Australasian Plant Pathology (1998) 27: 73-79

Histopathology of Ranunculus acris infected by a mycoherbicide,Sclerotinia sclerotiorum

S.Greerr'",R.E.Gaunr', Le. Han'eyB and G.W. Bourdot"

ADepartment of Plant Science, PO Box 84, Lincoln University, Lincoln, New ZealandBAgResearch, PO Box 60, Lincoln, New Zealand.epresent address: Agriculture and Agri-Food Canada Research Centre, Saskatoon, Saskatchewan, Canada.

Corresponding author: S. Green (Email greens@em.agr.ca)

Abstract

An anatomical study of the crown of Ranunculus acris (giant buttercup) and histopathological studies of infection ofthe crown by Sclerotinia sclerotiorum were carried out to assess the basis of crown resistance to this potential rnyco­herbicide. Resistance was largely related to morphological features of the crown, these being a thickened peripheralcortex, deposition of lignified material at the margin of infected tissue, a response to wounding, and the resistance ofthe crown's dense network of vascular tissues. Together, these non-specific defence mechanisms limited infectionwithin the crown of R. acris and enabled recovery of the plant by regeneration from the crown buds.

Introduction

Ranunculus acris subsp. acrisL., thegiantbuttercup,is a persistent, perennialweedin dairying regions ofNew Zealand. Resistanceto the herbicidesMCPAand MCPB(Bourdot et al. 1990) prompted researchinto alternative measures for control of this weed.Over the past 20 years, there has been increasinginterest in the use of fungi as biological controlagentsofweedsusing the mycoherbicide approach.Mycoherbicides are plant pathogenicfungiappliedinundativelyto controlweeds(Charudattan 1991).The fungal pathogen Sclerotinia sclerotiorum(Lib.) de Bary causes disease on a wide range ofherbaceous plants, including many weed species(Milleretal. 1989). 11tishasledtotheinvestigationof S. sclerotiorum as a potential mycoherbicideagainst a range ofweedspecies (Riddleetal. 1991;Waiparaetal. 1993;Bourdoterc/. 1995).

SuccessfulbiocontrolofR. acris withS. sclera­tiorum may be limited by the plant's perennialnature. In previous studies, plants recovered frominfectionbygrowthofbudson the crown, its under­ground storage organ, which remained substan­tially intact following inoculation with S. sclera­tiorum (Green et al. 1994and 1995).Conversely,Brostenand Sands (1986)and Bourdot et al. (1995)

Australasian Plant Pathology Vo!. 27 (2) 1998

foundthat S. sclerotiorum invadedthe root systemof Cirsium arvense (Canada thistle) and reducedregrowth capacityfromadventitious rootbuds, thusdemonstratingthe potentialfor control of this per­ennial species. The different responsesof R. acrisand C. arvense to S. sclerotiorum maybe linked tothe relative resistance ofthe undergroundorgans todegradation by this pathogen.

The histopathology and pathogenesis ofSclerotinia spp. on many hosts have been exten­sively documented becauseof the economicimpor­tance of this pathogen worldwide (Maxwell andLumsden 1970; Lumsden andDow 1973; Marcianoet al. 1983).However, host resistance to S. sclera­tiorum is less wellunderstood(Lumsden 1979). Anumberofnon-specific defence mechanismswhichlimit invasion have been reported for other patho­gens. These include anatomical features and hostresponses to infection and wounding (Perry andEvert 1983). Deposits oflignin, suberin,phenolics,waxes,cutins, tannins and other substances occurin the secondarywallsof hostcells as plant tissuesmature, or as a defence response to wounding orinvasion by a pathogen (Ride 1975; Vance et al.1980; Stockwell and Hanchey 1987).

This study was undertaken to increase our un­derstanding of the anatomical response of

73

crowns of R. acristo infectionbyS. sclerotiorum.This wasconsideredan essentialpart of the assess­ment of the mycoherbicidal potentialofthis patho­gen on this vigorous, perennial weed.

