PHYTOTOXIC ACTIVITY OF EUCALYPTUS GLOBULUS LEAF … · Udaipur. The Plant was identified by subject...

www.wjpps.com Vol 6, Issue 9, 2017.

1744

Mehta et al. World Journal of Pharmacy and Pharmaceutical Sciences

PHYTOTOXIC ACTIVITY OF EUCALYPTUS GLOBULUS LEAF

EXTRACTS ON GERMINATION AND SEEDLING GROWTH OF

TOMATO

Surbhi Mehta* and Kanika Sharma

Microbial Research Laboratory, Department of Botany University College of Science,

Mohanlal Sukhadia University Udaipur-31301, Rajasthan India.

ABSTRACT

The aim of this study was to evaluate the phytotoxic potential of 100%

alcoholic crude extract as well as petroleum ether fraction of young

leaves of Eucalyptus globulus on seed germination and seedling

growth of tomato (Solanum lycopersicon) as well as to determine the

growth bioassay. The crude extract was prepared by cold extraction

method and petroleum ether fraction was prepared by hot extraction

method with soxhlet assembly. The extraction was carried out with

young healthy leaves (in powder form) and organic solvent. 5, 10, 15,

20 and 25 mg/ml concentrations are used for phytotoxicity assay.

Phytotoxicity was done by standard blotter method. The Result showed

that 5, 10 and 15 mg/ml concentration of extract was non

phytotoxicand were further used for in vivo experiments using

chambered plastic tray. The phytotoxic potential of the crude as well as

partially purified extract of leaves of Eucalyptus globulus varied according to concentration

and it is called concentration depended inhibitory effect. 25 mg/ml concentration had the

greatest inhibitory effect on the seed germination that is 35% in crude extract and 30% in a

partially purified extract. Similarly, 25 mg/ml concentration observed to be toxic against a

seedling growth of tomato and indicating that the compound responsible for the phytotoxic

residue within these extracts. The major cause of performing this assay is related to develop a

safer and herbal formulation using this plant extracts to control plant pathogenic fungi.

KEYWORDS: phototoxic potential, crude extract, soxhlet assembly, herbal formulation,

*Corresponding Author

Surbhi Mehta

Microbial Research

Laboratory, Department of

Botany University College

of Science, Mohanlal

Sukhadia University

Udaipur-31301, Rajasthan

India.

Article Received on

18 July 2017,

Revised on 07 August 2017,

Accepted on 28 August 2017

DOI: 10.20959/wjpps20179-10137

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 6, Issue 9, 1744-1758 Research Article ISSN 2278 – 4357


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INTRODUCTION

Eucalyptus globulus Hook, the economically important genera belongs to family Myrtaceae

order Myrtales is a fast-growing species native to Australia and widely distributed in southern

China, such as Guangdong, Guangxi, Sichuan and Yunnan. They have been widely

introduced into countries throughout the world because of their rapid growth and the rising

demand for paper and plywood.[1]

It is commonly known as lemon scented tree due to lemon

type smell of aromatic substances in leaves and gum. Eucalyptus oil has great medicinal

values due to its anti-inflammatory, antispasmodic, decongestant and antiseptic properties- In

addition, also has anti-diabetic activity.[2]

Antibacterial and antifungal properties of

Eucalyptus extract and oil are also reported by some authors.[3-6]

Eucalyptus trees might

negatively impact seed germination and growth of native species.[7-8]

It is important to screen

for the lowest concentration and highest concentration effect on seed germination and growth

bioassay further, the productivity of eucalyptus might be enhanced when mixed with native

species under appropriate management.[9]

In the present study, we conducted a pro trays trial

as well as in vitro seed germination and seedling growth bioassay to examine the potential

phytotoxic effects of eucalyptus leaf extracts on tomato species. Our ultimate goal is to

develop suitable strategies for the establishment of the herbal biocontrol safe cheaper

alternative to chemical fungicides from Eucalyptus globulus extracts.

Eucalyptus is an evergreen tree of 24-40 mm in height with tall straight, solid cylindrical

woody and white shining stem. Leaf is petiolated expstipulate simple, lanceolated ovate

smooth aromatic with oil glands. The Inflorescence is umbellate in a cluster of three. The

flower of eucalyptus is pediceillate, complete actinomorphic and epigynous. Placentation is

axile. Seasonality of rainfall is not of critical importance to the species.

