Pertanika J. Trop. Agric. Sci. 19(2/3): 117-122 (1996)ISSN: 0126-6128

) Penerbit Universiti Pertanian Malaysia

Basidiomata Induction and Characterization of Ganoderma fromOil Palm (Elaeis guineensis) on Three Agrowaste Substrates

FARIDAH ABDULLAHBiology Department

Faculty of Science and Environmental StudiesUniversiti Pertanian Malaysia

43400 Serdangy Selangor, Malaysia

Keywords: pathogen, basidiomata induction, inoculum, solid substrates, laccate, relativehumidity, sporulation, Ganoderma

ABSTRAKGanoderma ialah patogen penyebab reput pangkal kelapa sawit. Ahli-ahli genus ini menunjukkan kepelbagaian

yang tinggi di tropika; tetapi adalah sukar untuk ditentukan pengertian kepelbagaian ini dari segi taksonomi keranakestabilan ciri-ciri tertentu kulat ini masih belum diketahui. Kertaskerja ini merangkakan kaedah pengkulturanyangmengaruhkan pembentukan basidiomata Ganoderma dibawah keadaan terkawal, dan seterusnya membolehkankestabilan ciri-ciri tertentu dinilaikan. Dengan menggunakan basidiomata aruhan, ciri-ciri terpilih dapat dihasilkanberulangkali tanpa perubahan diatas tiga jenis substrat, iaitu serabut kelapa sawit, serat kapas, dan habuk kayugetah. Dari segi kadar kolonisasi oleh miselia, kulat ini menunjukkan perbezaan bererti antara pertumbuhan diatasserat kelapa sawit', serat kapas dan habuk kayu getah; tetapi setelah basidioma mula terbentuk, kadar pertumbuhandan perkembangan basidioma adalah sama, dan tidak bergantung pada jenis substrat. Selain daripada kadarpertumbuhan Ganoderma; kajian inijuga memberi kefahaman keatas status warna basidioma matang, potensi ciriperlekatan stip digunakan sebagai nilai taksonomi, dan keperluan kelembapan bandingan persekitaran yang tinggiuntuk pengeluaran spora daripada basidiomata aruhan.

ABSTRACTGanoderma is a causal pathogen of basal stem rot of oil palm. Members of this genus are very diverse in the tropicsbut the significance of this diversity is difficult to relate to taxonomic levels in the genus, largely because of the lack ofknowledge about the stability of particular features of the fungus. This paper outlines a culture method that inducesthe formation of Ganoderma basidiomata under controlled conditions, and thus enables the stability of characters tobe evaluated. Using induced basidiomata, selected characteristics were found to be reproducible on 3 solid substrates,palm press fibres (PPF), cotton fibres (CF) and rubberwood sawdust (RSD). The rate of mycelial colonizationvaried significantly with different substrates but, once formed, the rates of basidioma growth and development werecomparable irrespective of substrate type. Besides the growth rates o /Ganoderma, this study also offers insight intothe status of colour in mature basidioma, the potential of stipe attachment as a taxonomic character, and therequirement for high ambient RH values for spore production of induced basidiomata.

INTRODUCTION

Species of Ganoderma are found worldwideas saprophytes on logs and stumps, andoccasionally as parasites on trees. InMalaysia and Sumatra, species of thisfungus cause serious root and basal stemrot of oil palm (Elaeis guineensis), for whichan effective means of control is still not

available. The bright and 'varnished'appearance of the fruiting body makes iteasily recognizable in the field, but identi-fication to species level is difficult.

Reports of pathogenic species on oilpalm in Malaysia range from 1 species (Hoand Nawawi 1985) to at least 4 (Steyaert1976) and as many as 8 (Turner 1981).

FARIDAH ABDULLAH

Conventional taxonomic approaches arebased on characteristics of the fruitingbody, which is also referred to as basidio-ma, basidiocarp, carpophore or sporo-phore. Current taxonomic keys are mostlyconcerned with temperate species, whilstthose for tropical species are mainly basedon dried specimens.

Members of this genus are extremelydiverse in the tropics and Ryvaarden(1995) has suggested, but not yet verified,that the taxonomic characters currentlyused in the identification of Ganodermaprobably vary under different growingconditions. Mycelial isolations can bemade from infected palm tissues or thesporophores themselves, but there is as yetno foolproof method of verifying that theseagar cultures belong to Ganoderma^ becauseGanoderma cultures on agar do not formfruiting bodies. In addition, all localcultures are white and remain as sterilemycelia, making hyphal characterization oflimited use. This study was undertaken toestablish a reliable method of inducing theformation and sporulation of Ganodermafruiting bodies on solid substrates to enablethe reliability of basidiomata characters tobe assessed.

