In the course of fungal attack on the cyanogenic rubber tree (Hevea brasiliensis Muell.-Arg.) HCN is liberated from infected tissue. The HCN interferes with plant host and fungal pathogen. It becomes inhibitory to active defense responses which are dependent on biosynthetic processes as far as a threshold concentration is transgressed

BIEHL, B.

GIESEMANN, A.

JUNQUEIRA, N. T. V.

LIEBEREI, R.

English

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Plant Physiol. (1989) 90, 33-360032-0889/89/90/0033/04/$01 .00/0Received for publication September 9, 1988and in revised form December 12, 1988CommunicationCyanogenesis Inhibits Active Defense Reactions in Plants'Reinhard Lieberei*, Bole Biehl, Anette Giesemann, and Nilton T. V. JunqueiraBotanisches Institut der Technischen Universitat Braunschweig, D-3300 Braunschweig, PF 3329 Federal Republicof Germany (R.L., B.B., A.G.) and EMBRAPA/CNPSD, C. P. 319, 69,000 Manaus, Amazonas, Brasil-(N.T.V.J.)ABSTRACTIn the course of fungal attack on the cyanogenic rubber tree(Hevea brasiliensis Muell.-Arg.) HCN is liberated from infectedtissue. The HCN interferes with plant host and fungal pathogen.It becomes inhibitory to active defense responses which aredependent on biosynthetic processes as far as a threshold con-centration is transgressed.Many plants liberate HCN after mechanical or chemicalinjury to their cells (2) or in the course of fungal infection(20). The role of this cyanogenic process in plants has beenwidely discussed but still remains unclear. There is consider-able evidence that HCN liberated during tissue destruction bygrazing animals acts as a feeding deterrent (10), but the dataon cyanogenesis are quite controversial in the case of fungaldiseases. Fry's working group (4) provided excellent evidencethat fungal pathogens of cyanogenic plants generally arehighly tolerant to hydrocyanic acid. From these findings itwas deduced that plant pathogenic fungi which lack a mech-anism to cope with HCN are incapable ofinfecting cyanogenicplants. Therefore, cyanogenic compounds were generally re-garded to be preformed resistance factors (8).In contrast to this conclusion, Ludtke and Hahn (18) re-ported that highly cyanogenic flax varieties are susceptible toColletotrichum lini, the causal agent of flax anthracnose,whereas moderately cyanogenic varieties are resistant to thesame isolates ofthe fungus. In addition, Lieberei (15) reporteda distinct correlation between a high capacity for HCN liber-ation and the high susceptibility of rubber tree leaves toMicrocyclus ulei, the causal agent of rubber tree leaf blight.M. ulei is tolerant to HCN, it even grows better in HCN-containing atmosphere (17). Obviously in flax and the rubberplant, HCN is not a simple resistance factor which can beovercome by pathogens adapted to it (16, 17); instead theHCN is impairing the defense response ofthese plants. Screen-ing studies revealed that all strongly cyanogenic Hevea vari-eties were susceptible to all existing isolates ofM. ulei, whereaslow cyanogenic plants respond in typical race-cultivar specificreaction patterns (11, 15).The objective of this study was to test directly, at themolecular level, to what extent the host plant's resistance is' Supported by the Deutsche Forschungsgemeinschaft and the Ger-man Agency for Technical Cooperation (GTZ).influenced by cyanogenesis. It could be shown that the activemetabolic defense response characterized by the productionof the Hevea phytoalexin scopoletin (6), is considerably im-paired by HCN confirming its role on a molecular level as asusceptibility factor.MATERIALS AND METHODSDefined Hevea clones were grown in standard garden soilin a glass house at 85 to 90% RH and 20 to 27°C, undernormal daylight with additional artificial illumination fromHQI lamps (800 uE m-2 s-'). Liquid fertiliser (Hoaglandsolution [9]) was applied once weekly. For the infectionstudies, young leaves of the defined developmental stage B (3)were chosen.The cyanogen content was measured indirectly by analyz-ing HCN after the enzymatic breakdown of the cyanogenicglycosides ( 14). Scopoletin was extracted in ethanol, partiallypurified by TLC, and quantified by fluorometry in a HitachiF-3000, according to Giesemann et al. (6). Culture of thepathogen Microcyclus ulei has been described by Lieberei etal. (17) as have the conditions for inoculation and