The essential oil extracted from the leaves of Eucalyptus globulus Labill is known to be a rich

source of traditional medicines with a variety of biological activities[10]

It is widely used to

treat pulmonary tuberculosis, diabetes[11-12]

, asthma and also used as disinfectant[13-14]

,

antioxidant agent and antiseptic agent especially in the treatment of upper respiratory tract

infections and certain skin diseases[15-16]

asthma. Antibacterial and antifungal properties of

Eucalyptus extract and oil are also reported by some authors.

Vegetables belonging to family Solanaceae are important due to their nutritional as well as

economical values. Tomato is very important solanaceous crops in India either for local

consumption and export.[17]

Tomato is considered as one of the highest nutritional crops


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because of its high contents of vitamin C.[18]

It is susceptible to infection by the blight disease

caused by Alternaria solani during the fruiting period[19-20]

which causes a great reduction in

the quantity and quality of fruit yield. Among the vegetables, tomato ranks next to potato in

world acreage and ranks first among the processing crops. Tomato is grown for its edible

fruits, which can be consumed either fresh or in processed form and is a very good source of

vitamins A, B, C and minerals.

Tomato is a herbaceous sprawling plant growing to 1-3 m in height with a weak woody stem.

The flowers are yellow in colour and the fruits of cultivated varieties vary in size from cherry

tomatoes, about 1–2 cm in size to beefsteak tomatoes, about 10 cm or more in diameter. Most

cultivars produce red fruits when ripe. Tomato is a native to the Peruvian and Mexican

region. Tomatoes are used for soup, salad, pickles, ketchup, puree, sauces and in many other

ways it is also used as a salad vegetable. Tomato has very few competitors in the value

addition chain of processing. The tomato fruit is classified botanically as a berry.

MATERIALS AND METHODS

Collection and Preparation of Plant powder: The healthy, infection free, mature and fresh

leaves of Eucalyptus globulus were used for the phytotoxicity assay. Test plant material

collected from University College of Science campus Mohanlal Sukhadia University

Udaipur. The Plant was identified by subject expert Mr. Amit Kotiya Assistant Professor

Rajasthan University Jaipur, Rajasthan, India and gave the acc.no. RUBL211446 to

Eucalyptus globulus. Plant part was disinfected with 0.1% HgCl2 and subsequently washed

with distilled water. Leaves were shade dried at room temperature and powdered

mechanically. The ground material was passed through sieve No. 240 so as to obtain powder

of mesh size 60, which was used to prepare extract.

Test Seeds: Tomato (Raja-1) seeds were collected from local market of Udaipur.

Extract preparation

Preparation of crude extract: Crude was prepared by cold extraction. It has been done in

alcohol. the cold extract was prepared according to modified method [21]

. 100% alcoholic

extract of leaf was prepared by dissolving 20 g dried and powdered plant material in 100 ml

of alcohol for 24 h. The mixture was then filtered and the supernatant was evaporated under

reduced pressure using a rotary evaporator. The dried residue was used an extract, which was

stored in an airtight jar in the refrigerator.


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Preparation of partially purified extract: Partially purified extract was prepared by hot

extraction method which involves successive extraction with solvents for the separation of

different phytochemical constituents from plant parts[22-23]

Solvent series used for successive

separation was non-polar to polar i.e.

Pet. ether Benzene Chloroform Acetone Methanol Water

This method involves continuous extraction of powdered dried plant material in soxhlet

apparatus with the above series of organic solvents. Extraction with next solvent was done

each time after the plant material was dried in an oven below 50°C.

In this study, only petroleum ether fraction is used for phytotoxicity assay because of their

inhibitory activity against test pathogen. 40gm dry plant powder was kept in soxhlet

extraction unit and extracted with 280 ml petroleum ether solvent till all petrol soluble

fractions were extracted. The extract was vacuum dried in the vacuum evaporator.

