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Laboratory of Plant Pathology, Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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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.
Key words: mating compatibility, somatic compatibility
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Carling et al 1999, 2002a). When isolates of R. solani are paired on water agar or glass slide fusion of hyphae is observed at the microscopic level between isolates from same AG. Isolates from same AG cause either a C2 or a C3 reaction (Carling 1996). A C2 reaction is a result in cell lysis after cell wall fusion, and occurs between genetically distinct isolates of same AG. A C3 reaction is complete somatic cell fusion with continuous flow of cytoplasm in the fusion point and occurs between clones or closely related individuals. In Rhizoctonia species paired isolates showing C3 reaction are somatically compatible, while paired isolates showing C2 reaction are somatically incompatible. To distinguish C2 and C3 reactions at the fusion point is useful for identification in context of population within AG 8 (MacNish and Carling 1993).
Several studies on the genetics of T. cucumeris reported that the observation of the tuft produced from the zone of contact between two different single basidiospore cultures based on pairing on potato-dextrose charcoal agar (PDCA) medium is a good indicator of heterokaryon formation (Whiteny and Parmeter 1963, Adams and Butler 1982). Previous studies have shown that field isolates of AG 1, 4 and 8 have a bipolar mating system (Whitney and Parmeter 1963, Anderson et al 1972, Yang et al 1992, Julian et al 1996, 1997). In these AGs the heterokaryon formation is controlled by two different factors (Bolkan and Butler 1973). However MAT (mating type) genes controlled by a single mating factor have not been identified in T. cucumeris. In addition somatic compatibility of single basidiospore isolates was not linked with mating compatibility (Julian et al 1996). In AG 4 the molecular marker based on RAPD (random amplified polymorphic DNA) analysis supported the occurrence of heterokaryotic mycelium between two different homokaryons (Cubeta et al 1993). In addition the data of AFLP (amplified fragment length polymorphism) analysis also supported the formation of heterokaryotic tuft and clarified that heterothallic single basidiospore progenies of AG 1-IC are genetically not identical (Julian et al 1999). Adams (1988) suggested that the mating system of R. solani AGs 2 and 3 differs from those of AGs 1, 4 and 8 and that isolates of AGs 2 and 3 might be homothallic. In this paper we present the study of mating systems of T. cucumeris AG 2-2 cultural type IV using field isolates and their single basidiospore progeny based on (i) the evaluation of heterokaryotic tuft formation by pairing on PDCA medium, (ii) microscopic hyphal interactions by pairing on glass slide and (iii) the confirmation of heterokaryon formation by phenotype of AFLP analysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Toda et al 2004) were used for production of basidiospores. Isolate Rh509 identified as AG 2-2 IV was provided from Hokkaido University, Japan. Among four isolates, three (SA-1, Rh509 and R94) were obtained from sugar beet (Beta vulgaris) in fields in Hokkaido or Ibaragi, Japan, and one isolate (92155) was obtained from burdock (Arctium lappa) in Hokkaido. Field isolates were maintained as parent isolates on 9 cm Petri dish containing 10 mL of potato-dextrose agar (PDA; Becton Dickinson & Co., Sparks, Maryland) and stored at 25 C.
