Plant and Soil 137: 223-227, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands. PLSO 839/)

Effect of eucalyptus oil on germination and growth of Phaseo lus aureus

Roxb.

DALJIT SINGH, R.K. KOHLI ~ and D.B. SAXENA 2 Allelopathy Research Laboratory Department of Botany, Pan jab University, Chandigarh-160 014, lndta. ~Dtvtston of Agrtcultural Chemicals, 1AR1, New Delht-llO 012, India. Correspondmg author

Received 3 August 1989. Revised November 1990

Key words: allelopathy, cineole, citriodora oil, Eucalyptus citriodora, E. globulus, eucalyptus oil, limonene, Phaseolus aureus, seed vigour

Abstract

Crude oil from Eucalyptus globulus and E. citriodora was extracted and the rich components, cineole and limonene were fractionated. The vapours of these oils and fractions were adsorbed onto the soil in one set of germination trials while in the other set a vapour column of volatile oils was maintained above the oil-treated soil. In both sets seed germination, seedling growth, relative growth rate, water content, height and number of leaves of Phaseolus aureus var. ML-267 were compared to those of controls. All parameters were found to be significantly affected. The effect was more pronounced with a combination of eucalyptus oil onto soil and a vapour-rich air column. There was a strong correlation between the vapour concentration and its inhibitory effect.

Introduction

Mature, undisturbed stands of Eucalyptus plan- tation are virtually devoid of other vegetation (A1-Mousawi and AI-Naib, 1975; Bhaskar and Dasappa, 1986; del Moral and Muller, 1969; 1970). This is attributed to allelopathy, the direct or indirect effect of one plant on another through production and liberation of chemical compounds into the environment. The leaves of Eucalyptus are aromatic, releasing a number of volatile terpenes into the environment (A1- Mousawi and A1-Naib, 1976; del Moral and Mul- ler, 1970). The released volatiles have vapour densities higher than that of air. These move downwards and are adsorbed on the soil particles (del Moral and Muller, 1970). The continuous release of oils from the leaves of the tree forms a continuous column from the leaves to the soil surface under the canopy. The possibility that these oil components from Eucalyptus might have a direct bearing on the growth of under-

storey vegetation led us to study this phenom- enon on Phaseolus aureus Roxb. var. ML-267 in soil alone or in combination with a vapour column.

Material and methods

Essential oils from E. globulus Labill. (referred to as eucalyptus oil) and E. citriodora Hook. (referred to as citriodora oil) were extracted by steam distillation• The oils obtained were dehy- drated over anhydrous sodium sulphate. Cineole and limonene (the relatively rich components in oil of E. globulus) of the eucalyptus oil were extracted using column chromatography over Alumina grade 1 (activated Aluminium oxide (Brockmann), pH 7.0 in aqueous medium). Eiu- tion with hexane first yielded cineole, while limonene was eluted later with benzene. The compounds were characterized by Co-GLC and IR spectroscopy.

For the bioassay, pure line seeds of Phaseolus

224 Daljit Singh et al.

aureus Roxb. (=Vigna radiata (L.) R Wilczek) var. ML-267 were procured from Seed Technolo- gy Unit, Punjab Agricultural University, Ludhiana. Washed sand and clay (4: I w/w; pH 7.2) was oven dried, autoclaved and sieved.

Experiment I

Effect of eucalpytus oil vapours and their pure components adsorbed on the soil alone or in combination with vapour column on germination behaviour of P. aureus The experiment was performed under two sets of conditions. In the first, the soil was treated by exposure to the volatile oils extracted from Eucalyptus leaves for 48 hrs. In the second set of conditions, the air column over the substratum of oil-adsorbed soil was filled with vapours of the respective oils. The soil (150gin) in each glass Petri dish (15cm diameter) for each concen- tration (10, 20, 30nLmL -1) of the volatile oils (eucalyptus oil, citriodora oil, cineole, limonene) was housed in separate environmental chambers (3m 3) maintained at normal temperature. Four Petri dishes were maintained for each control. Each chamber was fumigated with respective concentration of the volatile oils with the help of demothing vapourizer of Euroclean 200 (manu- factured by Eureka Forbes Ltd., Bombay) under an air displacement pressure of 126.2m 3h-1. The fumes were retained in the respective cham- bers for 48 hr with replacement by fresh fumes after 24 hr. At the time of replacement each Petri dish was thoroughly shaken to ensure the proper adsorption of the volatile oils on the soil particles. The soil of each of the Petri dishes was moistened with 40 mL of distilled water. Fifty healthy, uniform seeds of P. aureus were embed- ded equidistantly in the moistened soil in each of the Petri dishes.

For the first set, four Petri dishes for each treatment were transferred to a seed germinator maintained at 28 -+ 2°C temperature, 75 +- 3% re- lative humidity. For the second set, an equal number of Petri dishes were transferred to a seed germinator where chambers were fumigated after every 48 hr with fresh lots of the respective concentrations of respective oils. The number of seeds which had germinated were recorded daily in one or two representative Petri dishes for each

treatment until no more seed had germinated for a week. Final counts of the germinated seeds were made in all the Petri dishes after 15 days. The observations were made and expressed fol- lowing the method of Kumari et al. (1985) and Agrawal (1980), and ISTA (1976) rules. The water content of the seeds and their seedlings was measured directly on a Dean and Stark apparatus (Trease and Evans, 1983).