Extracts solubility test: Prior to phytotoxicity assay, crude as well as partially purified extract

were screened for the solubility testing in different solvents that is DMSO, DMF, Acetone,

Methanol, Alcohol and water. The solubility of the extracts and solvent in which maximum

extracts dissolved was selected for the further work. Residue remains after solubility of crude

and partially purified extract in respective solvent [24].

The extract showed lower residue had

higher solubility in the solvent. The determination of residual % was done by following

formula-

% residue (A) = (a)/ (b) ×100

[(a) =wt of residue of crude extract, (b) =wt of crude extract]

Study of germination bioassay: The Phytotoxicity of crude extracts as well as partially

purified extracts were assayed with respect to seed germination and seedling growth of

tomato by the Standard Blotter Method [25]

. 60 pre-sterilized seeds were soaked in 5mg/ml,

10mg/ml 15mg/ml 20mg/ml and 25mg/ml respectively for 30 minutes and kept on moistened

whatman no.1filterpaper in sterilizes petri plates. at 27°C.60 seeds were placed in each

labeled petri plate. Sterile distilled water was used at the place of extract for control. A

number of germinated seeds were recorded for different time intervals and radical length was

measured every 24 h up to 9 days. The germination percentage of seeds was calculated by

following formula:

Germination % = Germination seeds / Total Seeds x100


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Growth Bioassay: The growth bioassay of crude extracts as well as partially purified extracts

with respect to seed germination and seedling growth of tomato in pro tray was done by the

modified methods.[26]

5mg/ml, 10mg/ml and 15mg/ml concentration treated tomato seeds

were subjected to plastic chambered tray (27×52) containing a autoclaved soil and water. The

seeded pro tray was kept in a natural sun light. Because these concentrations had not any

toxic effect on tomato seed germination and seedling growth so that these concentrations

were selected for in vivo growth bioassay. Tomato seeds were soaked in their respective

concentration for 30 min. and then washed and sawed in a plastic tray. After 30-35 days small

seedlings came out and their root, shoot, and percent germination were recorded.

RESULTS AND DISCUSSION

The crude as well as partially purified extract was prepared by standard methods. Prior to

phytotoxicity assay, crude as well as partially purified extract was screened for the solubility

testing in different solvents that is DMSO, DMF, Acetone, Methanol, Alcohol and water.

Results of solubility testing suggest that DMF, DMSO and acetone showed 100%solubility of

both extracts. In vitro Phytotoxicity of crude extract as well as partially purified extracts were

assayed with respect to seed germination and seedling growth of tomato by the Standard

Blotter Method.

100% alcoholic crude as well as petroleum ether extract of Eucalyptus globulus leaves

showed a phytotoxic effect on the germination and growth bioassay of tomato seed. 5mg/ml,

10mg/ml 15 mg/ml, 20 mg/ml and 25 mg/ml concentrations were used for this assay. Results

showed that 20mg/ml and 25mg/ml of both types of extracts had a strong toxic effect on

tomato seed germination as well as seedling growth in comparison to control and other

concentrations. (table1 &2).The estimated maximum final germination time of tomato seeds

was 216 h in both types of extracts at a concentration of 20 mg/ml and 25 mg/ml and there

was a linear reduction in germinability. In 100% alcoholic crude extract % germination at 20

mg/ml and 25 mg/ml was 60% and 35% respectively and in petroleum ether extract %

germination at 20mg/ml and 25 mg/ml was 55% and 30% respectively( table 3&4). 5mg/ml,

10mg/ml and 15 mg/ml concentrations did not show any kind of phytotoxicity on tomato

seeds so that all these three concentrations were further used for In vivo plastic chambered

tray for testing the germination and growth ability of extracts on tomato under natural

conditions. The plantlet length, root length, shoot length and no. of leaves were calculated in

In vivo experiment. It is compared by control. (table 5-11&fig1).


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The results presented in this paper showed that E.globulus leaves contain phytotoxic

compounds that inhibit germination and growth of tomato thus showing a good allelopathic

potential. 100% alcoholic extract was lesser phytotoxic than the petroleum ether extracts, so

alcohol is a better solvent for extraction. As extraction method, classic reflux proved to be

better than cold extraction. We found that 25% concentration of extracts inhibited more than

50% of germination of tomato seeds and also the growth of seedling root and shoot.

