that genetic exchange can occur between homothallic and heterothallic isolates.

Heterokaryon formation in Thanatephorus cucumeris anastomosis group 2-2 IV

Thirty single basidiospore isolates (SBIs) obtsained from four field isolates of the basidiomycete fungus Thanatephorus cucumeris AG 2-2 IV were examined for heterokaryon formation. SBIs of three of four field isolates (Rh509, 92155 and R94) did not produce a tuft of mycelium in the hyphal interaction zone between paired isolates on 2% charcoal agar. Field isolates Rh509, 92155 and R94 indicated no death of interacting mycelium with their progenies on glass slide and microscopic examination. AFLP (amplified fragment length polymorphism) phenotypes of parent and their SBIs were identical. Field isolates Rh509, 92155 and R94 and their SBIs were homothallic. SBIs obtained from field isolate SA-1 were grouped into two mating types (SBI-M1 and SBI-M2), and a tuft of mycelium was formed between paired SBIs-M1 and -M2. SBIs of field isolate SA-1 indicated that no death and death of interacting mycelium were randomly observed. AFLP phenotypes among SBIs of isolate SA-1 were not identical and were also different from their parent isolate. AFLP phenotypes of tuft mycelia produced between heterothallic SBI-M1 and -M2 were heterokaryotic. The mating system of field isolate SA-1 and its SBIs was heterothallic. Both SBIs-M1 and -M2 further produced tuft mycelium with homothallic field isolates and their SBIs. AFLP banding patterns suggested that tuft mycelium was heterokaryotic produced from between heterothallic and homothallic isolates. Results from these experiments clarified that both homothallic and heterothallic isolates exist in population of T. cucumeris AG 2-2 IV, and that genetic exchange can occur between homothallic and heterothallic isolates.

Heterologous diploid nuclei in the violet root rot fungus, Helicobasidium mompa

Allelic genes hga1-1 and hga1-2, which encode G protein alpha subunit in the violet root rot fungus, Helicobasidium mompa, were sequenced and characterized. Restriction fragment polymorphism (RFLP) analysis determined that the gene is present as a single locus in the single basidiospore isolates, while strain V169 possessed both alleles of this gene. Therefore, although basidiospore isolates are dikaryon, they are homokaryotic. Field-isolated strain V169, the putative parent strain, is a dikaryotic heterokaryon. Allelic genes hga1-1 and hga1-2 segregated in almost a 1:3 ratio among single basidiospore isolates from the same fruiting body. Moreover, the copy number of hga1-1 was found to be less than that of hga1-2 in the V169 strain. These results suggest that one of the nuclei in the V169 parent strain is homozygous diploid and the other heterozygous diploid. This parent strain produced four homokaryotic and dikaryon basidiospores on each basidium.

Mycelial incompatibility operative in pairings between single basidiospore isolates of Helicobasidium mompa

Helicobasidium mompa is a clampless basidiomycete and binucleate both in secondary and primary hyphae. Single basidiospore isolates were paired in sibling and non-sibling combinations. We did not observe any alterations in colony morphology, such as tuft formation, but mycelial incompatibility, as indicated by the presence of a dark demarcation line between colonies, occurred in a unifactorial manner. Techniques using DNA molecular markers, such as internal transcribed spacer-restriction fragment-length polymorphism (ITS-RFLP), inter-simple-sequence-repeat polymerase chain reaction (PCR), and universally primed PCR, failed to identify nuclear migration to opposite single basidiospore isolates in all but one of the 92 pairings. These results imply that single basidiospore isolates of H. mompa may be incompetent to mate under laboratory conditions and that a single mycelial incompatibility factor operates in single basidiospore isolates.

Recovery of degenerate schizostatin production by screening single-basidiospore isolates in Schizophyllum commune

To recover degenerate schizostatin production in aSchizophyllum commune isolate, SANK 17785, we examined schizostatin production of 30 single-basidiospore isolates obtained from a basidiocarp of SANK 17785. One of the isolates showed high productivity (136–154 μg/ml) and maintained its high productivity for 26 mo. To explore other isolates with high schizostatin production, 76 single-basidiospore isolates from 7 wild basidiocarps were obtained and their schizostatin production was quantified. Four of the isolates produced more schizostatin than SANK 17785 did originally.

