2.2.54
MORPHOLOGICAL AND GENETIC SEGREGATION OF SMALL-SPORED ALTERNARIA SPECIES AND SPECIES GROUPS

RG Roberts and ST Reymond

USDA-ARS, Tree Fruit Research Laboratory, 1104 N. Western Ave., Wenatchee, WA 98801, USA

Background and objectives
Small-spored alternarias are cosmopolitan saprophytes, plant and animal pathogens, and allergens. Accurate identification of small-spored Alternarias is challenging to the nonspecialist because of morphological plasticity under non-standard conditions, and the common misapplication of the name Alternaria alternata in the scientific literature to a variety of closely related but morphologically distinct taxa. As currently applied, the name A. alternata has little meaning outside of a few well-characterized isolates in world culture collections identified and deposited by EG Simmons. Additionally, a system of naming phytotoxigenic alternarias as pathotypes of A. alternata, in use by some authors, has further clouded the meaning and usefulness of this specific epithet. Methods to distinguish sub-generic groups based upon sporulation patterns observable at X50 have been described [1] and independently verified [2]. This paper reports the use of RAPD analysis to evaluate the results of morphological segregation of small-spored Alternaria.

Materials and methods
About 260 isolates of small-spored Alternaria, primarily from but not limited to fruit substrates, were segregated into morphological groups by the method of Simmons and Roberts [1]. All isolates were also grown in shake cultures, from which total genomic extracts were prepared and quantified. Three primers, OPR-2, OPR-8, and OPR-12 were to initiate PCR. All PCR products were resolved by gel electrophoresis, then the gels were combined and analysed using band-based cluster analysis, fuzzy logic and the Ward algorithm in GelCompar 3.0

Results and conclusions
When cultured and observed under carefully defined and controlled conditions, morphological patterns of sporulation observable at X50 were powerful predictors of genetic relatedness as determined by RAPD analysis. In RAPD analyses, the following morphological groups were resolved as distinct branches of the dendrogram; Alternaria gaisen (=A. kikuchiana, A. alternata Japanese pear pathotype, Group 2), A. longipes (=A. alternata tobacco pathotype, also Group 2), the "tenuissima" group (Group 5), the "arborescent " group (Group 3), and the "infectoria" group (Group 6). Isolates in groups for which there were only a few representatives (e.g., Group 4, the "alternata" group and Group 1 (unnamed group, similar to Group 2 but generally not phytotoxic), were placed together into a branch which contained nearly all the underrepresented isolates. The RAPD analysis results confirm that when Alternaria isolates are cultured and observed under defined conditions, the phenotypic plasticity inherent to small-spored Alternaria cultures can be minimized, and that valid taxonomic separations can be reliably made based upon morphological characteristics. We conclude that the basic tenet of the A. alternata-pathotype system of naming these fungi; namely, that A. alternata, A. gaisen, and other related small-spored taxa producing phytotoxins are morphologically indistinguishable, is not correct. We further assert that the "pathotype" system of naming small-spored Alternaria taxa confers no predictive value relative to observable morphological and genetic characters, and should be abandoned.

References
1. Simmons EG, Roberts RG. 1993. Mycotaxon 48, 109-140.
2. Dickens JSW, Cook, RTA,1995. OEPP/EPPO Bulletin 25, 651-659.