Histological changes in roots of potato plants with the gene H1, which confers resistance to the potato cyst-nematode Globedera rostochiensis pathotype Ro 1, are described for periods of 24 h to 10 days after invasion by second-stage juveniles of G. rostochiensis. The changes are compared with those which occur in roots of a susceptible cultivar. Experiments were carried out on potato roots growing from sprouting tuber pieces on water agar. Root cells surrounding the invading nematode undergo a hypersensitive-like response and become necrotic. Cell walls of inner cortical and stelar cells adjacent to the stylet of the invading nematode break down to form a syncytial complex but cells surrounding the syncytium become necrotic, presumably limiting movement of nutrients from plant to nematode. Ultrastructural changes of cells in the cortex and the stele are illustrated and discussed.
Race 32 of Puccinia graminis f.sp. tritici sporulated in vitro on a chemically defined medium whose amino acid and carbohydrate composition corresponded to that of the susceptible wheat cultivar Little Club. Sporulation was affected by pH, heat-treatment of the medium and the spore-density of the inoculum. Good mycelia( growth was promoted at high inoculum density, low volume of culture medium, and when no heat treatment, or only limited heat treatment, was given during sterilization of the medium. When these conditions were reversed and at pH 5·2, sporulating colonies occurred. Under optimal conditions the first urediospore bearing colonies could be detected 10 days after inoculation of the medium. Several hundred fertile colonies could be detected up to day 20. Typical teliospores were usually found for the first time at day 30. Differentiation of infection structures had a negative effect on sporulation.
Phascollinisoflavan, an isoflavonoid phytoalexin produced by Phaseolus vulgaris, was metabolized by Fusarium solani f. sp. phaseoli. A major product of this reaction, designated metabolite-1 (M-1), was isolated from fungal cultures; whereas phaseollinisoflavan could not be recovered from the fungal culture medium 21 h after its addition, the level of M-1 rose steadily between nine and 24 h. Bioassays of radial mycelial growth or of dry matter accumulation involving F. solani f. sp. phaseoli, Pythium myrioylum, Phytophthora cryptogea and/or Rhizoctonia solani indicated that M-1 was less fungitoxic than phaseollinisoflavan. In addition to its production in liquid cultures treated with phaseollinisolflavan, M-1 was also isolated from Fusarium-infected bean tissues; trace amounts of M-1 were detected two days after inoculation and levels rose to at least I mg g−1 dry weight 8 days later. Purification of M-1 involved solvent partitioning, TLC, gel filtration and gas chromatography. Partial characterization indicated that the molecular weight of this metabolite was 342, 18 mass units greater than that of phaseollinisoflavan, suggesting the addition of the elements of water to the phytoalexin.
Combined treatment of cucumber protoplasts with actinomycin D and UV irradiation inhibited host RNA synthesis by more than 95%. Cucumber mosaic virus (CMV) replication was however, not affected in either susceptible, Ashley, or resistant, China (Kyoto), cucumber protoplasts, whether the treatment was administered before or immediately after inoculation, or whether the coculum was virus RNA or nucleoprotein. These inhibitors also failed to affect the expression of resistance in China (Kyoto) protoplasts suggesting the absence of an “active defence mechanism” operating at the single cell level.
Purine metabolism in the barley powdery mildew fungus (Erysiphe graminis sp. hordei) differs from that of its host. In the pathogen [3H]adenine and [3H]adenosine were extensively metabolised to inosine and to a lesser extent hypoxanthine but this was not the case in the host. This difference was correllated with the presence of adenosine deaminase in the mildew fungus but not leaves of the barley host. Infection brings together two pathways of purine metabolism with the result that purines are diverted from the host to pathogen. These changes are largely independent of any recognition events involved in determining compatibility/incompatibility. The importance of adenosine deaminase in directing these changes is discussed.
The effect of the host-specific toxin produced by Helminthosporium sacchari (HS-toxin) on cell membrane potential of susceptible sugarcane was investigated. Membrane depolarization was detected at concentrations as low as 50 nm HS-toxin. The onset of membrane depolarization was observed after a lag phase of 4–10 min depending on the toxin concentration.
