Pertanika 11(2), 175-189 (1988)

Evaluation of Potential Sources of Allelochemicals in Lalang(Imperata cylindrica)

A. RAJ AN1, J.V. LOVETT and A.C. ANDREWSDepartment of Agronomy and Soil Science,

University of New England,Armidale, MS. W. 2351, Australia.

Keywords: Allelopathy, Imperata cylindrica; lalang.

ABSTRAK

Luluh larut akua daripada serpihan daun lalang (in situ) yang mati menyebabkan percambahandalam bioasai kesemua enam spesies legum pastura yang diuji tertunda, manakala pertumbuhan radikeldan pucuk terencat bagi dua antara spesies itu. Ketoksidan fito bergantung kepada kuantiti sisa tumbuhanyang ada berhampiran dengan spesies 'fitometer* yang sedang bercambah. Luluh larut daripada rizomdan serpihan akar menunjukkan aktiviti yang merencat pada paras 5% (w/v), manakala luluh larutdaripada daun merencatkan aktiviti pada paras 10% ke atas. Bagi eksperimen-eksperimen dalam pasudengan menggunakan tanah yang mengandungi batang-batang lalang yang hidup atau bercampur denganrizom atau serpihan akar, perkembangan ketoksidan fito dalam ekstrak tanah ketara pada minggu keempatpertumbuhan atau pereputan. Bagi tanah yang mengandungi akar atau rizom yang reput, kehilanganaktiviti wujud dalam jangka masa inkubasi selama lapan minggu. Organisma-organisma tanah sepertiBacillus cereus (Frankland & Frankland), Alcaligenes faecalis (Castellani & Chalmers), dan satu spesiesTrichoderma didapati berhubung kait dengan pereputan rizom, menyebabkan bermulanya rangsanganpertumbuhan dalam spesies 'fitometer* dan diikuti dengan perencatan dan akhirnya aktiviti terhenti.Ekstrak tanah yang mengandungi batang-batang yang hidup menunjukkan aktiviti terhenti pada minggukeenam pertumbuhan, diikuti oleh respons perangsangan yang ketara pada minggu kelapan. Penggunaandua kawalan (kawalan -ekstrak tanah, dan kawalan air suling) membolehkan pengesanan aktiviti yangtetap dalam tanah kawalan. Perbandingan di antara kedua-dua kawalan bagi menghapuskan pengaruhpengaruh ini menunjukkan bahawa hanya batang-batang yang hidup, dan rizom serta akar yang reputdapat menjadi sumber yang berpotensi

ABSTRACTAqueous leachates from fragmented lalang (I. cylindrica (L) Raeuschel var. major,/ dead leaves

in situ, caused delayed germination in biossays of all six species of pasture legumes tested, while radicleand shoot growth were inhibited in two of the test species. Phytotoxicity was dependent on the quantityof plant residues available in the proximity of germinating phytometer species. Leachates from rhizomeand root fragments exhibited strong inhibitory activity even at the 5% (w/v) level while leaf leachateswere inhibitory at the 10% level and higher. In pot experiments with soil containing living lalang tillersor incorporated with rhizome or root fragments, phytotoxicity development in the soil extracts wasevident at four weeks of growth or decay. In soils with decaying roots or rhizomes there was a sub-

Present address : Department of Agronomy,Universiti Pertanian Malaysia,43400 UPM,Serdang,Malaysia.

A. RAJAN, J.V. LOVETT AND A.C. ANDREWS

sequent loss in activity over the eight week incubation period Extracts of soils with living tillers showedloss of activity at six weeks of growth followed by a significant stimulatory response at eight weeks. Theuse of two controls (control-soil extract and distilled water control) enabled detection of inherentstimulatory activity in the control soil Comparison with both controls to eliminate such influencesshowed only living tillers, and decaying rhizomes and roots as major potential sources of inhibitorysubstances. The common soil organism, Bacillus cereus (Frankland & Frankland), Alcaligenes faecalisCastellani & Chalmers} and a Trichoderma sp. were found to be associated with the decay of rhizomesresulting in an initial stimulatory growth reponse in the phytometer species followed by inhibition andsubsequent loss in activity. The nature of these organisms suggest that the release of phy to toxins throughdecay of rhizomes as a common, natural phenomenon in most soils. The dead leaves accumulating as litterin mature lalang vegetation may serve as an additional source of water teachable allelochemicah ifsufficient quantities were available in contact with the soil

four weeks following foliage removal in matureplants grown in pots, a phenomenon whichinhibitory potential of the different plant partsfoliage destruction by fire, herbicides, slashingor moving. However, the activity of the decayingresidues beyond the fourth week and the relativeinhibiroty potential of the different plant partsduring decay following soil incorporation are notknown. Studies with decomposing plant residuesindicate dissappearance of phytotoxicity after oneto several weeks (Cochran et al.y 1977; Elliotet al., 1978). Besides environmental factors,development and extent of toxic effects are alsodependent on the amount and type of residues(Toai and Linscott, 1979).

