Harry Wheeler

Ultrastructural study of penetration of maize leaves by Colletotrichum graminicola.

Abstract When maize leaves are inoculated with conidia of Colletotrichum graminicola the uninucleate, single-celled spores swell and undergo mitosis. In each spore a septum forms between the daughter nuclei and one cell of the resulting two-celled structure germinates to produce an appressorium with an electron-dense outer cell wall. Prior to penetration a new, inner, electron-lucent wall forms around the basal portion of the appressorium. During penetration, which occurs 6 to 9 h after inoculation, the outer dense wall of the appressorium disappears, without evidence of mechanical disruption, in the area of the developing infection peg. At all stages of infection the infection peg and the hypha which develops from it are enveloped by a distinctive, electron-lucent cell wall which is continuous with the inner wall of the appressorium. During penetration only the cuticular layers of the host cell wall are depressed inward. Dense host reaction products (papillae) are formed and the plasma membranes are disrupted before cell walls are completely penetrated and also in cells adjacent to those invaded by the fungus. The infection process is completed within 12 h by the formation of a septum between the appressorium and the swollen infection hypha.

PINOCYTOSIS AND MEMBRANE DILATION IN URANYL-TREATED PLANT ROOTS.

Electron-dense crystals formed in plant roots exposed to uranyl acetate have been used to identify binding sites and to follow the pinocytotic uptake of uranyl in the oat rootcap. Before uranyl enters the protoplast, the plasmalemma is greatly dilated. After uranyl is sequestered in vacuoles, the tonoplast is similarly dilated.

Factors affecting victorin-induced root cap cell death: temperature and plasmolysist

Abstract Victorin, the pathotoxin produced by Helminthosporium victoriae, causes false plasmolysis and death of root cap cells, and this effect is selective for oat cultivars susceptible to the fungus. The development of a procedure for the isolation of root cap cells made possible a study of victorins effects on populations of intact cells uniformly exposed to the toxin. Isolated root cap cells from resistant and susceptible cultivars were used to examine the effects of temperature and plasmolysis on victorin-induced cell death. Cell viability was determined by the ability to stain with fluorescein diacetate. Victorin-induced cell death was highly temperature dependent with a temperature coefficient of 3·5. At 10°C 96 to 98% of the cells remained alive for over 30 days when incubated in up to 50 units of toxin per ml. Plasmolysis also inhibited cell death; this protective effect was dependent upon solution tonicity. Non-permeating media, mannitol and sorbitol, protected more than sucrose in which cells slowly deplasmolyzed. Urea, a rapidly-permeating osmoticum, did not delay victorin-induced cell death. The modification of victorins lethal effects by temperature and plasmolysis may explain variability in reported effects of the toxin on isolated protoplasts.

Ultrastructure of penetration by Helminthosporium maydis

Abstract Penetration by Helminthosporium maydis race T into susceptible leaves from Texas male sterile and resistant ones from normal plants was studied with four different corn inbred lines. Six hours after inoculation, appressoria-like structures were present but were rare, and no evidence of penetration beneath such structures was found. All penetrations seen 6 h after inoculation took place between epidermal cells, and at least two-thirds of these occurred between cells adjacent to stomates. Breaks in the cuticle above subsidiary cells of the stomatal apparatus may account for the high frequency of penetration in this area. Eight to 10 h after inoculation, miniature infection cushions had formed at initial points of penetration, and from these, hyphae radiated out beneath the cuticle of epidermal cells. Haustoria-like branches produced by these subcuticular hyphae appeared to function in secondary infection. Hyphae of the fungus were seen occasionally within stomatal openings (apparent stomatal penetration), but these were consistently associated with penetrations which had occurred between nearby epidermal cells. No differences were observed at any stage of penetration or initial colonization between Texas male sterile and normal plants of the same inbred or among the four inbreds studied. These results suggest that resistance to this pathogen develops only after infection has occurred and that the selective pathotoxin produced by race T does not play an important role in the infection process.

Mechanisms of Pathogenesis

Pathogenesis can be viewed as a battle between a plant and a pathogen refereed by the environment. A small change in a single environmental factor can decide the outcome of the plant-pathogen struggle. For example, lines of wheat which carry a gene known as Sr 6 are highly resistant to race 56 of stem rust when grown at 20° C. If the temperature is increased to 25° C, the plants become highly susceptible to the same race of this pathogen (Daly, 1972). Single biological components of the environment can be equally decisive. Damage to tomato plants caused by Sclerotium rolfsii can be greatly reduced by adding a second fungus, Trichoderma harzianum, to the soil. In this case, biological control of the disease has been attributed to the ability of Trichoderma isolates to overgrow and kill S. rolfsii (Wells et al., 1972).

