C S I R O P U B L I S H I N G

AUSTRALIAN JOURNAL OF

PLANT PHYSIOLOGY

Volume 24, 1997© CSIRO Australia 1997

An international journal of plant function

w w w. p u b l i s h . c s i ro . a u / j o u rn a l s / a j p p

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Published by CSIRO PUBLISHINGfor CSIRO Australia and

the Australian Academy of Science

IntroductionIn the northern sugarcane (Saccharum interspecific

hybrid) growing regions of Australia the oomycete fungusPachymetra chaunorhiza Croft & Dick (Dick et al. 1989) isthe causal organism of the sugarcane disease PachymetraRoot Rot (Croft and Magarey 1989; Magarey 1994). Theonly means of control is through the use of resistantsugarcane cultivars in disease prone areas, as control byfungicide is impracticable (Croft and Magarey 1989). Croftand co-workers have identified resistant sugarcane cultivarsand developed a technique for assessing sugarcane cultivarsfor resistance, thus providing an effective method of control(Croft 1989).

When plants are attacked by pathogens they respond byactivating a variety of defence mechanisms. These includethe accumulation of antimicrobial compounds (Kosuge1969; Darvill and Albersheim 1984; Broglie et al. 1993) andthe physical strengthening of plant cell walls throughincreased production of hydroxyproline-rich glycoproteins,lignin and suberin (Vance et al. 1980; Espelie et al. 1986;Benhamou et al. 1991). Successful defence depends on thespeed with which the pathogen is detected, and the ability of

the plant to activate a response that is toxic or restrictive tothe invading pathogen. Conversely, susceptibility occurswhen the pathogen evades detection or the plantÕs responseis insufficiently rapid or effective.

Despite intensive investigations of plantÐpathogeninteractions in most major crops, sugarcaneÐpathogeninteractions have been little studied at either the biochemicalor molecular levels. Where studied, phenolic compoundshave been associated with increased plant resistance. Thecomposition of pre-formed phenolic compounds in sugar-cane cultivars resistant and susceptible to Colletotrichumfalcatum, the cause of red rot of stalks and mid-ribs, hasbeen investigated by paper chromatography (Singh et al.1993). Several phenolic compounds were present in resistantcultivars but absent in susceptible cultivars. Additionally, thestilbene piceatannol has been identified in sugarcane stemsinfected with C. falcatum (Brinkler and Seigler 1991) andaccumulated with the characteristics of a phytoalexin asdefined by Paxton (1981). Although piceatannol was firstdetected 2Ð3 days after infection of sugarcane stems with C. falcatum, piceatannol concentrations were not correlatedwith resistance in several sugarcane clones (Brinkler and

Aust. J. Plant Physiol., 1997, 24, 143Ð149

10.1071/PP96092 0310-7841/97/020143

Biochemical Responses of Suspension-cultured Sugarcane Cells toan Elicitor Derived from the Root Pathogen Pachymetra

chaunorhiza

Tony K. McGhieAD, Niall P. MaselA, Don MacleanB, Barry J. CroftC and Grant R. SmithA

ABureau of Sugar Experiment Stations, PO Box 86, Indooroopilly, Queensland 4068, Australia.BDepartment of Biochemistry, The University of Queensland, St Lucia, Queensland 4074, Australia.

CBureau of Sugar Experiment Stations, PO Box 566, Tully, Queensland 4854, Australia.DCorresponding author, present address: Division of Marine Science, CSIRO Marine Laboratories,

GPO Box 1538, Hobart, Tasmania 7001, Australia; email, Tony.McGhie@marine.csiro.au

Abstract. The fungus Pachymetra chaunorhiza Croft & Dick causes a root rot in sugarcane(Saccharum interspecific hybrid). Suspension-cultured sugarcane cells prepared from cultivars Q114(P. chaunorhiza resistant) and Q90 (P. chaunorhiza susceptible) were inoculated with a heat-derivedelicitor preparation from P. chaunorhiza and the cellular responses monitored by measuringphenylalanine ammonia-lyase (PAL) and peroxidase (POD) activity and the production of additionalphenolic compounds.