Eussen and Soerjani (1976) also implicatedphytotoxins from lalang leaves in the observedgrowth reductions in plants grown in soils sur-face-mulched or incorporated with the leaves;but the possible influence of other factors (e.g.nutrients) was not discounted.

The limitations mentioned above warrantedfurther study using alternative methods, to in-vestigate the relative importance of potentialsources of phytotoxins in lalang, and evaluatetheir significance.

Observations on an old lalang vegetationshowed considerable accumulation of leaf on thesoil surface, and an abundance of rhizomes invarious stages of decay to a depth of 15 cm inthe soil. Hence, under such vegetation, two mostlikely sources of inhibitory substances are thedead leaves accumulating and decaying on thesoil surface, and decaying rhizomes just belowthe surface. Further, two possible mechanismsmay be operative with the dead leaves (a) directleaching out of water soluble compounds and(b) release of substances during subsequent

INTRODUCTION

Lalang (Imperata cylindrica) (L.) Raeuschel var.major) is a noxious weed causing serious problemsin many countries (Holm etaLy 1977). It presentsmajor problems in plantation crops (Tempany,1951) and in pastures (Cabanilla, 1949; Yoshida,1953). Aided by fire, cultivation and other dis-turbance, the weed rapidly colonizes large areasto the virtual exclusion of other species, thusbecoming a dominant feature in many situationsin most countries where it is found.

The literature suggested that allelopathymay play an important role in the overall inter-ference of lalang with other species (Eussen andWirjahardja, 1973; Sajise and Lales, 1975; Eussenet al, 1976, Eussen, 1978). In the above studiesinvolving mixed and mono-cultures, allelopathywas implicated based on the observation that thespecies in mixtures were not mutually exclusive.However, the methods used present difficultiesin separating allelopathy from other neighboureffects (Trenbath, 1974; Dekker et al., 1983):Further, in the soil leachate experiments, con-tinous treatment of phytometer species withleachates for extended periods does not allow fora control where nutrient or other effects can becompletely eliminated.

The phytotoxic activity in extracts of lalangplant parts have also been investigated (Dhan-manonda, 1973; Eussen and Soerjani, 1976;Eussen, 1977). However, the use of maceratedtissue extracts in those studies raises the questionas to whether the biologically active substancespresent within tissues become available inleachates of intact or fragmented residues insufficient quantities to exert phytotoxic effects.

Eus'sen (1978) also demonstrated phytotoxicactivity in soil leachates collected at two and

176 PERTAN1KA VOL. 11 NO. 2, 1988

EVALUATION OF POTENTIAL SOURCES OF ALLELOCHEMICALS IN LALANG

microbial decay. Where renovation or improve-ment of lalang grasslands is attempted with cul-tivation, seeds of sown species may be exposedto three possible sources of inhibitory substances:(a) leachates from fragmented residues, (b)chemicals released following decay of the residues,or (c) exudates from plants regenerating fromrhizhomes. A similar situation would exist whenattempting to establish leguminous cover-crops inplantation agriculture. Seeds of species direct-drilled into chemically killed swards may beexposed to toxins leaching out of decayingrhizomes and roots, in addition to substancesleaching out of surface litter. Result of inves-tigations in phytotoxic activity in the lalangrhizosphere in relation to its growth, the influenceof soil factors under field conditions, and charac-terization of associated phytotoxincs will bereported later. This paper presents results ofstudies on the potential sources by which phy-totoxic substances may be introduced into thesoil environment.

MATERIALS AND METHODS

Information available on the nature of potentialinhibitors associated with the Poaceae (and thelevels of such inhibitors reported to be presentin the. natural environment) suggest delay or in-hibition in germination and early seedling growthas the most obvious effect of pyhtotoxins asso-ciated with most species, including lalang. Hence,in all bioassays phytometer species were treatedwith plant or soil leachates only for the firstfew days of growth. This also enabled the in-clusion of control treatments to differentiatenutrient and other chemical effects in bioassayswhere phytometer species were treated with soilleachates. In all bioassays indicator plants usedwere generally legume species commonly used asground covers in plantations or for improvingpastures.