incubation(14).RESULTSPlant-Pathogen CombinationsMicrocyclus ulei occurs as a variety of physiological raceswhich can be divided into two main groups according to theirvirulence for Hevea benthamiana and Hevea brasiliensis ( 11).Isolates of virulence group I infect Hevea plants carryinggenes of H. benthamiana. They normally do not infect H.brasiliensis, whereas the isolates of virulence group II infectpure H. brasiliensis especially but normally react incompati-bly with crossbreeds carrying genes of H. benthamiana. Thisgeneral relationship is broken, however, by those varieties ofH. brasiliensis which are characterized by a high HCN-poten-tial (Table I).HCN-Liberation from Infected LeavesThe amount ofHCN liberated in the course of leafinfectiondepends on the Hevea clone and the respective host reaction(14). In a comparative assay the amount of HCN liberated,as well as the amount of the phytoalexin scopoletin (6) pro-duced were estimated (Table II). The low cyanogenic, highlyresistant plants liberated only small amounts of HCN but33Plant Physiol. Vol. 90,1989Table I. Host Reaction to Four Isolates of the Different Virulence Groups of M. uleiHost Reaction toHevea Clones Species HON-Potential Virulence group Virulence group IIin Leaves _______________Isolate 1 Isolate 2 Isolate 3 Isolate 4mg CN-/g dry weightFx 3899 H. benthamiana x H. brasiliensis 3.4 ± 0.4 sa S R RF 4542 H. benthamiana 2.6 ± 0.7 S S R S/RF 985 H. brasiliensis 1.2 ± 0.3 R R S SMDF 180 H. brasiliensis 1.3 ± 0.4 R R S SRRIM 600 H. brasiliensis 13.7 ± 1.3 S S S SIAN 717 H. brasiliensis x H. benthamiana 12.4 ± 2.8 S S b Sa s, leaves susceptible or highly susceptible to M. ulei; R, Resistance reaction; S/R, intermedi-ate. b IAN 717 was not tested with this isolate 3, but was susceptible to 14 out of a series of 16isolates of M. ulei, collected from all growing areas of Brasil (11). For description of symptoms, seeJunqueira et al. (1 1).Table II. HCN Liberation and Scopoletin Accumulation in the Course of Leaf Infection of Hevea byM. uleiInfection was done with isolate 42 (virulence group 11) of the collection of N. T. V. Junqueira.Quantification of scopoletin was performed by fluorometry of TLC-purified samples using scopoletin asstandard. Although scopoletin accumulation was restricted to the border of the infection site, thecontent is calculated per leaf weight. Since less than 10% of the leaf area was infected, the scopoletinconcentration at the infection site was at least 10 times larger than given here.Reaction to HCN Liberation Scopoletin AccumulationHevea Olone Species Virulence Group II within 48 h within 48 hg 0CN-/g dry weight Og/g dry weightGT 1 H. brasiliensis Susceptible 33.2 ± 7.6 0.880RRIM 600 H. brasiliensis Susceptible 38.6 ± 2.4 0.480Fx 25 H. brasiliensis Intermediate 2.2 ± 0.2 1.375F 4542 H. benthamiana Resistant 2.3 ± 0.1 3.175accumulated large amounts of scopoletin around the penetra-tion points. The concentration of scopoletin around the le-sions on resistant reacting plants is high enough to causeinhibition of spore germination and of hyphal growth of M.ulei (6). In contrast, highly cyanogenic, susceptible plantsliberated relatively large amounts of HCN and revealed agreatly reduced accumulation of scopoletin (Table II).Enhancement of Scopoletin Production under LowCyanide ConditionsThe concentration of HCN in the atmosphere around theinfected leaf, liberated during pathogenesis, can be reducedby passing a continuous stream of moistened air through theincubation vessel which contains the inoculated leaves or byadding an alkaline trap for HCN to the incubation system.Both treatments lead to a significant reduction of the size andnumber of the lesions (14). Correlated with this enhancedresistance is the accumulation of scopoletin, impaired in thepresence of HCN, which takes place in the tissue adjacent tothe infection sites. This reaction was checked in a number ofclones (Fig. 1). Under conditions ofHCN depletion the highlycyanogenic, susceptible clones GTI and RRIM 600 (bothpure H. brasiliensis) accumulated increased amounts of sco-poletin. The low cyanogenic, resistant clone F 4542 (H.benthamiana) did not respond in this manner but did show asignificant decrease in scopoletin accumulation, when HCNwas artificially added to the incubation vessel containing theinoculated leaves. The artificial addition ofHCN to the tissuecorresponded in time (48 h) and in concentration to theamount of HCN which is liberated by infected, stronglycyanogenic plants. The liberation pattern of HCN from in-fected leaves has been described in detail (14). Apparently, inweakly cyanogenic plants the defense response, as measuredby scopoletin production, is not inhibited by the low amountsof HCN liberated during infection, whereas in highly cyano-genic plants the resistance phenomenon can only occur whenthe concentration of endogenously produced HCN is artifi-cially lowered.DISCUSSIONIn contrast to a wide range of other fungitoxic substancesproduced by plants, HCN does not accumulate around in-fected cells but easily diffuses throughout the host tissue. Asa volatile, highly soluble gas, it passes through a leaf blade inless than 30 s (13). Therefore, after liberation from its precur-sor, HCN rapidly becomes distributed throughout the neigh-boring tissue and comes into contact with the pathogen aswell as with the surrounding host tissue. This suggests thatboth the pathogen and the host will be poisoned by cyanideunless they are capable of effective detoxification reactions.34 LIEBEREI ET AL.CYANOGENESIS INHIBITS ACTIVE DEFENSE_ control,infectedinfected,m~ infected,GT 1susceptible+ + +uninfectedHCN added,HCN depletedRRIM 600 Fx 25susceptible intermediate+++ + +Figure 1. Enhancement of scopoletin production under low cyanide conditions. Inoculated leaves of developmental stage B2 (3) were incubatedin Lips-Conway diffusion plates, containing an inner chamber of 4 cm diameter and a concentric outer chamber of 8 cm diameter. The leaveswere located at the borders of the inner chamber. The outer chamber was filled with 5.0 mL of water (control and infected leaves) with 5.0 mLof 0.04 mm sodium cyanide solution (HCN added) or with 5.0 mL of 0.1 M NaOH (HCN depleted). In a second method, HCN depletion waseffected by flushing a stream of prewashed, moistened air over the surface of the incubating leaves. Both methods for HCN depletion led to thesame results. The control leaves often contained small amounts of scopoletin, which was induced by stress conditions in the greenhouse. Forevery clone leaves without scopoletin always occurred, when the plants were grown under semisterile conditions in growth chambers (6). (+++),Highly cyanogenic clones; (++), intermediate cyanogenic clones; (+), weakly cyanogenic clones.The pathogen M. ulei is highly tolerant to HCN (17) but, incontrast, in leaves of the plant host, HCN concentrationslower than 0.025 mmol/L are sufficient to inhibit the photo-synthetic C02 fixation in intact tissue; as a consequence ofthis inhibition, many synthesis associated reactions arestopped (R Lieberei, unpublished data).Successful pathogens on cyanogenic plants have beenshown to produce the enzyme formamide hydrolyase (FHL)which catalyses the hydration ofHCN to produce formamide(5). HCN metabolism in plants differs from that of fungi;FHL has not been detected in plants which contain insteadrhodanese and f3-cyanoalanine synthase. These enzymes arethought to be involved in HCN detoxification (1, 19).Weakly cyanogenic plants liberate only small amounts ofHCN against which their detoxification reactions providesufficient protection. Thus, the resistance reactions and theamounts of scopoletin produced in the course of these reac-tions are not inhibited. Therefore, scopoletin production willnot be enhanced when HCN in the atmosphere surroundingthe leaf is diminished experimentally. However, in stronglycyanogenic plants the liberation of HCN is rapid and theresulting HCN concentration in the leaves is too large to bedetoxified immediately by the plant. Consequently, the plant'sdefense responses will be hampered by HCN. This becomesevident when comparing the amount of scopoletin producedin highly cyanogenic plants under standard incubation con-ditions with the same plants under conditions of HCN-deple-tion (Fig. 1). Additionally, these data suggest that the suscep-tible, highly cyanogenic plants possess the same genetic poten-tial for defense reactions as the weakly cyanogenic plants butare inhibited by HCN produced endogenously in the courseof tissue colonization. These findings do not rule out a rolefor HCN as a defense factor against noncyanide adaptedmicroorganisms. However, the fact that most of the micro-organisms involved are able to detoxify HCN (12) suggeststhat HCN