Further studies are needed to determine the phytotoxic effects of E. globulus extracts in a

natural environment (field work) too, with the final purpose of application of our results in

agriculture. The results may also use to develop a strategy for using E.globulus plants as

sources of potential herbicides and herbal fungicides instead of destroying them.


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Table 1: In vitroPhytotoxicity assay of Crude extract of Eucalyptus globulus on tomato seed germination at various concentrations

Germination

period (h)

Mean length of seedlings(cm)±SD

Control 5mg/ml 10mg/ml 15mg/ml 20mg/ml 25 mg/ml

Radical Plumule Radical Plumule Radical Plumule Radical Plumule Radical Plumule Radical Plumule

24 - - - - - - - - - - - -

48 - - - - - - - - - - - -

72 1.50±0.04 - 1.42±0.02 - 1.00±0.01 - 0.30±0.01 - 1.00±0.01 - 0.10±0.1 -

96 1.66±0.02 0.20±0.01 1.50±0.01 0.54±0.02 1.18±0.02 0.33±0.01 0.45±0.02 0.42±0.01 1.15±0.01 0.55±0.02 1.00±0.01 0.21±0.01

120 2.08±0.01 0.25±0.02 1.78±0.02 0.87±0.02 1.33±0.02 0.67±0.01 0.59±0.01 0.52±0.01 1.25±0.01 0.60±0.02 1.02±0.02 0.25±0.02

144 2.41±0.03 0.32±0.02 2.50±0.01 1.09±0.01 1.87±0.02 0.92±0.02 1.00±0.01 0.62±0.02 1.38±0.02 073±0.02 1.05±0.01 0.32±0.01

168 2.67±0.01 0.41±0.03 2.39±0.01 1.15±0.01 2.50±0.01 1.08±0.02 2.32±0.03 0.71±0.02 1.46±0.02 0.81±0.01 1.09±0.02 0.35±0.01

192 3.33±0.02 0.53±0.02 3.52±0.01 1.55±0.01 3.20±0.02 1.13±0.01 2.55±0.02 0.82±0.01 1.69±0.03 0.89±0.01 1.13±0.01 0.38±0.02

216 5.42±0.03 0.73±0.02 4.20±0.02 1.68±0.02 3.89±0.02 1.15±0.01 3.00±0.02 1.00±0.01 2.02±0.02 0.91±0.01 1.17±0.02 0.46±0.03

Table 2: In vitro Phytotoxicity assay of Petroleum ether extract of Eucalyptus globulus leaves on tomato seed germination at various

concentration

Germination

period(h)

Mean length of seedlings(cm)±SD

control 5mg/ml 10mg/ml 15mg/ml 20mg/ml 25 mg/ml

Radical Plumule Radical Plumule Radical Plumule Radical Plumule Radical Plumule Radical Plumule

24 - - - - - - - - - - - -

48 - - - - - - - - - - - -

72 1.50±0.03 - 1.30±0.01 - 1.04±0.01 - 1.25±0.02 - 1.25±0.01 - 0.55±0.01 -

96 1.66±0.01 0.20±0.01 1.35±0.02 0.19±0.01 1.43±0.02 0.25±0.01 1.400.02± 0.10±0.01 1.32±0.01 0.09±0.01 0.60±0.01 0.02±0.01

120 2.08±0.01 0.25±0.02 1.85±0.01 0.22±0.01 1.67±0.02 0.30±0.01 1.60±0.01 0.15±0.01 1.40±0.01 0.05±0.01 0.75±0001 0.08±0.02

144 2.41±0.02 0.32±0.02 1.95±0.01 0.30±0.01 1.98±0.01 0.42±0.02 1.75±0.01 0.17±0.01 1.58±0.01 0.10±0.01 0.89±0.02 0.10±0.01

168 2.67±0.02 0.41±0.01 3.50±0.01 0.35±0.03 3.18±0.01 0.45±0.01 1.98±0.01 0.19±0.01 1.72±0.02 0.15±0.01 0.98±0.01 0.12±0.01

192 3.33±0.02 0.53±0.02 4.50±0.01 0.49±0.01 4.17±0.01 0.50±0.01 2.05±0.01 0.22±0.01 1.82±0.02 0.18±0.01 1.10±0.01 0.18±0.01

216 5.42±0.01 0.73±0.01 5.30±0.03 0.58±0.2 5.20±0.01 0.55±0.02 2.15±0.01 0.25±0.03 1.89±0.01 0.20±0.01 1.12±0.01 0.19±0.01


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Table 3: In vitro %Germination of tomato seeds treated with Crude extract of

Eucalyptus globulus laves.