Association between lowered kievitone hydratase activity and reduced virulence to bean in variants of Fusarium solani f. sp. phaseoli

Abstract The activity of kievitone hydratase (KHase)—the enzyme catalysing detoxification of the phytoalexin, kievitone, to kievitone hydrate (KH)—in extracts of Fusarium solani f. sp. phaseoli -infected hypocotyls of Phaseolus vulgaris increased for several days and reached a peak at 5 days after inoculation. Several variants of the virulent isolate of F. solani f. sp. phaseoli were obtained, either following exposure of the fungus to the mutagenic chemical, N-methyl-N′-nitro-N-nitrosoguanidine, or by making transfers from naturally occurring sectors in agar cultures. These variants were monitored for pathogenicity to bean, sensitivity to kievitone and ability to cause clearing of an opaque, kievitone-amended, modified Fries agar—an assay which indicated KHase activity. All variants with reduced KHase activity were more sensitive to kievitone and were less pathogenic to bean than the virulent strain from which they were derived. No virulent isolate that lacked KHase was found. The presence of KHase in Fusarium -infected bean tissues soon after inoculation, and for several days thereafter, as well as the association between lowered KHase activity and reduced virulence, suggest that this enzyme plays a role in the development of the fungus in its hosts tissues. Furthermore, these results are consistent with detoxification of the isoflavonoid, kievitone, by F. solani f. sp. phaseoli being important to the pathogenic behavior of this fungus.

Extracellular Sheaths on Hyphae of two Species of Helminthosporium

Transmission-electron micrographs indicate the presence of a welldefined extracellular sheath, approximately 0.5 /m thick, on more than 90% of hyphae of Helminthosporium victoriae on and in oat leaves 24 h after inoculation. Similar, but less well-developed sheaths were found on hyphae of H. maydis race T on and in corn leaves 48 h after inoculation and these findings confirmed previous scanning-electron-microscope observations. On inoculated oat leaves, hyphal sheaths on nonpathogenic isolates of H. victoriae were entirely similar to those on pathogenic isolates. Hyphal sheaths are primarily products of fungal metabolism since they formed on hyphae of H. victoriae grown in distilled water.

Pathotoxin-Induced Disease Resistance in Plants

Primary leaves of bean plants treated with nonphytotoxic concentrations of the pathotoxin victorin were rendered highly resistant to two plant viruses. Leaves treated with higher concentrations of victorin became necrotic. These effects on plants that are resistant to victorin and to the fungus that produces it lend support to the hypothesis that activation of a defensive self-repair mechanism may account for resistance to this highly selective pathotoxin.

The Role of Phytotoxins in Specificity

In his lecture, Brian indicated that Phytotoxins which selectively damage plants susceptible to the pathogen involved provide the clearest examples of agents which determine specificity in plant-pathogen interactions. Five years ago, a preceding Institute was devoted entirely to the role of Phytotoxins in plant disease. In the published proceedings of the Institute, Graniti (8) addressed problems of terminology and classification of toxins associated with plant diseases. Without retracing that ground, I should like to discuss briefly two terms, pathotoxin and host-specific toxin.

Prevention of effects of victorin on oat seed germination

Abstract The germination of dormant oat seeds, sensitive to the pathotoxin victorin, was reduced from 99% for controls exposed only to water to 20% for seeds treated with 5000 units/ml of victorin for 1 h and then washed for 1 h in running tap water. However, when victorin-treated seeds were washed for 16 h or post-treated for 10 min with alkali, germination increased to 90%. The alkali post-treatment was equally effective in reducing lethal effects of victorin when applied to seeds that had been dried and stored for 2 to 3 weeks after treatment with the toxin. These results do not support the conclusion, reached in a previous study, that victorin acts on dormant, resting tissues as well as on those with an active metabolism. Instead they indicate that residual adsorbed victorin, acting only after germination has begun, is chiefly responsible for the lethal effects which have been observed. Seeds with visible signs of damage and those deliberately damaged were much more sensitive to victorin than undamaged seeds. Such damage may account for the failure of post-treatments to nullify completely the effects of victorin and also for apparent discrepancies between these results and those of a previous report.