Showing papers in "Physiologial Plant Pathology in 1977"

Interaction of bacteria and host cell walls: its relation to mechanisms of induced resistance

Abstract: Cells of avirulent (B1) and incompatible (S210) strains of Pseudomonas solanacearum attach readily to the walls of tobacco mesophyll cells. By 4 h after infiltration of tobacco leaves with 10 8 bacterial cells/ml, fibrillar and granular material extruded from the host cell walls and bound by the outer wall layer envelop the attached bacteria. At the site of attachment, the host cell wall is frequently eroded, the plasmalemma separates from the cell wall and becomes convoluted and numerous membrane-bound vesicles accumulate in the space between the plasmalemma and the cell wall. With these bacteria, a hypersensitive reaction (HR) develops by 6 to 12 h after infiltration; as a result, the host cell collapses and organelles are deranged. In contrast, virulent strains of the pathogen (K60) are not attached and remain free to multiply in the intercellular fluid, causing no visible changes in organelle structure during the first 12 h after infiltration. Saprophytic bacteria ( Bacillus subtilis, Escherichia coli ) are attached and enveloped, but do not cause a visible HR. When heat-killed B1 cells are infiltrated into tobacco leaves, the dead bacteria attach in the same manner as avirulent, live cells and the initial host cell responses that envelop the bacteria are observed, but there is no cell collapse. After 24 h, challenge inoculation of the pretreated leaves with 10 8 live B1 cells/ml does not result in the HR and these bacteria do not attach to and are not enveloped by the host cell walls. Prevention of the HR appears to be related to this lack of attachment to the host cell walls.

Production and purification of syringomycin, a phytotoxin produced by Pseudomonas syringae

Abstract: Syringomycin (SR) production by Pseudomonas syringae in potato dextrose broth (PDB) still culture was approximately 20 to 136 times greater than that in potato dextrose agar (PDA) or in PDB shake cultures, respectively. Potato broth supplemented with 1·5% dextrose and 0·4% casamino acids was best for SR production when compared to other formulations of PDB, synthetic medium or biphasic media. Maximum production of SR corresponded with exponential cell growth of P. syringae in vitro . SR activity was completely partitioned into n -butanol from aqueous concentrates of acidified cultures. By means of ion-exchange chromatography (carboxy-methyl cellulose), thin-layer chromatography and Polyacrylamide gel electrophoresis, homogeneous preparations of SR were obtained with specific activities up to 47·9 units/μg. Eleven strains of P. syringae antibiotic toward Geotrichum candidum and representing a broad range of ecotypes were compared for the production of SR or other antibiotics. The only antibiotic isolated from ten of the strains was SR whereas the eleventh strain produced only syringotoxin (ST), a small biocidal polypeptide. Both SR and ST were phytotoxic and mimicked holcus spot on maize leaves at a threshold concentration of 50 units/ml. The threshold toxigenicity of SR to representative isolates of P. syringae ranged from 800 to 6400 units/ml compared with 100 units/ml for G. candidum .

Agglutination of avirulent strains of Pseudomonas sofanacearum by potato lectin

Abstract: Fifty-five virulent isolates and 34 avirulent variants of Pseudomonas solanacearum from different geographic regions and representing all major races and biotypes were tested for their ability to bind to potato lectin. The lectin was extracted from “Katahdin” potato tubers and purified to homogeneity. All avirulent isolates agglutinated strongly with the lectin, whereas virulent isolates either failed to agglutinate or agglutinated only weakly and at much higher lectin concentrations. Failure of the virulent cells to bind to the lectin was correlated with the presence of extracellular polysaccharide (EPS) which is formed by virulent, but not avirulent, cells. Virulent cells were agglutinated strongly by potato lectin after most of the EPS was removed by repeated washing and centrifugation. Agglutination of avirulent cells or washed virulent cells could be totally prevented by the addition of EPS to the cells prior to addition of lectin. The weak agglutination exhibited by some unwashed virulent cells in suspension could also be prevented by addition of EPS. This indicates that the amount of EPS formed in culture and released in water suspensions determines the degree of agglutination obtained when lectin is added. Potato lectin, conjugated with fluorescein isothiocyanate, bound to rabbit erythrocytes and to avirulent, but not to virulent, cells of P. solanacearum . Moreover, binding of lectin to avirulent cells was hapten-specific and could be inhibited by chitin oligomers. Purified lipopolysaccharide (LPS) from P. solanacearum K60-B1 cells precipitated when mixed with purified potato lectin. Initial evidence indicates that binding sites for the lectin are present in the lipid A portion of the LPS.

