Trans. Br. mycol. Soc. 91 (3),427-432 (1988)

[ 427 ]

Printed in Great Britain

INFLUENCE OF THE LEAF SURFACE OF HEVEA ONACTIVITY OF COLLETOTRICHUM GLOEOSPORIOIDES

By RADZIAH NOM ZAINUDDINRubber Research Institute of Malaysia, P. O. Box 10150, 50908 Kuala Lumpur, Malaysia

AND MOHAMMED OMARFakulti Sains Hayat, Universiti Kebangsaan Malaysia, 43600 Bangi Selangor, Malaysia

The susceptibility of some rubber clones to leaf disease caused by Colletotrichumgloeosporioides could not be related to the surface morphology of their leaves. Although minordifferences were observed in the time of deposition of wax and in its pattern and density, thesedid not affect spore germination or appressorial formation. Similar levels of germinationoccurred on both resistant and susceptible clones, but on slightly older and hence more waxyleaflets, spores tended to form longer germ-tubes. No directional response of germ-tubegrowth to any topographical features of the leaf surface was observed.

Colletotrichum leaf disease of rubber (Heveabrasiliensis Muell.-Arg.), caused by Colletotrichumgloeosporioides (Penz.) Penz. & Sacc., is a majorcause of secondary leaf fall affecting mature rubbertrees in Malaysia (Rao, 1975). During the annualrefoliation period, the fungus attacks young un-folding leaflets and causes defoliation. Trees con-tinuously attacked usually have smaller canopiesand subsequently suffer losses in latex yield (Rao,1972 ) .

Rubber clones vary in their susceptibility to thedisease (Wastie, 1973), but the mechanism ofdefence is not fully understood. This investigationwas undertaken to determine whether there aredifferences in the morphology of the leaf surface ofresistant and susceptible clones and the effects ofthese differences, if any, on germination andappressorial formation of the pathogen.

MA TERIALS AND METHODS

The selection of clones for this study was based ontheir susceptibility to Colletotrichum (Wastie,1973): PB 5/63 (highly susceptible), RRIM 600(moderately susceptible), FX 25 and PB 5/51(resistant).

The buddings were raised in polybags for abouta year and were pruned to encourage uniform leafproduction at the start of the experiment. Leafletswere tagged at budburst and only those aged5-16 d were used.

Variations in leaf surface morphology

Several methods were tested to find the mostsuitable way of preparing leaflets for scanning

electron microscopy (SEM) examination. Theseincluded freeze-drying, fixation followed byfreeze-drying or air-drying and fixation followedby dehydration with an acetone series and criticalpoint drying. All treatments except the last, whichwas preferred and described below, caused con-siderable shrinkage of specimens. Fresh specimenscould not be used as there was considerablecharging when they were examined in the SEM.

Leaflets of known age were detached andimmediately fixed in 6 % glutaraldehyde in phos-phate buffer at pH 6'8-1'0. Leaflets were cut intopieces approx. 3 x 3 mm, treated with 2 % osmiumtetroxide and dehydrated with increasing con-centrations of acetone. They.were then dried usingan Edwards Critical Point Drier (Polaron Equip-ment, Watford, England). Dried specimens weremounted on their adaxial or abaxial surfaces onaluminium stubs with colloidal silver. The mount-ed specimens were coated with gold (to 30 nmthick) with a Polaron Sputter Coater (PolaronEquipment, Watford, England). Observationswere made with a Cambridge Scanning ElectronMicroscope Model 150.

Effect of leaf surface morphology on sporebehaviour

The adaxial surface of intact leaflets which werekept in a horizontal position to prevent run-off ofthe droplets were inoculated. The petiole and thetip of the lamina were taped to a filter paper undereach leaflet. The filter paper was held up by stringstied to the roof of a rectangular enclosure madefrom pieces of polyethylene sheets sealed at theedges. This served as a humidity chamber. To

Colletotrichum on Hevea

~B1l~~Figs 1--6. Scanning electron micrographs of the adaxial (Figs 1, 3, 5) and abaxial (Figs 2, 4, 6) surfaces ofleaves of a susceptible clone PB 5/63. Figs 1, 2: 5-day-old leaflets, Figs 3, 4: 7-day-old leaflets, Figs 5, 6:9-day-old leaflets. Scale bars = 10 ftm.

Radziah Nom Zainuddin and M. Omar

Figs 7-12. Scanning electron micrographs of the adaxial (Figs 7, 9,11) and abaxial (Figs 8,10,12) surfacesofleaves of a resistant clone PB 5/51. Figs 7, 8: S-day-old leaflets, Figs 9, 10: 7-day-old leaflets, Figs 11, 12:9-day-old leaflets. Scale bars = 10 pm.