Introduction of the P. chaunorhiza elicitor induced marked changes in the biochemistry of bothsugarcane cell lines. Both cell lines produced additional phenolic compounds not present in untreatedcells and different compounds were produced by each cell line. Induced enzyme activities also differedbetween the cell lines with Q90 (susceptible) showing a large and transitory increase in PAL activity thatwas far greater than that observed for Q114 (resistant). POD activity increased more in Q114 than in Q90,although the differences between the resistant and susceptible cell lines were not as great as for PAL.

Keywords: plantÐpathogen interactions, phenolic compounds, peroxidase, phenylalanine ammonia-lyase, sugarcane, suspension-cultured cells, Pachymetra

T. K. McGhie et al.144

Seigler 1993). Stilbenes have been associated with increaseddisease resistance of grapevines to infection by fungi andincreased resistance of some pine species to soft-rot fungi(Schr�der et al. 1993).

Typically, sugarcane reacts to pathogen or pest attack byproducing an intense reddening of the affected tissue. Thisreddening is, in part, a result of the production of theanthocyanidins, luteolinidin and a luteolinidin glycoside(Godshall and Lonergan 1987). Although crude extracts ofchallenged sugarcane tissues containing theseanthocyanidins were shown to be toxic to C. falcatum,individual compounds were not tested and definitiveevidence of toxicity for the individual compounds has notbeen demonstrated.

Although sugarcane cultivars exhibit a wide range ofsusceptibility/resistance to P. chaunorhiza, the reasons andunderlying mechanisms have not been investigated. Thecurrent procedure for challenging sugarcane with P. chaunorhiza is suitable for rating cultivars for resistance(Croft 1989); however, the technique is not suitable fordetailed investigations of the responses of sugarcane toinfection because the time-course of the pathogen challengecannot be monitored. Here, we report the development of anexperimental procedure for investigating the reactions ofsugarcane to P. chaunorhiza, and the identification ofbiochemical responses of two sugarcane cultivars withdiffering resistance to P. chaunorhiza.

Materials and MethodsPlant Material

Sugarcane cultivars known to be highly susceptible (cv. Q90)and highly resistant (cv. Q114) (Croft 1989) were selected for thisstudy. Homogeneous suspension-cultured cells were produced forboth cultivars using techniques described previously (Taylor et al.1992). Cell lines of both cultivars readily formed stablehomogeneous suspension cultures consisting of clusters containing3Ð4 cells and were maintained by subculturing at 3Ð4 day intervals.All experiments were performed on cell cultures within 8 weeks ofstable homogeneous suspension cultures being obtained.

Elicitor Preparation

Isolate T120-1A of P. chaunorhiza was obtained from the TullySugar Experiment Station and cultured in 10% V8 broth withcontinuous shaking at 120 rpm. Cultures were grown forapproximately 7 days before the mycelium was harvested byfiltration. The elicitor preparation was obtained by grinding themycelium (3 g wet weight) to a fine powder in liquid nitrogen andextracting with 25 mL 100 mM Tris/HCl, pH 7.0. Followingcentrifugation (5000 g, 10 min, 4¡C), 20 mL methanol was addedto the pellet and the mixture shaken overnight. After furthercentrifugation, the pellet was washed once with 20 mL methanoland dried under vacuum for 2 h. After a further wash with 100 mM

Tris/HCl, pH 7.0, the pellet was resuspended in fresh 20 mL ofbuffer and the mixture autoclaved (120¡C, 30 min). Aftercentrifugation, the clear supernatant was used to elicit a responsefrom sugarcane cells.

Cell Culture/Elicitor Experiments

For each experiment a number of 50 mL sugarcane cellsuspension-cultures of each cell line were pooled (after 3 daysgrowth), fresh media was added, and the cultures were divided into10 mL aliquots in 50 mL conical flasks. The cultures wereincubated for a further 2 days to obtain a resumption of logarithmicgrowth. To commence the experiment 0.5 mL of filter-sterilisedelicitor solution or 100 mM Tris/HCl, pH 7.0 (for control) wasadded and the original growth conditions resumed. Individualsuspension-cultures were removed at 4, 8, 24 and 48 h and the cellswere separated by filtration and immediately frozen at Ð70¡C.