Ultrastructural and physiological properties of the host interfacial components of haustoria of Erysiphe pisi in vivo and in vitro

Abstract: The haustorium of Erysiphe pisi and its interfaces with the cytoplasm of Pisum sativum have been studied by light and electron microscopy of infected cells and haustorial complexes isolated from them. The term haustorial complex has been introduced for the haustorium, the extrahaustorial part of the host plasmalemma, and the intervening materials and structures. A method for the isolation and purification of haustorial complexes is described. It has been shown that the haustorial cytoplasm has one nucleus and is retained in isolated haustorial complexes by an amorphous plug in the septal pore between the haustorial neck and body. The body is invested by finger-like lobes arising near the neck and at the opposite end, the lobes being attached to the extrahaustorial membrane. Two annular structures, the A and B bands, in the neck wall are visible by light and electron microscopy. The A band adjoins the inner face of the host cell wall, and the extrahaustorial membrane and fungal plasmalemma are intimately associated with its surface even in the absence of both host and fungal cytoplasms. The B band is nearer the haustorial septum, comprises opaque, amorphous material and joins the extrahaustorial membrane to the neck wall; it is soluble in NaOH. The extrahaustorial membrane over the body is highly convoluted, being 20 to 25 nm thick, and osmotic experiments show that it is continuous and has high tensile strength. It is soluble in NaOH but is not destroyed by long periods in detergents, or by enzymes which degrade the host cell wall. The membrane is reduced in thickness to 15 nm and is rendered osmotically unstable by the enzymes. The extrahaustorial membrane stains with procedures specific for polysaccharides, but the remainder of the host plasmalemma does not show the special characteristics of this membrane. The extrahaustorial matrix is a fluid which reacts less intensely than the extrahaustorial membrane with the polysaccharide reagent and is removed by enzymes degrading the host cell wall. It is bounded by structures including the extrahaustorial membrane and the B neckband, none of which is permeable to its contents but some or all are permeable to water. The functional significance of these structures is discussed.

Vascular gelation: a general response phenomenon following infection

Abstract: Vascular gels that coat the walk and fill the lumina of infected vessels were found in nine species of plants that had been inoculated with non-host-specific forms of Verticillium dahliae Kleb, or with Fusarium oxysporum Snyd. & Hans. Gel formation is therefore a general phenomenon and represents one factor in the response of plants that provides for resistance to vascular infections. Gels arise from perforation plates, end-walls and pit membranes by a process of distension of primary wall and middle lamella constituents. Swelling of these pre-formed constituents can account for complete occlusion of vessel lumina. A protective wall layer located between the plasmalemma and the pit membranes was observed in parenchyma cells that lined the perimeter of infected vessels in several species of plants.

Competition for nutrients between epiphytic micro-organisms and germination of spores of plant pathogens on beetroot leaves

Abstract: When different numbers of leaf micro-organisms were applied in droplets to non-sterile beetroot leaves inhibition of germination of Botrytis cinerea conidia was positively correlated with uptake of amino acids. Of the micro-organisms tested two isolates of Pseudomonas and an isolate of Sporobolomyces were most effective at inhibiting germination and removing amino acids from droplets at comparatively low cell densities. Leaves bearing a natural microflora needed to be wetted for 24 h before appreciable uptake of amino acids occurred and conidia of B. cinerea were inhibited from germinating. Negligible quantities of 14C-labelled amino acids or glucose, added to droplets placed on the surface of sterile beetroot leaves, were taken up by the leaf. Most of the 14C label lost from sterile 14C-labelled beetroot leaves was detected in droplets shortly after placing them on the surface of the leaves. Germination of conidia of Phoma betae and Cladosporium herbarum was greatly inhibited on beetroot leaves in the presence of a leaf surface bacterium, Pseudomonas sp. isolate 14, and there was a close relationship with uptake of amino acids by the bacterium on leaves. Percentage germination of conidia of Colletotrichum dematium f. sp. spinaciae was not inhibited by the bacterium but length of germ tubes was reduced. Presence of the bacterium stimulated development of appressoria.