43° Colletotrichum on Hevea

Fig. 13. A conidium germinating with a short germ-tube and an appressorium on a 5-day-old leaflet of clonePH 5/63. Scale = 4 pm. a, Appressorium; gt, germ-tube; s, septum.Fig. 14. Direct penetration of the leaf surface of clone PH 5/63 by a germ-tube. Scale = 4pm. c, Conidium;gt, germ-tube.Figs 15, 16. Germination of conidia on waxy surfaces of 16-d-old leaflets of clone PH 5/51. Appressoria areformed in the vicinity of stomata but not on apertures (Fig. 15). Wax dissolution around fungal structuresis clearly shown in both micrographs. Scale = 10 pm. a, Appressorium; c, conidium; st, stoma.

maintain a high r.h. within the enclosure, the floorand sides of the enclosure were kept moist by watersprays applied in the morning and evening. Thetemperature within the enclosure was cooled to25°±2 °C during the day.

Suspensions containing 0'5 x 106 spores rnl:'were prepared from 2- to 3-day-old cultures of avirulent isolate grown on Czapek Dox agar. Two0'02 ml droplets were placed on each lamina of aleaflet and four leaflets of each age were inoculated

with spore suspension and another two with steriledistilled water. Incubation for specified periodsfollowed.

At the end of the incubation period each dropletwas drained of excess liquid by placing a piece offilter paper at the edge. The inoculated spots werecut out and specimens prepared for SEM examina-tion using the method outlined earlier. A minimumof six specimens (four inoculated and two unino-culated) of each age group were examined.

OBSERVATIONS AND DISCUSSION

Differences were apparent between the clones,especially in the time wax started to appear on theleaf surfaces, in the density of the wax and thearrangement of the wax lattices (Figs 1-12).

On the fifth day after bud-burst, wax did notappear to have been deposited on the adaxialsurface of the leaflets from the resistant orsusceptible clones (Figs 1, 7). There was someindication of wax deposition on the crests of theguard-cells surrounding the potential stomatalopenings on the abaxial surface of PB 5/51(Fig. 8).

The adaxial surface of 7-d-old leaflets did notundergo much change in wax deposition from thatof y-d-old leaflets (Figs 3, 9). On the other hand,wax started to form on abaxial surfaces of all theleaflets tested. Greater deposition and developmentwas observed on the lower surface of PB 5/51(Fig. to).

Between the seventh and the ninth day of leafletdevelopment, the rate of wax deposition increasedrapidly (Figs 5, 6, 11, 12). Wax lattices were wellformed on both surfaces of the leaflets from all theclones, although there were differences in theirpattern. At this age, stomata were alreadymature.

Wax lattice patterns on leaflets did not varymuch from the eleventh day from bud-burstonwards, except that the wax was denser on theresistant clone PB 5/51 than on the susceptibleclone PB 5/63. This period of 11 d coincides withthe appearance of the cuticle (Wastie & Sankar,1970).

Conidia germinated equally well on leaflets fromresistant and susceptible clones. This is notsurprising, as a number of studies on host-parasiterelationships have shown that the mechanism ofresistance to a pathogen does not come into playbefore penetration (Martin, 1964; Hashim, 1978).On 5-day-old leaflets on which cuticular waxes hadnot been deposited, conidia produced very shortgerm-tubes with appressoria (Fig. 13). There wasno difference in the number of appressoria pro-duced per conidium on susceptible and resistantleaflets.

Germinated conidia were septate, septa ap-pearing like a raised band (Fig. 13). Conidia didnot appear empty or collapsed when the appressoriawere mature, although this was noted with conidiathat germinated and formed appressoria on glassslides. After 18 h incubation, however, it wasobserved that a number of appressoria on thesusceptible clone, PB 5/63, had collapsed. It ispossible that the collapse was an indication ofpenetration having occurred. This phenomenon

15

Radziah Nom Zainuddin and M. Omar 431

was not observed on the resistant PB 5/51. Thegermination process at this stage of incubation wasvery similar to that observed on a glass slide.

Although penetration normally takes place afterthe formation of appressoria, direct penetration bygerm-tubes was occasionally observed (Fig. 14).Senechal et at. (1987) had also observed suchpenetration into 'vein stomata'. That penetrationactually occurred has been confirmed by trans-mission electron microscopy of sectioned speci-mens (Radziah, 1984).