Production of basidia and isolation of single basidiospore isolates (SBIs).— –
The soil-over-culture method was used to produce the perfect state (Ogoshi 1972). A single 3–5 mm agar plug of each parent (field) isolate from a stock culture was placed onto 9 cm Petri dish containing 10 mL of PDA at 25 C in the dark. After 3 d incubation, a 5 mm agar disk with mycelium was plated onto a center part of new 9 cm Petri dish containing 30 mL PDA containing 2.5% yeast extract (BactoTM yeast extract; Becton Dickinson & Co., Sparks, Maryland) (PDYA). When the mycelium covered the PDYA medium after 4–5 d sterilized soil used for seedling culture of rice (Kureha Chemical Industry Co. Ltd, Tokyo) was placed on top of the mycelium. Distilled water was supplied as needed to maintain soil moisture. Plates were incubated 5 d in growth chamber at 25 C. When a plate showed evidence of sporulation on soil surface, pieces of soil were taken with sterile forceps and placed on glass slide. For confirming basidia and basidiospore, pieces of soil were stained with 0.05% cotton blue for microscopic observation by 400x magnification. When basidia and basidiospore were observed a plate was inverted over Petri dishes containing acidified water agar (AWA) amended with 3% lactic acid (pH 4.0) to eject basidiospores. After 2–6 d single basidiospore isolates (SBIs) were isolated by examining of dissecting microscope from a hyphal tip of a germinated basidiospore on AWA, excising with a sterilized needle and transferring onto Petri dish containing 10 mL of PDA. SBIs were labeled as G1 (generation 1) progeny. Thirty SBIs as G1 progeny obtained from each parent (field) isolate were denoted 1* through 30*, and maintained on PDA plates at 25 C until use. For observing SBIs self-fertility, several SBIs as G1 progeny of each field isolates (SBIs Rh509-1*, -2* and -3*; 92155-1*, -3*, -4* and -5*; R94-1*, -2*, -3* and -5*; SA-1-1*, -2*, -4*, -6*, -7* and -8*) were randomly selected to produce the perfect state for next generation by soil-over-culture method. When basidia and basidiospore were observed single basidiospore progenies were isolated by same method described for G1 progeny. These second generation SBIs were labeled as G2 progeny, denoted 1* through 20* and stored on PDA plates at 25 C.
Staining of nuclei in basidiospore and hyphae.— – The number of nuclei in basidiospore and hyphae were observed microscopically. Pieces of soil showing the evidence of basidiospore obtained by soil-over-culture method were transferred on glass slide and stained with 0.05% DAPI (4',6-Diamidino-2-phenylindole, dihydrochloride, solution) (Dojindo, Kumamoto, Japan). The number of nuclei in basidiospore was counted with fluorescent microscope at 400x magnification. Field isolates and their SBIs as G1 and G2 progeny were grown on a 9 mm Petri dish containing 20 mL of PDA 3 d at 25 C in the dark. Mycelial plugs 5 mm diam were taken from the advancing margin of the pure culture of each isolate and transferred on glass slide. Mycelial plugs with glass slide were placed in a plastic box (100 x 200 x 30 mm3) containing with sterile paper towel moistened by sterile distilled water and incubated at 25 C in the dark. After 2 d grown hyphae were stained with 50x DAPI. The number of nuclei in 10 cells of each isolate was counted with fluorescent microscope at 400x magnification.
Evaluation of putative heterokaryon formation between SBIs.— –
Pairing incubation of field isolates and their SBIs as G1 and G2 progeny was performed to evaluate putative heterokaryon formation on PDA with 2% activated charcoal (PDCA), using a modified method of (Julian et al 1996). A single 3 mm agar plug of each isolate from PDA was placed 3 cm apart in a 9 cm Petri dish containing 20 mL of PDCA and incubated at 25 C in the dark. After 3 or 4 d incubation, tuft formation was evaluated in the hyphal interaction zone of two paired isolates by classification ratio. The categorization of tuft formation defined by (Julian et al 1996) was applied in this study. When the tuft was formed along the entire contact area of two colonies, paired isolates were considered to be mating compatible and this tuft was assigned as fibrous tuft (+). When tuft was formed at the several contact points, paired isolates also were considered to be mating compatible but tuft was assigned as compact tuft (+*). When tuft was not formed, paired isolates were considered to be mating incompatible and assigned as no tuft formation (–). When paired SBIs obtained from same parental field isolate formed the tuft, the two paired SBIs were distributed into two groups, referred to mating type 1 and 2 (SBIs-M1 and SBIs-M2). When these SBIs did not form tuft, they were distributed into same mating type.
Isolation of putative heterokaryons.— – Hyphae from each fibrous or compact tuft (putative heterokaryon) formed by several paired SBIs were picked with sterilized forceps and placed on each Petri dish containing 10 mL AWA. When hyphae appeared from tuft hyphal tips were transferred on PDA plates and stored as tuft isolates at 25 C in the dark. Several tuft isolates were selected randomly to produce basidia and basidiospore with the soil-over-culture method described in the previous section. Second generation SBIs obtained from tuft isolates were labeled as G2T progeny, denoted as 1* through 12* or 13*, and used for pairing incubation to observe tuft formation on PDCA.