The lalang materials used in all experimentswere obtained from a lalang-dominant pastureon a property at Enmore (30 45'50"S, 151 45'2CTE), 40 km south-east of Armidale, New SouthWales, located 1000 m above sea-level. All datawere statistically analysed and most parametersmeasured did not require transformations tostabilise the variance. The AOV comparisonspresented were generally based on original dataunless otherwise specified.

Experiment I: Inhibitory Activity in Dead LeafLeachates

The following experiment was conducted to studythe effect of leachates from dead lalang leavesaccumulating as surface litter, as a potentialsource of inhibitors during germination andearly growth of the test species.

The dead lalang leaves were cut into 4 cmpieces and 1 g (3.3% moisture) samples wereplaced at the bottom of 90 mm diameter petridishes. Ten ml distilled water was added and allow-ed to stand for 2 h. The leaves were than coveredwith Whatman 3 MM filter paper and 50 seeds ofeach species to be tested were placed on the paper.Another filter paper was placed on top of theseeds. All test species were thus placed in contactwith the leachates in the presence of the leafmaterial to simulate the field situation. Thecontrols had glass-wool (0.5 g) at the bottom ofthe dishes. All dishes were incubated at 24°Cin the dark for 5 d.

The study was conducted in two sets of biossays.

(a) The first set had 4 temperature and 4tropical pasture legumes and 2 crop species(Medicago sativa L. cv. Hunter River; Tri-folium repens L. cv. Huia; Trifolium semi-pilosum L. cv. Safari; Trifolium subterra-neum L. cv. Woogenellup; Desmodiumuncinatum Desv., Phaseolus atropurpureusL. cv. Siratro, Stylosanthes guyanenses Sw.cv. Schofield; Calapogonium mununoidesDesv.; Lycopersicon esculentum Mill. cv.Grosse Lisse; Lactuca sativa L. cv. GreatLakes).

(b) In the second set 7! subterraneum, T.repens, P. atropurpureus, D. uncinatum,M, sativa and L. sativa were used.

There were four replicates for each of thetest species in both biossays. In the first set, thedishes were not disturbed for the five days, at theend of which radicle and shoot lengths weremeasured. In the second set changes in germina-tion rate were recorded daily for the five days.

Experiment 2: Inhibitory Activity in GreenLeaf Dead Leaf Rhizomes and Root Leachates

The following experiment was conducted to assessthe relative phytotoxic potential of leachates ofthe different plant parts. The primary objective

PERTANIKA VOL. 11 NO. 2, 1988 177

A. RAJAN, J.V. LOVETT AND A.C. ANDREWS

was to study their relative importance and assessactivity at levels of materials that were realisticwith respect to quantities available in the field.

The experiment was conducted in petri-dishes using the procedure described in Experi-ment 1. Lalang materials collected fresh from thefield were separated into green leaves, dead leaves,rhizomes and roots; and cut into 4 cm pieces.Samples of green leaves, dead leaves and rhizomesof 0.5, 1.0, 1.5 or 2.0 g fresh weight and rootsamples of 0.5, 1.0 or 1.5 g were placed at thebottom of 90 mm petri dishes, 10 ml distilledwater added and allowed to stand for 2 h. Thecontrol had glass-wool (0.5 g) at the bottom ofthe dish. Fifty seeds of Trifolium subterraneumcv. Woogenellup were set in each dish using thesandwiched filter paper technique describedearlier. The dishes were incubated at 24 C for72 h, at the end of which radicle lengths weremeasured. There were eight replicates in a com-pletely randomised design. Moisture content ofplant parts were 45'2, 3.3, 62.5 and 10.6% forthe green leaves, dead leaves, rhizomes and roots,respectively.

The amount of plant materials available inthe field was determined from six random 60 x60 cm quadrat samples in an old uniform lalangvegetation at Enmore during mid-September.

Experiment 3 : Inhibitory Activity in Soilswith Living Tillers and Decaying Plant Parts.

The following experiment was conducted tostudy the changes in biological activity duringdecay of the various plant parts in a soil mediumand to determine the more likely source ofinhibitory substances. A living lalang tiller wasincluded as one of the treatments for observationof inhibitory activity in the rhizosphere soil.The effects of all treatments on seedling radiclegrowth were evaluated on a tropical and a tem-perate pasture legume.