per se can hardly be regarded as a resistance factoragainst them. It would seem that the HCN produced by theplant beyond a certain threshold level inhibits its own defensemechanisms, thus acting as a susceptibility factor in the plant.Like in many other plant-pathogen interactions (7), this ex-ample also supports the view that the speed of a resistancereaction is far more decisive for the outcome of an infectionthan whether it occurs or not. Considering the system de-scribed in this paper, it is not just the velocity of HCNliberation which is decisive, but rather the combination ofHCN liberation and HCN detoxification. The velocity andextent of HCN-liberation in cyanogenic plants are known tobe governed by several factors recently described by Selmar0.70.60.50.40.30.20.1so.a-Ba)ox~EzCC,)4-totoc;utF 4542resistant+35-0-mPlant Physiol. Vol. 90, 1989et al. (21) and Lieberei (15). However, little is known aboutthe rate of HCN detoxification in plants.ACKNOWLEDGMENTSGrateful acknowledgment is made to the CNPSD/EMBRAPA(Brazil) for administrative help and to Sabine Grocholewski for skilfultechnical assistance.LITERATURE CITED1. Chew MY (1973) Rhodanese in higher plants. Phytochemistry12: 2365-23672. Conn EE (1981) Biosynthesis of cyanogenic glycosides. In BVennesland, EE Conn, CJ Knowles, J Westley, F Wissing, eds,Cyanide in Biology. Academic Press, London, pp 183-1963. Dijkman MJ (1951) Hevea-Thirty years of research in the fareast. University of Miami Press, Coral Gables, FL, pp 155-1774. Fry WE, Evans PH (1977) Association of formamide hydrolyasewith fungal pathogenicity to cyanogenic plants. Phytopathol-ogy67: 1001-10065. Fry WE, Millar ML (1972) Cyanide degradation by an enzymefrom Stemphylium loti. Arch Biochem Biophys 151: 468-4746. Giesemann A, Biehl B, Lieberei R (1986) Identification of sco-poletin as a phytoalexin of the rubber tree Hevea brasiliensis.J Phytopathol 117: 373-3767. Gold RE, Aist JR, Stolzenburg MC, Marshall MR, Israel HW(1986) Effects of calcium nitrate and chlortetracycline on pa-pillae formation, ml-o resistance and susceptibility of barley topowdery mildew. Physiol Mol Plant Pathol 29: 115-1298. Hegnauer R (1986) Cyanogene Verbindungen. In R Hegnauer,Chemotaxonomie der Pflanzen, Vol 7, Nachtrage zu Bd 1 u.2. Birkhauser Verlag, Basel, pp 345-3749. Hoagland DR, Arnon DJ (1938) The water culture method forgrowing plants without soil. Univ Calif Coll Agric Circ No.34710. Jones DA (1981) Cyanide and coevolution. In B Vennesland, EEConn, CJ Knowles, J Westley, F Wissing, eds, Cyanide inBiology. Academic Press, London, pp 509-51611. Junqueira NTV, Chaves GM, Zambolim L, Gasparotto L, Al-fenas AC (1986) Variabilidade fisiologica de Microcyclus ulei.Fitopatol Bras 11: 823-83312. Knowles CJ, Bunch AW (1986) Microbial cyanide metabolism.Adv Microbiol Physiol 27: 73-11113. Lieberei R (1984) Cyanogenese und Resistenz. Habilitations-schrift, Techn Univ Braunschweig, pp 134-15614. Lieberei R (1986) Cyanogenesis during infection of Hevea bras-iliensis with Microcyclus ulei. J Phytopathol 115: 134-14615. Lieberei R (1988) Relationship of cyanogenic capacity (HCN-c)of the rubber tree Hevea brasiliensis to susceptibility to Micro-cyclus ulei, the agent causing South American leaf blight. JPhytopathol 122: 54-6716. Lieberei R (1988) The role of cyanide and cyanogenesis inplant-pathogen interactions. In D Evered, S Harnett, eds,Cyanide Compounds in Biology. Wiley, Chicester, pp 195-20017. Lieberei R, Schrader A, Biehl B, Chee KH (1983) Effect ofcyanide on Microcyclus ulei cultures. J Rubb Res Inst Malaysia31: 227-23518. Ludtke M, Hahn H (1953) Uber den Linamaringehalt gesunderund von Colletotrichum befallener junger Leinpflanzen.Biochem Z 324: 433-44219. Miller J, Conn EE (1980) Metabolism of hydrogen cyanide byhigher plants. Plant Physiol 65: 1199-120220. Myers DF, Fry WE (1978) Enzymatic release and metabolismof hydrogen cyanide in sorghum infected by Gloeocercosporasorghi. Phytopathology 68: 1717-172221. Selmar D, Lieberei R, Biehl B, Conn EE (1989) a-HydroxynitrileLyase in Hevea brasiliensis and its significance for rapid cyan-ogenesis. Physiol Plant 75: 97-10136 LIEBEREI ET AL.

Cyanogenesis inhibits active defense reactions in plants.

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Cyanogenesis inhibits active defense reactions in plants.

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