Germination was not affected by the presence ofcuticular wax on older leaflets. Germination waspossible even on leaflets more than 2 wk old (Figs15, 16). This could constitute further evidence forthe non-inhibitory property of wax. On these olderleaflets, however, penetration appeared to havebeen delayed because conidia tended to producelonger germ-tubes than on younger, waxless leaf-lets. Wax could have acted as a physical barrier,delaying contact with the host epidermal cellsnecessary for appressorial formation and pene-tration. At the same time, it is possible that olderleaflets produced more exogenous nutrients (vanBurgh, 1950; Skoropad, 1967) which encouragedgerm-tube growth. In this case, it may even bepossible that the wax itself could be a source ofnutrients to the fungus. Such a possibility has beensuggested by McBride (1972). Germ-tube growthdid not show directional response to any particulartopographical feature of the adaxial surface of theleaf. Stomata (Fig. 15), present only on the lowersurface of the leaf, also did not seem to stimulateappressorial formation at their openings. Appres-soria, however, were formed in their vicinity.

Cuticular wax lattices appear to have been alteredin the immediate vicinity of the fungus (Figs 15,16) and the wax crystals appear to have beendissolved. Dissolution of the wax crystals wasconsidered by Staub, Dahmen & Schwinn (1974)to be a critical step in the process of directpenetration of barley leaf by Erysiphe graminis DC.f. sp. hordei E. Marchal. It is not certain, however,that in this series of micrographs the alteration wasbrought about by the fungus. It could have beendue to the electron beams melting the cuticularwax (Holloway & Baker, 1974).

Preformed structures and substances have beenimplicated in the defence mechanisms of someplants against potential pathogens. Included in theformer are features of plants such as the compo-sition and arrangement of epicuticular waxes,cuticle composition and thickness and morphologyof stomata and guard cells. Although rubber clonesvary in their susceptibility to Colletotrichum leafdisease this investigation has shown that slightdifferences in the morphology of the leaf surfaces

MYC91

432 Colletotrichum on Hevea

of the clones tested are not related to differences inthe behaviour of the conidia of C. gloeosporioides. Itis, therefore, unlikely that the morphology of theleaf su rface is related to the resistance or suscepti-bility of the clone to infection by the fungus.

The authors wish to thank the Directorate of theRubber Research Institute of Malaysia for encour-agement and sup port .

REFERENCES

HASHIM, I. (1978). Histological and biochemical studieson South American LeafBlight ofHeuea species. Ph.D.Thesis, University of the West Indies, Trinidad.

HOLLOWAY, P . J. & BAKER, E. A. (1974) . The aerialsurfaces of higher plants. In Principles and Techniquesof S canning Electron Microscopy I (ed . M . A. Hayat),pp. 181-205. New York, U.S.A. : van NostrandReinhold Co.

McBRIDE, R. P . (1972). Larch leaf waxes utilized bySporobolomyces roseus in situ. Transactions of the BritishMycological Society 58, 329-331.

MARTIN, J. T . (1964) . Role of cuticle in the defence ofplant disease. Annual R eview of Phytopathology 2,

81-100.

RADZIAH, N . Z . ( 1984) . Colletotrichum diseases of rubber.Ph.D. Thesis, Universit i Kebangsaan Malaysia, M al-aysia.

RAo, B. S . (1972). Chemical defol iation of H euea brasil-iensis for avoiding secondary leaf fall. J ournal of theRubber R esearch Ins titute of Malaysia 23 , 248-256 .

RAo, B. S. (1975). Maladies of Hevea in Malays ia .Rubber Research In stitute of Malaysia.

SENECHAL, Y. , SANIER, c., GoHET, E. & D 'AuZAC, J.(1987). Differents modes de penetration du Col-letotrichum gloeosporioides dans les feuilles d' H eveabrasiliens is. Physiologie Vegetate.

SKOROPAD, W . P . (1967) . Effect of temperature on theability of Colletotrichum graminicola to form appressoriaand penetrate barley leaves. Canadian Journal of PlantS cience 47 , 431-434·

STAUB, T. , DAHMEN, H . & SCHWINN, F. J. (1974) . Lightand scanning electron microscopy of cucumber andbarley powdery mildew on host and non-host plants.Phytopathology 64, 364-372.

VAN BURGH, P. (1950) . Some factors affecting appres-sorium formation and penetrability of Colletotrichumphomoides. Phytopathology 40, 29.

WASTIE, R. L. (1973 ). Nursery screening of Hevea forresistance to Gloeosporium leaf disease. Journal of theRubber Research Institute of Malaysia 23, 339-350.

WASTIE, R. L. & SANKAR, G. (1970). Variability andpathogenicity of isolates of Colletotrichum gloeospori-oides from He v ea brasiliensis. Transactions of the Brit ishMy cological Society 54, 117-121.

(Received for publication 6 October 1984 and in rev ised f orm 25 February 1988)