Development and Characterization of a Genetic Linkage Map of Cryptococcus neoformans var. neoformans Using Amplified Fragment Length Polymorphisms and Other Markers

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.

Biological species concepts in eastern North American populations of Lentinellus

As part of a biosystematic study of the Lentinellus ursinus species complex in eastern North America, field and herbarium specimens from North America, Central America, and Europe were examined. Crossing single basidiospore isolates demonstrated tetrapolar (bifactorial) mating systems. Three biological species are recognized as L. ursinus, L. angustifolius, and L. vulpinus. The production of gloeocystidia and/or chlamydospores is considered taxonomically significant for identifying somatic cultures of these species. All three taxa produced laccase, tyrosinase, and peroxidase in culture and are white-rot fungi. This is the first confirmed report of L. angustifolius from North America.

Hyphal Morphology Associated with Strain Instability in the Commercial Mushroom, Agaricus bisporus

Light microscopy was used to examine the morphology of surface hyphae of strains of Agaricus bisporus that form “fluffy” or “stromatal” sectors on agar medium or in grain culture. Although differences were found between normal (U1 strain) and some sectoring isolates in the mean distance from the hyphal apex to the first branch, and in average internode length, these differences were not consistent under different growth conditions. Fluffy and stromatal isolates did show a high frequency of hyphae with irregular branching patterns. A correlation between high prevalence of hyphae with certain types of branching patterns and the ability to form sectors in grain culture was also observed in 35 single-basidiospore isolates derived from mushrooms produced by normal and sectoring cultures. It is suggested that the ability of any subculture to form a sector may depend on the prevalence of aberrent hyphae in the initial inoculum.

Spatial distribution of mycelia of the liana (L-) biotype of the agaric Crinipellis perniciosa (Stahel) Singer in tropical forest.

A detailed investigation of the Liana biotype (L-biotype) of Crinipellis perniciosa (Stahel) Sing, was conducted in Ecuador. Basidiocarps of the L-biotype were consistently found in association with stems or debris of the liana Arrabidaea verrucosa (Standl) A. Gentry, although no symptoms associated with witches'broom disease, were observed. Debris colonized by the L-biotype were often connected via pseudosclerotial pads to living sterns of A. verrucosa. Pairings between 87 isolates obtained from basidiocarp stipe tissues or liana bark cores from three sites permitted 37 somatic compatibility groupings (SCGs) to be defined. In all cases these were found to be very restricted in geographical distribution, a situation which contrasts markedly with the distribution of SCGs in the pathogenic and non-outercrossing eocoa biotype. Mating pairings between single basidiospore isolates confirmed chat the outercrossing mechanism of the L-biotype is muitialletic and that no significant biological barriers to gene flow exist between coastal and Amazonian populations. Several lines of evidence suggested that mycelia belonging to the same SCG (= genet) were isogenic. Correlations between field observations and the distributions of both SCGs and mating type factors art- discussed in relation to patterns of dispersal, establishment and spread of this fungus.

The breeding biology of biotypes of the witches' broom pathogen of cocoa, Crinipellis perniciosa

Investigation of the breeding biology of the agaric Crinipellis perniciosa (causal agent of witches' broom disease of cocoa in South America) found that two distinct breeding strategies exist among the four known biotypes of the fungus, which are specific to cocoa (C-biotype), solanaceous hosts (S-biotype), Bixa orellana (B-biotype) and liana vines (L-biotype). The homomictic (primary homothallic) behaviour of the C-biotype was confirmed and single basidiospore isolates (SSIs) of the S-biotype and B-biotype were found to behave in a similar manner. The mycelia of most SSIs of the L-biotype did not bear clamp connections and pairings between sibling SSIs established that an outcrossing mechanism under the control of two unlinked mating type factors was operating (i.e. bifactorial heterothallism). These matings also suggested that one of the mating factors has a two-gene structure. The occurrence of bi- and trinucleate basidiospores and SSIs bearing true or false clamp connections suggests that the L-biotype exhibits a limited degree of secondary homothallism.

