[ 117 ]

Trans. Br. mycol. Soc. 54 (1),117-121 (1970)Printed in Great Britain

VARIABILITY AND PATHOGENICITYOF ISOLATES OF COLLETOTRICHUM

GLOEOSPORIOIDES FROM HEVEA BRASILIENSIS

By R. L. WASTIE

The Rubber Research Institute of Malaya, Kuala Lumpur, Malaysia

AND GOMATHY SANKAR

Department of Biological Sciences, University of Malaya, Kuala Lumpur

(With Plate II and 1 Text-figure)

Twenty-seven isolates of Colletotrichum gloeosporioides from Hevea brasiliensisdiffered greatly in appearance, in intensity of sporulation, in the effect onsporulation of exposure to light and the addition of r.-asparagine to the medium,and in pathogenicity to young leaves of Hevea. The number of spores of eachisolate required to initiate infection in half the leaflets (the ED50) was deter­mined by inoculating leaflets with a range of spore concentrations and sub­jecting the data to probit analysis. Comparison of ED50 values on leaves ofdifferent ages indicated that susceptibility to infection decreased with age,with a well-defined increase in resistance between the eleventh and thirteenthday of leaf development. This coincided with the formation of the cuticle.

Several authors have described the differences in cultural characteristicswhich exist among isolates of Colletotrichum gloeosporioides Penz. and )tsperfect stage Glomerella cingulata (Stonem.) Spauld. & Schrenk, and haveattempted classification of the strains into morphological and geneticgroups (Shear & Wood, 1913; Burger, 1921; Ashby, 1931; Andes & Keitt,1950; Kaiser & Lukezic, 1966). Shear & Wood (1913) and Kaiser &Lukezic (1966) also reported differences in pathogenicity between strainsfrom the same host. This paper confirms the morphological variability ofisolates of C. gloeosporioides from the various diseases of Hevea with whichit is known to be associated, and compares their pathogenicity to Hevealeaves.

CULTURAL STUDIES

Twenty-two isolates of C. gloeosporioides were obtained from leavesaffected by Gloeosporium leaf disease, which is a major cause of secondaryleaf fall, a disease of economic importance when severe (Wastie, 1967).Three additional isolates were obtained from leaves of plants with anthrac­nose or Colletotrichum leafdisease, which are diseases of plants with nutrientshortage or water stress, and two from stems of plants showing severe die­back. No similarities in culture were observed between isolates from similarsituations.

Isolates were cultured on both potato-dextrose and Czapek-Dox agar

118 Transactions British Mycological Society

media at 26'5°C in the dark, and the 9 em Petri dishes were filled in3-4 weeks. The amount of aerial mycelium and intensity of sporulationvaried between isolates from negligible to extremely heavy. On threecultures sporulation was so vigorous as to completely cover the surfaceof the mycelium, and on several cultures sclerotia (perhaps representingsterile perithecia, as suggested by Shear & Wood, 1913) were abundant.On potato dextrose agar aerial mycelium and sclerotia were more abundantand sporulation much less. No detailed grouping of the isolates was madeowing to the great variability encountered.

Single-spore subcultures always repeated the morphological charac­teristics of the parent colony, as found by Burger (1921). However,exposure to continuous light greatly increased sporulation in five of ninecultures examined. Plates were incubated under fluorescent light (4306 Ix)for 6 weeks at a mean temperature of 28'5° and compared with adjacentcultures stored in light-proof canisters. The sporulation of four remainingcultures, including two lightly sporing isolates, was not increased by light.Fewer sclerotia were produced by cultures exposed to light. A similarlyvariable effect on the intensity of sporulation was obtained by supple­menting potato-dextrose agar with 0'2 % asparagine, as in the methodofMack,]ack, Harkness & Miller (1967), and comparing sporulation withthat on a non-supplemented medium. Of ten cultures examined, in onlytwo was a greater degree of sporulation shown on the asparagine-supple­mented medium; sporulation in the remaining eight cultures was un­affected.

