2.2.89
RELATIONSHIPS WITHIN TWO CYLINDROCLADIUM SPECIES COMPLEXES AS INFERRED BY ITS SEQUENCE ANALYSIS, RDNA HYBRIDIZATION AND GENERAL MORPHOLOGY

PW CROUS1, GRA MCHAU1, CL SCHOCH1 and BD WINGFIELD2

1Department of Plant Pathology, University of Stellenbosch, Private Bag XI, Matieland 7602, South Africa; 2Department of Genetics, University of Pretoria, Pretoria 0002, South Africa

Background and objectives

The genus Cylindrocladium, which has Calonectria teleomorphs, includes several well-known pathogens of agricultural and forestry crops in tropical and subtropical regions of the world. Species are primarily distinguished based on their vesicle morphology, conidium dimensions, septation, and Calonectria teleomorplis. In many instances, however, the Calonectria state is morphologically conserved, and vesicle and conidium morphology become the determining factors for species identification. Recently, sequence analysis and RDNA Southern analysis have been employed to supplement general morphology in determining the identity of new strains and the acceptable variation within species. The aim of the present study was to employ these techniques to identify several unknown isolates. and determine the acceptable variation within two species complexes to which they appeared most similar, namely Cylindrocladium pseudogracile and C. rumohrae.

Materials and methods
Isolates were compared on the basis of morphology, nucleotide sequences of their RDNA ITS regions, and rDNA digested with EcoRI, XhoI, DraI and SmaI, and probed with the 6.3-kb ribosomal DNA repeat unit of Neurospora crassa [1]. Morphological comparisons were made on causation leaf agar plates incubated at 25C under near-ultraviolet light. The internal transcribed spacer (ITS) regions 1 and 2 on opposing sides of the 5.8 S RDNA gene, as well as the gene itself, were sequenced and compared.

Results and discussion
C. ;pseudogracile is characterized by having clavate vesicles and 1-septate conidia that are longer than those of C. gracile. C. ;pteridis, C. colhounii var. colhouriii, C. colhounii var. macroconidiale and C. theae all have 1-3-septate condia, but either have distinct teleomorphs or different conidial dimensions. The unidentified strains in this complex had 1-3-septate condia and appeared intermediate between C. pseudogracile and C. theae. Based on the Southern and sequence analysis, these isolates grouped closely with C. pseudogracile, suggesting that although absent in the type culture, this species can have conidia with 1-3-septa. Furthermore, C. colhounii var. colhounii also clustered separately from C. colhounii var. macroconidiala, indicating that these two varieties would be better treated as separate species. C. rumohrae is characterized by having clavate vesicles and conidia which are primarily 5-septate and thus similar to those of C. quinqueseptatum and C. heptaseptatum, which primarily have 5- and 7-septate conidia, respectively. The unidentified strains examined in this complex were morphologically most similar to C. rumohrae and C. quinqueseptatum. Based on sequence analysis alone, isolates of C. quinqueseptatunl and C. heptaseptatum could not be distinguished. However. they appeared different based on their RDNA profiles. The unidentified strains also formed one cluster, appearing similar to C. rumohrae. Differences were, however, depicted between these isolates and C. rumohrae based on their RDNA profiles. It appears, therefore, that although these unidentified strains are closely related to C. rumohrae, they are not necessarily conspecific. The general similarity in sequence data between closely related species, and the clear differences depicted with RDNA Southern blot analysis, suggests that other, more variable areas of the genome will have to be sequenced in future studies.

References
1. Crous PW, Mchau GRA, van Zyiv M, Wingfield MJ, 1997. Mycologia 89, 653-660.