HPLC Analysis

Approximately 100 mg of cell material (fresh weight) wasweighed into a 1.5 mL test tube and mixed with 400 mL of 80%acetonitrile in Milli-Q (Millipore, Bedford, MA) water. The cellswere disrupted by ultrasonication for 2 min on ice and the mixturesincubated for a further 2 h at room temperature. Followingcentrifugation the supernatant was removed and analysed byHPLC. The HPLC system consisted of a gradient controller (model610), two pumps (model 510), an autosampler (model 510) and aphotodiode array detector (model 990+), all manufactured byWaters Chromatography (Milford, MA). Separations wereperformed using a Waters NovaPak C18, 4 mm column installed ina Waters RCM 8 ´ 10 module. Pump A delivered 5% methanol,95% Milli-Q water and pump B delivered 95% methanol, 5%Milli-Q water. The pH of both mobile phases was adjusted toapproximately 3 by the addition of 250 mL H3PO4/L. The mobilephase program started at 100% A, 0% B with a linear change to20% A, 80% B over 60 minutes; the composition was maintainedat 20% A, 80% B for a further 10 min before returning to 100% A,0% B for equilibration of the column and the next analysis. Mobilephase flow rate was 0.8 mL/min.

Phenylalanine ammonia-lyase (PAL) Activity

PAL activity was determined by measuring the production ofcinnamic acid (Cahill and McComb 1992). Approximately 100 mgof cell material (fresh weight) was mixed with 750 mL of buffercontaining 10% w/v polyvinylpyrrolidone (insoluble), 0.15% w/vDTT, 0.1% v/v Triton X-100 in 100 mM Tris/HCl, pH 6.5. Eachsample was briefly ground with a pestle in a 1.5 mL microtube andincubated on ice for 15 min. Following centrifugation (17000 g, 5min, 4¡C), PAL activity was determined in the supernatant byadding 50 mL enzyme extract to 1450 mL 10 mM D- or L-phenylalanine in 100 mM Tris/HCl, pH 8.8, and incubating at 35¡Cfor 60 min. The amount of cinnamic acid produced in the reactioncontaining L-phenylalanine was determined by measuring theabsorbance at 290 nm using the D-phenylalanine reaction as theblank. The extinction coefficient used for cinnamic acidquantification was 9180 MÐ1 cmÐ1. PAL activity was expressed asnmol cinnamic acid produced minÐ1 mg proteinÐ1.

Peroxidase (POD) activity

POD activity was determined by measuring the polymerisationof o-methoxyphenol (guaiacol). The reaction mixture (total volume1250 mL), contained 100 mM 2-[N-morpholino]ethanesulfonic acid(MES), pH 5.5, 6 mM o-methoxyphenol, 6 mM hydrogen peroxideand 2 mL of the extract. The enzyme extract, buffer and substratewere mixed together and the reaction started by addition of the

145Responses of Sugarcane Cells to Elicitor

hydrogen peroxide. The reaction rate was monitored by theincrease in absorbance at 430 nm on a Perkin Elmer Lambda 2spectrophotometer using the TimeDrive facility. Enzyme activitieswere expressed as DA430 minÐ1 mg proteinÐ1.

Protein Determination

The protein concentration of enzyme extracts was determinedaccording to Bradford using the BioRad protein reagent and bovineserum albumin as a standard (Bradford 1976).

ResultsInitial experiments showed that culture filtrates obtained

after growth of P. chaunorhiza in liquid culture appeared tocontain little or no elicitor activity, as no visual browningwas observed when added to sugarcane cells. In contrast, theautoclaved mycelial preparation elicited a rapid and intensebrowning reaction within sugarcane cells. To characterisethis reaction further, we performed time-course analyses ofthe suspension-cultured sugarcane cells for PAL, and POD

activity and the production of phenolic compounds after theaddition of this P. chaunorhiza elicitor preparation.