PATHOGENICITY

Variability among isolates

To determine if the variability in morphological characters among theisolates was paralleled by a similar variability in pathogenicity, as hasbeen reported by Dastur (1920) and Kaiser & Lukezic (1966), leaflets ofthe susceptible clone PB 86 were inoculated with spore concentrations ofone of four isolates of the fungus, using a method similar to that describedby Wastie & ]anardhanan (1970). Healthy leaves were picked after9 days from bud-burst, at which age the leaflet length was approximately5 em, Drops of size 0'01 ml (covering 7'0 mm" of leaf) containing five orsix different spore concentrations were placed in duplicate on each oftwenty-five leaflets with an 'Arnold' hand micro-applicator (Pi. I I). Twoadditional drops of distilled water were also applied. After inoculationleaflets were placed on wire trays in plastic boxes lined with dampblotting paper, and incubated for 72 h at 26'5° for infection to develop.The number of positive infections out of the total of 50 was then recorded,dark discoloration under the droplet being taken as evidence of penetra­tion of the leaf. The logarithm of the spore dose applied was plotted againstthe probit value corresponding to the percentage of infected sites, andthe regression line and ED 50 of each strain of spores calculated. TheED 50 (spores per drop) was used to compare the relative infectivities ofeach isolate (Lapwood & McKee, 1966). Table I shows that there wasa sevenfold difference in infectivity to 9-day-old leaves between the leastand most pathogenic isolates, i.e, between 86 and 643 spores per drop.

Colletotrichum. R. L. Wastie and G. Sankar 119

Table 1. ED 50 value (spores per 0'01 ml droplet) offour isolates ofC. gloeosporioides on Hevea leaves 9 days from bud-burst

5 % fiducial limitsA

Source of isolate

Secondary leaf fallTwig diebackSecondary leaf fallSecondary leaf fall

Lower Upper

II9326449

II 60

7r---------;---:---------,

6

CIl<iiv'".'=

.J:loli 5

3

3 5Spore concn, (Ioglo)

6

Text-fig. I. Relationship between concentration of spores of C. gloeosporioides andpercentage infection. Data from fifty leaflets of each age. Age of leaf (days from bud­burst): 0, 5; e, 7; 6, 9; "*', II; 0, 13; _, 15·

Effect of age of leaf

It has been suggested (Anon., 1968) that leaves are only susceptible toC. gloeosporioides during the period of bud-burst (about 5 days) and for10 days thereafter. To investigate the susceptibility of leaves of differentages, a range of spore concentrations was placed on leaves of six stages ofgrowth, differing in age in e-day steps from 5 to 15 days from the start ofbud-burst. Text-fig. I shows the relationship between the logarithm ofthe spore concentration and percentage infection, expressed as probits,

120 Transactions British Mycological Societytogether with the calculated regression lines. Very little infection wasobserved with the oldest leaf (15 days from bud-burst), and this stage hasbeen omitted from Table 2, which lists the ED 50 values, in terms of sporesper drop, for each age of leaf. Leaf resistance increased with age, with a25o-fold increase between the eleventh and thirteenth day, to give anED 50 of 88300 spores per drop. This is equivalent to 12600 spores permm" of leaf surface, a dose not likely to be reached very often under fieldconditions. The increase in resistance coincides with the development ofthe cuticle, since measurements of the thickness of the upper cuticle of11 -day-old leaves gave a mean of 3.6 pm while that of leaves 2 days olderwas 13'0 pm. Leaves 9 days old had no measurable cuticle.

