PerlanikaJ. of Trop. Agric. Sci. 16(1): 45-51(1993) ISSN: 0126-6128© Universiti Pertanian Malaysia Press

Comparative Morphology and Characterization of ColletotrichumIsolates Occurring on Cocoa in Malaysia

M.F. YEE and M. SARIAHDepartment of Plant ProtectionUniversiti Pertanian Malaysia

43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

ABSTRAK

Pemendlan daripada daun-daun koko yang menunjukkan gejala "shot-hole", hawar dan bintik daun, jugadaripada putik dan Imah yang reput menghasilkan Colletotrichum gbeosporioides. Tiada perbezaan jelas dari segiciri-ciri kultur dan morfobgi dicatatkan daripada pemencilan-pemencilan yang berbeza. Kulat didapati hidup danmembentuk spora dengan baik pada 30'C Agar Ekstrak Daun Koko (CLEA) merupakan medium yang sesuaiuntuk pertumbuhan miselium manakala Agar Kentang Dekstros (PDA) menggalakkan pensporulaan. Daun-daunkoko dan juga buah yang dicederakan mudah dijangkiti oleh C. gbeosporioides. Anak benih koko berumur tigaminggu dan putik menunjukkan peringkat yang paling rentan terhadap jangkitan.

ABSTRACT

Isolation from cocoa leaves showing symptoms of shot-hole, blight or irregular leaf spot and from cherelks and podrot yielded Colletotrichum gbeosporioides. No distinct differences in cultural and morphological characteristics werenoted between the various isolates. The fungus xoas found to grow and sporulate well at 30'C. Cocoa I\eaf ExtractAgar (CIJFA) zoos the best medium for mycelial growth zohile Potato Dextrose Agar appeared to favour sporulation.Both cocoa leaves and injured pods loere liable to infection by C. gbeosporioides. Three week old cocoa seedlings andcherelks were noted as the most susceptible stages.

Keywords: Colletotrichum gloeosporioides, cocoa, temperature, culture media, infectivity

INTRODUCTION

Leaf spot and pod rot of cocoa (Theobromaecocoa, L) incited by Colletotnchum gbeosporioideswhich were first reported to occur in Malaysia in1975 by Lin and Liew (1975), cause severe blight-ing and rotting of cherelles and immature pods.However, leaf spot disease and pod rot causedby C. gbeosporioides have yet to become a majordisease problem in Malaysia even though theyhave been recorded to be serious in severalcocoa growing countries in the world (Thorold,1975; Dakwa and Danquah, 1978). Althoughseveral workers have studied the taxonomy, mor-phology and biology of the pathogen (Stoneman,1898, Shear and Wood, 1913; Burger, 1921;Simmonds, 1965; McDonald, 1926; Wastie andShanker, 1970; Arx, 1970), very little informa-tion on the etiology of this disease under localconditions is available. Since this information isrequired for the formulation of a comprehen-sive disease control programme for Colletotrichum

leaf spot and pod rot of cocoa in this country,this study focuses on the occurrence of thedisease in Malaysia; and to establish the identityof the pathogen, its infectivity on different partsof the plant and its symptoms.

MATERIALS AND METHODS

Isolation of the pathogen

Cocoa leaves showing symptoms of leaf spotdisease were collected from both cocoa seed-lings and mature cocoa trees grown in the Co-coa Unit of Universiti Pertanian Malaysia. Inaddition to infected cocoa leaves, cherelles, im-mature pods as well as mature pods suspected ofbeing infected by Colletotrichum were also col-lected. Pieces of the infected leaf, 3mm X 3mmin area, and pod tissues were surface sterilised in5% sodium hypochlorite solution (NaOCl) for 5min., washed in two changes of sterile distilled waterand plated on potato dextrose agar (PDA). Theresulting cultures were sub-cultured onto fresh

M.Y YEE AND M. SARIAH

PDA plates until pure cultures were obtained.All cultures of the isolates were subculturedonto and maintained on PDA throughout thisstudy unless otherwise stated.