Both the wild-type isolate of Erwinia amylovora (Burrill) Winslow and a noncapsulated avirulent form, mutant S, when in contact with pear fruit slices, induced electrolyte leakage and grew rapidly. A capsulated form of the mutant S, induced by growing it in d-galactose medium, was found to have reduced capacity to cause leakage and grew less rapidly. Loss of capsule by the pre-capsulated mutant occurred simultaneously with increased leakage of pear electrolytes and bacterial growth. Another capsulated avirulent form, mutant P, did not induce electrolyte leakage from pear tissue and failed to grow. In mixed inocula, the capsulated mutant P partially inhibited the action and growth of both the wild-type and the non-capsulated mutant S on pear tissue. Prevention of contact between bacterial inoculum of the wild-type for the non capsulated mutant S, and the pear tissue by means of a membrane filter, also prevented loss of electrolytes and bacterial growth. Cell-free fluids from cultures of bacteria in artificial media or from inoculated pear tissue suspensions, failed to cause loss of electrolytes from healthy pear slices. Wild-type bacteria isolated from infected apple plants contained a proportion of cells wholly or partly deficient in outer surface components, in contrast to bacteria grown in culture, which were more uniformly coated. It is suggested that host damage is caused by contact with partially or wholly noncapsulated virulent bacteria and that the amount of damage is regulated by the properties of the outer coat of the pathogen. It is also suggested that the regulation of host damage is a requisite for infection and the progress of the disease.
Immunodiffusion, immunoelectrophoretic and crossed immunoelectrophoretic analyses of rice antigens in relation to sheath rot disease revealed a serological relationship between susceptible rice cultivars and isolates of the causal organism of sheath rot, Acrocylindrium oryzae. One precipitin band was observed when the antigen preparation of A. oryzae was cross-reacted with its own antiserum or against the antisera of four susceptible rice cultivars. No precipitin band was detected between the antiserum of the resistant cv. Mahsuri and antigen preparations from three isolates of A. oryzae or between the antigens of the resistant cultivars Mahsuri and Rupsail and the antiserum of A. oryzae. Crossed-immunoelectrophoretic tests confirmed that there was a common antigen between Mahsuri and Jaya, and between Mahsuri and CR-126-42-1. The precipitin band between the antigen preparation of Jaya and A. oryzae was found to be similar.
Hypocotyls of soybean (cultivar Altona) are resistant to race 4 of Phytophthora megasperma f.sp. glycinea at 25 °C but susceptible to the same race at 32°C. Lower concentrations of the phytoalexin, glyceollin, accumulate in the temperature-induced susceptible response at 32°C than in the resistant response at 25°C. The possibility that this is due to decreased production or activity of elicitors of glyceollin was examined. Following published procedures, elicitors were obtained from culture filtrates and cell walls of P. megasperma f.sp. glycinea race 4, and for comparison from similar preparations from the compatible P. megasperma f.sp. glycinea race 6. Cotyledon bioassays indicated that preparations obtained from cultures of P. megasperma f.sp. glycinea race 4 grown either at 25 or 32 °C were both active. Preparations obtained from P. megasperma f.sp. glycinea race 6 were active also. The cell-wall elicitor preparations obtained from cultures grown at 25 or 32 °C were active in bioassays performed both at 32 and 25 °C. The abiotic elicitor AgNO3 had similar activity at both temperatures. It is concluded that temperature-induced susceptibility to P. megasperma f.sp. glycinea race 4 is not due to inability to produce elicitors in culture, to inactivity at 32 °C of the elicitors examined or to inability of the host to produce glyceollin at the higher temperature.
The translational activity of total RNA and polyadenylated (polyA+) RNA isolated from susceptible leaves of Hordeum vulgare infected with Erysiphe graminis f.sp. hordei has been investigated. When equal quantities of total RNA from control and inoculated leaves were included in assays of protein synthesis using rabbit reticulocyte lysates, the amount of protein synthesised in assays programmed with RNA from infected leaves was reduced to 61% of the controls at 1 day after inoculation. This effect increased with infection until at 5 days after inoculation, RNA from infected leaves had only 11% of the activity of that of controls. Analysis of translation products by SDS-polyacrylamide gel electrophoresis and fluorography indicated reduced synthesis of most labelled polypeptides in assays of RNA from infected leaves. Analysis of total RNA on agaroseformaldehyde gels indicated no significant degradation of differences in RNA from control and inoculated leaves. The proportion of total RNA present as polyA+ RNA also declined during infection but a decrease in the translational activity of equal amounts of polyA+ RNA from control and infected leaves was not observed until 5 days after inoculation. This decreased activity was due mainly to reduced synthesis of the Mr 20000 protein which was the major product in assays of polyA+ RNA from controls. The reduced activity of the polyA+ RNA at this later stage of infection was not evident in the presence of greater than 0·5 mm 7-methyl guanosine 5′ phosphate and the translation products were identical in the presence of this inhibitor. These data suggest that infection of barley by the powdery mildew fungus caused a rapid and extensive decline in the amount of available host mRNA.
