Pathogen Hyphae Research Articles

INHIBITORY ACTIVITY OF YEASTS ISOLATED FROM FIG FRUITS AGAINST PENICILLIUM DIGITATUM

We report the isolation of 129 yeast from the carposphere of fig fruits capable of inhibiting Penicillium digitatum development on artificially wounded citrus fruits. The wounds were challenged with a pathogen suspension (10 5 or 10 6 conidia ml -1 ) after yeast inoculation (10 cells ml -1 ). The best results (96-100% inhibition) were obtained with the following strains: 5A of Pichia guilliermondii; 43E, 44E and 69F of Candida famata. One isolate, 43E, showing particularly high activity, was further tested in order to better characterise its mode of action against the pathogen. Mechanisms of action were primarily competition for space and nutrients through colonisation of pathogen hyphae, as seen in SEM photographs; it was not possible to detect antibiotic or toxic substance production by the yeast. The isolate 43E of C. famata was found to be compatible with high thiabendazole (TBZ) concentrations (5 g l -1 ). A test was carried out to determine the inhibitory activity at lower cell concentrations than usual (10 7 , 10 6 and 10 5 ml -1 ) and in the presence of TBZ. The fungicide was employed at about one/seventh and one/fifteenth of the concentration in commercial use. The results show that the 43E strain of C. famata, when used together with 0.1 g l -1 TBZ at the concentration of 10 6 cells ml -1 , gives significantly better disease control than either TBZ or the yeast alone.

Cell Defense Responses Associated with Localized and Systemic Resistance to Phytophthora parasitica Induced in Tomato by an Arbuscular Mycorrhizal Fungus

The arbuscular mycorrhizal fungus Glomus mosseae is able to confer bioprotection against Phytophthora parasitica in tomato roots. Localized and induced systemic resistance (ISR) have been demonstrated to be involved in pathogen control in mycorrhizal and nonmycorrhizal roots with a split root experimental system. Decreased pathogen development in mycorrhizal and nonmycorrhizal parts of mycorrhizal root systems is associated with accumulation of phenolics and plant cell defense responses. G. mosseae-containing cortical cells in the mycorrhizal tissues are immune to the pathogen and exhibit a localized resistance response with the formation of cell wall appositions reinforced by callose adjacent to intercellular hyphae. The systemically induced resistance in nonmycorrhizal root parts is characterized by elicitation of host wall thickenings containing non-esterified pectins and PR-1a protein in reaction to intercellular pathogen hyphae, and by the formation of callose-rich encasement material around P. parasitica hyphae that are penetrating root cells. PR-la protein is detected in the pathogen wall only in these tissues. None of these cell reactions are observed in nonmycorrhizal pathogen-infected root systems, where disease development leads to host cell death. The cellular and molecular basis of bioprotection by an arbuscular mycorrhizal fungus is discussed in relation to that induced by other nonpathogenic microorganisms.

Cytochemical Investigation of the Antagonistic Interaction Between a Microsphaeropsis sp. (Isolate P130A) and Venturia inaequalis

ABSTRACT In an attempt to better understand the mode of action of the antagonistic fungus Microsphaeropsis sp., the interaction between this fungus and Venturia inaequalis was studied, using both light and electron microscopy. Cytological observations indicated that the antagonistic interaction between the two fungi likely involves a sequence of events, including (i) attachment and local penetration of Microsphaeropsis sp. into V. inaequalis hyphae; (ii) induction of host structural response at sites of potential antagonist entry; (iii) alteration of host cytoplasm; and (iv) active multiplication of antagonistic cells in pathogen hyphae, leading to host cell breakdown and release of the antagonist. The interaction was investigated further by gold cytochemistry. The use of gold-complexed beta-1,4-exoglucanase and wheat germ agglutinin/ovomucoid-gold complex to localize cellulosic beta-1,4-glucans and chitin monomers, respectively, resulted in regular labeling of V. inaequalis cell walls. This finding supports other studies refuting the classification of ascomycetes as only a glucan-chitin group. At an advanced state of parasitism, the labeling pattern of cellulose and chitin, which clearly showed that the level of integrity of these compounds was affected, suggested the production of cellulolytic and chitinolytic enzymes by Microsphaeropsis sp. Wall appositions formed in V. inaequalis in response to the antagonist's attack contained both cellulose and chitin. However, penetration of this newly formed material frequently succeeded. This study provides the first detailed picture of the cytological events associated with mycoparasitism in V. inaequalis.

Biocontrol by yeasts of blue mould of citrus fruits and the mode of action of an isolate of Pichia guilliermondii

SummaryNineteen yeast isolates, from different fruits, were tested for antagonistic activity against blue mould (Penicillium italicum) on artificially wounded citrus fruits. Strains 1 A, 5A, and 29A of Pichia guilliermondii, strains 21D, 22D and 30F of Candida famata and strain 13 L of C. sake reduced infection from 86 to 98% compared with the control. Isolate 5 A of P. guilliermondii was the most effective antagonist and its mode of action was investigated further. Its activity against the pathogen declined when the number of yeast cells inoculated in the wound was reduced although it showed good ability to colonize the wound site and grow rapidly, cell numbers increasing by up to 60-fold within 24 h. Scanning electron microscopy of the wound also showed attachment of the yeast cells to the pathogen hyphae. The high antagonistic activity of isolate 5A appeared to be related to its capability to occupy space and use nutrients available in the wound, thus depriving the pathogen of them. It also induced in the wound the biosynthesis of the phytoalexins (scoparone and scopoletin); however this mode of action seems not to play an essential role in the biocontrol of the pathogen.