When the P. chaunorhiza elicitor preparation was addedto the suspension-cultured sugarcane cells Q90, a browncolouration first became visible about 4 h after the addition.The intensity of the colouration increased over the time-course and by 48 h the Q90 cells had turned dark red-brown.Addition of elicitor to the Q114 suspension-cultured cellsalso resulted in a colour change starting at about 4 h. Incontrast to Q90, the cells changed to a yellow-orange colourafter 48 h. In both cell lines, the medium also accumulatedcoloured components similar to those observed in the cells.Analysis of the acetonitrile extracts of both cell lines showedthat UV-absorbing compounds, additional to those producedby untreated cells, were accumulated after addition of the P.chaunorhiza elicitor preparation. Chromatogram tracesshowing the changes over time for both cultivars arepresented for Q114 in Fig. 1(a) and Q90 Fig. 1(b). In cells of

Fig. 1. HPLC chromatogram traces (310 nm) of acetonitrile extracts from suspension-cultured cells of (a) resistant cv. Q114 and (b) susceptiblecv. Q90 following the addition of the heat-derived elicitor from P. chaunorhiza.

(a) (b)

T. K. McGhie et al.146

Q114 (resistant cultivar), four major new peaks wereobserved. The UV spectra of these peaks had maximabetween 280 and 340 nm (Fig. 2a), indicating that they arelikely to be phenolic compounds. Peak Q114-a had aretention time and UV spectrum identical to chlorogenicacid, a cinnamic acid derivative known to be present insugarcane (Paton 1992). In contrast, cells of Q90(susceptible cultivar) produced three major new UV-absorbing components (Fig. 1b), none of which correspondto the components produced in Q114 cells. The UV spectraof these compounds (Fig. 2b) were similar to chlorogenicacid although the longer retention times indicate that thesecompounds are less polar, suggesting that they could beoxidation products of chlorogenic acid. Enzymatic oxidationproducts resulting from the polymerisation of chlorogenicacid by polyphenol oxidase have spectra resemblingchlorogenic acid and had longer retention times whenanalysed using a similar HPLC system to that used here(Paton and Duong 1992). Oxidative polymerisation ofchlorogenic acid, immediately on formation, would accountfor the absence of a major peak corresponding tochlorogenic acid in Q90 cells. This explanation would alsobe consistent with the visual increases in colour as theappearance of new peaks in the HPLC chromatograms werealso first detected at 4 h after addition of the P. chaunorhizaelicitor preparation.

Concomitant with the increase in phenolic content of thesuspension-cultured cells were increases in POD (Fig. 3a)and PAL (Fig. 3b) activities. POD activity increased in boththe Q90 and Q114 suspension-cultured cells, although theQ114 POD activity increased more rapidly after addition ofelicitor and attained a higher level than in Q90 cells. Incontrast, PAL activity showed a large and sustained increasein activity only in the susceptible cultivar Q90 compared

with the resistant cultivar Q114, reaching approximately 25times the control activity at 24 h after addition of elicitor. InQ114 cells, PAL activity only showed a small, transientincrease 4 h after addition of elicitor.

DiscussionThe results presented here demonstrate that an elicitor

solution obtained by autoclaving cell walls from the fungalroot pathogen P. chaunorhiza was able to stimulate defenceresponses in suspension-cultured suspension cells byinducing increases in both POD and PAL activities, andcausing the production of, as yet unidentified, phenoliccompounds.

Phenolic phytoalexin compounds are often positivelyassociated with increased disease resistance (Kosuge 1969;Nicholson and Hammerschmidt 1992). In particular,increased production of isoflavones by many legumes canincrease resistance (Blount et al. 1992). Other phenoliccompounds such as cinnamic acid derivatives (Bernards etal. 1991; Assabgui et al. 1993) and stilbenes also possesantimicrobial activity (Schr�der et al. 1993). During thecurrent experiments we attempted to determine if extractsfrom elicited sugarcane cells were toxic to P. chaunorhiza byincorporating the extracts into agar media and measuring theincrease in colony diameter. Although P. chaunorhizagrowth in vitro was inhibited when authentic chlorogenicacid was added to the medium, no toxic effects weredetected for the sugarcane cell extracts (data not presented),even though HPLC analysis indicated that chlorogenic acidwas present in extracts of Q114 cells. Failure to observe arelationship between plant resistance and the tissueconcentration of antimicrobial compounds accumulated byplants in response to a pathogen has been reported, andattributed to the fact that antimicrobial concentrations of

Fig 2. UV spectra of the major elicitor-induced components produced by (a) Q114 and (b) Q90 suspension-cultured cells. Components areidentified in Fig. 1 .