MATERIALS AND METHODS

Fungal Inocula

Two Ganoderma isolates were selected basedon the different morphological appearanceand growth habit of their respectivesporophores in the field.

The first sample was EGSP 03, isolatedfrom a sporophore collected from a recentlyinfected eighteen-year-old oil palm at SriPelangi Estate, a coastal area in TelukIntan, in the state of Perak. At the time ofcollection the EGSP 03 basidioma had ablackish-brown stipe measuring 1.7 cm inlength. The main body, or pileus, measured6.3 cm wide at the broadest part and 4.2 cmlong from the distal end to the base of the

pileus. The dorsal surface was a brightreddish-brown with tinges of yellow, andhad conspicuous concentric growth rings.The margin was light brown. The under-surface, or pore layer, was a light creamcolour. The dorsal surface of the pileus wassmooth and highly laccate ('varnished')with an index of '5' on a scale of 0 to 5,where 0 indicates a complete absence and 5the highest degree of lacca deposition.EGSP 03 was identified as G. boninensebased on CMI Descriptions of PathogenicFungi and Bacteria Paper No. 444.

The second sample, EGUJ 02, was fromUlu Jempul, an inland area in the state ofPahang, where the sporophore was col-lected from an oil palm stump which hadbeen in the oil palm plantation for nearly 3years. The sporophore of EGUJ 02 wassessile and 8.5 cm at the widest part and 5.6cm in length from the distal margin to thebase. The sporophore shape was subungu-late; the basal portion was approximatelyeight times thicker than the distal margin.The basal end measured 3.8 cm from top tobottom and 0.5 cm at the distal end. Thedorsal surface was thick, corrugated anduneven with the presence of ridges, whichalso made barely discernable concentricgrowth rings. The pileus was uniformly dullbuff brown, while the ridges on it wereblackish. The pileal surface was matt, withan index of '0' on a scale of 0-5, where 0indicates a complete absence of laccadeposition. The pore layer was a lightcream colour. Based on CMI Descriptions ofPathogenic Fungi and Bacteria Paper No. 443, itis probably G. cf applanatum.

Fungal mycelia isolated from the con-textual tissues of Ganoderma basidioma wereused as the source of inoculum. Pieces oftissues measuring 2 x 2 x 4 mm each werecut out from its contextual layer andsurface-sterilized in 10% chlorox (NaOCl)for 5 min. A single piece was picked with aflamed scalpel or tweezers and placed in the

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centre of a 2% malt extract agar (MEA)culture plate, which had earlier beensteam-sterilized at 120°C, 25 psi for 15min. The inoculated plates were incubatedat 28°C (± 1.5°C). Pure mycelia obtainedfrom these isolations were subcultured onMEA slopes in universal bottles as stockcultures and stored at 10°C until required.

Substrate Preparation and InoculationThe 3 substrates used were empty fruitbunches of oil palm commonly called palm-press fibres (PPF), rubber wood sawdust(RSD) and cotton fibres (CF). PPF wasobtained from oil palm plantations, CFfrom textile industries and RSD fromfurniture-processing plants. PPF weresoaked overnight to saturate the fibres.Rubberwood sawdust and cotton fibreswere mixed with 1:1 volume of water. All3 substrates were then separately drainedon fine wire mesh so that the water contentwas just at saturation point, and packedseparately into 15 x 33 x 0.05 mm heat-resistant polypropylene autoclavable bags.The substrate was pressed into compactblocks of approximately 20 cm in height, 12cm in circumference and 960-1000 g inweight. The free end of the bag was putthrough a PVC pipe 5 cm long and 3 cmwide to secure the bag, and a cotton ballwrapped in gauze was put through the pipeto close it. These packed bags of substratewere then steam-sterilized at 121°C, 25 psifor 45 min, and were ready for inoculationwhen sufficiently cooled.

The inoculum starters were preparedby subculturing the isolate from slopecultures on 2% MEA culture plates,incubated at 28°C. A 7-day-old culturecolony was selected and aseptically macer-ated with a flamed scalpel. The PVC tubewas pulled off, the bag opened, and themacerated mycelia transferred on to theupper surface of the compacted substrate.The tube was put back in place and the

culture bag subsequently closed with thecotton plug, ready for incubation.

