James R. Aist

Hyphal tip cell ultrastructure of the fungus Fusarium: improved preservation by freeze-substitution.

Ultrastructure of freeze-substituted hyphal tip cells of Fusarium acuminatum was compared to that after conventional chemical fixation. As a result of improved preservation by a modified freeze-substitution procedure, we document here: (i) an extensive system of smooth membrane cisternae with electron-dense contents, including tubules and single, fenestrated cisternae; (ii) two types of apical vesicles with a homogeneously electron-dense interior; (iii) an extensive system of microtubules which extend well into the apical dome; (iv) four distinct layers in the apical “primary” cell wall, with basipetal thickening due almost exclusively to enlargement of the outer cell wall layer; (v) hexagonal microvesicles; and (vi) a smooth profile of all cellular membranes and most organelles.

Invasion of Plants by Powdery Mildew Fungi, and Cellular Mechanisms of Resistance

Cereal powdery mildews are among the most intensely studied of plant diseases. Consequently, much of what is known about early host-parasite interactions has come from studies of these diseases. Most of this information has already been adequately reviewed within the past decade (Aist, 1981, 1983; Aist and Gold, 1987; Bushnell, 1982; Kunoh, 1982, 1987; Sherwood and Vance, 1982) and will not be emphasized here. We will focus, instead, on results of more recent research into early interactions between Erysiphe graminis and its cereal hosts, especially those interactions related to disease resistance, to provide an update on the state of our knowledge in this host-pathogen system.

Alternative disease control agents induce resistance to blue mold in harvested 'red delicious' apple fruit.

ABSTRACT Alternative control agents, including UV-type C (254 nm) irradiation, yeasts antagonistic to fungal growth, chitosan and harpin, were evaluated for their ability to induce resistance in cv. Red Delicious apple fruit against postharvest blue mold caused by Penicillium expansum. Freshly harvested and controlled atmosphere (CA)-stored fruit were treated with these agents at different doses and concentrations or with paired combinations of the agents. Treated fruit were inoculated with P. expansum 24, 48, or 96 h following treatment, and stored at 24 degrees C in the dark. The fruit were evaluated for development of disease every 2 days for 14 days by measuring the diameter of lesions that formed. The area under the disease progress curve (AUDPC) was calculated and analyzed statistically. All treatments were effective in reducing the AUDPC; UV-C was most effective, followed by harpin, chitosan, and the yeasts, respectively. Regardless of treatment, fresh fruit were more responsive to treatments than CA-stored fruit. There was a clear time-dependent response of the fruit to the treatments, in which treatments applied 96 h before inoculation provided the best results. In a few situations, the combinations of agents did provide an additive effect, but no synergistic effects were detected. Moreover, disease severity in fruit treated by any combination was markedly better than that in the controls. Although the combinations of treatments was overall less effective than the single treatments, they did provide significant reductions of the progress of disease in comparison with the controls. Because the fungus did not come into contact with any of the control agents, this study showed conclusively that the agents studied were able to induce resistance in the fruit rather than merely inhibit the pathogen directly. It also showed, for the first time, that harpin is able to induce resistance in harvested apple fruit. The use of these control agents may minimize the costs of control strategies and reduce the risks associated with the excessive use of fungicides in harvested apple fruit.

Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser

We have compared two different laser-induced optical light traps for their utility in moving organelles within living animal cells and walled fungal cells. The first trap employed a continuous wave neodymium-yttrium aluminum garnet (Nd-YAG) laser at a wavelength of 1.06 micron. A second trap was constructed using a titanium-sapphire laser tunable from 700 to 1000 nm. With the latter trap we were able to achieve much stronger traps with less laser power and without damage to either mitochondria or spindles. Chromosomes and nuclei were easily displaced, nucleoli were separated and moved far away from interphase nuclei, and Woronin bodies were removed from septa. In comparison, these manipulations were not possible with the Nd-YAG laser-induced trap. The optical force trap induced by the tunable titanium-sapphire laser should find wide application in experimental cell biology because the wavelength can be selected for maximization of force production and minimization of energy absorption which leads to unwanted cell damage.

Inhibition by 2-deoxy-D-glucose of callose formation, papilla deposition, and resistance to powdery mildew in an ml-o barley mutant☆

Abstract Resistance to powdery mildew in the ml - o mutant barley line, Riso 5678/3 ∗ Carlsberg II, is characterized by a high frequency of early formed, callose-containing papillae. Treatment of ml - o resistant barley coleoptiles with 10 −5 m 2-deoxy-D-glucose, an inhibitor of callose formation in plants, resulted in a decrease in papilla frequency from 92-41% and an increase in penetration efficiency from 15–78%. Time-course analysis showed that papilla formation was also delayed by 2-deoxy-D-glucose treatment and that sites with late-forming papillae were penetrated by the fungus whereas sites with early-forming papillae were not. These results support previous evidence that early papilla formation is the mechanism of ml - o resistance. Use of the callose-specific dye, sirofluor, showed that nearly all papillae in both the controls and the 2-deoxy-D-glucose-treated cells contained callose, suggesting that callose formation may greatly facilitate papilla deposition in this ml - o barley mutant. Papilla frequency also decreased in the susceptible barley isoline, and an increase in penetration efficiency occurred at 4 × 10 −5 m 2-deoxy-D-glucose. We infer that the mutation at the ml - o locus enhanced a constitutive, but originally ineffective, resistance mechanism, namely papilla formation.

