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Coumestrol produced by alfalfa (Medicago sativa L.) can reduce the ovulation rate of ewes. This study isolated agronomic factors that affect coumestrol levels in alfalfa. Fungal diseases explained most differences in coumestrol. Alfalfa inoculated with Stemphylium vesicarium contained 169 ± 25.1 mg kg -1 dry matter (DM) compared with 3.4 ± 0.84 mg kg -1 DM in controls. However, in field-grown alfalfa there was a low relationship (R 2 = 0.388) between coumestrol content and visual fungal damage score which indicates a need for alternative methods of prediction. In the field, a range of cultivars all reached coumestrol levels reported to pose a risk to ewe reproductive performance (>25 mg kg -1 DM). In S. vesicarium-inoculated leaves, the modern cultivar 'Stamina 5' had 396 ± 82.4 mg kg -1 DM compared with the 40-yr-old industry standard 'Wairau' at 143 ± 35.6 mg kg -1 DM. Flowering in isolation did not produce a coumestrol response, with increases detected or not detected simultaneously in vegetative and flowering plants. Pea aphids had a minor effect on coumestrol content. When alfalfa was subjected to ~5 aphids per stem for 4 wk coumestrol increased from 2.4 ± 0.39 to 5.3 ± 0.65 mg kg -1 DM. This study highlights fungal pathogens traditionally not considered problematic in alfalfa stands due to limited effects on stand persistence and yield, as important causal agents of elevated coumestrol levels. Plant breeding to reduce their impact is recommended.

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