Growth and Development on Solid SubstratesBoth EGSP 03 and EGUJ 02 isolates wereused in this experiment. Data collectionwas confined to EGSP 03 only, but inducedbasidiomata from both cultures were usedfor the morphological study. Mycelialinoculum was prepared as outlined above.One culture was used as inoculum starterfor one bag. Ten replicates were made perisolate, per substrate type, and all wereincubated at 28°C (±1.5°C). The bagswere placed upright to allow maximumcolonization of the surface area andinspected daily for mycelial extensionsfrom the top to the lower portion of thebag. When fully colonized, a 2 x 4 cm slitwas made in the side of the plastic bag toexpose the mycelia, which was thentransferred to the mushroom nursery tostimulate basidioma development.

The mushroom nursery consisted of anenclosure surrounded by fine-mesh plasticnetting to keep out insects and pests. Theceiling was fitted with a fine-volume watersprinkler, which produced a mist-likespray. The sprinkler automatically turnedon for 15 min at 6-hour intervals tomaintain the nursery at 90-95% relativehumidity (RH). A thermohygrograph wasplaced in the nursery to check that theambient temperature was 27-28°C and theRH value was 90-95% throughout the dayand night. Once spores were produced, thewhole bag was placed in a 20 x 30 x 15 cmperspex box, in the bottom of which wasplaced a white paper lining to gauge sporeproduction. The top was taped with amuslin cloth to confine spores within thebox.

Data collection of EGSP 03 develop-ment included rates of fungal growth on 3substrate types at 3 distinct developmentalstages; namely, (i) mycelial establishment

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on the substrate, (ii) time taken forbasidioma formation and (iii) duration ofspore production until senescence sets in.

Morphological CharacteristicsThree morphological characteristics wereexamined in induced basidiomata of EGSP03 and EGUJ 02. They were: colour ofbasidioma from stipe to pileus margin,degree of lacca deposition, and attachmentof the stipe. Comparisons of these charac-teristics were made among replicates onPPF, between replicates on different sub-strates, and between a representative ofinduced EGSP 03 and EGUJ 02 basidiomaand the original fruiting bodies obtainedfrom the field. Another series of observa-tions was made to compare these charac-teristics in mature basidioma (i.e. aged 3-7weeks) with young basidioma (aged under3 weeks, which had not yet producedspores).

RESULTS AND DISCUSSION

Growth on Solid SubstratesEGSP 03 completely colonized all 3 solidsubstrates at a rate of 13.8a mm/day onPPF, 12.5b mm/day on CF and 9.2C mm/day on RSD (figures with different lettersdenote a significant difference at p — 0.05,using Duncan's multiple range test(DMRT) in the analysis of variance). Thecolonies were smooth, white and dense onall 3 substrates but on exposure turnedbrown and crustose and primordial budsarose. The total incubation time neededbefore the basidioma buds (or primordium)started to appear was fastest on PPF (with amean of 21.5a days), followed by CF (at27.3b days) and slowest on RSD (35.5C

days).

The buds appeared as a raised, dome-shaped, velvety white structure measuring5-10 mm at the widest point. The timetaken for the primordium to elongate into aslender stipe and to reach a constant length

from its initial formation was 3.5a days onPPF, 3.7a days on CF and 3.7a days onRSD. The stipe lengths reached a constantlength of 2.5-3.1 cm, and each bore a whitetip, the primordium, now reduced in size.From this point onwards the tip expandedto form a flat, bracket-like pileus. The timetaken for the basidioma to reach a constantsize was 19.2a days on PPF, 19.3a days onCF and 19.3a days on RSD.

Spore Production

Basidioma maturity was marked by sporeproduction, which occurred once the fruit-ing body reached a constant size. Sporeswere strongly ejected from the pore layerand fell on the box lining, as well as all overthe basidioma surface. The 'spore deposits'were light brown and powder-like on thepaper lining; they were also found on theupper surface of the basidioma, giving it avelvety brown appearance.

Spore discharge was light in the first 2days, becoming very heavy from the thirdday onwards. Spores were produced be-tween midnight and 3 a.m. Sporulation wasprofuse for an average of 7 weeks, irrespec-tive of substrate type. By the 8th week,spore production started to decrease andhad stopped completely by the 10th week.

Morphological CharacteristicsInduced EGSP 03 basidiomata were stipi-tate for all replicates on all 3 substrates. Amean reading for 5 induced EGSP 03mature basidiomata showed that stipelength was 1.7 (±0.02) cm. The pileusmeasured 5.3 (±0.13) cm at the widest partand was 3.4 (±0.08) cm long from the distalmargin to the base of the pileus. Themargin was slightly rounded ('flabelli-form'), measuring 8 (±0.12) cm in thick-ness increasing to 1.75 (±0.07) cm at thepileus base.