There were six soil treatments: four with thedifferent plant parts, one with a living tiller andone control (soil only) with four replicates ofeach treatment. The phytotoxicity due to treat-ments was evaluated after 2, 4, 6 and 8 weeks ofdecay or growth in bioassays treated with therespective aqueous soil extracts. The levels ofplants parts were 10 g per pot for fresh leaves,dead leaves or rhizomes and 2 g per pot for roots.

The tillers were established from three-noderhizome cuttings, grown to a height of 5 cm inthe glasshouse, and transplanted ( 1 tiller per pot).

The fresh plant parts were preparedas described in Experiment 2, mixed into 200 gof soil and placed into 8 cm pots lined withpolyethylene film. The lining had six equallyspaced 2 mm perforations at the bottom topermit drainage. The soil was a 1:1 mixture ofsandy loam and sand, sieved (2 mm) prior tomixing.

The pots were set in 4 blocks, with treat-ment pots randomized within blocks, in a growthchamber at 29°C (12 h, day) and 23°C (night).Relative humidity was maintained between68-76% and the light density flux was 300uE m'2 s*1 . Pots were watered daily with 40 mldistilled water and no nutrients were provided.

At the completion of each incubation/growth period, 1 pot of each treatment wassampled from every block. The contents weretransferred into 500 ml beakers, 200 ml distilledwater added and allowed to stand for 30 min,stirred for 2 min and allowed to stand for afurther 30 min. With the living tillers, plantswere gently shaken to loosen soil adhering to rootsand removed, prior to stirring the remainingcontents. The aqueous soil extracts were filteredthrough glass-wool (to remove larga debris), chilledto 4°C and centrifuged at 3000 rpm for 30 min.

Biossays for phytotoxic activity wereperformed using the sandwiched filter papertechnique described in Experiment 1, onboth Trifolium subterraneum and Calopogoniummununoides, with 40 seeds per dish. Two dishesper extract (pot), for each of the six treatments inreplicates, were set at each sampling time. A dis-tilled water control ( 2 x 4 dishes) was includedin the biossays to differentiate the inheret bio-logical activity in the control soil. Five ml extractor distilled water was used per dish with T. sub-terraneum incubated for 72 h, and 7 ml wasused with C. mucunoides incubated for 96 h,both incubations being performed at 24°C in thedark. (Preliminary studies suggested a 96 hourbioassay to be suitable for Calopogonium bioassaydue to its slower radicle emergence, and also alarger volume of water was found to be requiredfor that period). At the end of the incubationperiod dishes and contents were frozen and heldfor radicle length measurement. Electrical con-

178 PERTANIKA VOL. 11 NO. 2, 1988

EVALUATION OF POTENTIAL SOURCES OF ALLELOCHEMICALS IN LALANG

ductivity and pH values of fresh extracts werealso determined.

On a dry weight basis, the levels of plantparts used in this study were 2.7, 4.9, 2.0 and0.9% (residue wt/soil wt) for the green leaves,dead leaves, rhizomes and roots, respectively.However, taking into account the 1:1 extraction,the soil extracts for the decaying green leaves,dead leaves and rhizomes were equivalent to the5% leachates (fresh weight/volume) used in ex-periment 2, while the decaying root extractswere equivalent to a 1% leachate.

Experiment 4: Inhibitory Activity in Leachatesof Decaying (Surface-Sterilised) Rhizomes

It was noted earlier that a large proportion ofrhizomes under field conditions were at variousstages of decay. The decaying rhizomes couldconstribute a continuing source of phytotoxins.However, the nature of the organisms responsiblefor the decay is not known. The following ex-periment was conducted primarily to (a) obtainsupporting evidence for the responses observedduring decomposition of rhizomes in soils(Experiment 3), and (b) to isolate and identifythe microorganisms involved. Leachates of surfacesterilised rhizomes allowed to decay for 1, 2, 3,5, 9 or 13 weeks, were evaluated for phytotoxicactivity in seedling biossays. There were a totalof seven treatments in the biossays including adistilled water control, with four replicates pertreatment.