Effect of Environmental Factors on Growth, Sporulationand Cultural Characteristics of Colletotrichumgloeosporioides isolatesFor this study and subsequent studies, the fourColletotrichum isolates as shown in Table 1 wereused as the test fungi. To study the effect oftemperature, each PDA plate was centrally in-oculated with 5mm diameter fungus plug takenfrom the advancing margin of a 5-day old cul-ture and incubated in the dark at 20, 25, SO and35°C for five days.

TABLE 1Isolates of Colletotrichum gloeosporioides from infected

cocoa leaf and pod associated with leafspot and pod rot disease

Isolate Source

Sh cocoa leaf with shot hole symptom

Lb cocoa leaf with blight symptom

Is cocoa leaf with irregular spot symptom

Pr cocoa pod with pod rot symptom

The effect of culture media on the growth,sporulation and cultural characteristics were de-termined by centrally inoculating PDA, CDA(Czapek Dox Agar), MEA (Malt Extract Agar),OMA (Oat Meal Agar), Cooks (Cooks medium)and CLEA (Cocoa Leaf Extract Agar) plates witha 5mm diameter fungus plug taken from theadvancing margin of a five-day old culture andincubated at 30°C in the dark for five days.

Each treatment was replicated four times.Growth measurements, degree of sporulationand cultural characteristics were assessed at theend of the experiment. Mycelial growth wasassessed by taking the average of the two per-pendicular distances across the centre of thecolony. Spore concentration was determinedwith the aid of a Neubauer haemocytometer.

Infectivity Studies

Cocoa seedlings of mixed hybrid of 2, 3 and 4-week old were inoculated with spore suspensionof 10" conidia/ml until run-off. The spore

suspension was prepared by flooding a five-dayold culture; three drops of Tween 80 were thenadded to the resultant spore suspension beforespraying. Sterile distilled water was used as thecontrol. Seedlings were maintained in a moistenvironment for infection to occur. The experi-ment was replicated ten times, arranged inrandomized complete block design (RCBD) witheach replication consisting of a single seedling.Assessment of percentage leaf area infected wasconducted on the tenth day.

Infectivity studies were also carried out ondetached cocoa pod; Cherelle (25-30 mm long),young pod (40-50 mm long) and green maturepod (85-100 mm long) of mixed hybrid whichwere surface sterilised with 5% NaOCl and rinsedwith sterilised water. Pods were inoculated di-rectly or injured with a 0.5mm diameter steri-lised inoculating needle prior to inoculation.Pods were spot inoculated with IOJJLI of sporesuspension of 106 conidia/ml. The inoculatedpods were incubated in a moist chamber at 30°Cwith 98% relative humidity. Ten pods of eachsize were used for each treatment. The controltreatments were inoculated with sterile distill*water. Lesions were assessed on the tenth day):taking the average of the two perpendiculardistance across the centre of the lesion.

Data were statistically analysed and thdifference between individual means waitested using Duncan Multiple Range Te(DMRT).

RESULTS

The Pathogen

Isolation made from the three types of foliisymptoms viz: shot-hole, leaf blight and irrelar spot and a pod rot symptom consist*yielded the fungus Colletotrichum. Cultural stiies on the pure cultures obtained showedthe cultures isolated from infected tissuesing the same type of symptom, were similar itone other; however, they were different fn*those isolated from other types of sympttResults as shown in Table 2 suggest the ideof the four cultures was close to thatgloeosporioides (Penz.) Sacc. The identifies!was confirmed by IMI, Kew, England. No othfdistinct characteristics were noted which coulfacilitate strain differentiation within &species.

PERTAMKAJ. TROP. AGRIC. SCI. VOL. 16 NO. 1, 1993

COMPARATIVE MORPHOLOGY AND CHARACTERIZATION OF COUJiTOTRICHUM ISOLATES

TABLE 2Cultural and morphological characteristics of 5-Day old

C. gloeosporioides isolates from cocoa grown on PDA

Isolate Culturalcharacteristics

Morphologicalcharacteristics

Sh Colony appeared white andgradually turned greyishsalmon in colour as the culturegrew older. Aerial myceliumslightly flocculose with orangeconidial pustules evident at thecentre of the colony. Reverse ofcolony appeared smoky greyin colour.

