Cytology Studies On Effects Of Metalaxyl On A Wild-Type Isolate And A Metalaxyl-Resistant Mutant Of Phytophthora Sojae

Phytophthora root rot of soybean (Glycine max(L.) Merr.), caused by Phytophthora sojae, is a major disease of soybean worldwide. P. sojae was A1 import quarantine fungus and quarantine fungus in China. Metalaxyl ((N-(2,6-dimeth ylphenyl)-N-(methoxyacetyl)-DL -alanine methyl ester), an acylalanine phenylamide fungicide exhibiting systemic properties, was used extensively world-wide to control a broad range of diseases caused by Oomycetes. While, because of the highly selective action at a single target site, a mutation changing the target site affinity might easily lead to a metalaxyl resistance. Thus, the research was focurs on the morphological and structural differential between the wild-type isolate and the metalaxyl-resistant mutant of Phytophthora sojae and the effects of metalaxyl on the both two isolates in vitro; the paper also reported on the infection process of the susceptible and the resistant isolate in hypocotyls of soybean seedlings grown from untreated and metalaxyl treated seeds using light and electron microscopical methods. The results were obtained as follows:1. Siginificant morphological and structural differences between of the wild-type isolate Ps-411 and the metalaxyl-resistant mutant Ps-411-M from the control. Hyphae from Ps-411 appeared smooth and slender, branching of hyphae always occurred at some distance from the hyphal tips; Hyphal cells showed dense cytoplasm with mitochondria and small vacuoles, cell wall appeared as clear distinguishable layer of uniform thickness. Hyphae of Ps-411-M appeared to grow somewhat irregularly with more branches. Thickening or twisting at some sites and the diameters of hyphae were somewhat thicker compared to Ps-411. And Ps-411-M showed dense cytoplasm with more leucoplastids and mitochondria compared to the Ps-411.2. Ultrastructral observations in the wild-type isolate Ps-411 revealed marked alterations at a low concentration of metalaxyl (1μg/mL), such as uneven growth of hyphae, excessive branching of hyphal tips, cessation of hyphal growth and bulb-like swelling of hyphal tips; mitochondria lost their distinct structure, number and size of vesicles increased, cell walls became thickened and hyphal cytoplasm grievously degenerated in presence of a high concentration of metalaxyl (10μg/mL). While, hyphae of the metalaxyl-resistant mutant Ps-411-M displayed little morphological changes after treatment with the high concentration of metalaxyl (10μg/mL). Observations indicated hyphae tips just appeared little excessive branching, hyphal cell contained a high number of lipid bodies, but fungal cell walls did not become thickened. Hyphae maintained their growth and get across the filter treated with 10μg/mL metalaxyl.3. Pathogenicity tested on hypocotyls of soybean seedlings indicated: the infection process of the wild-type isolate Ps-411 and the metalaxyl-resistant mutant Ps-411-M were approximately the same. Hyphae penetrated epidermis by infection hyphae at the junction between periclinal host cell walls. The cortex was very important for colonization and development of fungus. Intercellular hyphae penetrated adjacent cell and formed haustoria. The pathogenic fitness of Ps-411-M in hypocotyls of soybean seedlings was slightly lower compared to Ps-411. In hypocotyls grown from metalaxyl treated seeds, disease development by Ps-411 was markedly inhibated, hypocotyls of soybean seedlings resembled an incompatible interaction, whereas when infected with the metalaxyl-resistant mutant Ps-411-M derived from the compatible isolate the compatible interaction was not changed.4. Ultramicroscopical studies of the infection process showed that metalaxyl seedcoating treated caused a series of changes to the wild-type isolate Ps-411. Cysts formed malformed germ tubes which were not able to invade the host tissue. The time of germination lasted about 0.5 h longer compared to the control. Few hyphae continued to spread slowly in the intercellular space of hypocotyls. Numerous profound ultrastructural alterations in hyphae were observed: separation of plasmalemma from hyphal wall, disintegration of the cytoplasm and organelles, increasing of mitochondria and large vacuoles, pronounced thickening of fungal cell walls and encasement of haustorium-like bodies. Electron dense material was deposited in host cell walls in contact with hyphal cells, sometimes enveloped the while haustorium-like bodies, the colonization in the hypocotyls was strongly inhibited. While, the penetration or spread of hyphae in the hypocotyls of the resistant isolate Ps-411-M were not affected in metalaxyl treated compared to the control. Hyphae in fungicide treated hypocotyls showed an increased number of lipid bodies, large vacuoles and seemed to form more haustorium-like bodies than in nontreated hypocotyls. However, no thickenings of hyphal cell walls were observed, electron dense material was hardly deposited on host cell walls adjacent to hyphal cells.