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The pre-penetration sequence involving zoospore taxis, encystment, cyst germination and germ-tube orientation was studied by video microscopy when roots of wheat, cress or tomato were placed in suspensions of zoospores of Pythium aphanidermatum (Edson) Fitzp. The sequence was also studied in vitro, to identify the responsible factors.Several amino acids, sugars or volatile compounds elicited one of more stages of the sequence, but only glutamic acid and aspartic acid elicited all stages and thus could account for the observed behaviour on roots. Polyuronates of root surface slime also may be needed for encystment and orientation of spores, because germ-tubes emerged towards roots from a fixed point near the former position of the water-expulsion vacuole. However, identical patterns of zoospore accumulation and encystment on untreated roots and roots encased in alginate gel indicated that neither polyuronates nor specific sugar residues of root slime caused localized encystment on roots.Based on these results, a 'simplest case' model of the pre-penetration sequence on roots is proposed.

Newly-formed zoospore cysts of Pythiurn aphaniderrnaturn adhered to glass and germinated without a further trigger, but both adhesion and germination were suppressed by EGTA. Older (10 min) cysts adhered and germinated poorly unless supplied with Ca2+. Adhesion and germination were linked but separable, because young cysts germinated when dislodged from glass after 10 min, and older cysts kept in suspension germinated in response to high (7-10 mM) concentrations of Ca2+, Mg2+ or Sr2+. Other cations (Li+, Na+, K+, Ba2+, Mn2+, Fe3+, Cu3+) had no effect up to 25 mM concentration or were toxic. The naturally low germination of suspended cysts was suppressed by EGTA (chelator), La3+ or verapamil (Ca2+ entry blockers), amiloride (Ca2+ flux inhibitor), trifluoperazine or dibucaine (calmodulin antagonists), TMB-8 (intracellular Ca2+ antagonist) or A23187 (Ca2+ ionophore). Suppression by TMB-8 or A23187 was not relieved by any post-treatment. Suppression by EGTA was relieved only by divalent cations. Suppression by all other compounds was relieved only by specific L-amino acids that stimulated germination of control cysts (asparagine, aspartic acid and glutamic acid but not lysine or glutamine, where tested). Alanine relieved suppression by verapamil or dibucaine in Pythiurn dissotocum but not P.aphanidevmaturn. The findings indicated a central role of Ca2+ (presumably released during encystment) in adhesion and germination, such that zoospores would germinate autonomously by a Ca2 +-mediated cascade when they dock and encyst on roots with the fixed point of germination adjacent to the host. Amino acids are not essential but apparently synergize this process by species-specific receptor-binding which facilitates Ca2+ uptake or Ca2+ signalling.

SUMMARY Nineteen L‐amino acids, the corresponding D‐amino acids and 10 sugars were tested as elicitors of zoospore taxis, encystment and cyst germination in Pythium aphanidermatum (Edson) Fitzp., P. catenulatum Matthews and P. dissotocum Drechs. Selected compounds were also tested for promotion of germ‐tube growth and competition in zoospore taxis. Each fungus responded to a different spectrum of compounds at different stages of development; for example, in P. aphanidermatum L‐glutamine elicited taxis but not germination, whereas glucose elicited germination but not taxis. At each stage of development the fungi showed common responses to some compounds but different responses to others. For example, all fungi showed positive taxis to L‐aspartic acid, L‐asparagine, L‐glutamic acid, L‐glutamine and L‐alanine, but differed in taxis to sugars. Nevertheless, all three fungi responded to the same broad range of compounds at one stage or another. The compounds that elicited germination did not always promote growth, and D‐amino acids had no effect at any stage, indicating that taxis, encystment and germination were receptor‐mediated. However, some non‐attractant amino acids interfered with chemotaxis, to different degrees in the three fungi. The findings are discussed in relation to host‐location mechanisms, including host‐differential taxis to roots. Variations in chemoresponses of Pythium are suggested to involve receptors for a common range of compounds, differentially active at different stages of the pre‐infection sequence.

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