Lb Colony appeared white andgradually turned olivaceousgrey in colour as the culturegrew older. Aerial myceliumflocculose with orange conidialpustules apparent at the centreof the colony. Reverse ofcolony appeared smoky greyin colour.

Is Colony appeared white and graduallyturned greyish white as the culturegrew older. Aerial mycelium slightlyflocculose with orange conidialpustules evident at the centreof the colony. Reverse of colonyappeared smoky grey in colour.

Pr Colony appeared smoky grey incolour with thick floccose aerialmycelium and orange conidialpustules at the centre. Reverse ofcolony appeared in the form ofdistinct olivaceous grey zonationalternated with rosy buff zonation.

Conidia cylindrical with obtuse ends, hyaline, aseptate,uninucleate, 5-22 [im x 2-6 jum, formed in setose orglobose acervuli or on solitary phialides on mycelium.Acervulus round to elongated to irregular 60-240 (imin diameter. Setae sparse to profuse, dark brown toblack, straight to slightly curved, 1-4 septate, swollenat the base and tapering towards the apex, 50-170 Jimlong.

Conidia cylindrical with obtuse ends, hyaline,aeptate, uninucleate, 4-24 |Um x 2.5 jLlm, formedin setose or globose acervuli or on solitary phialideson mycelium. Acervulus round to elongated toirregular, 70-250 um in diameter. Setae sparseto profuse, dark brown to black, straight to slightlycurved, 1-4 septate, swollen at the base and taperingtowards the apex, 50-80 |Hm long.

Conidia cylindrical with obtuse ends, hyaline,aseptate, uninucleate, 4-23 \im x 2-6 jlm, formedin setose or globose acervuli or on solitary phialideson mycelium. Avervulus round to elongated toirregular 65-190 jam in diameter. Setae were sparseto profuse, dark brown to black, straight to slightlycurved, 1-4 septate, swollen at base and taperingtowards the apex, 70-165 jam long.

Conidia cylindrical with obtuse ends, hyaline,aseptate, uninucleate, 4-22 Jim x 2-5 jum, formed insetose or globose acervuli or on solitary phialideson mycelium. Acervulus round to elongated toirregular, 80-230 Jim in diameter. Setae sparse toprofuse, dark brown to black, straight to slightlycurved, 1-4 septate, swollen at the base andtapering towards the apex, 80-170 (im long.

Effcct °f Environmental Factors on Growth andSporulation of Colletotrichum gloeosporioides IsolatesStudies on the effect of temperature on themycelial growth and sporulation (Fig. 1A & IB)of the four Colletotrichum isolates proved to besignificant at P = 0.05. All the four isolates werefound to grow better at 30°C over an incubationperiod of 5 days Sh and Lb isolate attained amycelial growth of 51 mm. in diameter Is isolate37 mm. and Pr isolate, 30mm. The fungi werealso found to sporulate better at 30°C with the

highest number of spores being harvested fromIs isolate (9xlO6 spores/ml), followed by Pr iso-late (8xlO6 spores/ml), Sh isolate (2xlO6 spores/ml) and Lb isolate (lxl0tv spores/ml).

Growth media influence the growth rateand sporulation of Colletotrichum isolates. How-ever, the variation in growth and sporulationwas insignificant at P = 0.05. CLEA was noted asthe best medium for mycelial growth followedby OMA, Cook's, PDA, MEA and CDA. On theother hand, CLEA, failed to maintain favourable

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 16 NO. 1, 1993 47

M.Y. YEE AND M. SARIAH

I SH

25 30TEMPERATURE ( ° C )

• I IS H i Lb iPr

Fig. 1A: Effect of Temperature on the Linear Growth of C.gloeosporioides Isolates from Cocoa on PDA(LSD(m= 8.47)

sporulation. PDA was the best sporulation me-dium followed by CDA, OMA, MEA, Cook's andCLEA.