Microscopic hyphal interaction.— –
The four field isolates and their single basidiospore progeny as G1 and G2 also were examined microscopically for somatic compatibility (hyphal anastomosis reaction). The field isolates and their SBIs as G1 and G2 progeny were grown 3 d on a 9 mm Petri dish containing 20 mL PDA at 25 C in the dark. Mycelial plugs 5 mm diam were taken from the advancing margin of the pure culture of each isolate and paired 3–4 cm apart on glass slide. Paired mycelial plugs with glass slide were placed in a plastic box (100 x 200 x 30 mm3) containing a sterile paper towel moistened by sterile distilled water and incubated at 25 C in the dark. Three replicates were prepared from each pairing combination. After 2 d the overlapping portion of hyphae, which was growing on glass slide, was stained with 0.05% cotton blue in acetic acid and observed initially for hyphal fusion at 100x magnification. The type of hyphal anastomosis reactions (C2 and C3 reactions) categorized by (Carling 1996) was determined by observation at 400x magnification. Paired isolates that formed no death of interaction cells at five contact points were somatically compatible and categorized as C3 reaction. Paired isolates that showed hyphal cell death after the fusion event were somatically incompatible and categorized as C2 reaction. When paired isolates showed each C2 and C3 reaction at the different points on same glass slide, they were categorized as C2/3 reaction.
DNA extraction.— –
Total genomic DNA of field, single basidiospore and putative heterokaryon isolates was extracted with a modified method of (Yoder 1988). Three 3 mm agar plugs on PDA plate with 3 d old colonies were transferred onto a 9 mm diam Petri dish containing 10 mL potato-dextrose broth at 25 C in the dark. After 5–6 d mycelial mats were harvested by filtration, washed with sterile distilled water, blotted dry and stored at –80 C. Frozen mycelial mats were ground in liquid nitrogen, suspended in 0.6 mL of extraction buffer (10 mM Tris-HCl, pH 7.5, 100 mM EDTA, 0.5% SDS and 100 mM LiCl) and heated at 50 C for 15 min. The supernatant obtained by centrifugation (15 000 rpm, 10 min) was extracted in same volume of phenol-chloroform-isoamyl alcohol (25:24:1). The DNA was precipitated with 0.6 mL of isopropyl alcohol, rinsed with 1 mL of 70% ethanol and redissolved in 0.5 mL of 1x TE buffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA). After treatment with 2 µL RNase A (5 mg mL–1; Sigma) at 37 C for 1 h, the DNA was extracted sequentially with 0.5 mL of Tris-saturated phenol, phenol-chloroform-isoamyl alcohol, chloroform-isoamyl alcohol (24:1) and diethyl ether. Same volume of isopropyl alcohol was added to resulting solution and placed on ice for 10 min. The DNA precipitate was collected by centrifugation in 15 000 rpm at 4 C, washed with 1 mL of 70% ethanol, dried in a desiccator and dissolved in 50 µL TE buffer.
AFLP analysis.— –
AFLP analysis was performed with extracted genomic DNA. Genomic DNA concentration was adjusted to10 ng/µL and used for AFLP analysis according to the procedure described by (Vos et al 1995). DNA digestion and ligation reactions of AFLP were performed with the AFLP kit (Applied Biosystems, Foster City, California). The primer pair used for the selective amplifications was Eco RI-AG/Mse I-CA. Eight µL of AFLP product was subjected to electrophoresis in a 15% poly-acryl-amid gel. The gel was stained sequentially with 0.1% AgNO3 and 1.5% NaOH containing formaldehyde for 10 min respectively and viewed with an acryl board light. The reproducibility of PCR products by AFLP was observed by repeating procedure three times with same genomic DNA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Evaluation of putative heterokaryon formation between SBIs.— –
Pairing of SBIs as G1 progenies of parent (field) isolates Rh509, 92155 and R94 showed no tuft formation in any of the pairings (TABLE I). G1 progenies also did not form the tuft with their parent isolates (TABLE I). SBIs G2 progeny obtained from G1 progenies (Rh509-1*, -2* and -3*; 92155-1*, -3*, -4* and -5*; R94-1*, -2*, -3* and -5*) did not form tufts in any of combinations, and they also did not form tufts with their parent isolates and G1 progenies (TABLE I). Furthermore tuft formation was not observed among different parent (field) isolates and SBIs as G1 and G2 progenies obtained from different field isolates.