Methods

Experiment1. Crownanatomyand histopathologyof crown infection Inoculumconsisted ofkibbledwheat infested with isolate S13ofS. sclerotiorum,originally isolated from Cucurbita moschata(squash) (Green et al. 1995).Under asepticcondi­tions,6-mm-diameter mycelial diskswereremovedwith a cork borer from the growing margin of3-day-old colonies on malt-extract agar (MEA;Oxoid). Colonies were grown at 25°C with a 12 hphotoperiod. Twenty mycelial disksweremixedthor­oughlywith 500 g of sterilekibbledwheat in trays.The trayswere coveredwith sterile aluminiumfoiland incubated in thedarkat 25°Cfor 14days. Inocu­lumwasair-driedfor3daysat 27°Cand thengroundand passedthrough a 2 mm sieve. PlantsofR. acriswere grown from seed in a glasshouse in potscontaining a peat/bark potting mix with 8- to9-month-release Osmocote fertiliser containingNPK(16:3.5:1O).

Fifty plants of R. acris (4-months-old) wereusedin thisexperiment. Inoculum (1g perplant)wasplaced at the petiolebases adjacent to the crownofeachplant. Plants were mistedbeforeand immedi­atelyafter inoculationwith steriledistilledwatertoaid adherence of inoculum, and incubated for21 dayson a glasshouse bench under a mistingunitwhichmaintainedalmostconstantleafwetness. Theglasshouse had supplementary lightingfor 12hldaywith daylength extended to 16 hlday supplied by6 x 400Whalogenbulbs spaced at approximately 1mintervals above the plants. The temperature wasmaintained between 17and 22°C. The plants werearranged in five blocks,and ten harvests werecar­ried out, the first at 3 days after inoculation (DAI),and then every second day until 21 DAI. At eachharvest,one plant wasselected randomlyfromeachblock and the leaves and roots removed. Thecrowns were cut into longitudinal and transversesections and fixed under vacuum for 24 h in FAA(90 mL 70% ethanol; 5 mL glacial acetic acid;5 mL formalin). They were then washed in twochanges of 50% ethanol, dehydrated overnight inthree changes of 70% ethanol, four changes of100%ethanol and two changes of toluol (Shannon

74

Citadel 1000 processor), and embedded in wax(paraplast). Longitudinal and transverse sections(4 mmthick) werecutand stainedwith thionin andorange G (Stoughton 1930), which stained fungalhyphae violet-purple, cellulose walls yellow,lignified tissueblue,amyloplasts purple,and meris­tematic cells deep purple. Five non-inoculatedcrowns werealsoprepared. Sections wereexaminedwithan Olympus BH2compoundmicroscope fittedwithan Olympus OM2camera.

Experiment 2. Effect of wounding on crowninfection Twenty-sixplantsofR. acris (6-months­old)wereusedin this experiment. Four tofiveadja­centleaves wereremoved fromeach plant to exposea sectionofthe crown surface for inoculation. Iso­late S13ofS. sclerotiorum was grown on PDA for3 daysat 25°C with a 12 h photoperiod. Inoculumdisks(3 mmdiameter) wereremoved fromthegrow­ing marginofcolonies, dippedin 0.3% wateragar toaid adherenceand placedonto the crown surfaceofeach of eight plants in the following treatments:

i) Surfacewounding; a 3-mm-diameter sterilecork borer was used to mark the crown, and theexternal layeroftissue within this area removedbygently scraping the surface with a sterile scalpel.Inoculumwas placed on top of this wound.

ii) 'kscul.ar-wounding; crowntissue(3 mmdiam­eter and 2 mm deep, to ensure that the wound wasdeeper than the ring of vascular tissue) was re­moved withthe corkborer,and the inoculumplacedinside the wound.

Hi) Control; surface-wounded plants wereinocu­lated with disks of uninfested PDA.

Afterinoculation, plantswereplacedinsideplas­ticbagswhichhadbeenpre-moistened on the inside,and incubatedfor 21 daysin a growth cabinet with16 h of daylight-fluorescent light at a photon fluxdensity of221EMlm2/s, and day/nighttemperaturesof22°Cand 15°C,respectively. The bags remainedon the plants for the duration of the experiment toprovidehigh humidityand plants were mistedwithsteriledistilled water dailyduring the experiment.The experimental design was a randomised blockwith fourreplicates.

