Ecochemical interactions between plants and phytopathogenic Aphanomyces cochlioides zoospores

Members of oomycete genera Phytophthora, Pythium and Aphanomyces are the most devastating pathogens of plants, animals, fishes and humans. Their zoospores are believed to locate the host plant by chemotaxis and change morphologically before penetrating root tissue. The phytopathogenic oomycete, Aphanomyces cochlioides is a serious pathogen of sugar beet, spinach and some other members of Chenopodiaceae and Amaranthaceae. Understanding the mechanism of ecochemical interactions between zoospores and host and nonhost plants are fundamental requirements for developing an alternative biorational control measure for low input sustainable agriculture. Therefore, the objectives of this study were to clarify the mechanism of interactions between zoospores of the A. cochlioides and host- and nonhost-plants via their secondary metabolites. To achieve the research objectives, several independent but related experiments were carried out and important findings are summarized as follows: A. Behavioral and morphological features of Aphanomyces cochlioides Zoospores The morphological diversity of zoospores and their dynamic changes when interacting with the host materials (roots and cotyledons of spinach) were investigated by light and electron microscopy. The reniform-ovate zoospore has two heterokont flagella both inserted in a ventral groove and ornamented with characteristic hairs. The anterior flagellum possesses two rows of tripartite tubular hairs (TTHs) distributed throughout its length, whereas the posterior flagellum was ornamented with two rows of fine hairs, except its tapered terminal part. The tip of posterior flagellum has a bunch of similar fine hairs like those on the flagellum shaft. The zoospores quickly aggregated on the specific sites of host root, adhere to the root surface, encysted by shedding the flagella, germinated at a fixed point to form germ tubes and finally invaded the root tissue via the appressoria within 50-60 min. B. Interaction of Aphanomyces zoospores with host plant tissues The mode of contact, aggregation, attachment and differentiation of zoospores when interacting with the host root was investigated by SEM and light microscopy. When a zoospore approached very close to the host root, it seemed to halt, and then coiled its anterior flagellum on its body. The halted zoospore appeared to contact the host surface with its posterior flagellum, which gradually drew the encysting zoospore onto the root surface. The spore then docked precisely on the root surface at its ventral face with the help of posterior flagellum and anchored itself by releasing some adhesive materials. The adherent spore became a spherical after shedding its flagella and rapidly turned into an expanded cyst forming a smooth cyst coat around it, and finally changed into a smaller cystospore covered with a wrinkled surface. These results Indicate that A. cochiloides zoospores may use their posterior flagella for successful docking on the host surface. Some host-specific triggers may be involved in rapid developmental transitions of a zoospore to a germinated cystospore on host surface. C. Host-specific attractant, cochliophilin A triggers differentiation of zoospores Investigation on the host signal regulating developmental transitions in zoospores on spinach roots revealed that the gradient of a host-specific attractant, cochliophilin A (5) triggers encystment and germination of zoospores at a concentration likely to occur within the rhizosphere. Particle bioassay method was modified and improved to evaluate the effect of compound 5 on the differentiation of zoospores. Bioassay demonstrated that compound 5 attract zoospores at a minimum concentration of 3 x 10-to M and causes their encystment at 1 x 10E-8 to 1 x 10E-60 M. Cystospores adhering to the surface of particles germinated within 30-35 mm and those close to the particles exhibited germ tube tropism toward the particles. SEM observation revealed that the differentiation of zoospores triggered by cochliophilin A (5) is identical to that observed on spinach roots: first they shed flagella and become round smooth-surfaced cysts; then those develop a rough surface and germinate. These observations suggest that cochliophilin A (5) is indeed a host-specific plant signal, which plays essential roles in both locating host roots and initiating encystment and germination. D. G-protein mediated signal transduction pathway in Oomycete zoospores Mastoparan is a cationic amphipathic tetradecapeptide isolated from wasp venom and act as a generic activator of animal heterotrimeric GTP-binding regulatory proteins (G-proteins). Mastoparan agonised differentiation of zoospores, which was suppressed by PLC inhibitors and Ca2-f1ux regulators. Furthermore, the synthetic peptide analog Masl7, predicted not to form an amphipathic helix at the lipid interface because of the replacement of leu-6 by lys, is totally devoid of agonist activity. Taking together, these results indicate that zoospore may perceive host signal by a G-protein-coupled receptor and then translate into responses (taxis and differentiation) via phosphoinositide-Ca signaling cascades. E. Nonhost plants possess "chemical weapons" to defend Oomycetes A hypothesis of "non-susceptible plants contain chemical defences against oomycetes" was investigated by screening extracts of 200 non-susceptible plants. Almost half of the extracts were affected motility and viability of zoospores. Two plants viz., Lan nea coromandelica (halting and lytic factors) and Amaranth- us gangeticus (attractant and halting factors) were selected for further investigations to identify their active principles. The active priciple of L. coromandelica was characterized by MALDI-TOF-MS as a mixture of angular polyflavonoid tannins. Both Lannea and industrial polyflavonoid tannins showed motility inhibition followed by characteristic lysis of zoospores. In addition, the mammalian estrogens which are found in plants, for example, 17beta-estradiols (14) displayed potent repellent activity toward the trivial zoospores. Structure-activity relationships study revealed that aromatization of the A ring with a free hydroxyl group at C-3 position of a steroidal structure is necessary for higher repellent activity. Interestingly, methylation of a synthetic estrogenic compound, diethylstilbestrol (DES) (11) (yielded completely different activity i.e., both mono- and di-O-methyl ethers of DES (31 and 32) showed attractant activity. The estrogenic and repellent activities of known estrogenic compounds revealed to be correlated. F. Triggering developmental transitions in zoospores by host and nonhost metabolites The compounds responsible for attractant and halting activities in A. gangeticus extracts were revealed by chromatography to be N-trans-feruloyl-4-O- methyldopamine (6) and nicotinamide (8) respectively. Compound 6 had no inhibitory effect on zoospores whereas compound 8 immediately halted motility and caused encystment. Interestingly, the cysts induced by compound 8 regenerated zoospores instead of germinating. Compounds 6 and 8 together produced cysts that germinated within 30-35 mm just like those exposed to cochliophilin A (5). When an A. gangeticus root was immerged in a zoospore suspension, zoospore development around the root tip was halted; the spores then encysted and regenerated after 3h, suggesting that A. gangeticus root exude nicotinamide. G. Conclusion Chemotaxis and subsequent differentiation of zoospores by a host-specific plant signal (cochliophilin A, 5) appears to be essential factor for A. cochlioides pathogenesis. The nonhost plants contain some diverse "chemical weapons" to protect themselves directly from the attack of oomycetes. Discovery of some potential antagonists/inhibitor from the natural sources, and elucidate the signal transduction mechanism of the developmental transitions of zoospores by host and nonhost metabolites would offer attractive targets for alternative methods of disease control. The easy production of zoospores and their synchronous developmental changes to cystospores and subsequent transition to hyphae by host-specific plant signal in laboratory condition would allow using A. cochlioides as an excellent model system to study all aspects of cell differentiation at single-cell level as well as to elucidate molecular mechanisms involved in plant- pathogen interactions.