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). The determination of mating compatibility was performed with isolates 4 * as SBI-M1 and 2* as SBI-M2, which were paired in all possible combinations with 30 SBIs (17 were M1 and 13 were M2) of parent isolate SA-1 (TABLE II). All pairings between SBIs-M1 and -M2 produced tuft formation categorized as fibrous but not from any pairings of isolates of the same mating type. Both SBIs-M1 (SA-1-1*, -4* and -6*) and -M2 (SA-1-2*, -7* and -8*) produced basidia but did not produce basidiospores.
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). Field isolate SA-1 produced compact tuft with its progenies SBIs-M1 and SBIs-M2 but did not produce tuft with field isolate Rh509, 92155 and R94 and their SBIs (TABLE I).
Isolation of putative heterokaryons.— –
Hyphae from fibrous and compact tufts formed by several paired isolates were successfully collected as fibrous tuft isolates ([SA-1 1* x 2*], [SA-1 4* x 2*], [SA-1 4* x 7*]) and compact tuft isolates ([SA-1-1* x Rh509], [SA-1-2* x Rh509], [SA-1-1* x 92155-1*], [SA-1-2* x 92155-1*]). All tuft isolates produced basidia and basidiospores. Based on pairing incubation on PDCA, second generation progenies (G2T progenies) obtained from three tuft isolates were analyzed: 13 SBIs as G2T progenies obtained from (SA-1 4* x 7*) segregated into two groups of eight M1 SBIs and five M2 SBIs exhibiting the same mating behavior as the first generation SBIs-M1 and SBIs-M2 (shown in TABLE I) respectively; 13 SBIs as G2T progenies obtained from (SA-1-1* x 92155-1*) segregated into two groups of seven M1 SBIs and six SBIs exhibiting the same mating behavior as the first generation SBIs-M1 deriving from SA-1 and 92155 (or the SBIs issued from it, TABLE I) respectively; 12 SBIs as G2T progenies obtained from (SA-1-2* x 92155-1*) segregated into two groups of six M2 SBIs and six SBIs exhibiting the same mating behavior as the first generation SBIs-M2 deriving from SA-1 and 92155 (or the SBIs issued from it, TABLE I) respectively. In addition G2T progeny in accord with SBIs-M1 and -M2 produced basidia but not basidiospore by soil-over-culture method, while G2T progeny in accord with isolate 92155 produced basidia and basidiospores.
Microscopic hyphal interaction.— –
All parent (field) isolates Rh509, 92155 and R94 and their SBIs as G1 and G2 progenies were somatically compatible and no death of interacting cells was observed in all three replicates when their parents were same (TABLE III). Somatic incompatibility, hyphal cell death after the fusion event, was observed between parent isolates Rh509, 92155 and R94 and between their G1 and G2 progenies obtained from different parent isolate in all three replicates.
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and III). Several SBIs-M1 and -M2 showed both somatically compatible and incompatible interactions at the different fusion points on the same glass slides in all three replicates (TABLES II and III). Parent isolate SA-1 also showed somatic compatibility and incompatibility with its progenies SBI-M1 and -M2 randomly in all three replicates (TABLE II). Isolate SA-1 and its progenies SBIs-M1 and -M2 were somatically incompatible with all isolates of Rh509, 92155 and R94 and their G1 and G2 progenies in all three replicates (TABLE III).
