SUMMARY
Many secondary metabolites found in plants have a role in defence against herbivores, pests and pathogens. In this review, a few examples are described and discussed, and some of the problems in determining the precise role(s) of such metabolites highlighted. The role of secondary metabolites in defence may involve deterrence/anti-feedant activity, toxicity or acting as precursors to physical defence systems. Many specialist herbivores and pathogens do not merely circumvent the deterrent or toxic effects of secondary metabolites but actually utilize these compounds as either host recognition cues or nutrients (or both). This is true of both cyanogenic glucosides and glucosinolates, which art discussed in detail as examples of defensive compounds. Their biochemistry is compared and contrasted. An enormous variety of secondary metabolites are derived from shikimic acid or aromatic amino acids, many of which have important roles in defence mechanisms. Several classes of secondary products are ‘induced’ by infection, wounding or herbivory, and examples of these are given. Genetic variation in the speed and extent of such induction may account, at least in part, for the difference between resistant and susceptible varieties. Both salicylates and jasmonates have been implicated as signals in such responses and in many other physiological processes, though their prescise roles and interactions in signalling and development are not fully understood.

https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1469-8137.1994.tb02968.x

Secondary metabolites in plant defence mechanisms

Antibiotic phenols have been found in all plants investigated to date. Some occur constitutively and are thought to function as preformed inhibitors associated with nonhost resistance (84, 94, 128, 134). Others, which are the subject of this review, are formed in response to the ingress of pathogens, and their appearance is considered as part of an active defense response. Since the first suggestions that phenolic intermediates have a role in the active expres­ sion of resistance, an underlying problem in ascertaining that such secondary metabolites are of primary (rather than secondary) importance has been the localization and timing of the host response. In this review we address the problem of response localization and localization of phenolics relative to the sequential development of stages of disease that lead ultimately to resistance expression. Initial demonstrations that phenols are significant components of the host

Phenolic Compounds and Their Role in Disease Resistance

Many plants produce low-molecular-weight compounds which inhibit the growth of phytopathogenic fungi in vitro. These compounds may be preformed inhibitors that are present constitutively in healthy plants (also known as phytoanticipins), or they may be synthesized in response to pathogen attack (phytoalexins). Successful pathogens must be able to circumvent or overcome these antifungal defenses, and this review focuses on the significance of fungal resistance to plant antibiotics as a mechanism of pathogenesis. There is increasing evidence that resistance of fungal pathogens to plant antibiotics can be important for pathogenicity, at least for some fungus-plant interactions. This evidence has emerged largely from studies of fungal degradative enzymes and also from experiments in which plants with altered levels of antifungal secondary metabolites were generated. Whereas the emphasis to date has been on degradative mechanisms of resistance of phytopathogenic fungi to antifungal secondary metabolites, in the future we are likely to see a rapid expansion in our knowledge of alternative mechanisms of resistance. These may include membrane efflux systems of the kind associated with multidrug resistance and innate resistance due to insensitivity of the target site. The manipulation of plant biosynthetic pathways to give altered antibiotic profiles will also be valuable in telling us more about the significance of antifungal secondary metabolites for plant defense and clearly has great potential for enhancing disease resistance for commercial purposes.

Fungal Resistance to Plant Antibiotics as a Mechanism of Pathogenesis

One of the best and longest-studied defense response of plants to infection is the induced accumulation of antimicrobial, low-molecular-weight secondary metabolites known as phytoalexins. Since the phytoalexin hypothesis was first proposed in 1940, a role for these compounds in defense has been revealed through several experimental approaches. Support has come, for example, through studies on the rate of phytoalexins in relation to cessation of pathogen development, quantification of phytoalexins at the infection site, and relationship of pathogen virulence to the phytoalexin tolerance. Evidence in support of phytoalexins in resistance as well some recent advances in phytoalexin biosynthesis are reviewed. Criteria for evaluating a role for phytoalexins in disease resistance are also discussed.

PHYTOALEXINS: What Have We Learned After 60 Years?