A complete inventory of fungal kinesins in representative filamentous ascomycetes

Complete inventories of kinesins from three pathogenic filamentous ascomycetes, Botryotinia fuckeliana, Cochliobolus heterostrophus, and Gibberella moniliformis, are described. These protein sequences were compared with those of the filamentous saprophyte, Neurospora crassa and the two yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Data mining and phylogenetic analysis of the motor domain yielded a constant set of 10 kinesins in the filamentous fungal species, compared with a smaller set in S. cerevisiae and S. pombe. The filamentous fungal kinesins fell into nine subfamilies when compared with well-characterized kinesins from other eukaryotes. A few putative kinesins (one in B. fuckeliana and two in C. heterostrophus) could not be defined as functional, due to unorthodox organization and lack of experimental data. The broad representation of filamentous fungal kinesins across most of the known subfamilies and the ease of gene manipulation make fungi ideal models for functional and evolutionary investigation of these proteins.

Evidence that molecular components of papillae may be involved in ml-o resistance to barley powdery mildew☆

Abstract We demonstrate that papillae in a resistant barley isoline, Riso 5678 (R), contain a light-absorbing component that is not characteristic of papillae in the corresponding susceptible isoline. In resistant coleoptiles incubated on a standard Ca(NO 3 ) 2 solution, penetration attempts by the powdery mildew fungus were typically unsuccessful and papillae contained the light-absorbing component, whereas in the susceptible coleoptiles the reverse occurred. Chlortetracycline strongly inhibited both the resistance to penetration and the incorporation of the light-absorbing component into papillae in the resistant isoline but had little or no effect in the susceptible isoline. When data from all treatments and genotypes were combined, the penetration efficiency was 5% where papillae contained the light-absorbing component, 99% where they did not, and 47% where the amount of light absorbance was intermediate. Autofluorescence, UV absorbance and lacmoid staining all suggested that the light-absorbing component is rich in phenylpropanoids. Acid fuchsin revealed a basic staining material in papillae in resistant but not in susceptible coleoptiles, and in resistant coleoptiles the basic staining material was specifically associated with penetration failures. Thus, the light-absorbing component and basic staining material in papillae may be molecular components of the ml-o resistance mechanism.

Pre- and Post-harvest Harpin Treatments of Apples Induce Resistance to Blue Mold

Harpin was studied for its ability to induce resistance in apple fruit to blue mold caused by Penicillium expansum after harvest. Red Delicious fruit were harvested and sprayed with harpin at 0, 40, 80, and 160 mg/liter applied as a commercial formulation. At 48, 96, and 144 h after treatment, fruit were wound inoculated with spore suspensions of P. expansum at 103, 5 × 103, or 104 spores/ml. The diameters of the resulting lesions were directly proportional to the inoculum concentration. Fewer fruit treated with harpin became infected relative to the controls, and disease progress was considerably reduced. In a second experiment, apple trees of the cultivars McIntosh, Empire, and Red Delicious were sprayed with different concentrations of harpin 8 or 4 days before harvest. Fruit were harvested, wounded, inoculated with the fungus, and stored in a commercial cold room. Fewer fruit treated with harpin became infected compared with the controls. Greater control resulted from the higher concentrations of harpin, but no difference in control occurred as a function of interval between the spray time and harvest. Spraying apple trees with harpin a few days before harvest is a promising strategy for reducing blue mold decay in storage.

Effects of calcium nitrate and chlortetracycline on papilla formation, ml-o resistance and susceptibility of barley to powdery mildew

Penetration sites of Erysiplie graminis f. sp. hordei on partially dissected coleoptiles of near-isogenic resistant and susceptible ml-o barley were examined in vitro to study the effects of Ca(NO 3 ) 2 and the Ca 2+ chelator and ionophore, chloretracycline, on penetration efficiency and papilla formation. Compared to water controls, incubation on 10 m M Ca(NO 3 ) 2 allowed a marked expression of ml-o resistance. Time-course studies of coleoptiles incubated on Ca(NO 3 ) 2 showed that papillae, on the average, were initiated in the resistant isoline 1·0 h before penetration pegs; in the susceptible, 0·7 h after. These differences were coincident with a penetration efficiency of 5% and 62% and a papilla frequency of 87% and 77% in the resistant and susceptible coleoptiles, respectively. Growth rates of papillae in the two isolines were not significantly different during the first 2 h of development on Ca(NO 3 ) 2 ; however, papillae in the resistant isoline were twice the size of those in the susceptible isoline at the time of penetration peg initiation. In comparison to Ca(NO 3 ) 2 , incubation on Chlortetracycline delayed papilla initiation relative to penetration peg initiation by 2·2 h on average, decreased papilla frequency nearly 30% and increased penetration efficiency to 74% in the resistant isoline, but caused no significant effects on these parameters in the susceptible isoline. These results suggested that earliness of the papilla response in the resistant isoline is an important component of the disease resistance mechanism, that Chlortetracycline neutralized the mechanism by delaying the average time of papilla initiation, and that ml-o resistance may be calcium-dependent.

The role of papillae in resistance to powdery mildew conditioned by the ml-o gene in barley. I correlative evidence

Abstract Resistance to powdery mildew conditioned by the ml-o gene in barley is expressed mainly during primary penetration by the pathogen. Since barley epidermal cells usually respond to attempted penetration by the deposition of a papilla at the encounter site, the possible association of papilla formation with ml-o resistance was examined. Information on penetration efficiency, papilla frequency and papilla diameter was obtained using coleoptiles and leaves of near-isogenic resistant and susceptible barley breeding lines. Increased papilla frequency and, usually, increased papilla diameter were associated with a much lower penetration efficiency on resistant lines. Moreover, penetration failures in the presence of papillae were more frequent on resistant than on susceptible lines. These results establish a clear coincidence between resistance to penetration and an enhanced papilla response in ml-o barley and set the stage for the companion experimental study.