2.9.2
USING ENDOPHYTIC FUNGI IN AMARANTHUS HYBRIDUS AS BIO-INDICATORS OF HOST VIGOUR

WJ SWART1, JT BLODGETT1, SVDM LOUW2, WJ WEEKS3 and CM BENDER1

Departments of 1Plant Pathology, 2Zoology and Entomology, University of the Free State, Bloemfontein, South Africa; 3North-West Agricultural Research Institute, Potchefstroom, South Africa

Background and objectives
The genus Amaranthus includes species cultivated for their grain and/or leaves in several developed and developing countries [1]. Amaranthus hybridus is a nutritious, fast-growing vegetable crop, with the potential for increased use in semi-arid areas. A sustainable pest and disease management approach for the cultivation of A. ;hybridus requires an understanding of the abiotic and biotic factors that affect this crop. Plant-fungal relationships are especially relevant, since they are known to play an important role in the biology of cultivated plants. Endophytic fungi can affect various ecological and physiological processes [2]. True endophytic fungi live entirely within living plant tissues but do not elicit disease symptoms. They can have a symbiotic relationship with their host by acting as bioregulators of plant health or by providing protection from pathogenic fungi and herbivorous insects. Latent-infecting fungi are endophytic fungi that elicit disease symptoms only when induced by environmental conditions or host maturity. The objectives of this study were to determine how endophytic fungi infect Amaranthus and their colonization pattern within leaf tissues, quantify frequencies of endophytic fungi associated with A. ;hybridus, identify individual species of fungi, and determine the influence of soil amendments and irrigation on fungal occurrence.

Materials and methods
For surface observations, leaf sections from 5-month-old A. ;hybridus plants were examined using scanning electron microscopy and the presence of fungal mycelium in the tissue was confirmed using light microscopy. Five-month-old A. ;hybridus plants were also sampled from each of four plots comprising the following treatments: the addition of wood ash and no irrigation manure and irrigation, or fertilizer and irrigation; control plots were not amended nor irrigated. Five plants were collected from each treatment and their biomass determined. Ten asymptomatic leaves, petioles, and roots from each of five plants per treatment were surface disinfected to remove exterior microorganisms. Ten small sections of tissue were removed from each leaf, and five from each petiole and root. Sections were placed on corn-meal agar plates containing streptomycin sulphate, incubated for 5 ;days and resulting fungal colonies counted and transferred onto separate agar plates for identification.

Results and conclusions
Light microscopy revealed Alternatia alternata spores germinating on the surface and only entering leaves through stomata. The leaves had an internal net of hyphae surrounding mesophyll tissue, with no host cell penetration, and no visible defence response by the plant. Significant differences in recovery of fungi occurred among the four soil treatments (P<0.001 for all tissues). The highest recovery rate occurred from the fertilized and watered treatment for all tissues (mean 97% for all tissues). Recovery rate from leaves was positively correlated with biomass (r=0.60, P<0.001). Differences in recovery rates also occurred among plant tissues (P<0.001). Leaf sections yielded fungi 98% of the time, petioles 84%, and roots 61%. Differences in species composition occurred among plant tissues (P< 0.001) and among soil treatments (P<0.001). The most common species isolated was Alternaria alternata comprising 90% and 70%, respectively, of species isolated from leaves and petioles. Different fungal genera predominated in the roots with A. ;alternata comprising only 27% of the fungi recovered. These results confirm that A. ;alternata is able to infect and colonize A. ;hybridus leaf tissue in a manner consistent with other endophytic fungi. They also indicate that endophytic colonization is influenced by soil conditions and that their frequencies may serve as a useful bioindicator of host vigour in sustainable crop production.

References
1. Harlan JR, 1992. Crops and Man, 2nd edn.Madison: American Soc. Agronomy.
2. Petrini O, 1996. Endophytic Fungi in Grasses and Woody Plants. St Paul: APS Press, pp. 87-100.