(a) (b)

147Responses of Sugarcane Cells to Elicitor

whole tissue do not necessarily reflect the localisedconcentrations present at the site of infection (Brinkler andSeigler 1993). Using UV microspectroscopy, Snyder and co-workers demonstrated that anthocyanidin phytoalexinsaccumulated in sorghum tissues to a localised concentrationgreatly exceeding that required for toxicity toColletotrichum graminicola (Hipskind et al. 1990; Snyder etal. 1991), clearly demonstrating that phytoalexins canaccumulate to toxic concentrations within plant cells and canbe a direct cause of increased plant resistance.

Increased activity of both POD and PAL were observed insugarcane cells following addition of the P. chaunorhizaelicitor preparation. POD activity increased in both cellslines, especially at 24 and 48 h after addition of the elicitor.The increase was greater and became evident sooner in theresistant Q114 cultivar compared with the susceptiblecultivar Q90. In contrast, the activity of PAL was greatlyincreased both in magnitude and duration in cultivar Q90,compared with cultivar Q114 which showed a smalltransient increase at 4 h. Increases in POD activity arethought to lead to resistance to fungal attack by increasingcell wall lignification, producing biotoxic hydrogenperoxide and contributing to the propagation of the stresssignals leading to increased intensity of the plant defence.POD activities have often been associated with increasedresistance; a particularly relevant example is the interactionbetween Phytophthora cinnamomi and Eucalyptus

calophylla (Cahill and McComb 1992). PAL activity isbelieved to determine the biosynthetic flux through thephenylpropanoid pathway and so higher PAL activityproduces greater concentrations of phenolic phytoalexins intissues (Orr et al. 1993). In the susceptible cultivar Q90,PAL activity increased substantially as did the concentrationof new phenolic compounds that were not detected inunchallenged cells. In the resistant cultivar Q114, PALactivity showed a relatively small, transient increase at 4 h;however similar to Q90, an increase in phenolic compoundswas also observed. The compounds accumulated in Q114cells were different to those accumulated in Q90 cells asindicated by differences in UV spectra and retention time. Insome plants, PAL activity is positively related to theproduction of phenolic phytoalexins and to resistance but, inother systems, PAL activity shows no apparent relationshipto resistance (Nicholson and Hammerschmidt 1992). Therelationship between PAL activity and pathogen resistancecan be influenced by additional factors and the interpretationof PAL activity levels requires caution. For example, in theinteraction between P. chaunorhiza and sugarcane, theaccumulation of phenolic phytoalexins may be controlled byan alternative pathway such as the hydrolysis of pre-formednon-toxic glycosylated phenolic compounds (Moniz de Sa etal. 1992) which may not have been detected by this HPLCanalysis. It is also possible that resistance may not be relatedto the accumulation of phytoalexin phenolic compounds but

Fig. 3. Enzyme activities of Q90 (susceptible) and Q114 (resistant) suspension-cultured cells following the addition of the heat-derived elicitorfrom P. chaunorhiza. Ð Ð Ð unchallenged. ÐÐÐ challenged. Á Q114. ª Q90. (a) POD and (b) PAL. All points represent to two independent samples.The mean difference between sample duplicates was 12.2 AU430 ´ 10Ð3 minÐ1 mg proteinÐ1 and 2.9 nmol cinnamic acid min-1 mg proteinÐ1 for PODand PAL assays respectively.

(a) (b)

T. K. McGhie et al.148

due entirely to other mechanisms such as hydrolytic enzymeproduction or the strengthening of cell walls.

