INTRODUCTION 



After a plant pathogen, such as a rust, enters its host, colonization and establish- 

 ment are important to the continuation of its life cycle. Cronartium ribicola, the 

 blister rust fungus, enters its pine host through needle stomata and from the substomatal 

 cavity grows passively within intercellular spaces to colonize the host tissues. 

 Intercellular fluids may provide substances required by this heterotroph, which would 

 negate the necessity to enter the host cells. Instead, the fungus establishes itself 

 by adhering to the exterior surfaces of host cells and then developing special hyphae, 

 haustoria, that penetrate the host cell walls. Successful haustoria penetration occurs 

 only when the rust fungus produces enzymes that are capable of hydrolyzing the cellulose, 

 pectin, and protein constituents of the host cell. 



Rust fungi are obligately parasitic and consequently dependent on the survival 

 of their hosts, therefore, teleologically they must be weak pathogens with sensitively 

 controlled host destroying mechanisms (Wood 1967). It follows that any enzyme systems 

 used by the pathogen to modify and to penetrate the host cell walls must be highly 

 discriminatory and operative only in a local area (Albersheim and Anderson-Prouty 

 1975). Studies using the electron microscope have shown the wall texture to be 

 modified in the vicinity of contact between rust mycelium and pine cell walls (Welch 

 and Martin 1975). Apparently the enzymes neither attack constituents that would kill 

 the pine cell nor produce products in quantities that exceed detrimental threshold 

 values for either the pathogen or host. 



Earlier work illustrated the circumstantial evidence for the involvement of 

 pectinases in the blister rust disease of western white pine (Martin 1967) . Analysis 

 of infected bark revealed a significant depression of pectic substances when compared 

 to noninfected bark of the same tree (Welch and Martin 1974). The objective of the 

 study reported here is to assess in vitro the tolerance of pine cells or rust mycelium 

 to pectic compounds and the role of these substances in the host parasite relationship. 



MATERIALS AND METHODS 



Cultures of pine tissue were established on an agar medium as described by 

 Harvey (1967). All explants used in this study were selected for uniformity in size, 

 green color, and evidence of cell proliferation. Seven three-week-old cultures were 

 submerged in 5 ml each of glass-distilled water solutions (0.001, 0.005, 0.01, 0.025, 

 0.05, 0.1, 0.5 percent concentrations) of citrus pectin, sodium polypectate, or 

 pectic acid. The cultures were then observed at weekly intervals for continued 

 proliferation, color changes, and signs of organ primordia. 



Numerous isolates from 36 different infections of C. ribicola were established 

 on agar media according to the procedures used by Harvey and Grasham (1969). Two 

 isolates (the fastest and slowest growing) were selected for study (fig. 1) . 



The isolates were challenged with pectic compounds representing different molecular 

 structural complexity by placing solutions of each compound in a small well cut into 

 agar media (Dingle and others 1953). Six wells were positioned in a circle and 

 spaced equidistant from the center of a petri dish and from each other. The wells 

 were filled in the order of decreasing structural complexity with 1 percent aseptic 

 solutions of citrus pectin, sodium polypectate, polygalacturonic acid, pectic acid, 

 and glass-distilled water. Twenty-four hours later a 2 mm piece of agar cut from the 

 periphery of an isolate was transferred to the center of each of 20 dishes. Radial 

 growth from the center of the inoculum toward each well was measured weekly for 

 8 weeks. All cultures were incubated at 20°+2°C under 3,400 lux of fluorescent light 

 in growth chambers. 



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