AFLP analysis.— –
AFLP phenotype of field isolates Rh509, 92155 and R94 were identical with their G1 and G2 progenies, but AFLP phenotype were distinguishable among field isolates Rh509, 92155 and R94 (FIG. 1). AFLP phenotype of isolate SA-1 and its SBIs were not identical, and 13 multiple AFLP products were evaluated from isolate SA-1 and its 12 SBIs (FIG. 2). AFLP phenotypes of fibrous tuft isolates contained the specific markers of each paired SBI-M1 and SBI-M2 of isolate SA-1 (FIG. 3). AFLP phenotypes of compact tuft isolates also contained the specific markers of SBI-M1/M2 and isolates of Rh509 and 92155 (FIG. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The results of this study show that T. cucumeris AG 2-2 IV exhibits homothallic and heterothallic mating systems. Field isolates Rh509, 92155 and R94 and their progeny were homothallic. The field isolate SA-1 and its progenies were heterothallic with a bipolar mating system that is similar to those of AG 1-IC. SBIs as G1 progeny obtained from Rh509, 92155 and R94 produced basidia and basidiospores as G2 progeny (self-fertile). G1 and G2 progeny did not produce tuft mycelium when they were paired on PDCA medium. In addition these G1 and G2 progeny were genetically similar; SBIs obtained from the same parent showed C3 reactions in all pairings and their AFLP phenotypes were identical. Homothallic strains of T. cucumeris have been hypothesized to conserve the genetic characters of their parents (Adams 1988, Cubeta and Vilgalys 1997). Ceratobasidium bicorne J. Erikss, a species of uninucleate Rhizoctonia, has a homothallic mating system, but single basidiospores obtained from the same parent are somatically incompatible (Hietala et al 2003). Although single basidiospores of C. bicorne were genetically dissimilar (Hietala et al 2003) isolates Rh509, 92155 and R94 are clonally related to their progeny and have fulfilled the predicted definition of homothallism. On the other hand the field isolate SA-1 had a heterothallic mating system because progeny obtained from isolate SA-1 grouped into two mating types (SBIs-M1 and -M2). However not all hyphal fusion among SBIs of isolate SA-1 were somatically compatible, and AFLP phenotypes among SBIs differed from each other. Because one nucleus was observed in each basidiospore we considered that recombination of chromosome must have occurred during meiosis in the basidium. In addition the genetic diversity observed from progeny of isolate SA-1 suggests that SBIs of isolate SA-1 are haploid.
The fibrous tuft mycelium formed by paired SBIs-M1 and SBIs-M2 on PDCA was confirmed as a heterokaryon by AFLP analysis. Fibrous tuft isolates produced basidia and basidiospores, while SBIs of SA-1 produced the basidia but never produced basidios-pores. We considered that diploidization was caused by fusion between haploid SBI-M1 and -M2. The results of field isolate SA-1 and its SBIs were consistent with results of (Julian et al 1996, 1999) for single basidiospore progenies of AG 1-IC. Fibrous tuft isolates could fruit and their SBIs as G2T progeny were divided into two groups, which were in accord with SBIs-M1 and -M2 as G1 progeny. This suggests that mating type of each haploid strain in the heterothallic mating system could be conserved after crossing between two haploid strains.
The existence of heterothallic isolates as well as homothallic isolates was confirmed in T. cucumeris AG 2-2 IV. We further discovered an interesting mating system of T. cucumeris. Either heterothallic SBIs-M1 or SBIs-M2 derived from SA-1 fused with homothallic isolates Rh509, 92155, R94 and their progenies and produced tuft mycelium. The shape of this tuft mycelium was compact, while heterothallic SBIs-M1 and M2 produced fibrous tuft. The AFLP phenotype consistently supported that compact tuft is heterokaryotic. The G2T progenies obtained from compact tuft isolates were distributed into heterothallic and homothallic types, which showed same mating behavior with their parental heterothallic and homothallic G1 progeny, respectively. From these our results provide the evidence of genetic exchanges between strains having homothallic and heterothallic mating systems of T. cucumeris. In other basidiomycete fungi there is evidence of crossing between different mating systems. Ullrich (1973) reported that Sistotrema brinkmannii is an aggregate of species processing homothallic, bipolar heterothallic and tetrapolar heterothallic mating systems. Crossing between bipolar heterothallic and homothallic strains were confirmed from S. brinkmannii, but this crossing has been forced with nutritional markers (Ullrich and Raper 1975). In addition Agaricus bisporus (Lange) Imbach var. bisporus has bipolar heterothallic and pseudohomothallic mating systems (Callac et al 1998) and A. bisporus var. eurotetrasporus has homothallic mating system (Callac et al 2003). The crossing between the two varieties was confirmed under field conditions, and genetic recombination was observed from the next generation progenies (Callac et al 1998, 2003). In the present study the crossing of homothallic and heterothallic strains of T. cucumeris AG 2-2 IV was not forced. We considered that such crossing of T. cucumeris could easily occur under field conditions and might generate genetic diversity by recombination.