β-1,3-glucanase activity of two saprophytic yeasts and possible mode of action as biocontrol agents against postharvest diseases

Abstract The yeasts Rhodotorula glutinis (isolate LS-11) and Cryptococcus laurentii (isolate LS-28), showing different levels of antagonistic activity against a range of postharvest pathogens, were examined for their possible mode(s) of action, in order to highlight the reasons for the higher activity of isolate LS-28. Competition for nutrients appeared to play a role in the activity of both yeasts, especially in the case of isolate LS-11. Direct interaction with pathogen hyphae was shown only by cells of this same isolate, whereas no interaction with fungal hyphae was observed for the more active antagonist LS-28. The latter isolate was able to produce in vitro significantly higher levels of extracellular β -1,3-glucanase activity than LS-11 when grown in the presence of hyphal cell walls of the pathogens Penicillium expansum and Botrytis cinerea as sole carbon sources. In our experimental conditions, antibiosis did not appear to be involved in the activity of either antagonists.

Mycoparasitism of Pythium ultimum by Antagonistic Binucleate Rhizoctonia Isolates in Agar Media and on Capsicum Seeds

AbstractHyphal interactions between two antagonistic binucleate Rhizoctonia isolates (BNR) and the seedling dampingoff pathogen, Pythium ultimum var. sporangiiferum, were observed by both light‐and scanning electron microscopy (SEM), on agar media and on capsicum seeds in sterilized potting mix. Both BNR isolates displayed similar mycoparasitic behaviour towards P. u. sporangiiferum on agar media. This included parallel growth along the pathogen hyphae, formation of hook‐shaped hyphal tips and coils on the surface of P. u. sporangiiferum hyphae and penetration and growth within pathogen structures. Disruption of cytoplasmic streaming and disorganisation of pathogen cytoplasm were also observed. SEM observations revealed alterations in P. u. sporangiiferum cell wall structure and the presence of penetration holes apparently due to digestion by the BNR. P. u. sporangiiferum was also parasitised by both BNR isolates on capsicum seed coats, with parallel growth, hook formation and coils commonly observed. The above observations indicated that mycoparasitism is a possible mode of action of BNR against P. u. sporangiiferum.

Ultrastructural and Cytochemical Investigations of the Antagonistic Effect of Verticillium lecanii on Cucumber Powdery Mildew

ABSTRACT Chronological events of the intercellular interaction between Verticillium lecanii and cucumber powdery mildew, caused by Sphaerotheca fuliginea, were investigated at different times after inoculation by transmission electron microscopy. V. lecanii hyphae colonized host structures by tight binding, apparently mediated by a thin mucilaginous matrix. As early as 24 h after application of the antagonist, increased vacuolation and disorganization of the cytoplasm of the pathogen hyphae were easily detected. By 36 h after treatment, plasmalemma retraction and local cytoplasm aggregation were typical features of damage. Labeling chitin with the wheat germ agglutinin (WGA)/ovomucoid-gold complex showed that intracellular invasion of S. fuliginea by V. lecanii did not cause extensive host cell wall alterations, except in the area of hyphal penetration. By 48 h after inoculation, further cytoplasm disorganization was observed, as evidenced by the loss of cell turgor and contortion of the cell wall. Such deformation suggests that penetration of the antagonist results from mechanical pressure or localized enzymatic hydrolysis through the action of chitinases, as confirmed by the pattern of labeling obtained with the WGA/ovomucoid-gold complex. By 72 h after contact between the fungi, S. fuliginea cells were markedly collapsed, depleted of their protoplasm due to extensive multiplication of the antagonist, and totally encircled by the antagonist. Based on the current observations, the antagonism of S. fuliginea by V. lecanii appears to involve the following events: (i) attachment of the antagonist to the powdery mildew fungus; (ii) mechanical pressure and production of cell-wall degrading enzymes such as chitinases; (iii) penetration and active growth of the antagonist inside the pathogen hyphae; and (iv) digestion of host tissues and release of the antagonist from dead cells of S. fuliginea. The interaction between V. lecanii and S. fuliginea also affected the morphological and structural features of the haustorial bodies, as shown by increased vacuolation, distortion, and necrotization of the haustorial lobes. These observations provide the first experimental evidence that V. lecanii, primarily known as an entomopathogenic fungus, also has the potential to colonize mycelial structures of S. fuliginea. V. lecanii, therefore, may become a valuable alternative to current management of cucumber powdery mildew in greenhouses.