At 7 and 21 DAI, four plants within each treat­ment were selected at random. The crowns weresectioned longitudinally through the inoculatedregion and the extent of infection assessed as fol­lows: 0 =no infection, 1= necrosis of tissue imme­diatelysurroundinginoculumdiskwith nopenetra­tion of vascular layer, 2 == infection spreading to

Australasian Plant Pathology Vol. 27 (2) 1998

causenecrosis oftheperipheral cortexwithnopen­etrationofthevascularlayer, 3=infection penetrat­ing and causing necrosisof thevascularlayerwithinner cortex showingnecrosis, 4 =25-90% crownnecrotic, 5 = over90%crownnecrotic.

The extent of crown infection at 7 DAI and21 DAl wasanalysed withanalysis ofvariance. Thecrown sectionswerepreparedfor light microscopyas described in Experiment 1.

Experiment3. Effect ofwoundingseverity oncrowninfection PlantsofR. acris (6-months--Qld) wereused in this experiment. Inoculumwaspreparedasdescribed in Experiment 2. All leaves except theyoungest were removed to expose the crown sur­face. The plants were sorted into three blocksaccording to crown size, and the following eighttreatments appliedto the crownsofsixplants.Thetreatments were designedto test the relative resist­ance of vascular and cortical tissuesto infection:

A. No wound + inoculationonto outer surface.B. Wound to3 mmdepthwitha 3-mm-diameter

cork borer + inoculationinto wound.e. Woundtoinnercortexwitha 3-mm-diameter

cork borer+ inoculationinto wound.D. Crownsreduced toa central Io-mm-diameter

plugofcortical tissue, cut witha corkborer, retainingapexand rootsystem + inoculation ontosideofplug.

E. As in D but apexexcised.F. As in D but rootsexcised.G. As in D but roots and apex excised.H. Asin G + inoculationwith non-infested agar

plug as control.Tominimise theriskofcontamination, plastic film

was placed across the pot surface around eachcrown,and a sterilecorkborerand scalpelusedforwoundingand inoculating. In Treatments Band C,the inoculum plug was pushed into positionthrougha sterilecorkborertopreventmycelial con­taminationof the outercortex. Theplugsofcorticaltissue in Treatments F, G and H weresupportedinan upright position abovethe layerof plastic filmwitha wireloopattachedto a wooden pegat thesideof the pot. This was done to ensure that all treat­mentswere incubatedsimilarly.

Afterinoculation, plantswereplacedinsidepre­moistenedplastic bags, and incubatedfor 21 daysin a growthcabinetas in Experiment 2.Theexperi­mental design was a randomised block with sixreplicates. All treatments weremisted twice daily forthe first 3 days of the experiment, and then dailyuntil harvest at 21DAI. At harvest, the percentage

Australasian Plant Pathology Vo!. 27 (2) 1998

ofcrowntissueper plant that had rotted wasdeter­minedvisually and the95%confidence limitsofthemeanswerecalculated.

Results and Discussion

Experiment 1. Crownanatomyand histopathologyofcrown infection

Crown anatomy Examination of uninoculatedcrowns of R. acris revealed that it has four mainregions oftissue. Aperipheral cortical layer surroundsa ring ofvasculartissueswhich almostcompletelyencircle an inner cortical region. At the tip of thecrownis the apical zone.

The peripheral cortex,which formed the outerlayer of the crown, generally consisted of matureparenchyma cells with thickened, darkly stainedwalls (Figure 1).The vascular tissue comprised adense cylindrical network ofinterconnected vascularbundles. Theseappearedin cross sectionas a con­tinuous, complex ringofbundles at about0.5-2 mmfrom the outer crownsurface, surroundedon bothsidesby densely packed starch-containing paren­chymacells. The inner cortexconsistedof starch­containing parenchyma cells with thin, lightlystainedwalls, suggestinga predominanceof cellu­lose rather than secondary wall depositions(Figure I). Thesecellsbecame lessdensely packedtowardsthe centreofthe inner cortex.Each crowncontained a largenumber ofaxillarybudsconnectedto the crown vascular network, thus providing aconsiderable numberofpotential regrowthpoints.

Histopathology ofcrown infection Large,granu­lar,vacuolated hyphae, similar to infectionhyphaedescribed by Lumsden and Dow (1973) andLumsden and Wergin (1980), were commonlyobserved accumulating on the outside of theperipheral cortical layerofthe crown,withmacera­tion of the surface cells of the peripheral cortex(Figure 2). Completemacerationof the peripheralcortical cells was rarely observed. Many axillarybudson thecrownremaineduninfectedand devel­opedinto regenerating shoots (Figure 3).