Compact tufts were observed constantly from all pairings among field isolate SA-1 and its progenies (SBIs-M1 and -M2) on PDCA. The shape of this tuft was similar to the compact tufts produced between heterothallic and homothallic strains. We expect that a hybrid heterokaryon was produced between heterokaryotic field isolate SA-1 and its homokaryotic progenies and that this compact tuft would be the evidence of Buller phenomenon (Buller 1931). Unfortunately we failed to produce basidia and basidiospores from these compact tuft isolates. But if sporulation from compact tuft isolates produced between field isolate SA-1 and its progeny is successful, we will be able to know whether progenies derived from a hybrid heterokaryon could be accordant with SBIs-M1 and -M2.
The merging reactions categorized as "merge" by (MacNish et al 1997) were actually observed at several pairings, especially between the same clones. Ceresini et al (2002) reported that this phenomenon is related to somatic compatibility in T. cucumeris AG 3. In this study some reactions between paired SBIs were not well defined and were not in accordance with the categorization of (MacNish et al 1997). Therefore we focused on whether tuft formation was observed from each pairing.
The genetics of somatic incompatibility has been considered to be an independent phenomenon from mating incompatibility in T. cucumeris AG 1-IC (Julian et al 1996) and in most fungi, except for Neurospora crassa (Leslie 1993, Philley et al 1994). The relationships between mating and somatic compatibility of T. cucumeris AG 2-2 IV SBIs were similar with those of AG 1-IC SBIs; when paired SBIs-M1 and SBI-M2 produced fibrous tuft, not all pairings were somatically compatible and some pairings were categorized as "C2/3" showing both C2 and C3 reactions. Carling et al (2002b) categorized the C2 reaction in more details as C2– and C2+. Our finding of "C2/3" was different from C2– and C2+ and would be a new categorization. In addition all anastomosis reactions between heterothallic and homothallic isolates were somatically incompatible. Nevertheless a compact tuft, which was confirmed as a new heterokaryon by AFLP analysis, was produced in all pairings between heterothallic and homothallic isolates. However it is a contradiction that heterokaryon was produced at the contact points of paired isolates which showed the somatic incompatibility. Considering the evidence of heterokaryon formation, the nuclear migration must occur from the cell of either paired isolate to the fused cell of the recipient isolate. We considered that the nucleus could move faster to the next cell before C2 reaction and heterokaryotic hyphae could be produced from that next cell. The hyphal cell death of C2 reaction in T. cucumeris was completed within 48–120 min after contact (Kim et al 1989, Yokoyama and Ogoshi 1986). Because the rate of nuclear movement of basidiomycete fungus Schizophyllum commune was estimated to be 6 mm/h (Snider and Raper 1958) the basidiomycete T. cucumeris could show similar nuclear migration to the next cell within 10–20 s. However the width of dolipore septa generally does not allow for nuclei passing. In Coprinus cinereus the degradation of the dolipore results when two monokaryotic hyphae fuse, and reforming of septa between the cells was observed after nuclear migration (Casselton and Olesnicky 1998). T. cucumeris may have the ability to cause the degradation of the dolipore for nuclear migration. Mating systems of both S. commune and C. cinereus are heterothallic but tetrapolar (Casselton and Olesnicky 1998), while T. cucumeris is bipolar heterothallic. Although there are differences between bipolar and tetrapolar, T. cucumeris belongs to the same class (homobasidiomycete) and possibly has a similar nuclear migration process as S. commune and C. cinereus. Further research on the mechanisms involved in heterokaryotic formation in T. cucumeris is required by observing the cells around contact points of paired colonies and degradation of septal pores. We also think that it is important to understand the genetic basis of mating and somatic compatibility phenomena, which were confirmed in this study.
To our knowledge this research represents the first documentation of genetic exchange occurring between isolates of T. cucumeris with homothallic and heterothallic mating systems. The similar phenomena of AG 2-2 IV are not confirmed from other AGs of T. cucumeris.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Current address: Department of Plant Pathology, North Carolina State University, Box 7616, Raleigh, North Carolina 27695-7616; E-mail: ttoda{at}ncsu.edu; TEL: (919) 513-4840; FAX: (919) 513-0024.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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