Effect of Taxol and Related Compounds on Growth of Plant Pathogenic Fungi

Taxol, currently in use as therapy for refractory ovarian cancer, has no known function in the plant (Taxus brevifolia) from which it is isolated. In an effort to explore its biological role, we investigated the effect of Taxol and related compounds on the growth of plant pathogenic fungal hyphae. Taxol and cephalomannine inhibited the growth of several fungi, especially Ptytophthora and Pythium species (Oomycetes) and Rhizoctonia solani (Basidiomycetes). Baccatin III had no effect on the growth of any fungus tested. Strains of Aspergillus (Deuteromycetes) and Fusarium (Ascomycetes) were not inhibited by any taxane. As little as 1.0 nmol of Taxol or cephalomannine severely retarded radial growth of Phytophthora species on solid medium

In Vivo and in Vitro Nuclear Conditions in the Hyphae of Fusarium solani and Aspergillus flavus

Aspergillus flavus and Fusarium solani are opportunistic agents of mycoses in immunosuppressed patients. While species of both genera can be readily identified in culture, they are indistinguishable in histological sections. In this report an attempt was made to differentiate between them in vivo on the basis of nuclear condition. The results showed that in vivo, as well as in vitro, hyphae of both pathogens consisted of multinucleate cells. Furthermore, the nuclear condition in Fusarium species differs from one strain to another within the same species. Differentiation on this basis is discussed as a possibility among fungal pathogens that are indistinguishable in vivo.

Growth and sporulation of the dikaryons of the dwarf bunt fungus in wheat plants and in culture

In the wheat plants infected with dwarf bunt, sporulation occurs only in developing kernels. The dikaryon was isolated from infected kernels and from the rachis of infected spikes. The development of pathogenic hyphae and teliosporogenesis in vivo and in vitro is described. Ultrastructural studies indicated that in vivo teliospore primordia detach from sporogenous hyphae in the hymenium layer and develop into mature teliospores. Teliospores were borne terminally on sporogenous hyphae in vitro. Substances that stimulate hyphal branching and inhibit teliospore formation of the dikaryon were isolated from vegetative wheat tissues but not from bunt-infected spikes containing the sporulating dikaryon. The dikaryon resumed sporulation only when transferred to rich nutrient media without extracts. Extracts from infected wheat spikes containing the sporulating dikaryon induced the vegetative dikaryon to sporulate. The bioregulators involved in these phenomena were not identified. Bioactive extracts had no apparent effect on the growth of the monokaryon. A fluorochrome, mithramycin, was used to detect nuclei in monokaryotic and dikaryotic hyphae. New approaches to bunt control are discussed. Key words: Tilletia caries, Tilletia controversa, monokaryon, teliospores, bioregulators.

Biological control of air-borne pathogens

Some pathogens are partly controlled by microorganisms that occur naturally on aerial surfaces of plants, and many attempts have been made to improve control by applying selected antagonists to such surfaces. Antagonists often compete for nutrients with the pathogen, and antibiotics may be formed that reduce germination of its spores and subsequent growth. Hyphae of fungal pathogens may be killed on contact with the antagonist or by direct penetration. The plant’s defences may be stimulated before challenge by a pathogen. Apart from killing the pathogen, an antagonist may reduce its reproductive capacity. The examples given illustrate the operation of these different mechanisms in the control of a wide variety of diseases. For diseases of foliage, flowers or fruit, glasshouse crops offer more attractive possibilities for control than field crops because the population level of antagonists is easier to maintain. In some cases plants can be protected by inoculation before transplanting them to the field. Foliage and canker diseases of forest trees present problems too intractable for successful control, but in orchards the prospects are better; for example, methods are available for combining pruning with application of inoculum. Similarly, in some circumstances tree stumps can be inoculated to prevent colonization by a pathogen. Where biological methods are as effective as chemical ones and comparable in cost, they are to be preferred on environmental grounds. In some cases they can be combined with advantage; for example a lower concentration of fungicide may suffice if applied with an antagonist.

Ultrastructural Comparison of Microbodies in Pathogenic and Saprophytic Hyphae of Fusarium oxysporum f. sp. lycopersici

Ultrastructural Comparison of Microbodies in Pathogenic and Saprophytic Hyphae of Fusarium oxysporum f. sp. lycopersici

The mechanisms associated with the antagonistic relationship between corticium solani and Gliocladium virens

Laboratory tests showed that direct parasitism was unimportant compared with antibiosis in the antagonistic activities of Gliocladium virens against the pathogen Corticium (Rhizoctonia) solani . Apart from evidence of antibiotic production on agar plates and in liquid media, lethal concentrations of antibiotics were demonstrated on antagonist-infested sclerotia borne on potato tubers but not on other parts of the tuber surface. Culture filtrates of the antagonist contained both gliotoxin and viridin, were active against the pathogen hyphae and killed the sclerotia even at low concentrations. The compensating effect of continuous antibiotic production for the instability of the antibiotics produced by the progressively developing colonies of the antagonist on stored potato tubers is discussed with regard to the control of C. solani by biological methods.