The use of protoplasts for the preparation of homokaryons from heterokaryotic isolates of Rhizoctonia solani

A technique for preparing homokaryotic strains from heterokaryotic isolates of Rhizoctonia solani is described. Protoplasts were released from the heterokaryotic mycelium and mononucleate protoplasts were concentrated by low-speed centrifugation. The protoplasts were regenerated and homokaryons were selected on the basis of their ability to form a heterokaryon with known homokaryotic single-basidiospore isolates. The homokaryotic nature of these isolates was confirmed by pairing them with each other in all possible combinations. The method was used to prepare homokaryons from a dikaryon synthesized from two homokaryotic single-basidiospore isolates and from a heterokaryotic field isolate. This technique should prove to be useful for preparing homokaryons from isolates of R. solani which cannot be induced to sporulate.

Morphological Characteristics of Incompatibility Reactions and Evidence for Nuclear Migration in Armillaria Mellea

A new technique was developed to distinguish the four incompatibility interactions in Armillaria mellea sensu stricto. The technique involves confronting two single-basidiospore isolates on oak wood extract agar and then transplanting a strip from the colonies including the interaction zone onto squeezed orange juice agar. A compatible (A#-B #) interaction results in a change of the colony morphology along the entire agar strip. Morphological characteristics of compatible interactions are highly variable. They may be expressed as appressed or crustose secondary mycelium. In a common B (A¢B=) interaction, such a change occurs only in the interaction zone. The ends of the agar strip remain white and fluffy. Both the common A (A=B-#) and common AB (A=B=) interactions remain fluffy and unchanged. The genotype can be deduced after identifying the A# B and A# =B= interactions. In addition, pairing mycelium produced by the confrontations with known monosporous tester isolates showed that nuclear migration occurs in A=B-# and A-# B# interactions but does not occur in A #B= interactions.

Morphological Characteristics of Incompatibility Reactions and Evidence for Nuclear Migration in Armillaria mellea

A new technique was developed to distinguish the four incompatibility interactions in Armillaria mellea sensu stricto. The technique involves confronting two single-basidiospore isolates on oak wood extract agar and then transplanting a strip from the colonies including the interaction zone onto squeezed orange juice agar. A compatible (A≠B≠) interaction results in a change of the colony morphology along the entire agar strip. Morphological characteristics of compatible interactions are highly variable. They may be expressed as appressed or crustose secondary mycelium. In a common B (A≠B=) interaction, such a change occurs only in the interaction zone. The ends of the agar strip remain white and fluffy. Both the common A (A=B≠) and common AB (A=B=) interactions remain fluffy and unchanged. The genotype can be deduced after identifying the A≠B≠ and A≠B= interactions. In addition, pairing mycelium produced by the confrontations with known monosporous tester isolates showed that nuclear migration occurs in A=B≠ and A≠B≠ interactions but does not occur in A≠B= interactions.

Patterns of nuclear migration and heterokaryosis in pairings between sibling homokaryons of Heterobasidion annosum

Hyphal and mycelial interactions within progeny sets of single basidiospore isolates derived from Heterobasidion annosum fruit bodies from various parts of the northern hemisphere were studied microscopically using microculture chambers and with pairing tests, respectively. The behaviour within different sets was highly variable with respect to mating, nuclear exchange, morphogenetic patterns and somatic rejection responses. Evidence was obtained that rapid nuclear migration can occur between certain strains. An unusually large proportion of pairings (84 %) produced stable heterokaryons, as determined from back pairings with original homokaryons, of which 54 % possessed clamp connections and 46 % did not. Studies of isozyme patterns of pectinases of one selected pairing confirmed the presence of clampless heterokaryons. The genetic control of heterokaryosis is complex, with subsidiary systems being superimposed on those which govern formation of clamp-connections and mycelial morphogenesis. The implications of these findings in understanding gene flow within and between natural populations of H. annosum are discussed.