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Studies On the Culturing and Parasitism of Plant-Parasitic Nematodes, in Particular Ditylenchus Dipsaci and Aphelenchoides Ritzeiwabosi On Alfalfa Tissues

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Ditylenchus dipsaci and Aphelenchoides ritzemabosi reproduced rapidly on alfalfa callus tissue grown on a nutrient agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Cultures inoculated with 50 nematodes and kept at 22° C contained 40,000 to 80,000 individuals after two months. Pratylenchus zeae and P. penetrans reproduced well and Tylenchorhynchus capitatus and Hoplolaimus coronatus slowly on callus. Four of these species were maintained alive in such cultures for two years without subcultures. The histopathology of D. dipsaci and A. ritzemabosi on alfalfa seedlings grown in soil, seedlings grown aseptically on nutrient agar medium, and callus tissue grown aseptically on nutrient agar medium plus 2,4-D was examined. D. dipsaci penetrated directly through the epidermis in the bases of cotyledons of alfalfa seedlings in soil and formed cavities in cortical parenchyma within 12 hours of inoculation. Infected stems were noticeably swollen at 36 hours after inoculation. Many parenchyma cells were destroyed and others distorted in galled tissue, particularly in young, actively growing shoots. Only cavities were formed in roots of infected seedlings growing on agar medium with no galling; shoot galls were identical with those of infected plants in soil. A. ritzemabosi fed primarily as an ectoparasite on shoots of alfalfa seedlings inhibiting apical growth and resulting in severely stunted and malformed plants, but a few lived in the mesophyll of cotyledons. Both nematode species fed and reproduced in the loose parenchyma of callus tissue, with the nematodes moving from the callus to the agar and test tube walls in older cultures. Sugars, organic acids and amino acids were examined in alfalfa tissues as used in the histological studies. Neither nematodes nor the conditions under which plants or tissues were grown affected qualitatively the sugars or organic acids, and affected only two of the 23 major amino acids identified. Gall tissue of plants infected by D. dipsaci and all callus tissue samples contained free tryptophane whereas healthy plants or those parasitized by A. ritzemabosi did not. Tissue parasitized by A. ritzemabosi contained abundant free tyrosine whereas nematode-free or D. dipsaci-infected tissues contained little or none.

Affiliations: 1: Rothamsted Experimental Station, Harpenden, Herts., England


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