Table 2. ED 50 value (spores per 0'01 ml droplet) of a secondary leaffall isolateofC. gloeosporioides on Hevea leaves ofdifferent ages

Age of leaf(days frombud-burst) ED 50

5 427 2309 357

II 35013 88 300

5 % fiducial limits~__J.~_-----,

Lower Upper

29 60186 285284 449191 439

9010 373°00

DISCUSSION

Isolates of Colletotrichum gloeosporioides from Heoea appear to be asvariable in morphology and pathogenicity as those from other hosts. Thusthe variation in the severity of secondary leaf fall from year to year maybe a reflexion of the variability of the pathogen as well as that of theclimate. Variations in host reaction are also important in determining theseverity of leaf disease, for clones vary widely in their susceptibility toC. gloeosporioides, PB 86 being one of the most susceptible. Although thedevelopment of the cuticle is indicated as being responsible for the increasein resistance as the leafages, it is not yet clear if this is a factor ofimportancein determining clonal susceptibility; indications are that cuticle develop­ment occurs at about the same time in several clones, and that otherfactors determine the degree of resistance of the youngest leaves.

The ED 50 of spores on the youngest leafstudied was 42 spores per drop,approximately equivalent to 6 spores per mm'' of leaf. The most virulentisolate was four times as pathogenic as this to older leaves, suggesting thatthe ED 50 on young leaves of such a virulent isolate would be less than2 spores per mm", and for still younger leaves, too small to be used withthe droplet inoculation technique, perhaps even less. The ubiquitousdistribution of C. gloeosporioides throughout the country, together with thehigh pathogenicity of at least certain strains of the fungus, helps to explainthe ease with which outbreaks of Colletotrichum leaf fall occur. The simul­taneous development of uniformly aged new leafafter the annual winteringaffords ideal conditions for local epidemics to occur.

Trans. Br. mycol. Soc. Vol. 54. Plate I I

(Facing p. lin)

Colletotrichum. R. L. Wastie and G. Sankar 121

REFERENCES

ANDES, J. O. & KEITT, G. W. (1950). Variability of Glomerella eingulata (Stonem.)S. & v. S. from apples. Phytopathology 40,915-925.

ANON. (1968). Gloeosporium. Plrs' Bull. Rubb. Res. Inst. Malaya, no. 97, pp. 110-113.AsHBY, S. F. (1931). Notes on 32 isolations of Gloeosporium from bananas in Trinidad.

Trap. Agrie., Trin.8, 322-325.BURGER, O. F. (192 I). Variations in Colletotriehum gloeosporioides.'], agrie. Res. 20, 723-736.DASTUR,J. F. (1920). Glomerella eingulata (Stoneman) Spauld. and v. Sch. and its conidial

forms, Gloeosporium piperatum E. and E. and Colletotrichum nigrum E. and Hals., onchillies and Carica papaya. Ann. appl. Bioi. 6, 245-268.

KAISER, W.J. & LUKEZIC, F. L. (1966). Occurrence, sporulation and pathogenicitystudies with Glomerella eingulata associated with crown rot of boxed bananas.Myeologia 58, 397-400.

LAPWOOD, D. H. & McKEE, R. K. (1966). Dose-response relationships for infection ofpotato leaves by zoospores of Phytophthora infestans. Trans. Br, myeol. Soc. 49, 679--686.

MACK, J. H., JACK, R. C. M., HARKNESS, S. H. & MILLER, L. P. (1967). Increasedconidial production of Glomerella cingulata by L-asparagine. Contr. Boyce ThompsonInst, Pl. Res. 23, 381.

SHEAR, C. L. & WOOD, A. K. (1913)' Studies offungous parasites belonging to the genusGlomerella. Bull. U.S. Dep, Agrie. Bur. Pl. Ind. 252.

WASTIE, R. L. (1967). Gloeosporium leaf diseasejof rubber in West Malaysia. Planter, KualaLumpur 43, 553-565.

WASTIE, R. L. & JANARDHANAN, P. S. (1970). Pathogenicity of Colletotriehum gloeo­sporioides, C. dematium and C. crassipes to leaves of Hevea brasiliensis. Trans. Br, myeol.Soc. 54, 150-152.

EXPLANATION OF PLATE I I

Colletotrichum gloeosporioides from Hevea brasiliensis. Applying spore droplets to Hevea leaflets.

(Accepted for publication 5 August 1969)