Infectivity Studies

Each isolate could produce more than one typeof symptom depending on the leaf age and the

Sh

PDA

OMA

I t

CULTURE MEDIA

WM CDA

O COOK'S

Lb

• 1 MEA

CLEA

Pr

25 30

TEMPERATURE (°C)

Sh Lb UI1 Pr

tig Hi: Effect of temperature on Sporulation of C.gloeosporioides Isolates from Cocoa on PDA(LSDMr 8.47)

Fig. 2A: Effect of Culture Media on the Linear Growth o/Cgloeosporioides Isolates from Cocoa on PDA

Sh

PDA

OMA

IS Lb

CULTURE MEDIA

WBH CDA

1 : COOK'S

• 1~3

MEA

CLEA

2R'f^ °f Culiure Med™ ™ the Sporulation.#gloeosporioides Isolates from Cocoa on FDA

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 16 NO. 1, 1993

COMPARATIVE MORPHOLOGY AND CHARACTERIZATION OF COLIKIOTRICHUM ISOIATES

extent of the infection. Blight and shot-holesymptoms were more frequently observed onyounger leaves although they were also seen onolder leaves. Irregular spot symptom, however,dominated on the older leaves with blight symp-toms being occasionally observed. The firstevidence of infection was noted after four daysof incubation. The symptoms caused by thevarious isolates of Colletotrichum wereundistinguishable at the initial stage of infec-tion. The lesion first appeared as minute yellow-ish specks, and later discernible as circular red-dish brown lesions with a chlorotic halo. As theinfection progressed, three clearly distinguish-able foliage symptoms viz, shot-hole, irregularspot and blight were apparent on the affectedseedlings. The results presented in Table 3indicate that Pr isolate was the most virulent onetested on the cocoa seedlings, recording a meanpercentage leaf area infection of 2.2%, 12.2%and 11% on 2-week, 3-week and 4-week oldcocoa seedlings respectively. Isolate Is was theleast pathogenic, exhibiting a mean percentageleaf area infected of 1.1%, 6.5% and 6.0% on 2-week, 3-week and 4-week old seedlings respec-tively.

Lesions incited by the various cocoa isolatesof Colletotrichum on detached injured pods areshown in Table 4. The severity of infection oncherelle was significantly different from that ofgreen mature pod. Isolate Pr was noted to bethe most pathogenic strain followed by Sh, Isand Lb. Infection was first observed on theinoculation spot 5 days after incubation in theform of a small brownish round spot with ayellow halo. The affected area later becamedarker and formed a depressed lesion, followed

TABLE 3Severity of infection incited by Colletotrichum

gloeosporioides isolates on cocoa seedlings

of different ages

Treatment Mean percentage leaf area infected*

Seedling Age Is Lb Sh Pr Control

TABLE 4Lesion development caused by Colletotrichum

gloeosporioides isolates on injured detached cocoapods of various sizes

2 weeks3 weeks

4 weeks

1.1"6.5b

6.0b

1.7'8.8b

8.0h

1.8"7.4b

6.8U

2.2;i

12.2"

11.0b

0"

Treatment

Pod size Is

Cherelled

Young pod

Green maturepod

4.8Li

4.7"

0h

Lesion size

Lb Sh

3.£» 10.4"

0h 8.3l)

0b 0*

(mm)*

Pi

13.0

12.5

7.5

Control

• ()••

• 0"b 0 ,

Any two means within the column followed by thesame letter are not significantly different at 5%level based on Duncan Multiple Range Test.

Any two means within the column followed by thesame letter are not significantly different at 5%level based on Duncan Multiple Range Test.

by the production of greyish white myceliumwith pink coloured masses of conidia. Lesionscaused by the various isolates of Colletotrichumappeared identical and reisolation from podlesion yielded only the isolate with which theparticular pod was inoculated. No infection wasnoted in any of the non-wounded inoculatedpods.

