2.2.37
TAXONOMICAL STUDY OF MICROSPHAERA PULCHRA ON CORNUS SPP. BASED ON MORPHOLOGICAL CHARACTERS AND PCR-RFLP ANALYSIS OF THE RDNA ITS REGION

Y SATO1, S. TAKAMATSU2 and Y. YAMAMOTO1

1College of Technology, Toyama Prefectural University, Kosugi-machi, Toyama 939-0398, Japan; 2 Faculty of Bioresources, Mie University, Tu-City, Mie 514-8507, Japan

Background and objectives
Microsphaera pulchra is divided into two varieties: var. pulchra (MPP) and var. japonica (MPJ) [1]. However, when many powdery mildew specimens on Cornus spp. were examined, we found that some of them could not be placed in either of the two varieties based on the morphological characters of the cleistothecial stage. Therefore, we considered that the taxonomy of M. pulchra on Cornus spp. should be reevaluated by using other characters. In this study, we examined the morphological characters of the conidial stage of M. pulchra on Cornus spp. collected in Japan and also their molecular characters by using the PCR-RFLP analysis of the rDNA ITS region.

Material and methods

Two varieties of M. pulchra can be identified based on the number and the length of appendages, and the number of ascospores [1]. We examined 33 powdery mildew specimens isolated from three species of Cornus in Japan. The morphology of conidia and conidial germ tubes were examined using 16 powdery mildew specimens on three species of Cornus. The nucleotide sequences of the rDNA ITS region of the two varieties of M. pulchra were determined according to the procedure described by Hirata and Takamatsu [2]. In the PCR-RFLP, the rDNA fragment amplified by the two sequential cycles of PCR using nested primer set was digested with HindIII and subjected to agarose gel electrophoresis. For the PCR-RFLP analysis, 11 powdery mildew specimens on three species of Cornus were used. Among the 11 specimens used in this analysis, one from Cornus controversa was identified as MPJ and one each from Cornus kokusa and Cornus florida was identified as MPP, based on the morphology of the cleistothecial stage.

Results and conclusion
Among the 33 specimens used in this study, 14 specimens were placed in either of the two varieties of M. pulchra based on the morphological characters of the cleistothecial stage [1]. The number and length of appendages were considered to be useful for identification of the fungi but the number of ascospores was not. The number of appendages per ascocarp of MPP was greater ((9-)11-19(-22)) than that of MPJ ((7-)9-14(-16)). The length of the appendages of MPP was (115-)133-200(-220) Ám and somewhat longer than that of MPJ (103-143(-160) Ám). The other 19 specimens could not be identified by the cleistothecial characters because the characters of the specimens did not completely coincide with those of the two varieties in either number or length of appendages. The conidial dimension of the specimens on C. controversa (21.6X13.8 Ám) were smaller than that of the specimens on C. kokusa (34.2X16.8 Ám) and C. florida (33.7X15.9 Ám). Although all of the specimens on C. controversa developed a single-celled germ tube with a simple lobed appressorium (Erysiphe polygoni type), all of the specimens on C. kokusa and C. florida developed a morphologically distinct germ tube with two appressoria: a simple lobed appressorium at their top and a complicated appressorium at their base (Uncinula type). The length of the rDNA ITS region of MPP on C. florida and C. kokusa was 554 bp, and that of MPJ on C. controversa was 563 bp. There was 94% identity between the nucleotide sequences of MPP and MPJ, suggesting that the difference between the two varieties was at the interspecies level. The PCR-RFLP analysis of the rDNA ITS region showed that the DNA fragments of all the specimens from C. kokusa and C. florida were cleaved by HindIII, but those of the specimens from C. controversa were not. It was concluded that M. pulchra should be divided into two species or two varieties based on the conidial dimensions, the features of germ tubes, the nucleotide length and PCR-RFLP analysis of the rDNA ITS region of the fungi. It was also concluded that the specimens with ambiguous cleistothecial stage morphological characters could be placed in taxonomical position by using the morphological characters of the conidial stage or the molecular characters of the rDNA ITS region.

References
1. Braun U, 1982. Mycotaxon 15, 121-137.
2. Hirata T, Takamatsu S, 1996. Mycoscience 37 283-288.