I. Spore Attachment and Invasion.- 1. Adhesion of Fungi to the Plant Surface: Prerequisite for Pathogenesis.- 2. Signaling for Infection Structure Formation in Fungi.- 3. The Plant Cell Wall as a Barrier to Fungal Invasion.- 4. Rust Basidiospore Germlings and Disease Initiation.- 5. Attachment of Mycopathogens to Cuticle: The Initial Event of Mycoses in Arthropod Hosts.- 6. The Fate of Fungal Spores in the Insect Gut.- 7. Candida Blastospore Adhesion, Association, and Invasion of the Gastrointestinal Tract of Vertebrates.- 8. Infectious Propagules of Dermatophytes.- II. Fungal Spore Products and Pathogenesis.- 9. Melanin Biosynthesis: Prerequisite for Successful Invasion of the Plant Host by Appressoria of Colletotrichum and Pyricularia.- 10. The Plant Cuticle: A Barrier to Be Overcome by Fungal Plant Pathogens.- 11. Appearance of Pathogen-Related Proteins in Plant Hosts: Relationships between Compatible and Incompatible Interactions.- 12. The Role of Cuticle-Degrading Enzymes in Fungal Pathogenesis in Insects.- 13. Potential for Penetration of Passive Barriers to Fungal Invasion in Humans.- 14. Dihydroxynaphthalene (DHN) Melanin and Its Relationship with Virulence in the Early Stages of Phaeohyphomycosis.- III. Host Response to Early Fungal Invasion.- 15. Invasion of Plants by Powdery Mildew Fungi, and Cellular Mechanisms of Resistance.- 16. Induced Systemic Resistance in Plants.- 17. The Plant Membrane and Its Response to Disease.- 18. The Fungal Spore: Reservoir of Allergens.- 19. Conidia of Coccidioides immitis: Their Significance in Disease Initiation.- 20. Cell-Mediated Host Response to Fungal Aggression.- 21. Suppression of Phagocytic Cell Responses by Conidia and Conidial Products of Aspergillus fumigatus.- IV. Molecular Aspects of Disease Initiation.- 22. Molecular Approaches to the Analysis of Pathogenicity Genes from Fungi Causing Plant Disease.- 23. Current Status of the Molecular Basis of Candida Pathogenicity.- Taxonomic Index.

The Fungal spore and disease initiation in plants and animals

A radioimmunoassay specific for glyceollin I was used to quantitate this phytoalexin in roots of soybean (Glycine max [L.] Merr. cv Harosoy 63) after infection with zoospores of either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin. The sensitivity of the radioimmunoassay and an inmmunofluorescent stain for hyphae permitted quantitation of phytoalexin and localization of the fungus in alternate serial cryotome sections from the same root. The incompatible interaction was characterized by extensive fungal colonization of the root cortex which was limited to the immediate vicinity of the inoculation site. Glyceollin I was first detected in extracts of whole roots 2 hours after infection, and phytoalexin content rose rapidly thereafter. Significant concentrations of glyceollin I were present at the infection site in cross-sections (42 micrometers thick) of such roots by 5 hours, and exceeded 0.6 micromoles per milliliter (EC90in vitro for glyceollin I) by 8 hours after infection. Longitudinal sectioning (14 micrometers thick) showed that glyceollin I accumulated particularly in the epidermal cell layers, but also was present in the root cortex at inhibitory concentrations. No hyphae were observed in advance of detectable levels of the phytoalexin and, in most roots, glyceollin I concentrations dropped sharply at the leading edge of the infection. In contrast, the compatible interaction was characterized by extensive unchecked fungal colonization of the root stele, with lesser growth in the rest of the root. Only small amounts of glyceollin I were detected in whole root extracts during the first 14 hours after infection. Measurable amounts of glyceollin I were detected only in occasional cross-sections of such roots 11 and 14 hours after infection. The phytoalexin was present at inhibitory concentrations in the epidermal cell layers, but the inhibitory zone did not extend appreciably into the cortex. Altogether, these data support the hypothesis that the accumulation of glyceollin I is an important early response of soybean roots to infection by P. megasperma, but may not be solely responsible for inhibition of fungal growth in the resistant response.

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Quantitative Localization of the Phytoalexin Glyceollin I in Relation to Fungal Hyphae in Soybean Roots Infected with Phytophthora megasperma f. sp. glycinea

Race:cultivar-specific induction of enzymes related to phytoalexin biosynthesis in soybean roots following infection with Phytophthora megasperma f. sp. glycinea.