A comparative study of non-host interactions with rust fungi

Abstract: The interactions of non-host plants with sunflower, corn and cowpea rusts were compared with those described previously for other cowpea rust-non-host combinations. Compared with the situation on the respective host plant, all three rusts made fewer attempts at penetration into the leaf on half or more of the non-hosts examined. Nevertheless, in no case could such surface behavior completely account for non-host resistance since at least a few attempts at penetration were observed for all the non-host-rust combinations. In only one non-host, cabbage, was linear growth inside the leaf significantly less than that in the respective susceptible plant during the first 24 h after inoculation. Since, for cowpea and sunflower rusts at least, growth in this non-host was also less than that achieved on artificial membranes, it was concluded that non-host resistance of cabbage leaves involved an inhibition of fungal growth. In contrast, a detailed study using cowpea rust showed that the extent and pattern of growth in most of the other non-hosts was similar, apart from a greater frequency of haustorial mother cells, to that on the membranes. Thus it seemed that these non-hosts had little influence on the development of the fungus except in the induction of the haustorial mother cell septum. Susceptible plants, however, appeared to stimulate the formation of secondary hyphae, and it is postulated that the resistance of many non-hosts to this rust, and others, may be due to the absence of any similar stimulation by the plant. The importance of the haustorium in this stimulation could not be clearly determined. The possibility that the non-host plant may actively inhibit haustorium formation, however, was suggested by the fact that their absence in many non-host-rust combinations could be correlated with the darkening of the plant wall adjacent to the haustorial mother cell. The general lack of specificity of non-host responses to rust fungi was indicated by the observation that where a given non-host-rust combination was characterized by a particular feature, such as the presence of wall darkening or the relatively high frequency of haustoria, then a similar feature was often seen following infection of the same non-host with the other two rusts. The observation of a few instances where this was not the case, however, suggested that the nature of the fungus may have a modifying effect on the plant response.

Changes in phytoalexin concentrations in tissues of the broad bean plant (Vicia faba L.) following inoculation with species of Botrytis

Abstract: Six phytoalexins, the furano-acetylenes wyerol, wyerone, wyerone acid and wyerone epoxide, an unidentified wyerone derivative (M+ 290) and the isoflavanoid medicarpin, have been recognized as the major components of the phytoalexin response of Viciafaba. All of the phytoalexins examined were more active against germ tubes of Botrytis cinerea than B. fabae; differential toxicity was much more marked with wyerone derivatives than with medicarpin. The inhibitors were placed in the following order of antifungal activity against B. cinerea at pH 4-0: wyerone epoxide > wyerone acid > wyerone > medicarpin > wyerol. Activity of the acid was less at pH 500. Activities of wyerone acid and epoxide were additive. Phytoalexins accumulated in limited lesions caused by Botrytis. Time-course studies showed that the rapid accumulation of wyerone or wyerone acid could alone account for the restriction of fungal growth in cotyledons or leaves and pods respectively. Wyerone appeared to be deposited on pod endocarp cell walls and converted to wyerone acid in vivo. Results suggested that the weakly active wyerol was a precursor of other wyerone derivatives. An initial increase in phytoalexin concentrations in leaves and pods after inoculation with B. fabae was followed by a decrease as tissues became completely blackened and colonized by the pathogen. The biochemical mechanisms underlying the specificity of B. fabae to V. faba are discussed.

Association of coumestanss with tee hypersensitivity of Lima bean roots to Pratylenchus scribneri

Abstract: Roots of Lima bean (Phaseolus lunatus L.) exhibited a hypersensitive responsess to Pratylenchus scribneri Steiner, concomitant with the accumulation of at least four coumestants. Roots of snap bean (Phaseolus vulgaris L.), on the other hand, did not exhibit a visible response to inoculation, allowed rapid multiplication of the nematode and did not accumulate significant quantities of the coumestans, despite the presence of small amounts in healthy roots. One of the Lima bean compounds that accumulated was identified as coumestrol and another, more tentatively, as psoralidin. Although insufficient quantities of the latter compound were isolated for biological experiments, coumestrol inhibited the motility of P. scribneri above concentrations of 5 μg/ml in vitro. No phytoalexins active against fungi were detected in extracts from hypersensitive Lima bean roots. The evidence supports the hypothesis that the coumestans are phytoalexins active against nematodes and may be causally related to the expression of resistance of Lima bean roots to P. scribneri.

The fungitoxicity of isoflavanoid phytoalexins measured using different types of bioassay