Distributions of the hydrolytic enzymes acid phosphatase and non-specific esterase in compatible barley coleoptiles inoculated with Erysiphe graminis fsp. hordei were investigated histochemically by light and electron microscopy. The four different enzymes investigated-acid β-glycerophos-phatase, acid naphthol AS-TR phosphatase, indoxyl acetate esterase and naphthol AS-D acetate esterase—had similar localization patterns. Reaction products were distributed generally in the fungal and host cells, but distinct accumulations were found in certain regions and structures at the encounter sites, including appressoria, penetration pegs, haustoria, papillae, and host cytoplasm. Most papillae showed conspicuous amounts of reaction products, suggesting that papilla formation includes depositions from secretory vesicles derived from the lysosomal system in host cells. There was a marked difference in the apparent concentration of hydrolytic enzyme activity between normal-size papillae accompanied by haustoria (sites of successful penetration) and oversize papillae (sites of unsuccessful penetration). Hydrolytic enzymes, localized most intensively in a lAyer of the oversize papillae, may confer resistance to fungal ingress by degrading components of the penetration pegs.
Stem inoculation of Burley tobacco with sporangia of the blue mold pathogen, Peronospora tabacina, elicited high systemic resistance in the foliage against disease caused by subsequent inoculation with the pathogen. Girdling the stem above the site of inoculation prior to 9 days after stem inoculation prevented the elicitation of resistance, whereas girdling below the site did not. Increased growth (height, number of leaves, reduced time to flowering) was observed in ungirdled, stem-inoculated plants, plants stem-inoculated and girdled above the site of inoculation at day 6 or later and in plants stem-inoculated and girdled below the site of stem inoculation. The factor responsible for resistance did not precent penetration by the fungus, and it may or may not be the factor responsible for enhanced growth. The resistance factor was graft transmissible between rootstock and scion. Induced scions (10 cm in length), when grafted onto control rootstocks, developed into fully grown plants that remained systemically protected against blue mold.
Extractable levels of phenylalanine ammonia-lyase (E.C. 4.3.1.5) (PAL) increased in a susceptible combination of the maize mesocotyl with Helminthosporium maydis, but not in resistant combinations with either H. maydis or H. carbonum. The increase in extractable PAL was detected only in tissue incubated in the dark and was prevented or significantly reduced by cycloheximide and cinnamic acid. The in vivo effect of H. maydis on the mesocotyl was compared with that of the PAL inhibitor a-aminooxyacetate (AOA). AOA caused an increase in the level of extractable PAL and prevented the expression of resistance of the mesocotyl to H. carbonum. H. maydis also altered the expression of resistance to H. carbonum, in that it allowed a significant increase in post-penetration growth of H. carbonum. We suggest that susceptibility of maize to H. maydis is due to the fungus interfering with the plant's resistance response.
Extracts of three virulent strains of Endothia (Cryphonectria) parasitica and three hypovirulent strains derived from them were assayed for enzyme activities that would produce oxalate. Oxaloacetate acetylhydrolase (E.C. 3.7.1.1), the enzyme that converts oxaloacetate to oxalate and acetate, was at least four times higher in extracts of the virulent strains than in extracts of the hypovirulent strains. The level of activity of this enzyme could account for the formation of all the oxalate produced by the virulent cultures. When extracts of hypovirulent strains were added to extracts of virulent strains, the measured rate was less than the sum of the two rates. The inhibition did not change when the hypovirulent extract was either dialysed or boiled. No enzyme activity for the conversion ofglycolate or glyoxylate to oxalate could be detected in any of the extracts.