Induced EGSP 03 had a blackish-brown stipe. The pileus was smooth, flat

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and dimidiate. The pileus was reddish-orange with a very wide white margin. Thiswhite margin disappeared completely inmature basidioma. The dorsal surface ofmature basidioma was a bright reddish-brown with a band of yellow on theoutermost part. It had conspicuous con-centric growth rings and a light red-brownmargin. The pore layer was light cream.The young basidioma (pre-spore produc-tion) was more rounded in shape. Thedorsal surface in both young and maturebasidiomata was highly laccate with anindex of 5. The colour of mature basidio-mata was similar for all replicates on PPF,was reproducible between replicates on all3 substrates and matched those collectedfrom the field. The basidioma graduallydarkened in colour once spore productionhad stopped. By the 12th week, thesporophore had lost its original colour andturned uniformly blackish-brown; the dor-sal surface became dull, and the laccateindex decreased from 5 to 1. The deadbasidioma was dull, dry and brittle.

Induced EGUJ 02 basidiomata weresessile and applanate (i.e. of uniformthickness from base to pileus) on all 3substrates. The young basidioma (pre-sporeproduction) was a buff light brown colourfrom base to pileus with a distinct whitemargin all round. In mature basidioma, thepileus was the same buff, light-browncolour, but the white margin had disap-peared. The dorsal surface was matt,scoring '0' on the laccate index in bothyoung and mature basidiomata. EGUJ 02was of approximately uniform thicknessfrom base to margin in both young andmature basidiomata and measured 8.6(±0.05) mm in thickness in the latter. Thebasidiomata colour of induced EG UJ 02did not vary significantly within replicatesand between replicates on different sub-strates, nor in young and mature basidio-mata, and matched those collected from the

field. However, there was a completemismatch in shape for EGUJ 02. Allinduced EGUJ 02 were applanate irrespec-tive of substrate type, but the originalspecimen collected from the field wassubungulate with the basal portion ap-proximately 8 times the thickness at themargin. Except for the reduction in size forEGSP 03, no such morphological differ-ences were observed.

CONCLUSION

All 3 substrates supported the growth anddevelopment of Ganoderma basidiomata.The rates of mycelial colonization variedsignificantly from one substrate to the otherbut the rate of basidiomata growth anddevelopment was relatively uniform andindependent of substrate type.

Morphological examinations showedthat size and shape of Ganoderma basidio-mata are not good taxonomic characters.Basidioma colour holds potential but has tobe treated with caution as the character isnot stable in young, pre-sporulating Gano-derma boninense, whilst it is homogeneouslyblackish in old, post-sporulating specimens.

However, in mature and actively-sporulating Ganoderma boninense basidioma(i.e aged 3-7 weeks), colour is a constantand reproducible characteristic, regardlessof substrate type. Attachment of stipe holdspotential as a taxonomic character.Steyaert (1976) observed that attachmentof stipe may be dependent on point ofinsertion, whilst Ryvaarden (1994) ob-served that it seemed to be a consistentcharacter in the Ganoderma lucidum group inEurope.

Spore production in induced basidio-carps occurs only once in its life span.Under normal circumstances, it shows adiurnal pattern of spore release, whichcontinued for approximately 7 weeks. AnRH value exceeding 90% at an ambienttemperature of 28°C was essential to

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FARIDAH ABDULLAH

stimulate spore production.

In conclusion, development of a culture

technique to induce basidioma formation

on solid substrates allows investigation into

the growth habits of Ganoderma and pro-

vides a means of verifying whether parti-

cular characters are stable and are of

taxonomic value.

REFERENCES

HO, Y.W. and A. NAWAWI. 1985. Ganodermaboninense (Pat.) from basal stem rot of oil palm(Elaeis guineensis) in Peninsular Malaysia.Pertanika 8: 425-428.

RYVAARDEN, L. 1995. Can we trust morphologyin Ganoderma? In Ganoderma: Systematics, Phyto-pathology and Pharmacology•, ed. P.K. Buchanan,R.S. Hseu and J.M. Moncalvo, p. 19-24.Taipei, Taiwan: National Taiwan University.

STEYAERT, R.L. 1976. The concept and circum-scription of Ganoderma tornatum. Transactions ofBritish Mycological Society 65: 451-467.

TURNER, P.D. 1981. Oil Palm Diseases andDisorders, Kuala Lumpur: Oxford UniversityPress.

(Received 5 July 1996)

(Accepted 10 November 1996)

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