Abstract: The effects of the phytoalexins phaseollin, phaseollidin, phaseollinisoflavan, kievitone, medicarpin and pisatin on fungi were assessed in five different bioassays The assays measured spore germination on agar and in liquid media, the growth of 1- and 3-day-old sporelings in liquid medium and the growth of mycelium on agar medium Assays using sporelings in liquid media produced the most reliable indications of the phytoalexins' fundamental activity In tests using 1-day-old sporelings in liquid media, concentrations of phaseollin, phaseollidin and phaseollinisoflavan between 5 and 25 (μg/ml completely inhibited the growth of all the tested fungi Medicarpin and kievitone were totally inhibitory at levels of 37·5 or 50 and 50 or 100 μg/ml respectively At doses causing complete inhibition all these compounds were fungicidal Pisatin rarely killed cells and frequently failed to prevent growth at 100 μg/ml The results of other tests were less uniform and often complete inhibition of growth did not occur For most fungi, spores and 1-day-old sporelings were equally sensitive However, the spores of Alternaria brassicicola, Aspergillus niger, Botrytis cinerea and Glomerella cingulata were markedly resistant to phaseollin, phaseollidin and phaseollinisoflavan, often germinating in assays using 100 μg/ml Similarly, 3-day-old sporelings were sometimes able to grow in relatively high levels of these phytoalexins The growth, which was produced after prolonged incubation, arose from a few cells which survived the potentially fungicidal effects Mycelial growth on agar media containing phytoalexins was very variable and was rarely linear throughout the incubation period The results obtained were thus very difficult to interpret and it was not possible to use them to measure the toxicity of these compounds

Localized infection with tobacco necrosis virus protects cucumber against Colletotrichum lagenarium

Abstract: A cultivar of cucumber susceptible to Colletotrichum lagenarium race 1 was systemically protected against disease caused by the fungus by prior infection with tobacco necrosis virus (TNV) Infection of one cotyledon with the virus protected the opposite cotyledon and the first true leaf against disease caused by a subsequent infection with C lagenarium Autoclaved sap from TNV-infected plants or sap from non-inoculated plants did not elicit protection Infection with the virus prior to infection with C lagenarium caused a reduction in the number and size of lesions caused by the fungus at low spore concentrations, but was less effective against higher spore concentrations The number of lesions formed on a TNV-infected cotyledon was not affected by prior infection of the opposite cotyledon with TNV or C lagenarium

Cytological studies of early stages of powdery mildew in barley and wheat leaves: (II) significance of the primary germ tube of Erysiphe graminis on barley leaves☆

Abstract: Early stages of the infection processes of Erysiphe graminis hordei, race I (compatible with barley cv. Kobinkatagi) and of Erysiphe graminis tritici, race t2 (incompatible with barley cv. Kobinkatagi) were observed on barley (Hordeum vulgare L., cv. Kobinkatagi) by scanning electron microscopy. Behavior of both fungi was similar and nearly 99% of the germinated conidia of both races had more than one germ tube 12 h after inoculation. Among the germ tubes, one, designated as the primary germ tube, induced a host papilla or cytoplasmic aggregate below it 4 to 6 h after inoculation, and another swelled to become an appressorium. Almost every conidium which had induced a papilla below an appressorium had earlier induced a papilla below the primary germ tube. The results suggest that the induction of a papilla by the primary germ tube may be critical to attempted penetration by the appressorium from the same conidium. Micromanipulation indicated that the papilla below the epidermal cell wall was continuous with or attached firmly to the inner host wall layer, and also that the wax crystals on the leaves were dissolved by the fungal structures which attempted to penetrate, i.e. the primary germ tube and appressorium.

Rôle of syringomycin in holcus spot of maize and systemic necrosis of cowpea caused by Pseudomonas syringae

Abstract: Syringomycin (SR) is a phytotoxin produced by isolates of Pseudomonas syringae which cause holcus spot of maize. When purified to homogeneity and administered to maize leaves or cowpea seedlings. SR induced disease lesions typical of those produced by P. syringae. However, SR was not recovered from either diseased maize leaves or cowpea seedlings infected with P. syringae or in significant amounts from plants treated with SR. Treatment of excised maize leaves with a preparation of [14C]SR (sp. act. 475 ct/min or 20 units/μg) suggested that SR was metabolized to several substances during development of disease lesions. Growth of P. syringae in host tissue corresponded to bacterial populations in vitro which produced concentrations of SR (1600 to 3200 units/ml) many times the threshold concentration necessary to cause disease lesions on maize (approx. 50 units SR/ml). These results indicate that SR may be a primary determinant of disease caused by host-specific strains of P. syringae.

Ultrastructural histopathology of tomato plants infected with Corynebacterium michiganense

Abstract: Spread of Corynebacterium michiganense in tomato plants is restricted initially to the xylem vessels; the phloem elements remaining bacterium-free until advanced stages of pathogenesis. Where vessels abut, the adjoining primary walls may swell, shred and completely degrade; however, where an invaded or non-invaded parenchyma cell adjoins an invaded vessel initially only the primary wall of the vessel undergoes these changes, the wall of the parenchyma cell remaining apparently unaffected. At advanced stages of pathogenesis the secondary thickening may also become degraded, even in the absence of bacterial cells, indicating that the incitant relies for its pathogenicity on an active enzyme complement, particularly extracellular cellulases and, possibly, hemicellulases. The bacteria eventually pass from the disrupted vessels into the intercellular spaces of the adjacent xylem parenchyma. The cementing substances between these cells are degraded causing the cells to separate, plasmolyse and collapse. The bacteria migrate through the disrupted ground tissue and eventually enter the phloem elements which are rapidly destroyed. It would seem that wilting of the host is due to degradation of the conducting tissue by bacterial enzyme action rather than to the action of a toxin produced by the pathogen or to the accumulation of plugging substances in the vessels.