Experiment II

Effect of eucalypt oil vapours on growth of young P. aureus plants Plants of P. aureus, twenty days old, grown in plastic pots containing washed sand and clay (4:1w/w) and maintained under continuous light were transferred to the fumigation cham- bers (3m~). These chambers were maintained at 28 -+ 2°C temperature, 75 -+ 3% relative humidity and 16hr photo-period at 270 p, W cm 3 energy (approx 60001ux). The chambers were flushed with 13.5 nL mL -I of the oils extracted from E. globulus or E. citriodora. The oil vapour con- taining chambers were refumigated at 5-day in- tervals with vapours of the respective oils in the same concentration replacing the earlier fume- rich air. Seedling length, number of leaves, RGR (relative growth rate) with respect to dry weight and water content were recorded after 15 days of exposure to the vapours of the two oils. Plants maintained under the same conditions, but with- out any oil vapours in the air, served as the control. A sample size of 50 plants was main- tained for each treatment. Duncan's Multiple Range Test (DMRT) was used to compare the differences between the control and the various treatments (Duncan, 1955).

Results

Soil which had adsorbed eucalyptus oil (Set I) and soil which had adsorbed eucalyptus oil in combination with oil vapours in the air (Set II) lowered the percentage of germination compared with the controls. The effect was relatively more pronounced in the conditions of the latter set. No significant difference was noticed in case of lowest concentration (10 nL mL- ~) of any of the

Effect of Eucalyptus oil on Phaseolus aureus 225

a a a a

B0 15.0

o z 10 20 30 'r0 20 30 10 "20 30 10 20 30

EUCALYPTUS CITRIOOORA LIMONENE CINEOLE o ,.=, OIL OIL e

,z z 100 a a _ _ FIa.TB

OIL ADSORBED 0 10 20 30 10 20 30 10 20 30 10 20 30 SOIL

COLUMN OFOIL 0 10 20 30 10 20 30 10 20 30 10 20 30

EUCALYPTUS CITRIODORA LIMONENE CINEOLE OIL OIL

CONCENTRATION (nLmL 11

Fig. 1. Effect of eucalyptus oil vapours and their pure components adsorbed on the soil alone (a) or in combination with vapour column (b) on seed germination, seed vigour and water content of seeds and seedlings of P. aureus var. ML-267. Means of per cent germination have been shown in the graph. Those having same superscript symbols in Fig. la and lb, collectively, are not significantly different at 5% level applying DMRT (Duncan, 1955).

soils tried in the Set I (Fig. la). In contrast, in the case of Set II (Fig. lb) the similar concen- tration of cineole or citriodora oil t reated seeds showed significantly lower germination percen- tage while those treated with eucalyptus oil or l imonene registered insignificant difference over that of control. However , a relatively low value of seed vigour was seen in these t reatments . With every increase in concentrat ion of the oil tried under both sets of conditions, the germina- tion and seed vigour values steeply increased. None of the seeds germinated in Set II which had been t reated with the highest concentration (30 nL m L ~ of eucalyptus oil and citriodora oil or cineole. The water content of the treated seeds or their seedlings in both sets decreased gradually with increasing concentrat ion of the oil vapours, compared to the control. However , the effect was more pronounced in the second set of conditions.

The lengths of the seedlings (radicle and plumule) developed from seeds germinated from both sets were shorter than those of the controls (Figs. 2a and 2b). With increasing concentration of the oil vapour in both sets, the seedling lengths decreased with almost the same trends.

However , there was a more pronounced de- crease even at low concentrations for Set 1I (Fig. 2b). The value of the correlation coefficient (r) in all cases was negative and close to 1 (Fig. 2a and 2b).

The relative growth rate, average number of leaves per plant, height of plant and water con- tent were significantly less than the control, in response to vapours ( 1 3 . 5 n L m L -~) of oil ex- tracted from E. globulus and E citriodora (Table 1). Plants t reated with oil vapours extracted from E. citriodora had significantly lower values for each of these parameters compared to those t reated with oil vapours extracted f rom E. globulus. An exception, however, was found in the case of number of leaves where the differ- ence was not significant among the t reatments of the two oils.