The extent of infectionof the crown peripheralcortex by S. sclerotiorum may be limited by thethickenedcellwalls in this region; thesewallsweredeeply stained (dark blue/purple) indicating thepresenceof lignin (Figure1).Phenolicsubstances,suchas lignin, whichare deposited as plant tissues

75

Figures 1-7 Photomicrographs of infection by Sclerotinia sclerotiorum in 4-month-old crowns ofRanunculus acris. Figure 1 Cross section through a healthy crown showing position of vasculartissue (V) relative to the peripheral cortex (P) and inner cortex (IC). Note the abundance of amyloplasts(starch granules) within all cortical cells (Bar =100 1JlIl). Figure 2 Accumulation of hyphae (By) outsideperipheral cortex (P), 11 days after inoculation. Note maceration of peripheral cortex to several cellsdepth (Bar = 100 urn). Figure 3 Eleven days after inoculation. Note infection ofleaftissue (L) andperipheral cortex (P). B, axillary bud (Bar = 100 urn). Figure 4 Dark staining of cortical tissue at thelesion margin, indicating phenolic deposition, 21 days after inoculation (Bar = 100 urn), Figure 5 Deepstaining of cell walls and protoplast at the infection site, indicating phenolic deposition, 15 "days afterinoculation (Bar = la urn). Figure 6 Rotting of peripheral cortex (P) down to vascular bundles (V),11 days afterinoculation (Bar =100 um). Figure 7 Seven days after vascular wounding + inoculationwith S. sclerotiorum. De, deposition of lignin, suberin, and dead cells on injured surface; W, wound tissue(Bar= 1001JlIl)·

76 Australasian Plant Pathology Vot 27 (2) 1998

mature, can bean effectivebarrier to invading patho­gens since they are not readily degraded by fungalenzymes (Fahn 1990; Cooke and Whipps 1993).

Cells of R. acris at infection sites, or surroundingcrown lesions, often exhibited dark staining of theprotoplast and walls (Figures 4 and 5) indicatingphenolic deposition (Stoughton 1930). These

Experiment 2. Effect of wounding on crowninfection Surface wounding resulted in signifi­cantly (P<0.05) greater crown infection than non­wounding at 7 DAI, but by 21 DAI no treatmentsdiffered (Table 1). In general, wounding and inocu­lating CrO\\l1S did not cause extensive crown infec­tion. The disease severity scores were low for alltreatments (Table 1).

Infection hyphae invaded longitudinallythrough the peripheral cortex of surface woundedcrowns, rather than transversely through vasculartissue and inner cortex. Infection in vascularwounded crowns was limited to parenchyma cellsimmediately surrounding the inoculation wound.By 7 DAI in all wounding treatments, a region oflarge, often elongated parenchyma cells had devel­oped around the wound, the surface of which wascovered with a deeply stained, thick layer of deadcells (Figure 7). This was observed in the peripheralcortex and in the inner cortex ofcontrol crowns aswell as infected crowns; thus it appeared to be aresponse to wounding itselfrather than as a specificresponse to the pathogen. Wounds in tubers ofSolanum tuberosum (potato) healed by deposition

findings were similar to those of Stockwell andHanchey (1987), who, during an investigation ofinfection of hypocotyls of Phaseolus vulgaris byRhizoctonia solani, suggested that the symptomswere due to deposition of lignin and phenolicswithin the cells at the lesion border, and that thiscreated an impermeable barrier to pathogen­produced enzymes. Auld et al. (1994) found thatresistance ofXanthium occidentale (Bathurst burr)to the potential mycoherbicide Colletotrichumorbiculare, included the formation of lignin-likesubstances around leaf infections. The present re­sults indicate that a similar resistance reaction couldbe operating in crown tissues ofR. acris, to limit thefurther spread of infection by S. sclerotiorum .

