Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa

Background and objectives
Pre- and post-harvest losses due to disease are of great economic importance to the South African citrus industry. Compared to local distribution, long transit periods during shipping of fruit often lead to greater post-harvest losses [1]. Although it is difficult to determine the full extent of post-harvest losses due to disease, losses have been estimated to be 5-50% or more of the harvest [2]. Fungicides are the primary means of controlling post-harvest diseases [3]. Limited control can be achieved by pre-harvest protective sprays and post-harvest hot-water dips incorporating commercially used fungicides. Protective pre-harvest fungicides often leave unacceptable residues on fruit, and this may result in rejection of export consignments [1]. This, together with an increasing number of fungicide-resistant strains, and the potentially detrimental effect on the environment and human health, necessitate alternative disease measures [2]. Several antagonistic microorganisms capable of controlling a wide spectrum of citrus post-harvest pathogens are known [1], and therefore biological control is a viable alternative. Bacterial antagonists include several Pseudomonas spp., Bacillus subtilis, Bacillus pumilis and yeasts which include Pichia guiliermondii (Wickerham), erroneously identified as Debaryomyces hansenii (De Bary) Arnaud [2]. This study was conducted to screen bacteria and yeasts, originally isolated from citrus leaf surfaces, for antagonism against pre- and post-harvest citrus pathogens and to select potential strains for future biocontrol.

Materials and methods
Leaves of citrus (Citrus sinensis) from a chemically unsprayed orchard at Letaba Estates, Tzaneen were used to isolate potential bacterial and yeast angatonists. Each isolate was preliminarily screened in vitro against Alternaria citri, Colletotrichum gloeosporioides, Diplodia natalensis, Fusarium oxysporum, Guignardia citricarpa, Penicillium digitatum, Penicillium italicum and Trichoderma viridae. Isolates with in vitro antagonistic abilities against all these pathogens were selected for secondary in vivo screening against P. digitatum. Valencia fruit were prick-wounded with micro-needles and pre-inoculated with the pathogen by dipping in pathogen spore suspension (106spores/ml), 24 h prior to dipping in antagonist suspension (107cells/ml). Disease development was determined after 3 weeks' incubation in humid chambers.

Results and conclusions
In vitro and in vivo evaluation of bacteria and yeasts as potential antagonists from citrus have been described [2]. This is, however, the first report in which bacteria and yeasts from citrus leaf surfaces have been isolated and their antagonistic potential evaluated against eight citrus pathogens in South Africa. The most inhibitory bacteria belonged to the genus Bacillus, with the most effective bacterial isolates identified as B. licheniformis. B. licheniformis was previously shown to be antagonistic in vitro against a range of plant pathogens, but this is the first report on its antifungal activity against A. citri, C. gloeosporioides, D. natalensis, F. oxysporum, G. citricarpa, P. digitatum, P. italicum and T. viridae. In addition, B. licheniformis significantly controlled artificial infections of P. digitatum on Valencia citrus fruit.

This investigation has shown that citrus leaf surfaces are inhabited by various bacterial and yeast epiphytes inhibitory towards citrus pathogens. Bacillus spp. formed a major component of the microflora and proved to be highly effective as a potential biocontrol against P. digitatum.

1. Korsten L, De Villiers EE, Wehner FC, Kotze JM, 1993. Post harvest handling of tropical fruit, pp. 172-185.
2. Chalutz E, Wilson CL, 1991. Postharvest News and Information 2, 169-173.
3. Wilson CL, Wisniewski ME, 1989. Annual Review of Phytopathology 27, 425-441.