Pectin lyase, produced by Erwinia chrysanthemi strain EC183 following mitomycin C treatment, was purified to near homogeneity from the culture lysate. The purification procedure involved ultrafiltration, ammonium sulphate fractionation and ion exchange chromatography on DEAE-BioGel A and CM-sepharose. The molecular weight and pI of the pectin lyase were estimated to be 34 500 and 9·45, respectively. The enzyme was most active on Link (98% esterified) pectin and least active on nonesterified pectic acid. Pectin lyase cleaved Link pectin in an endo manner, with optimum pH of 8·3 and optimum temperature of 33 †C. The Km and Vmax of the enzyme with Link pectin as the substrate were estimated to be 7·3±2·0 mg ml−1 and 47·6±10·7 A235 min−1, respectively. Antipectin lyase antibody reacted with pectin lyases from 11 of 12 E. chrysanthemi strains and all four Erwinia carotovora subsp. atroseptica strains tested but not with enzymatic preparations from E. carotovora subsp. carotovora, Erwinia milletiae, Erwinia rhapontici or Aspergillus sojae. These data indicate that pectin lyase of E. carotovora subsp. carotovora is immunologically distinct from that produced by E. chrysanthemi.
Growth of Erysiphe graminis hordei and Erysiphe pisi on the same cells of barley coleoptiles was observed in detail by light microscopy to determine significant factors conditioning host cells toward susceptibility. When E. pisi attempted penetration more than 60 min earlier than E. graminis on the same coleoptile cell, E. pisi never succeeded in penetration (0% penetration efficiency) and the penetration efficiency of E. graminis was lowered from 75·0 to 28·6%, suggesting that the resistance to E. graminis invasion might be enhanced under this condition. When both fungi attempted penetration of the coleoptile cell almost simultaneously (within 30 min of each other), the penetration efficiency of E. pisi increased to 11·8%. Moreover, the penetration efficiency of E. graminis was 55·8%. When E. graminis attempted penetration 60 min or more earlier than E. pisi, the mean penetration efficiency of E. pisi was 29·2% and that of E. graminis to 75·0%. These observations suggest that the coleoptile cells are conditioned toward susceptibility by prior-penetration of E. graminis or even by its post-penetration if it penetrates after E. pisi E. pisi developed haustoria only in coleoptile cells where E. graminis formed haustoria. The induced susceptibility and enhanced resistance states in coleoptile cells had not transferred to the adjacent cells within 3 h.
A low molecular weight (less than 3000) phytotoxic polypeptide fraction was partially purified from the protein-lipopolysaccharide (PLP) complexes produced in culture by a strain of Verticillium dahliae pathogenic on potato. The toxic fraction was host-specific and antigenically identical to the high molecular weight PLP. Immunodiffusion and immunofluorescence assays showed that a substance is present in stems and tubers of infected potato plants which was antigenically related to the low molecular weight toxin. When used as a tool for screening new potato varieties for tolerance to Verticillium wilt disease, the low molecular weight toxin was more reliable than the PLP because non-specific sensitivity to macromolecules was not observed.
Application of digitonin to the leaf surface of potato plants was demonstrated to activate O2− generation of the leaf tissues as determined by extracellular cytochrome c reduction which was inhibited by superoxide dismutase (SOD). Protoplasts prepared from stem shoots of potato enhanced NADPH-dependent reduction of extracellular cytochrome c immediately after being treated with digitonin. The reduction was also inhibited by SOD. The similar activation of SOD-sensitive cytochrome c reducing activity was observed in leaves or protoplasts of some other plants treated with digitonin.
Relationships between the ability to produce indole-3-acetic acid (IAA) and cytokinins in culture and pathogenicity were studied in different strains of Pseudomonas syringae pv. savastanoi from olive and oleander and in their α-methyltryptophan resistant mutants. Wild type olive and oleander strains of pv. savastanoi produced both IAA and cytokinins in culture but the amount produced varied between strains. The α-methyltryptophan resistant mutants produced little or no IAA but about the same amount of cytokinins as their parent strains.
The growth of Sclerotinia sclerotiorum in white beans (Phaseolus vulgaris) was much slower in the tolerant cv. Ex Rico-23 than in the susceptible cvs. Kentwood and Seafarer. The difference was paralleled by the rate of diffusion in leaf tissue of oxalic acid, a phytotoxin of the white mold fungus (Sclerotinia sclerotiorum). Uptake of oxalic acid into excised leaves through the petiole resulted in more severe brown-rot-like symptoms in the leaves of cv. Kentwood than in those of Ex Rico-23. When [14C] oxalic acid was fed through petioles, radioactivity in the interveinal tissue of Kentwood was about three times higher than that in Ex Rico-23. Autoradiographs of leaves showed that radioactivity in Ex Rico-23 was confined to major veins and that activity in the interveinal tissue was low, whereas in Seafarer and Kentwood radioactivity did not accumulate in the veins but was distributed uniformly throughout the leaf tissue, with a slight accumulation along leaf margins.
