652 ( I ) Direct effects of non-vector herbivores 653 (2) Indirect effects, mediated through the host 654 IV. Root-shoot interactions 657 ( I ) Above-ground insects and below-ground fungi 657 (2) Above-ground fungi and below-ground insects 658 . . . . . . 11. The effect of plant pathogenic fungi on herbivorous insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (c) Effects on insects . . . . . . . . . . . . 648 . . . . . . 111. The effect of herbivorous insects on plant pathogenic fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Induced resistance a common defence against pest and pathogen ? . . . . 660 ( I ) The hypersensitive response and lignification . . . . . . . . 661 ( 2 ) Phytoalexins . . . . . . . . . . . . . . 662 (3) Proteinase inhibitors 663 (4) Lipoxygenase 664

Three‐way interactions between plant pathogenic fungi, herbivorous insects and their host plants

SUMMARY
Phytoalexin synthesis is a defence-response- that is characterized by a requirement for a number of distinct elements, all of which must be present for the response to be expressed fully. These same elements: a signal, a cellular receptor, a signal transduction system and a responsive metabolic system, are also used to describe a stimulus-response system. A number of molecular species can function as signal molecules or elicitors of phytoalexin synthesis, including poly- and oligosaccharides, proteins and polypeptides, and fatty acids. Few receptors for elicitors have been identified but those that have been are proteins located on the plasma membrane of the plant. Induction of phytoalexin synthesis involves selective and co-ordinated activation of specific defence response genes, including those encoding the enzymes of phytoalexin synthesis, and these genes constitute the responsive metabolic system. The separate, and distant, locations of the receptor and the responsive genes means that the event in which the signal is perceived by the receptor must be relayed to the genes by means of a second messenger system. Several second messengers are candidates for such a coupling- or signal transduction-system, including udenosine-3′,5′-cyclic monophosphate, Ca2+, diacylglycerol and inositol 1,4,5-trisphosphate, active oxygen species and jasmonic acid. Each has been examined as a possible component of the signal transduction system mediating between the elicitor receptor interaction and the phytoalexin synthesis it induces. Analysis of the signalling events is made complex by the simultaneous solicitation by the invading micro-organism of several defence responses, each of which might involve elements of a different signal system. The same complexity is evident which the role of phytoalexin accumulation in resistance is analysed. Evaluation of the contribution made by phytoalexin accumulation towards resistance has been attempted by the use of various inhibitors and enhancers of the process. Transgenic and mutant plants with specific alterations in one or more ot those elements necessary for the plant to respond to the signals for phytoalexin synthesis and other defence responses, are beginning to aid resolution of the complex pattern ot signalling events and the respective roles of the inducible defence mechanisms in resistance.

Tansley Review No. 86 Accumulation of phytoalexins: defence mechanism and stimulus response system.

Phytoalexin biosynthesis is part of the defense mechanism of soybean (Glycine max) plants against attack by the fungus Diaporthe phaseolorum f. sp. meridionalis (Dpm), the causal agent of stem canker disease. The treatment of soybean cotyledons with Dpm elicitor or with sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in a high accumulation of phytoalexins. This response did not occur when SNP was replaced by ferricyanide, a structural analog of SNP devoid of the NO moiety. Phytoalexin accumulation induced by the fungal elicitor, but not by SNP, was prevented when cotyledons were pretreated with NO synthase (NOS) inhibitors. The Dpm elicitor also induced NOS activity in soybean tissues proximal to the site of inoculation. The induced NOS activity was Ca(2+)- and NADPH-dependent and was sensitive to the NOS inhibitors N(G)-nitro-L-arginine methyl ester, aminoguanidine, and L-N(6)-(iminoethyl) lysine. NOS activity was not observed in SNP-elicited tissues. An antibody to brain NOS labeled a 166-kD protein in elicited and nonelicited cotyledons. Isoflavones (daidzein and genistein), pterocarpans (glyceollins), and flavones (apigenin and luteolin) were identified after exposure to the elicitor or SNP, although the accumulation of glyceollins and apigenin was limited in SNP-elicited compared with fungal-elicited cotyledons. NOS activity preceded the accumulation of these flavonoids in tissues treated with the Dpm elicitor. The accumulation of these metabolites was faster in SNP-elicited than in fungal-elicited cotyledons. We conclude that the response of soybean cotyledons to Dpm elicitor involves NO formation via a constitutive NOS-like enzyme that triggers the biosynthesis of antimicrobial flavonoids.

Nitric oxide synthase-mediated phytoalexin accumulation in soybean cotyledons in response to the Diaporthe phaseolorum f. sp. meridionalis elicitor.

The normal (25° C) response of parsley (Petroselinum crispum Mill.) cell suspension cultures to ultraviolet (UV) light was suppressed by a simultaneous 37° C heat-shock treatment, as indicated by the loss of the inducibility of two enzymes of flavonoid biosynthesis, phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone synthase. The effects on enzyme activity and on enzyme synthesis in vitro and in vivo were similar, indicating that regulatory control is at an early step of gene expression, presumably transcription. When heat shock was given during the course of an ongoing UV induction, both enzyme synthesis and enzyme activities ceased rapidly. Likewise, the induction of phenylalanine ammonia-lyase by an elicitor from Phytophthora megasperma f. sp. glycinea was terminated upon transfer from 25° C to 37° C. Based on these and previously published data, it is concluded that stress responses in this system are preferentially expressed in the order of heat shock, fungal elicitor and UV light.

The induction of phenylpropanoid biosynthetic enzymes by ultraviolet light or fungal elicitor in cultured parsley cells is overriden by a heat-shock treatment.

Laboratory studies showed that the soybean phytoalexin glyceollin, an isoflavonoid previously investigated as an inhibitor of fungal pathogens, is an effective antifeedant for some insect species. Glyceollins extracted from soybean, Glycine max Merrill, cotyledons were applied to the surface of common bean leaves, Phaseolus vulgaris L., in five concentrations, including physiological concentration, for feeding preference tests. Leaves treated with glyceollin at concentrations below physiological levels were less acceptable to the southern corn root worm, Diabrotica undecimpunctata howardi Barber, and the Mexican bean beetle, Epilachna varivestis Mulsant, than untreated leaves. Feeding deterrence was positively correlated with increasing concentrations. The bean leaf beetle, Cerotoma trifurcata Forster, was not affected even by very high doses. An ethological concentration (EC 50) was computed based on the log dose–reduction of acceptance of treated disks. The EC 50 for the Mexican bean beetle was 6.1 μg/mg leaf dry weight and 3.5 μg/mg for the southern corn rootworm. Results indicate that soybean phytoalexins may represent a common defense against microorganisms and insect herbivores.

Effect of Glyceollin, a Soybean Phytoalexin, on Feeding by Three Phytophagous Beetles (Coleoptera: Coccinellidae and Chrysomelidae): Dose versus Response

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 AgNO 3 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.

D. Classen

Papers

Temperature-induced susceptibility of soybeans to Phytophthora megasperma f. sp. glycinea: production and activity of elicitors of glyceollin