The interaction of yellow rust (Puccinia striiformis) with winter wheat cultivars showing adult plant resistance: macroscopic and microscopic events associated with the resistant reaction

Abstract: The development of race 104 E 137 of Puccinia striiformis was observed in leaves of four winter wheat cultivars which were susceptible at the seedling stage but showed a graded series of reactions at later growth stages. The degree of resistance, measured by the reduction in area of sporulating tissue, increased gradually with successively appearing leaves on the main axis of Cappelle Desprez, Holdfast and Maris Widgeon. Resistant reactions were associated with reduced fungal colony growth rates, the appearance of scattered cells showing hypersensitive necrosis and with more general areas of chlorosis and browning of host tissue. The differences in resistance between cultivars were correlated with different frequencies of hypersensitively necrotic cells but the gradual increase of resistance in successive leaves of Maris Widgeon did not correspond with the occurrence of hypersensitive necrotic cells which appeared suddenly in the second leaf and remained similar in successive leaves. By contrast the rate of fungal colony growth during the latter half of the infection process and the development of tissue chlorosis and browning were well correlated with increasing resistance of successive leaves, final reaction type and relative differences in resistance between cultivars. The significance of these observations in terms of resistance mechanisms is discussed.

Isolation of two toxins produced by Pyrenophora teres and their significance in disease development of net-spot blotch of barley

Abstract: Two toxins, designated toxin A and B, were isolated from a cell-free, sterile culture filtrate of Pyrenophora teres by means of extraction, gel filtration and ion-exchange chromatography. Without the presence of the pathogen each of the purified toxins incites the most important symptoms of the disease when introduced into healthy barley leaves. Toxin A is the most potent, producing symptoms in concentrations as low as 25 μg/ml. The most virulent isolates produce the highest concentration of toxin in vitro . A good, although not complete, correlation was found between the host range of the pathogen and that of the toxins. Both toxins were isolated from infected but not from healthy barley leaves. The results indicate that P. teres produces two toxins which may be involved in the disease syndrome of net-spot blotch of barley. The toxins do not seem to determine pathogenicity of P. teres but contribute to the virulence of individual isolates.

The rôle of cytokinins in the systemic acquired resistance of tobacco hypersensitive to tobacco mosaic virus

Abstract: Cytokinin content increased in Xanthi tobacco leaves systemically resistant to challenge inoculation by tobacco mosaic virus (TMV). Subsequently, root formation was strongly suppressed and shoot formation was promoted in leaf cultures derived from resistant leaves. All of these events coincided with the time of development of systemic acquired resistance, i.e. 8 days after the primary inoculation of the lower leaves. In spite of a reduction in the number of visible local lesions in resistant upper leaves, infectivity of ultracentrifuged TMV did not change markedly. Following a 1 day treatment at high temperature (32°C), when necrotization is inhibited and TMV becomes systemic, this type of resistance was not manifested. It is suggested that only the necrotization is suppressed in challenge-inoculated resistant leaves, and the production of the virus is not reduced. Chloramphenicol and ethrel, which promote local lesion production, were counteracted in leaves in which systemic acquired resistance had been induced.

Accumulation of antibacterial isoflavonoids in hypersensitively responding bean leaf tissues inoculated with Pseudomonas phaseolicola