Freshly collected rhizomes (65% moisture)were washed thoroughly with water, rinsed indistilled water, cut into 4 cm pieces, surfacesterilised in 10% calsium hypochlorite solution for3 min, and washed thoroughly with sterile distilledwater. Eight g of surface sterilised rhizomes wereplaced in 90 mm petri dishes, 10 ml sterile dis-tilled water was added and incubated at 28°C.The water was replenished as required at weeklyintervals. A sufficient number of dishes wasprepared to enable sampling at 1, 2, 3, 5, 9 and 13weeks from the start of the experiment. At eachsampling time 10 dishes from each of the fourreplicates were removed, the contents filteredthrough glass-wool and the leachates pooled.The dishes and rhizomes pieces were washed byapplying sterile water dropwise to extract as muchof the original leachate as possible, and the volume

made up to 100 ml (total of ten dishes). Thepooled leachates were filtered through 0.2 urnMillipore filters.

The phytotoxicity of the leachates wasevaluated, using the sandwiched filter papertechnique on T. subterraneum and C. mucu-noides with 40 seeds per dish. Five ml leachateor distilled water (control) was used per dishwith T. subterraneum incubated for 72 h and 7 mlwith C. mucunoides incubated for 96 h, bothincubations being performed at 24°C in the dark.At the end of the incubation period, radiclelengths were measured.

At the end of the second week, small piecesof rhizome tissues and droplet samples of leachateswere taken and cultured on Oxoid tryptone soyagar (4%) or malt-yeast extract-agar (17:1:15 g/L)containing aureomycin (5 mg/L) to obtain purecultures of bacteria and fungi, respectively. Theisolated organisms were identified by theCommonwealth Mycological Institute, England.

RESULTS AND DISCUSSION

Experiment I: Inhibitory Activity in DeadLeaf Leachates

Inhibitory effects of dead leaf ieachates on germi-nation of the six species tested are presented inFigure 1. Germination was found to be significant-ly delayed in all species tested but the degree ofinhibition varied between species. Percent in-hibition in germination on the second day variedfrom 9.4% in M. sativa to 86.5% in T. subterra-neum. Four of the six legume species testedshowed complete recovery in percent germinationby the fifth day. M. sativa and D. uncinatumrecovered completely after three days and P.atropurpureus and L. sativa recovered after fivedays. However, T. repens and T. subterraneum,while showing trends towards recovery were stillinhibited on the fifth day.

The effects of the dead leaf leachates onseedling growth are presented in Table 1. Theleachate treatment had no effect on seedlinggrowth in six of the ten species tested. In tomatoseedlings both radicle and shoot growth werestimulated. Shoot and radicle growth were bothsignificantly inhibited in the temperate legumeT. subterraneum and in the tropical legumeC mucunoides. In D. uncinatum, shoot wasinhibited while radicle growth was not affected.

PERTANIKA VOL. 11 NO. 2, 1988 179

A. RAJAN, J.V. LOVETT AND A.C. ANDREWS

repens b) T. Subterraneum

(c) M. Sativa

100

d) D. uncinatum

100

Daya

e) P. Atropurpureus

100

f) L. sativa100

0 1 2 3 4 6Daya

/ : Effect of lalang dead leaf leachates on germination of six test species ( Control;Leaf Leachates)1

— Dead

Vertical bars denote S.E.180 PERTANIKA VOL. 11 NO. 2, 1988

EVALUATION OF POTENTIAL SOURCES OF ALLELOCHEMICALS IN LALANG

TABLE 1Effect of leachates from dead lalang leaves on seedling growth of test species after 5 days

M, sativa(Hunter River)

T. semipilosum(Safari)

T. repens(Huia)

T. subterraneum(Woogenellup)

D. uncinatum

P. atropurpureus(Siratro)

S. guyanensis(Schofield)

C. mucunoide

L sativa(Great Lakes)

L esculentum(Grosse Lisse)

RadicleLength (mm)

Control Leachate

26.1a

15.4a

14.9a

21.3a

16.9a

20.9a

21.7a

21.4a

31.4a

9.1b

25.5a

15.3a

15.0a

I8.21b

16.7a

21.3a

19.8a

16.6b

31.9a

15.2a

Probability

n.s.

n.s.

n.s.

<0.05

n.s.

n.s.

n.s.

<0.05

n.s.

< 0.001

ShootLength (mm)

Control Leachate

25.8a

18.0a

21.5a

21.5a

19.2a

10.6a

27.1a

18.0a

25.6a

5.9b

25.3a

15.3a

20.3a

b13.9

I4.4b

11.4a

26.0a

13.5b

23.8a

8.5a

Probability

n.s.

n.s.

n.s.

