Robert N. Goodman

Host-Specific Phytotoxic Polysaccharide from Apple Tissue Infected by Erwinia amylovora

A toxin isolated from apple fruit tissue infected by Erwinia amylovora is 98 percent galactose in polymeric form, 0.375 percent protein, and has an average molecular weight of approximately 165,000. Young shoots of rosaceous, but not nonrosaceous, species wilt in a manner characteristic of the disease when placed in toxin solutions with concentrations as low as 10 micrograms per milliliter. Varieties of apple and pear susceptible to Erwinia amylovora wilt in 1 to 3 hours, whereas resistant varieties display symptoms 12 to 24 hours after treatment.

Ultrastructural changes in tobacco undergoing the hypersensitive reaction caused by plant pathogenic bacteria

Abstract The hypersensitive reaction induced by either Erwinia amylovora or Pseudomonas pisi is indicative of widespread damage to membranes of subcellular organelles and particulate cellular components in tobacco leaf tissue. Plasmalemma, tonoplast, as well as the bounding and internal membranes of chloroplasts and mitochondria and the external membrane of microbodies, are profoundly deranged in 7 h. Cytoplasm, groundplasm of chloroplasts, mitochondria and microbodies and membrane-attached and free ribosomes are similarly affected. The damage coincides with and is probably the cause of the observed tissue collapse and rapid loss of electrolyte-laden water. The presence of few bacterial cells (not more than 4 × 106 cells/leaf disk 1 cm in diameter) cause the observed symptoms which appear, at the ultrastructural level, to be unique.

Resistance mechanisms of apple shoots to an avirulent strain of Erwinia amylovora

Abstract Avirulent bacteria of Erwinia amylovora were localized within 5 mm from the point of inoculation on Jonathan apple petioles. The failure of the bacteria to translocate from petiole to stem was due to three host defense mechanisms. In the first, bacteria that entered the intercellular spaces of ground tissue were localized by a hypersensitive reaction characterized by degeneration of subcellular organelles in parenchyma cells, which prevented further spread of the bacteria. In the second, bacteria that entered petiolar xylem vessels were prevented from proceeding to and entering the xylem vessels of stem tissue by an agglutination reaction in situ. In the third, avirulent bacteria observed in xylem parenchyma cells were digested within the vacuoles, a phenomenon that seemed analogous to phagocytosis commonly observed in animal cells.

Prevention of bacterially induced hypersensitive reaction by living bacteria

Abstract Infiltration of tobacco leaf with the low concentration (5 × 10 5 cells/ml) resulted in decreased capacity of the tissue to develop hypersensitive reaction (HR) against higher concentration of the same bacterium. Development of HR against 5 × 10 6 cells/ml (the lowest HR-inducing concentration) was prevented as early as 3 h after inoculation with 5 × 10 5 cells/ml. HR induction with bacterial concentration up to 10 8 cells/ml could be prevented by preinoculation of the tissue with 5 × 10 5 cells/ml twice at 24-h intervals. Preventive effect was light dependent and is translocatable to the untreated surrounding tissue.

Erwinia amylovora cell wall receptor for apple agglutinin

Lipopolysaccharide (LPS) was isolated from an acapsular strain of Erwinia amylovora . This “crude LPS” was divided into 2 fractions by elution from a Sepharose CL-6B column. These were designated the low molecular weight form (retained by the column) and the high molecular weight form (not retained by the column). When assayed by rocket electrophoresis, only the low molecular weight form served as a receptor for the apple agglutination factor (AF) and cross-reacted with antiserum prepared to E. amylovora . Mild acid hydrolysis of crude LPS gave a milky suspension that was divided into a pellet (Lipid A) and a supernate (O-side chain plus core) by centrifugation. The supernate, which contained all of the receptor for AF, was further divided into O-side chain and core by elution from a Bio-Gel P-30 column. Only those fractions which would be expected to contain core region of LPS interacted with AF in agarose gel to form precipitant peaks.

The effect of salts on the viscosity and wilt inducing capacity of the capsular polysaccharide of erwinia amylovora

Water solutions of the capsular polysaccharide (EPS) of Erwinia amylovora exhibit long flow times (t) in a kinematic viscometer. Addition of salts to the solutions greatly decreases the flow times. The salt-induced decrease in flow time is directly related to the ionic strength of the solution and independent of the ionic species present. The salt effect does not result from a change in the molecular weight of the EPS. Addition of NaCl, at concentrations sufficient to reduce the flow time, decreases or eliminates the capacity of EPS solution to cause wilt in the cut shoot assay. Treatment of EPS with either of two depolymerase phages decreases both t and the molecular weight of the EPS (from 100 × 106 D to less than 4 × 104 D). Such phage-produced fragments (ψdp) retain their capacity to cause wilt in the cut shoot assay but, like EPS, lose this ability in the presence of salts. Radiolabeled EPS and ψdp is retained at the end of the cut shoot when wilt occurs but is distributed throughout the shoot when wilt is inhibited by salt.

Comparison of the extracellular polysaccharides produced by Erwinia amylovora in apple tissue and culture medium

The extracellular polysaccharides (EPS) produced by three isolates of Erwinia amylovora during growth on apple fruit (amylovorin) or semisolid medium were found to be indistinguishable upon elution from ion-exchange and gel filtration columns. The polysaccharide-preparations carried the same complement of antigenic determinants and all were substrates for the enzymes produced by two depolymerase phages. The amylovorins and EPS were distinguished by the shape of the peaks formed when each was subjected to crossed immunoelectrophoresis. The amylovorins produced a broader, shouldered peak and exhibited a characteristic distortion of one precipitate band. The amylovorins and EPS produced different standard curves with a colorimetric reagent. The amylovorins and EPS were found to be antigenically indistinguishable from lipopolysaccharide (LPS) when compared by tandem crossed immunoelectrophoresis. The amylovorins and EPS were shown not to be contaminated with LPS because their antigenic determinants were present in a substrate for the extracellular polysaccharide-specific depolymerase phages.

Ultrastructural changes in tobacco thylakoid membrane protein caused by a bacterially induced hypersensitive reaction

Abstract Structural proteins (SP) were obtained from chloroplast thylakoid membranes of tobacco leaf tissues 6 h after infiltration with either water or 108 cells/ml of Erwinia amylovora. Lipids were extracted from the thylakoid membranes of normal tobacco leaf tissues. Each of these three preparations was solubilized with sodium dodecyl sulfate and dialyzed against dilute beta buffer containing 20 m m -Mg2+. The SP from water-infiltrated tissues formed spherical aggregates whereas the SP from bacteria-infiltrated tissues aggregated into irregular clumps. The extracted lipid coalesced to form globules. Thin sections of the lipid revealed alternating light and dark bands. When solubilized lipid and the solubilized SP from water-infiltrated tissues were mixed and dialyzed, membrane-like structures approximating those of thylakoids in thickness were formed. Very few membrane-like structures were formed when the lipids were mixed with SP from bacteria-infiltrated tissues. Since the lipid used in these two experiments was identical, the inability to form membrane-like structures was attributed to the alteration in SP properties by hypersensitive reaction-induding bacteria.