Abstract: Red Kidney bean ( Phaseolus vulgaris L.) plants inoculated with Pseudomonas phaseolicola (isolate HB-36) showed water-soaked infection centers on the inoculated leaf surfaces about 48 h after inoculation. In contrast, resistant bean plants (GN Nebraska 27) inoculated with the same isolate responded by developing a hypersensitive reaction (HR) after roughly the same period after inoculation. High concentrations of phenolic compounds were detected in the ethanolic extracts of leaf tissues showing HR. Among these were identified the isoflavonoid phytoalexins phaseollin (pterocarpan), phaseollinisoflavan (isoflavan), coumestrol (coumestan) and kievitone (isoflavanone). These isoflavonoids were detected in substantial quantities 24 h after inoculation and their concentrations increased further up to 72 h, after which time they began to decrease. In susceptible Red Kidney leaves, isolate HB-36 induced much lower concentrations of these compounds at all stages of disease development. In repeated in vitro bioassays the above four compounds consistently restricted colony formation by P. phaseolicola isolates. However, the race 2 isolates, HB-36 and G-50, were found to be comparatively more tolerant to these compounds than the race 1 isolates, HB-33 and HB-20. Colony formation in race 1 isolates was completely inhibited by phaseollin and phaseollinisoflavan at 42 and 21 μg/ml; colony formation of race 2 isolates was retarded (90 to 98%) but not completely suppressed at 52·5 μg/ml of phaseollin and 52 μg/ml of phaseollinisoflavan. Coumestrol and kievitone at 21 and 33 μg/ml respectively were totally toxic to race 1 isolates but they were relatively less toxic to race 2 isolates even at higher test concentrations. All four phytoalexins also restricted the growth of P. glycinea isolates, R-2 and 724 (Leben). Isolate 724, however, exhibited a greater degree of tolerance than isolate R-2. P. fluorescens , a saprophyte, was relatively insensitive to the phytoalexins and showed only 26 to 30% retardation in colony formation at the maximum test concentrations used. These studies show that the virulent isolates of P. phaseolicola are not as sensitive to bean phytoalexins as the relatively less virulent isolates of P. phaseolicola .

Barley epidermal apoplast structure and modification by powdery mildew contact

Abstract: Fine structure of the outer epidermal wall and cuticle of Hordeum vulgare is described prior and in response to contact by Erysiphe graminis. Halo formation occurs independently of fungal penetration and is not a site of fungal enzymatic degradation, host cuticle and wall substructure remaining largely intact, but a region of wall encrusted with opaline silica in association with cuticular lipid deposition. In agreement with current physiological concepts, the halo is thought to provide a seal against excessive water loss following contact. A new and specific technique for fine structural localization of silica is described.

The effect of the Lr20 resistance gene in wheat on the development of leaf rust, Puccinia recondita

Abstract: Interactions between primary leaves of the cultivar Thew carrying the Lr20 gene for resistance and avirulent and virulent races of Puccinia recondita fsp tritici were followed by light-microscopy At 20·5°C, physiological changes in protoplasts of cells surrounding avirulent hyphae were first detected 20 h after inoculation Protoplasts changed in their response to stain after 28 h and collapsed after 36 h Growth of the avirulent mycelium was affected after about 48 h The subsequent slow growth of avirulent colonies resulted in a low infection type The exponential growth of virulent colonies resulted in a high infection type The resistance determined by the Lr20 gene was partly effective at 26°C and completely ineffective at 30·5°C The temperature-sensitivity of the interaction seemed to lie with the host Resistance appeared to be associated with the changes in host cells leading to the widespread collapse of protoplasts

Toxic effects of phaseollin on plant cells

Abstract: Plant cells, from bean and tobacco suspension cultures and from pods and hypocotyls of bean, were rapidly killed following immersion in solutions containing 30 μg phaseollin per ml. These concentrations inhibited the respiration of bean cells within 2 min of treatment and also reduced the growth of bean suspension cultures during a 3 week incubation period. Thereafter, as a result of a few cells surviving, the rate of growth of the suspension cultures increased and significant amounts of dry matter were produced. The total amount of phaseollin present in the suspension cultures and its concentration in the growth medium decreased during the incubation period. These findings are discussed in relation to the similarities between the effects of phaseollin on plant and on fungal cells, and to their relevance to current concepts about the formation and role of phaseollin in infected plant tissues.

The use of amino acid fungal auxotrophs to study the predisposition phenomena in the root-knot-wilt fungus disease complex of tomato

Abstract: Root-knot nematode ( Meloidogyne incognita ) infections of tomato plants resistant to the wilt fungus ( Fusarium oxysporum f. sp. lycopersici ) often predispose the tomato to this fungus. The role of free amino acids, which are abundant in nematode-galled tissue, in the predisposition of a resistant host to the fungus was investigated. Amino acid levels in the exudates of healthy and nematode-galled tomato cultivars were determined. Single step amino acid mutants (auxotrophs) were induced in the wild type (race 1) wilt fungus. Those auxotrophs that lost their relative pathogenicity as compared to the wild type were selected for the study of the influence of increased or decreased levels of the corresponding free amino acids in the exudates of host cultivars. Auxotrophs requiring threonine, proline, methionine, histidine and glycine were pathogenic only when the relevant amino acid was present above a certain threshold level in the exudates of genetically susceptible, nematode-galled cultivars. However, these caused negligible pathogenicity in nematode-inoculated cultivars that are genetically fungal resistant. A similar trend in pathogenicity was observed when threonine, proline, methionine and histidine were applied exogenously to the tomato cultivars before inoculation with the fungal auxotrophs. The role of these and other amino acid auxotrophs are discussed in the light of the nutritional hypotheses of Lewis and Garber [ 12, 18 ]. These experiments show that amino acids may play a significant role in predisposing fungal-resistant plants in a disease complex.