<0 .01

<0 .05

n.s.

n.s.

<0 .01

n.s.

<0 .05

Between treatment means with the same subscript are not significantly different at p < 0.05 (Duncan's Multiple RangeTest).n.s. • non-significant at p < 0.05.

The results suggest delay and inhibition ofgermination and early seedling growth as im-portant mechanisms by which establishmentof some species will be affected when seeds arelocated around lalang surface litter. On a perunit surface area basis, the quantity of deadleaves found under established lalang standsat Enmore was 20 to 60% higher than that usedin this study. In addition, the concentration ofleachates used in the study was only 10% (i.e.1 g/10 ml). Under lower soil moisture conditionsin the field, significantly higher leachate concen-trations would occur and increased phytotoxicactivity can be expected. Thus, under matureundisturbed lalang vegetation, the litter leachateswould constitute a significant source of inhibitorsthough actual activity may fluctuate with environ-mental factors and microbial activity.

Levels of accumulation of inhibitory com-pounds within plant tissues are also known to varydepending on environmental factors (Koeppe etal9 1970; Hall et ai, 1983). Winter killed foliagewould have a different chemical compositioncompared to foliage dying through normal senes-cence. Hence, some variation in responses observedabove may be expected depending on source ofsamples.

Experiment 2: Inhibitory Activity in GreenLeaf, Dead Leaf, Rhizome andRoot Leachates

Rhizomes and root leachates caused sig-nificant root reductions even with the 5% leachateconcentration (0.5 g/lOml), whereas the green anddead leaf leachates were inhibitory with the 10%concentration and higher (Table 2).

PERTANIKA VOL. 11 NO. 2, 1988 181

A. RAJAN, J.V. LOVETT AND A.C. ANDREWS

TABLE 2Effect of increasing levels of Imperata leaf, rhizome and root leachates on Trifolium subterraneum radicle growth (mm)

Plant Part

Green leaves

Dead leaves

Rhizomes

Roots

0(control)

14.86a

13.27a

14.59a

15.05a

Levels of Imperata leachates (g/10 ml water)

0.5 g(5%)

15.09a

11.46ab

11.62b

9.7 l b

1.0 g(10%)

11.29b

9.81b

9 3 8 b c

8.36bc

1.5 g(15%)

10.26b

9.61b

8.79C

6.68C

2.0 g(20%)

9.03b

9.40b

8.44C

-

Means within rows with the same subscripts are not significantly different at p < 0.05 (Duncan's Multiple Range Test).

With the 5% leachates the percent inhibition(% of control) obtained with the green leaves,dead leaves, rhizomes and roots were —1.6, 13.6,20.4 and 35.5%, and with the 10% leachates thecorresponding values were 24.0, 26.0, 35.7 and44.5%, respectively. Thus, relative importance inorder of decreasing phytotoxicity over these twolevels was roots, rhizomes, dead leaves and greenleaves.

The mean fresh weights of rhizomes, roots,dead leaves (including surface litter) and greenleaves available in the field per 60 x 60 cm groundsurface were 655, 55, 459 and 314 g, respectively.Thus, taking into account both the relativeamounts present in the field, and the relativephytotoxic effect of each component, and assum-ing uniform distribution of the various plantsparts within a 20 to 25 cm soil depth in thefield following cultivation, the rhizomes wouldbe the plant part most likely to exert inhibitoryeffects on germinating seeds. However, residuesare never uniformly distributed in soils (Patrick,1971; Cochran et aL, 1977), Elliot et al., 1978).Hence, in concentrated 'pockets', dead and greenleaves could also be potential sources of inhibitoryleachates. Root leachates were relatively morephytotoxic than the other leachates, but due tothe small amounts available in the field the rootmaterial by itself will not be of any significance.

Experiment 3; Inhibitory Activity in Soils withLiving Tillers and Decaying Plant Parts

Radicle growth in T subterraneum in general,showed strongest phytotoxic effects in thefourth week (Figure 2). Compared to the control

182 PERTANIKA VOL

soil (CS), the decaying dead leaves (DL), rhizomes(RZ) and roots (RT) all showed suppressed rootgrowth in Trifolium in the fourth week, whilethe green leaves (GL) showed growth inhibitionat the second aud sixth week. With the exceptionof GL, the other plant parts were not inhibitoryat the second, sixth or eighth week. Extracts ofsoils with the living tiller (LT) showed strongphytotoxic effects in the fourth week, and to alesser degree in the second week.