A prerequisite for the study of plant responses to infectionby a pathogen is a suitable experimental system which mustbe highly reproducible and capable of controlled assessmentof plant responses to challenge by the pathogen. This hasbeen difficult to achieve for the interaction between P.chaunorhiza and sugarcane. Croft (1989) developed a pot-based method for infecting sugarcane plants to determine theresistance rating for individual sugarcane cultivars thatinvolved planting germinated sugarcane setts into pottingmixture containing P. chaunorhiza spores and assessing theinfection as the proportion of rotted roots after 6Ð8 weeks.This method was not suitable for this study because of longexperiment cycle times of 2Ð3 months and, moreimportantly, because the infected material recovered hasbeen infected by P. chaunorhiza for unknown (and varying)lengths of times. We therefore attempted to develop aninoculation technique more amenable to laboratory basedexperimentation. As an alternative to sugarcane plants, weattempted to infect excised sugarcane roots maintained onagar and small sugarcane plantlets grown aseptically intissue-culture conditions. Both attempts were unsuccessful(data not presented). Excised roots could not be infectedbefore the roots showed signs of senescence and, althoughinfection of several tissue-culture plantlets was eventuallyachieved, the technique was not sufficiently reproducible. Itis possible that P. chaunorhiza growth was retarded by therelatively high salt concentration required for plant growthin vitro. Both the above experiments were constrained byour current lack of knowledge of how to control thegermination of P. chaunorhiza oospores, and our consequentinability to obtain consistent infection of sugarcane by P.chaunorhiza under controlled conditions.

As a result of the difficulty in obtaining synchronousinfection of sugarcane under controlled conditions, weinvestigated the use of suspension-cultured sugarcane cellschallenged with elicitor preparations from P. chaunorhiza.Similar techniques have been widely and successfully usedto detect and identify plant responses to the presence ofpathogens; however, the approach has a number ofadvantages and disadvantages which need to be consideredwhen evaluating results. Advantages are: a high degree ofreproducibility; rapid experiment cycle times; and mostimportantly, as each cell in the culture is uniformly exposedto the elicitor, the response of cells is relatively uniform andintense and therefore can be more readily detected andquantified. This contrasts to tissue or organ infection wherecells are exposed to variable amounts of a pathogen-generated elicitors and therefore respond to different degrees.The main disadvantage of using suspension-cultured cellswith a pathogen-derived elicitor is that the system is farremoved from the natural setting where differentiated plant

cells respond to the presence of an invading pathogen.However, despite this, it has generally been found thatresponses detected in suspension-cultured cells/elicitorsystems parallel those found in whole plants (Schmelzer etal. 1989; Campbell and Ellis 1992). We believe the use of asuspension-cultured cells/elicitor system is valid for initiallydefining plant responses to pathogen challenge provided thatthe parameters identified are subsequently characterised in amore natural plantÐpathogen system.

Concluding RemarksWe have demonstrated that a combination of cell-

suspension cultures and heat-derived elicitor preparationscan be used to monitor and investigate the reaction ofsugarcane to the presence of the pathogen P. chaunorhiza.The primary objective of this study was to identifydifferences in the biochemical reaction of resistant andsusceptible sugarcane cultivars in order to identify potentialbiochemical markers for resistance. Differences weredetected in all three parameters monitored (accumulation ofindividual compounds, POD, PAL), some considerable.Increases in POD activity and accumulation of phenoliccompounds are frequently associated with increasedresistance to fungal pathogens and were observed in theresistant sugarcane cultivar Q114 but also in the susceptiblecultivar Q90. These results suggest that the expression ofsugarcane defence systems are complex and further researchwill be required to characterise the defence response in awider selection of resistant and susceptible sugarcanecultivars and to compare responses in both cultured cells andnormal root tissue, in seeking useful markers for resistanceto P. chaunorhiza.

AcknowledgmentsThe authors thank R. C. Magarey for his interest in this

work and valuable suggestions. Financial support for thisresearch was provided by the Sugar Research andDevelopment Corporation, grant BS79S. This research waspartly supported by the Co-operative Research Centre forTropical Plant Pathology.

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Manuscript received 14 August 1996, accepted 14 November 1996