Purification and characterization of a phytotoxin produced by Phoma tracheiphila, the causal agent of mal secco disease of citrus

Abstract: Cultures of Phoma tracheiphila produce an extracellular phytotoxic glycopeptide capable of inciting symptoms similar to those of mal secco disease in lemon shoots. The toxin was purified by Sepharose 6B column chromatography and affinity chromatography on a Concanavalin A-Sepharose 4B column. The estimated molecular weight of the toxin is 93 000 and it possesses an isoelectric point of 4·3. The carbohydrate portion (29·5%) consists of mannose, galactose and glucose. The peptide portion (36%) contains most of the common amino acids with aspartate, glutamate threonine and serine as major amino acids. Beta-elimination studies of the peptide followed by a reaction with sodium sulphite indicate that threonine and serine are involved in the sugar-peptide linkages. Anionic substances of the hydrolysed toxin reacted positively with carbazole reagent and suggest the presence of sugar acids in the toxin structure. The toxin is heat stable and is not host specific. Injection of pure toxin into lemon leaves caused necrosis and vein clearing followed by wilt and leaf shedding. Proteolytic enzymes such as pronase, papain or pancreatin abolished its biological activity. Production of the toxin increased during the stationary phase of growth.

Induced resistance to Botrytis cinerea in root slices and tissue cultures of carrot (Daucus carota L.)

Abstract: Pretreatment of carrot tissue culture CRT 1 with heat-killed conidia for 4 days induced partial resistance to inoculation with live spores of Botrytis cinerea . Induced resistance was associated with an increase in peroxidase activity and lignin synthesis in the surface cells of the callus and a delay in germination of spores, accompanied by a reduced rate of germ tube elongation. Heat-killed conidia and cell-free germination fluid induced partial resistance in slices of carrot root, the optimum pretreatment period prior to inoculation with live spores being 6 h. In slices, resistance was associated with an acceleration of suberin synthesis above that occurring as a response to slicing (wound healing), and reductions both in percentage germination of spores and in the rate of germ tube elongation on the pretreated tissue. The results are discussed in relation to various active defence mechanisms operating in plant tissue, e.g. anatomical barriers and phytoalexins. It is concluded that whereas the callus and carrot slices displayed different mechanisms of induced resistance, the carrot root slices, after induction, behaved in a similar way to whole carrot roots, localizing infections after wound inoculation with B. cinerea . They therefore merit further study.

Stimulation of appressorium formation in Colletotrichum acutatum by phylloplane bacteria

Abstract: Two isolates of bacteria, Pseudomonas spp. isolates 14 and UV 3, known to compete strongly for amino acids on leaf surfaces, stimulated formation of appressoria by Colletotrichum acutaturn on both glass and sugar beet ( Beta vulgaris ) leaves. Appressoria reached maturity more frequently on leaves than on glass. Two other isolates of bacteria, Pseudomonas fluorescem isolate 15 and Flavobacterium isolate 11 b, known to compete less actively for amino acids, stimulated formation of appressoria to a lesser extent. In the presence of nutrients Pseudomonas sp. isolate 14 failed to stimulate formation of appressoria. Two chitinoclastic bacteria or the enzyme chitinase had no effect on numbers of appressoria. Leaching conidia of C. acutatum on either cellulose acetate or polycarbonate membranes, to simulate the possible action of bacteria, increased numbers of appressoria formed. Mature appressoria formed more readily on polycarbonate membranes. Results suggest that nutrient competition by phylloplane bacteria stimulates formation of appressoria by species of Colletotrichum on leaf surfaces.

Penetration of carrot roots by the grey mould fungus Botrytis cinerea Pers. ex Pers

Abstract: Infection of carrot roots by Botrytis cinerea at 11 to 14°C was studied by light and electron microscopy. Conidia of the fungus, in nutrient solution on the surface of uninjured carrots, germinated and produced dome-shaped infection cushions in 48 h. Infection cushions were formed by the development and aggregation of hyphae produced by several conidia. Penetration was effected by infection pegs that arose from swollen hyphal tips on the undersides of infection cushions. Occasionally, infection by single germinated conidia in the vicinity of infection cushions was observed; entry was direct via a narrow infection peg. Prior to penetration the peridermal wall layers of the host became swollen and changes occurred in their ultrastructural staining properties. A multivesicular body was present in the fungus near the point of attachment to the host wall where it may have been involved in the enzymic penetration of the host periderm. Evidence for degradation of suberized host walls during penetration is presented and discussed.