DISCUSSION

Isolation of the disease pathogen from the re-spective types of foliar symptoms viz., shot-hole,leaf blight and irregular spot and pod rot yieldedColletotrichum isolates. These isolates were iden-tified as Colletotrichum gloeosporioides (Penz.) Sacc.and subsequently confirmed by the InternationalMycological Institute. Cultural and morphologi-cal studies showed no distinct differences incharacteristics among the C gloeosponoides iso-lates which could facilitate strain differentiationwithin the species, except for slight variation inculture colour and consistency of the mycelium.All the C. gloeosporioides isolates produced cylin-drical conidia with obtuse ends, hyaline, aseptate,uninucleate and measured 4-24jnm x 2-6um whichwere formed in setose or globose acervuli. Theshape of the acervuli ranged from round toelongated to irregular and measured 60-250|imin diameter. Setae were sparse to profuse, darkbrown to black, straight to slightly curved, 1-4septate, swollen at the base and tapering towardsthe apex.

Studies on the species of Colletotrichum else-where have shown them to be very variable intheir morphological (Arx, 1970) and cultural

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 16 NO. 1, 1993 49

M.Y. YEE AND M. SAR1AH

characteristics (Stoneman, 1898; Shear andWood, 1913; Burger, 1921). Mohanan (1983)was able to classify the isolates of C. gloeosporioidesfrom cocoa associated with irregular spot, blightand shot-hole symptom into white, dark andlight types. Dakwa and Danquah (1978) alsoobserved distinct variation in morphological andcultural characteristics among the isolates of C.gloeosporioides which cause leaf blight of cocoa inGhana.

Studies on the effect of temperature onmycelial growth and sporulation of C.gloeosporioides isolates proved to be significant atP = 0.05. The isolates were found to grow andsporulate better at 30°C on PDA. Similar obser-vations were made by Quimio (1974) and Scattarand Malik (1938) on C. gloeosporioides frommango, and Lii (1972) on C. gloeosporioides fromguava. Earlier work by Wastie and Shanker(1970) and Muthappa (1971) were confirmedby our studies that growth media could influ-ence the growth rate and sporulation of C.gloeosporioides. However, the effects were insig-nificant (P = 0.05). CLEA was observed to bethe best medium for mycelial growth followedby OMA, COOK'S, PDA, MEA and CDA. In ourstudy, however, CLEA failed to maintain itsstatus in favouring sporulation; we noted thatPDA was the best sporulation medium followedby CDA, OMA, MEA, COOK'S and CLEA.Radziah (1985) claimed that PDA and CDAfavour sporulation while working on C.gloeosporioides from rubber.

Results obtained from our infection studiesreveal that C. gloeosporioides could infect bothcocoa leaves and pods. Wilting of cherelles andyoung pods were generally considered to be dueto physiological factors, but recent observationsby Mohanan and Kaveriappa (1983) showed thata considerable percentage of pod rot of cherellesand young pods was due to Colletotrichum infec-tion. Similar observations were reported byBailey (1966) from Nigeria and Reddy andMohanan (1976) from India. Mohanan andKaveriappa (1983) in their studies on thesymptomatology of Colletotrichum disease of co-coa reported that the occurrence of three differ-ent types of symptoms on the foliage of cocoaplant caused by C. gloeosporioides could be attrib-uted to the existence of different varieties of thesame species or pathological strain. However, inour studies, we found that each isolate couldproduce more than one type of symptom de-

pending on the leaf age and the extent ofinfection. Wastie and Shankar (1970) claimedthat apart from climatic factors, leaf age has aninfluence on the severity of Colletotrichum infec-tion. Blight and shot-hole symptoms were moreprominent on younger leaves although they werealso spotted on older leaves. On the otherhand, irregular spot symptoms dominated onthe older leaves with blight symptom being occa-sionally observed. Dakwa and Danquah (1978)reported similar observations. Spotting of olderleaves caused little damage; however, infectionon newly formed young leaves could result inimpairment of the functional photosynthesis(Sarma and Nambiar, 1976) and leaf fall, pro-ducing bare tips which could subsequently beinvaded by Botryodiplodia theobromae Pat. (ShellChemicals Technical Bulletin).