However, the CS extract showed significantstimulation of Trifolium root growth in thesecond, sixth and eighth week when compared tothe distilled water control (CW). In these instancesthe absolute inhibitory potential of a treatmentwill certainly be over-estimated if quantified withrespect to CS particularly if the activity inherentin the soil was due to stimulatory levels of poten-tially inhibitory substances; and the reverse willalso be true where the control soil exhibits inhibi-tory activity relative to the distilled water control.The use of the two controls was thus useful! indifferentiating inherent factors that may influencethe growth response in the soil medium. Compari-son with both CS and CW clearly suggest only theLT, RT and RZ treatments to have the potentialfor inhibitory activity.

The LT extract in the second week wasinhibitory with respect to CS, but stimulatorywith respect to CW. The inhibitory effect wasmore pronounced and significant compared toboth CW and CS extracts at the fourth weekwhen the plants were visually observed (duringsoil extraction) to have attained near maximumroot development. There was a marked difference. 11 NO. 2, 1988

EVALUATION OF POTENTIAL SOURCES OF ALLELOCHEMICALS IN LALANG

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PERTANIKA VOL. 11 NO. 2, 1988 183

A. RAJAN, J.V. LOVETT AND A.C. ANDREWS

in root growth between the second and fourthweek tillers. Over the six and eight weeks thefoliage of tillers showed nutrient deficiencysymptoms, and there was no marked increasein root development after four weeks. At eightweeks LT extract showed significant stimulationrelative to both CW and CS extracts. These ob-servations strongly suggest that inhibitory activitywith the living klang plants was associated withthe plants growth acitivity. Release of organicsubstances via root exudation has been reportedto be directly related to growth of the root system(Prikryl and Vancura, 1980). Over the eightweeks, it appears that the level of inhibitorysubstances produced was low initially, increasedto a maximum at four weeks and subsequentlydeclined to levels sufficient to exert stimulatoryeffects. The response was characteristic of thegeneral concentration effect often observed withmany growth-regulatory chemicals, i.e., stimula-tory at low and inhibitory at higher concentrations(Kaindl, 1956;Lovett, 1982).

In relation to the amount of materialsavailable in the field, the results of the ex-periments in this study suggest that the deadleaves (including surface litter) and rhizomesare the major sources of inhibitory substancesin mature lalang vegetation. While the effectsare short-lived with residue incorporation, theamounts of decaying materials under old undis-turbed lalang stands is not a fixed quantity. Theaccumulation and decay of surface litter, andthe death and decay of old rhizomes and rootsis a dynamic process. Further, effects in the fieldwould be the net or combined effect of all possiblesources of inhibitors.

In Calopogonium mununoides, inhibitionwas observed only with the decaying root trea-ment at four weeks (Figure 3). The decayingrhizomes at four weeks appeared to have someinhibitory effect on growth, but the effect wasnot significant. The LT extracts also had noeffect. Compared to T. subterraneum, the tropicallegume appeared to be more tolerant of theinhibitory extracts.

The change in pH of the aqueous soil ex-tracts showed a general increase at the fourthweek, both with treatments involving decayingmaterials and the living tillers (Figure 4). Themaximum pH range observed was from 7.2 (CSextract) to 8.0 (RZ extract) in the fourth week of

decay or growth, but the pH increase was notdirectly related to inhibitory activity (Figure 2,3 and 4). Patrick and Koch (1958) reported moreacid conditions during decomposition of plantresidues incorporated into soils, but the crockscontaining the soil and residues were coveredafter watering to saturation to prevent moistureloss during the decay process. The production ofinhibitors in their experiment could have been dueto more anaerobic conditions under which volatileorganic acids can be expected to accmulate (Gotohand Onikura, 1971). Further, they observed lossin activity of extracts when stored without a layerof toluene on the surface, suggesting that in-hibitors were volatile substances.

The pH increases observed in this study arein agreement with those observed by Kanchan andJayachandra (1979). The change in pH of extractsobserved by Patrick and Koch (1958) were on theaverage from 6.5 to 5.0, and that observed byKachan and Jayachandra (1979) was from 7.5to 8.3. In both these studies it was demonstratedthat pH changes had no direct effect on growthof the test species.