The effect of morphogenically active factors from host and nonhost plants on the in vitro differentiation of infection structures of Puccinia graminis f. sp. tritici

Abstract: Morphogenically active factors from the leaf surface of host plants control early developmental processes of Puccinia graminis f. sp. tritici. Two different biochemical fractions induced the differentiation of complete infection structures (appressorium, peg and substomatal vesicle) at a high rate in vitro when they were supplied simultaneously to germinating uredospores at 22°C. It is suggested that in vivo the active factors are part of the biochemical environment prevailing at the stomata where the fungus is induced to produce the appressorium. Similar fractions from various host and nonhost plants were found to cause different effects on the pathogen isolate. In agreement with earlier reports in the literature treatment at increased temperatures also induced the formation of infection structures in the absence of the biochemical fractions.

Phaseotoxin suppresses bacterially induced hypersensitive reaction and phytoalexin synthesis in bean cultivars

Abstract: The hypersensitive reaction (HR) of leaves of bean seedlings of cultivars GN Nebraska 27 (GN), P.I. 150414 (PI) and U.I. 34 (UI) to incompatible isolates of Pseudomonas phaseolicola was accompanied by accumulation of antibacterial phytoalexins, phaseollin, phaseollinisoflavan, coumestrol and kievitone. When GN and PI plants were treated with phaseotoxin prior to inoculation with the incompatible isolate HB-36 of the pathogen, HR was suppressed and typical susceptible symptoms were produced in inoculated leaves. Concomitant with the suppression of HR a significant reduction in the accumulation of phytoalexins was observed. Furthermore, in toxin-treated tissues bacterial multiplication was several orders of magnitude greater than in hypersensitively responding controls. A similar pattern of HR suppression, phytoalexin accumulation and increase in bacterial multiplication was observed in UI plants inoculated with the toxigenic strain HB-36 (which elaborated phaseotoxin “in vivo”). UI plants inoculated with the non-toxigenic isolate HB-33 of P. phaseolicola which served as controls showed HR, accumulation of phytoalexins and restriction of bacterial growth. These studies indicate that phaseotoxin-induced susceptibility of bean cultivars to P. phaseolicola may result from the suppression of synthesis of antibacterial compounds.

Wilt-inducing antibiotic compounds produced by Cephalosporium gregatum

Abstract: Five metabolites were isolated from the culture filtrate of a clone of Cephalosporium gregatum from diseased adzuki beans. The phytotoxicity and antibiotic activity of these compounds were tested. Among the compounds, which were characterized as derivatives of tetronic acid and designated Gregatin A, B, C, D and E, Gregatin A, C and D produced wilting, death of leaves and vascular browning of adzuki bean and mung bean cuttings. These same compounds produced leaf wilting but no vascular browning in soybean and kidney bean cuttings, thus confirming the results of earlier pathogenicity trials on specificity of the adzuki bean pathogen to adzuki beans. The results suggest a possible role for Gregatin A, C and D in pathogenesis. Gregatin A, and to a lesser extent C and D, were also inhibitory to a wide range of fungi and bacteria in antibiotic tests.

Effect of blasticidin S on development of potential of potato tuber cells to react hypersensitively to infection by Phytophthora infestans

Abstract: It was reported previously that the hypersensitive death of surface cells of discs infected by the incompatible race 0 of Phytophthora infestans occurred late, when inoculated soon after preparation (cutting) of the discs. When other discs were inoculated 17 to 20 h or more after cutting, on the contrary, hypersensitive cell death occurred very quickly. These results indicated that the cells at the cut surface lack the ability to undergo immediate hypersensitivity, but they acquire this ability by 20 h after cutting. When tuber discs were treated with 10 parts/10 6 of blasticidin S (BcS) for 3 h beginning 3 h after cutting, the cells did not acquire the potential to react hypersensitively. This indicated that de novo protein synthesis was necessary for cells of the cut surface of tubers to acquire this potential. On the contrary, treatment with BcS had no effect on the potential, when the treatment was done after the potential had been allowed to build up after cutting. However, hypersensitive cell death in tissues which were treated with BcS 20 h after cutting was delayed when inoculation with incompatible race 0 was made 14 h after BcS treatment. Potato cells infected by compatible race 1 survived for a long time. In this case, too, cell death was greatly delayed by treatment with BcS, resulting in an increase in aerial mycelium and spores production. This suggested that de novo protein synthesis is not necessary for the establishment of a compatible relation between the host and the parasite.