Laboratory studies with detached cocoa podsshowed that only injured cocoa pods were liableto infection by C. gloeosporioides conidia and thatcherelles and young pods were more susceptiblethan green mature pods. Although pathogenicitytests were only carried out in the laboratory, thisinformation suggests a possible mode of pen-etration for field infection. Various factors havebeen linked to the initiation of spore germina-tion and disease spread. Mohanan (1983) sug-gested that free water available on leaf surfacecould facilitate conidial germination and infec-tion. Sarma and Nambiar (1976) reported thatalthough shot-hole incidence was found through-out the year in Kasaragod district of Kerala, theintensity was higher when a temperature rangeof 19 - 33°C and relative humidity of 77 - 98*prevailed. On the other hand, Dakwa andDanquah (1978) were of the opinion that thehigh incidence of leaf blight in Ghana could beattributed to the availability of tender leavescoupled with the high relative humidity andmoisture on leaf surface. Although Colletotrichundisease of cocoa is not very serious at present inthis country, its wide distribution and occurrence makes it necessary for effective controlmeasures to be taken to prevent the fungusfrom attaining epiphytotic proportions as ha.(

happened in Ghana.

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Gloeosporium. Cramer, Lehre. p. 203p.

Bailey, A.G. 1966. A check list of plant diseases ifNigeria. Federal Republic of Nigeria Memorandum, No. 96, 1-33.

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COMPARATIVE MORPHOLOGY AND CHARACTERIZATION OF COLLETOTRICHUM ISOLATES

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Ln,J.L. 1972. Identification and occurence of per-fect stage and cultural and morphological vari-ants of Colletotrichum gloeosporioides from guavain Puerto Rico. Puerto Rico Univ. J. Agric, 56:171-180.

LIN, P.S.W. and W.C. LEW. 1975. Diseases of cocoain Sabah. Dept. of Agric. Sabah.

McDonald, J. 1926. A preliminary account of adisease of green coffee berries in Kenya colony.Trans Bri. Mycoi Soc, 11: 145-154.

Mohanan, R. 1983. Colletotrichum species of cocoain South India. Ph.D. Thesis. Mangalore Uni-versity.

Mohanan, R. and K.M. Kaveriappa. 1983.Symptomatology of Colletotrichum disease ofcocoa in India. Planter, 59: 333-338.

Muthappa, B.N. 1971. Studies on Colletotrichumcoffeanum in India. II. Cultural studies andthe factors affecting spore germination. J. Cof-fee Res., 1: 3-8.

Quimio, T.H. 1974. Mango anthracnose and lowtemperature storage. Phil. Agric,, 58: 192-199.

Radziah, Z. 1985. Colletotrichum diseases of rubber.Ph.D. Thesis, Universiti Kebangsaan Malaysia.

Reddy, M.K. and R. Mohanan. 1976. Occurrenceof pod rot of cocoa caused by Colletotrichumgloeosporioides in India. /. Plant Crops, 4: 14-15.

Sarma, Y.R. and K.K.N Nambiar. 1976. Leaf spot ofcocoa. Curr. Sri., 45-466.

Scatter, A. and S.A. Malik. 1938. Some studies onanthracnose of mango caused by Glomerellacingulata in the Punjab. Indian J. Agric. Sc.t 9:511-521.

Shear, C.L. and A.K. Wood. 1913. Studies onfungus parasites belonging to the genusGlomerellay United States Dept. Agric. Bureau ofPlant Industry Bull. 252: 1-110.

Simmonds, J.H. 1965. Study of the species ofColletotrichum causing ripe fruit rots in Queens-land. Queensland J. Agric. Anim. Sri., 22: 437-459.

Stoneman, B. 1898. A comparataive study of thedevelopment of some anthracnose. Bot. Gaz.,26: 69-120.

Thorold, C.A. 1975. Diseases of cocoa. ClarendonPress. Oxford. 423 pp.

Wastie, R.L. and G. Shankar. 1970. Variability andpathogenicity of isolates of Colletotrichumgloeosporioides from Hevea brasiliensis. Trans. Bri.Mycol. Soc, 54: 117-121.

(Received 15 May 1992)

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