The electrical conductivity (EC) valueswere measured to monitor salinity and ionicconcentrations in the extracts (Giffin andJurinak, 1973) in order to ensure that it was wellwithin levels that would not exert any directeffects on the bioassays. Significant changes inEC values were observed with all plant residuesand there was a general rise in EC values from twoto eight weeks of decay or growth (Table 3).Highest values were obtained in extracts of soilscontaining the green leaves. In all cases, thevalues recorded were well below the levels report-ed to have any direct effect on seedling growth(Patrick et al, 1964). The LT extracts which werestrongly inhibitory to 71 subterraneum radiclegrowth during the fourth and stimulatory duringthe eighth week) showed no difference in ECfrom corresponding CS extracts at two, four,six or eight weeks. The increase in EC valueswas also not directly related to relative inhibitoryactivity of decaying plant parts.

Experiment 4: Inhibitory Activity in Leachatesof Decaying (Surface-Sterilised) Rhizomes

Leachates of surface sterilised decaying rhizomesshowed initial stimulation, followed by inhibition

184 PERTANIKA VOL. II NO. 2, 1988

EVALUATION OF POTENTIAL SOURCES OF ALLELOCHEMICALS IN LALANG

TABLE 3Influence of decaying lalang plant parts and living tillers on electrical conductivity values of soil extracts

Time(weeks)

2

4

6

8

ControlSoil(CS)

0.107c

0.134C

0,177d

0.278c

DeadLeaves(DL)

0.160b

0.233b

0.199cd

0.298c

GreenLeaves(GL)

O.32Oa

O.323a

0.361a

0.445a

Rhizomes

(R2)

0.121bc

0.277ab

0.236C

O.39Ob

Roots

(RT)

0.292a

0.280ab

O.3O5b

0.381b

Livingtillers(LT)

O.123bc

0.140C

0.197^

0.266c

Electrical Conductivity in mScm -1

Between treatment means within rows with the same subsripts are not significantly different at p < 0.05 (Duncan'sMultiple Range Test).

as decay progressed to the ninth week, and loss inactivity at 13 weeks in both test species (Table 4).The results suggest a gradual increase in growth-regulatory substances to inhibitory levels ataround nine weeks, and subsequent inactivationof these substances within the next four weeks.The growth effects on T. subterraneum over thefirst nine weeks was typical of the concentrationeffect on biological reponses by growth- regulatorysubstances including allelochemicals (Kaindl,1956;Lovett, 1982).

The longer time taken for the productionof substances at inhibitory levels, compared to theexperiment 3 is probably due to the need for themicrobial population to build-up to sufficientlevels from the inoculom within the tissues.

In the Calopogonium bioassay, significantstimulation in radicle growth was only obtainedwith the fifth week leachates, while Trifoliumresponded earlier. This slight delay in the stimula-tory response suggests that slightly higher concen-trations were required to exert the growth effecton Calopogonium . The greater tolerance ofCalogonium to the inhibitory substances was alsosuggested by the results with decaying plant partsin soils.

The organisms isolated from the decayingrhizomes were Bacillus cereus (Frankland andFrankland) and Alcaligenes faecalis (Castellaniand Chalmers), both non-pathogenic bacteria,very common in soils; and a fungus, Trichodermasp.f a very common saprophyte in soils and on

TABLE 4Effect of leachates from decaying rhizomes on root growth response in

mucunoides l

1

T. subterraneum 9.13

C. mucunoides 3.06

2

-16.10

- 2.67

Period of decay

3

-25.73*

- 2.19

Trifolium subterraneum and Calopogonium

(weeks)

5

-15.24

-32.61*

9

31.74**

39.81**

13

-5.89

-3.49

deduction (+) or Stimulation (-) in radicle length expressed as percent of control.

* Significant at p < 0.05 (compared to distilled water control).** Significant at p <C 0.01 (compared to distilled water contiol).

PERTANIKA VOL. 11 NO. 2, 1988 185

A. RAJAN, J.V. LOVETT AND A.C. ANDREWS

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A. RAJAN, J.V, LOVETT AND A.C. ANDREWS

woody tissues. Hence, the production of phytoto-xic substances through decay of rhizomes is likelyto be a common and natural phenomenon in mostsoils.

ACKNOWLEDGEMENT

The sponsorship offered by Universiti PertanianMalaysia to the senior author, and the researchfacilities provided by the Department of Agro-nomy and Soil Science, University of NewEngland, Australia are gratefully acknowledged.The authors also wish to thank Puan RahimahHj. Abdullah for typing the manuscript.

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