1Department of Microbiology and Plant Pathology; 2Department of Botany, University of Pretoria, Pretoria 0002, South Africa

Background and objectives
Colletotrichum dematium (Pers) Grove is the causal agent of a new anthracnose disease of cowpea (Vigna unguiculata) in South Africa [1]. Stem symptoms on cowpea begin as tan brown discolorations that enlarge, becoming dark to purplish-brown. Sunken, necrotic lesions and small black acervuli are visible on the stems. As yet, no studies have been conducted on the infection process and structures of Colletotrichum dematium on cowpea. The main aim of this study, therefore, was to gain knowledge about the nature of the infection processes of C. dematium on cowpea stems.

Materials and methods
Inoculum was prepared by pouring sterile distilled water over C. dematium cultures and agitating. Stems of 3-week-old cowpea seedlings were painted to run-off with a spore suspension of 1x105 conidia ml-1. Inoculated plants were covered with plastic bags to maintain a high humidity and kept at 22C in a greenhouse. To study the infection process and quantify pre-penetration structures, stems were cut into 20-mm-long segments at various time intervals after inoculation. The epidermis of these segments was stripped, mounted on microscope slides, stained with lactophenol blue and studied under a photomicroscope. To study pre-penetration behaviour, 5-mm segments were cut from the inoculated plants at various time intervals after inoculation, and prepared for scanning electron microscopy using the traditional chemical method.

Results and conclusions
Conidia began germinating at 6 h post-inoculation (h.p.i.), forming appressoria directly or at the ends of germ tubes. Upon germination, two or three septae became visible on the previously aseptate conidia. Germ tubes developed from random points on the conidia. The majority of conidia formed only one germ tube, but occasionally more than one was formed. Appressoria and germ tubes formed directly above epidermal cells with no specific orientation to stomata, whether open or closed. Appressoria were bulbous and either sessile, or subtended from the tips of short or longer germ tubes. By 14 h.p.i. appressoria had melanized and penetration of host tissue had begun. Penetration by infection pegs occurred directly through the plant cuticle and cell wall. At 20 h.p.i., infection vesicles formed in epidermal cells. Thick, knotty primary hyphae formed from these vesicles and entered adjacent cells. The primary hyphae branched and grew rapidly until the initially infected cells were packed with convoluted mycelium. At ca 40 h.p.i. C. dematium produced secondary hyphae which were highly branched and grew extensively inter- and intra-cellularly. After 48 h.p.i., light brown lesions began to appear on the stem, due to the invasion of secondary hyphae into cells and subsequent cell necrosis. Acervuli with one or two melanized setae were visible on lesions by 70 h.p.i. Mature, sporulating acervuli became visible at ca 80 h.p.i. By 100 h.p.i., brown lesions also began to appear on leaves of inoculated plants. This study reveals that C. dematium is a hemibiotrophic pathogen. An initial symptomless biotrophic phase is followed by a necrotic phase during which light brown lesions appear on the stem. The infection process of C. dematium on cowpea stems is generally consistent with that of other Colletotrichum spp. on various hosts, including cowpea. Much is known about the cytology of infection of other Colletotrichum spp., even on cowpea, but in contrast this is one of few studies on the infection process of C. dematium and will broaden the knowledge of infection processes of Colletotrichum spp., especially on legumes.

1. Smith JE, Aveling TAS, 1997. Plant Disease 81, 832.