Primary roots of soybean (Glycine max (L.), Merrill, cv. Harosoy 63) seedlings were inoculated with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea and total callose was determined at various times after inoculation. From 4 h onward, total callose was significantly higher in roots showing the resistant rather than the susceptible response. Local callose deposition in relation to location of fungal hyphae was determined in microtome sections by its specific fluorescence with sirofluor and was quantified on paper prints with an image-analysis system. Callose deposition, which occurs adjacent to hyphae, was found soon after inoculation (2, 3 and 4 h post inoculation) only in roots displaying the resistant response, and was also higher at 5 and 6 h after inoculation in these resistant roots than in susceptible roots. Early callose deposition in the incompatible root-fungus reaction could be a factor in resistance of soybean against P. megasperma.

Race cultivar-specific differences in callose deposition in soybean roots following infection with Phytophthora megasperma f.sp. glycinea.

The activities of the following enzymes in soybean roots were determined at early times after infection of the roots with zoospores of an incompatible or a compatible race of Phytophthora megasperma f.sp. glycinea: dimethylallyl-diphosphate : 3,6a,9-trihydroxypterocarpan dimethylallyltransferase (prenyltransferase), an enzyme specific for glyceollin biosynthesis; NADPH-cytochrome reductase and hydroxymethylglutaryl-CoA reductase, enzymes related to the glyceollin pathway; and isocitrate dehydrogenase. Already at 4 h after infection there was a higher activity of the prenyltransferase in the incompatible interaction than in the compatible interaction, and enzyme activity in the incompatible interaction increased considerably between 4 and 8 h after infection. In the compatible interaction prenyltransferase activity was only slightly higher than in uninfected roots. The activity of the other enzymes in infected roots was not significantly different from that in the uninfected roots. No qualitative differences could be detected between the two-dimensional patterns of unlabelled proteins or proteins labelled with L-[35S]methionine of infected and uninfected roots at early times after infection. We conclude from these and earlier results (A. Bonhoff et al. (1986) Arch. Biochem. Biophys. 246, 149-154) that infection of the soybean roots with an incompatible race of the fungus leads to selective induction of the phytoalexin pathway and presumably to induction of other as yet unknown defense mechanisms.

Further investigations of race:cultivar-specific induction of enzymes related to phytoalexin biosynthesis in soybean roots following infection with Phytophthora megasperma f.sp. glycinea.

Phytoalexin production is one of a number of inducible plant defense reactions which is thought to confer disease resistance against microbial infections in plants. Soybean (Glycine max) tissues produce and accumulate isof lavonoid phytoalexins (glyceollins) following either inoculation with a soybean pathogen, the fungus Phytophthora megasperma f.sp. glycinea, or treatment with a β-glucan elicitor isolated from the fungal cell walls. Studies on the regulation of phytoalexin biosynthetic enzymes suggested that the phytoalexin defense response in soybean is controlled by temporary gene activation. The activities of several of the glyceollin biosynthetic enzymes have been studied during race-cultivar specific interactions between P. megasperma zoospores and intact soybean primary roots, a natural site of attack by the fungus. Only in the incompatible (host-resistant) interaction, is there an early enhancement of the enzyme activities starting at 2–4 h after inoculation, which correlates with the onset of glyceollin accumulation and expression of the hypersensitive response. Elicitor treatment of soybean cell suspension cultures causes major changes in the population of total translatable mRNA, which indicates large metabolic changes associated with phytoalexin synthesis and possibly other defense responses of the challenged cells.

Anne Bonhoff

Papers

Quantitative Localization of the Phytoalexin Glyceollin I in Relation to Fungal Hyphae in Soybean Roots Infected with Phytophthora megasperma f. sp. glycinea

Race:cultivar-specific induction of enzymes related to phytoalexin biosynthesis in soybean roots following infection with Phytophthora megasperma f. sp. glycinea.

Race cultivar-specific differences in callose deposition in soybean roots following infection with Phytophthora megasperma f.sp. glycinea.

Further investigations of race:cultivar-specific induction of enzymes related to phytoalexin biosynthesis in soybean roots following infection with Phytophthora megasperma f.sp. glycinea.

Phytoalexin Synthesis in Soybean Following Infection of Roots with Phytophthora Megasperma or Treatment of Cell Cultures with Fungal Elicitor