A consistent observation throughout these ex­periments was the resistance of the crown vasculartissue to infection by S. sclerotiorum (Figure 6).Infection hyphae and cell maceration rarely devel­oped past the crown's dense network of vascularbundles and into the inner cortex; this indicates thatvascular tissue could play an important role inlimiting the spread of infection within crowns. Priorand Owen (1964), Lumsden and Dow (1973) andTariq and Jeffries (1986) also observed resistance ofhost vascular tissues to degradation by Sclerotiniaspp.

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Australasian Plant Pathology Vo!. 27 (2) 1998 77

of suberin on healthy cells adjacent to the injuredsurface, followedby the formation of wound peri­denn cellsbeneath the suberisedlayer; this reducedinfection by Fusarium oxysporum and Erwiniacarotovora pv.carotovora (Morriset al. 1989). Thepresent study has highlighted the capacity ofcortical parenchyma cells in the crown of R. acristo heal after wounding, and prevent entry ofS. sclerotiorum.

Experiment3. Effectofwoundingseverity oncrowninfection The percentage of infected crowntissues21 DAI in treatments A (no wound),B (wounded to3 mmdepth)and H (non-inoculated controlplug)didnot differ, havingzeroor minimal infection. Dissec­tion and visual assessmentofcrowns in treatmentsA and B showed that infection was limited to cellsimmediately adjacentto the inoculationsite.Treat­ments C (wound to inner cortex), D (plug of innercortical tissue + apex + roots),E (plug of inner cor­tical tissue- apex+ roots),F (plugof inner corticaltissue + apex - roots) and G (plug of inner corticaltissue- apex- roots)didnot differ, exhibitingexten­sive or 100% necrosis of tissue 21 DAI (Table2).

These results indicate that the inner pith tissueof crowns of R. acris was susceptible to infectionby S. sclerotiorum. The cells in the inner corticalregion are not densely packed, and have thin, pri­marywallswithoutsecondary thickening(Figure 1).This tissue probably presents little resistance tointercellular infection hyphae ofS. sclerotiorum.

S. sclerotiorum completely invaded crowns ofR. acris onlyrarely, probably becauseofthe difficul­ties of accessing the easily rotted inner pith tissue.Most infections stopped at the peripheral cortex,probablybecause ofthe greater cellular resistancein this region of the crown and a further barrier toinfection provided by the vascular tissues. Theresulting containment of infection enabled theplants to recoverbyvigorous growth of uninfectedaxillarybuds. Therefore, results from these studiesindicate that the resistanceofcrowns ofR. acris toS. sclerotiorum maybe linked to the morphologicalfeaturesofthe crown.This resistancemay limit thefieldefficacy ofS. sclerotiorum as a mycoherbicidefor R. acris when appliedto the foliageof the weed.Efficacy may, however, be improvedbysomesort ofsevere wounding of the upper part of the crown.

Acknowledgements

The authorsthank DaveSavillefor statisticaladviceduring this study. Financial support for this studywas providedby the Commonwealth Scholarshipsand FellowshipsPlan.

Mean severity ofcrown infection"

Table1 The effectofwoundingoncrowninfectionin Ranunculus acris inoculated with Sclerotlniasclerotiorum

Table 2 Effect of increased levelof wounding onpercentage infection of crown tissues ofRanunculus acris 21 days after inoculation withSclerotinla sclerotiorum

7DAIB 21DAI

Treatment" Per cent crown tissuesinfected 21 DAIB

AData refer to infection severity index outlined inthe methods section.BDays after inoculation.'The controltreatmentwasexcluded fromtheanaly­sis since all data values were zero.

AThe 95% confidence limitforeachmeanis giveninbrackets.BDays after inoculation.

Non-woundingSurfacewoundingVascularwoundingControl?LSD(P<O.05)

0.3 0.31.8 0.80.8 1.0

(0.0) (0.0)1.0

A: No woundB: 3 mm woundC:VVoundtoinnercortexD: Inner cortex+apex + rootsE: Inner cortex- apex + rootsF: Inner cortex +apex - rootsG: Inner cortex- apex- rootsH: Non-inoculatedcontrol

6.7 (±8.0)A17.5 (±32.8)50.8 (±50.6)

100.0 (±O.O)70.8 (±48.6)

100.0 (±O.O)100.0 (±O.O)

0.0 (±O.O)

78 Australasian Plant Pathology Vo!. 27 (2) 1998

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Manuscript received 25 June 1997. accepted 2 February1998.

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