1Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa; 2The Mycology Group, Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark

Background and objectives
Alternaria dry core rot of Red Delicious apple cultivars is a serious post-harvest disease, not only in South Africa, but in most of the apple-producing areas of the world. Losses of 5-8% may result, as well as a marked reduction in consumer confidence. Alternaria species are most often the largest fungal contributors to this disease complex [1]. At present, Alternaria alternata is known as the main culprit, but because of the difficulties involved in the correct identification of this species, authors often only identify this fungus to genus level. During a previous study several hundred Alternaria strains were isolated as endophytes at different stages of fruit development throughout the season. Our aim was to characterize 30 of these isolates as well as reference isolates of A. alternata and A. infectoria, and to identify them to species level.

Materials and methods
Isolates were characterized based on various features. The morphological study concentrated on the three-dimensional sporulation patterns under X50 magnification [2]. In the pathogenicity test, Top Red apples were inoculated by the different Alternaria species and lesion size and type recorded after 2 weeks. A growth study was carried out on five different media: Campbell’s V8 juice agar, dichloran rose-bengal yeast extract sucrose agar (DRYES), malt extract agar, nitrite sucrose agar and potato carrot agar. Colony size and colour on DRYES were determined after 1 week. All isolates were tested for production of 59 secondary metabolites, using TLC. RAPDs as well as sequencing of the ITS1 and ITS2 regions of rDNA were also determined.

Results and discussion
Four different Alternaria species could be identified according to their three-dimensional sporulation patterns under X50 magnification. These inluded A. alternata, A. infectoria, A. tenuissima and an arborescent group that has not yet been formally described. Results from the pathogenicity test and growth study showed the same segregation of four species. A. infectoria produced no lesions, and formed white colonies on DRYES. Both A. alternata and the arborescent group produced small, black, spongy lesions and dark, olive-green colonies. Large, wet, light-brown lesions were caused by A. tenuissima isolates which formed colonies of various shades of green. Colony diameter and lesion size were of less significance. A. infectoria did not produce any mycotoxins, while all other species formed alternariol, alternariol monomethyl ether, altertoxin 1 and tenuazonic acid. A dendrogram of the 32 isolates tested for the production of 59 different secondary metabolites, including the four mycotoxins, clearly separated A. infectoria, A. tenuissima, A. alternata and the arborescent group from one another. RAPD anyalysis showed no clear groupings. Sequencing of the ITS1 region consistently separated A. infectoria from the rest owing to an insert of 29 base pairs, which was lacking in the other species. A few exceptions were found, however, which emphasizes the importance of taking all characters into account during the identification of Alternaria species. Furthermore, this study revealed that A. tenuissima, and not A. alternata as formerly believed, is primarily associated with dry core rot of apples.

1. Combrink JC, Kotze JM, Wehner, FC, Grobbelaar, CJ, 1985. Phytophylactica 14, 81-83.
2. Simmons EG, Roberts RG, 1993. Mycotaxon 48, 109-140.