D i s c u s s i o n

The allelopathic potential of volatile oils from Eucalyptus grown under agroforestry and social forestry p rogrammes in India was repor ted by various workers (Al-Mousawi and AI-Naib,

226 Daljit Singh et al.

a e Plumule length • Radicle teng~ch

110. ae -- Eucatyp'tus oil ,Ra L!mone,ne . . . . Citriodora oil a ~ . . . . Cmeote

'°° a~ o 9 =-o.828 ,\~,, r, =-o.,5o E" I, % r'':-°''ss80- \ ''4b r3"=-°'9'6

r 2 = 0 8 4 ~ r 4 = oses

C r2/=-0 @71 r~=-0 964

_~ 40- fg

20- .~. g~ -'.-4.h.i j

0 r r [ I rill ' J t~"%"~=~ J O~t abSorbed soit 0 '~0 20 30 0 10 20 $0

b EuCalyptus air - - Limonene

110- - - - - Ci~triodora oil l a . . . . Cineote

100- a a ~ \ r 7 =-0.$13 r 5 = -0 925 E E rS"= - 0 931 ~ ~ r?" = -0 960

8 ~ - 0 810 ~ 80- , r 6 . _ 0 8 4 B " ~ 8 /

60- \ \ r d = - 0 930 = -0 831

h

I 9

20' ghk. × x . ~ , ~ j hi . .~t.h i ~ X - ~ h i

Oil absorbed salt 10 20 30 0 10 20 30 Column of oil 10 20 30 0 10 20 30

CONCENTRATION (nLrnL 1)

Fig. 2. Effect of eucalyptus oil vapours and their pure com- ponents (cineole and limonene) adsorbed on the soil alone ( la) or in combination with vapours column ( lb) on the seedling growth (plumule and radicle) or P. aureus var. ML-267. Means of lengths of radicle and plumule have been shown separately in graph. Those having same superscript symbols on respective parameter (radicle or plumule) are not significantly different at 5% level of significance applying DMRT (Duncan, 1955). r and r ' represent the correlation coefficient between concentrations and mean length of radi- cle and plumule, respectively in response to eucalyptus oil (r] or rs), citriodora oil (r_, or %), l imonene (r 3 or rT) and cineole

(r 4 or rs).

1975, 1976; Kohli, 1987; Kohli et al., 1987; del M6ral and Muller, 1969, 1970). However, direct evidence involving pure allelochemics is scarce. del Moral and Muller (1970) showed that volatile oils from E. camaldulensis reduced germination

Table 1. Effect of eucalyptus oil (cineole rich) and citriodora twenty days old P. aureus vat. ML-267 plants*

of Bromus rigidus seeds. The volatile oils from Eucalyptus leaves as in E. camaldulensis (del Moral and Muller, 1970) are adsorbed on the soil under the tree canopy.

The present investigation shows that the oil vapours, when adsorbed on the soil particles, retain their activity and inhibit germination of seeds, speed of germination (Fig. la) and seed- ling growth (Fig. 2a). The inhibitory effect of the oils from Eucalyptus leaves or that of their rich components (cineole and limonene) is increased if the air above the substratum has also the vapours of these oils adsorbed or suspended. This is apparent from the highly reduced germi- nation, seed vigour, and radicle and plumule lengths of P. aureus seeds and their seedlings (Fig. lb, 2b). The water content of the seeds or their seedlings was highly reduced in treatments with oil adsorbed on the soil particles or in combination with oil suspended in the air col- umn, compared with the control. Reduced water content in response to vapours of different oils had also been observed in another plant, Parth- enium hysterophorus by Daizy (Personal com- munication). The values of the correlation coeffi- cient (r), negative in character and close to 1 especially in case of radicle and plumule growth in response to any of the oil vapours clearly demonstrate a strong reciprocal relationship be- tween the concentration of the oils and the growth of the seedling. This is an important observation which explains, at least in part, why densely grown Eucalyptus trees are devoid of other vegetation under them. Bhaskar and Dasappa (1986) studied 7-yr-old plantations of several tree species. They found that Grevillea robusta had an understorey of 54 plant species with a density of 2400 plants m -2, followed by Casuarina equisetifolia with 30 species, Acacia

oil (citrionellal rich) in concentration of 13.5 nl m L 1 on growth of

Treatment R G R Mean No. of Plant height Water w.r.t, leaves per (ram) content dry wt. plant (%)

Control 0.122 ~ 6.10 a 192.8" 85.0 ~' Eucalyptus oil 0.079 b 4"20b 152.6 b 63.4" Citriodora oil 0.068 ~ 3.70h 142-6c 53"0c

* Means of observed parameters are given in columns, those having same superscript are not significantly different from each other applying Duncan's multiple range test (Duncan, 1955) at 5% level of significance.

auriculiformis with 27 species and Eucalyptus tereticornis with only 14 species and only 9 plants m 2. The soil surface under Eucalpytus planta- tion compared to that of other trees is thus very sparsely. It is not only the germination or seed- ling length, but also the further growth of the plant that is affected adversely when exposed to volatile oil from Eucalyptus. Our studies showed that the volatile oils from Eucalyptus trees have a direct inhibitory impact on the vegetation in their vicinity.

The crude anhydrous oil from E. globulus or E. citriodora significantly reduced the RGR, leaf differentiation and the height of the young plants. Their water content is also reduced in response to these volatile oils. Citriodora oil, was more inhibitory than the Eucalyptus oil.

Acknowledgements

The financial support from the Department of Environment, Government of India, is gratefully acknowledged.

References

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Effect of Eucalyptus oil on Phaseolus aureus 227

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