Germination

Period (h)

Total no. of seeds

incubated

% Germination

Control 5

mg/ml

10

mg/ml

15

mg/ml

20

mg/ml

25

mg/ml

24 60 - - - - - -

48 60 - - - - - -

72 60 52.3 53.4 51.3 50.2 25 10.21

96 60 65.0 62.1 60.12 54.3 30.9 15.25

120 60 76.0 73.2 78.12 60.9 38.8 18.1

144 60 87.3 80.00 80 63.8 43.9 22.5

168 60 95.3 94.0 80.66 68.9 50.8 26.2

192 60 100 100 95 72.3 53.2 30

216 60 100 100 100 78.80 60 35

Table 4: In vitro %Germination of tomato seeds treated with partially purified extract

of Eucalyptus globulus laves.

Germination

Period (h)

Total no. of seeds

incubated

% Germination

Control 5

mg/ml

10

mg/ml

15

mg/ml

20

mg/ml

25

mg/ml

24 60 - - - - - -

48 60 - - - - - -

72 60 52.3 60.00 50.00 42.00 29.00 15.00

96 60 65.0 65.0 55.12 48.21 30.1 20.00

120 60 76.0 70.00 60.30 52.20 45.21 22.15

144 60 87.3 80.51 62.10 58.11 48.13 26.29

168 60 95.3 85.21 75.20 60.65 50.19 28.29

192 60 100 95.17 80.90 65.00 52.00 29.89

216 60 100 100 100 75.00 55.00 30.00

Table 5: Effect of crude extract at 5mg/ml concentration.

Plant per

cavity

Various parameters of tomato growth bioassay

Total length of

tomato (cm)

Length of shoot

(cm)

Length of root

(cm)

No. of leaves

per plant

1. R1 10.5 5.5 3 15

R2 8 4 3 9

R3 7 3 1 9

2. R1 6 4 1 8

R2 6 3.5 2 9

R3 6.5 4 1.5 12

3. R1 7 4.5 1.5 8

R2 9.5 5.5 4 15

R3 5 3 2 11

4. R1 9 5 4 13

R2 9 5 4 14

R3 4 2 1 7

5. R1 6 5 1 15

R2 3 6 1 4

R3 9 5 3 11


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Plant per

cavity


Total length of tomato

(cm)

Length of shoot

(cm)

Length of root

(cm)

No. of leaves

per plant

1. R1 7 5 2 11

R2 3 2 1.8 8

R3 2 1.5 1 2

2. R1 7.5 5.5 2 14

R2 9.5 5 4 19

R3 4 1.5 2 2

3. R1 10 5 4 13

R2 12 6 4 10

R3 9 5 3 11

4. R1 12 5 4 16

R2 13 6 5 12

R3 12 5 3 12

5. R1 15 5 6 14

R2 8 2 4 7

R3 10 5 4 9


Plant per

cavity



(cm)

Length of shoot

(cm)

Length of root

(cm)

No. of leaves

per plant

1. R1 10 6 2 13

R2 13 5 5 13

R3 8 4 3 9

2. R1 6 2 2 5

R2 8 5 1 11

R3 7 4 2 9

3. R1 12 5.5 3 12

R2 9 5 2 13

R3 10 4 3 12

4. R1 11 6 2.5 12

R2 10 5 4 12

R3 10 5 3 10

5. R1 11 5.5 2.5 13

R2 8 5 2 11

R3 8 6.5 2 6


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Table 8: Effect of partially purified extract (PE) at 5mg/ml concentration.

Plant per

cavity



(cm)

Length of shoot

(cm)

Length of root

(cm)

No. of leaves

per plant

1. R1 7.5 4.5 1 11

R2 8 4 1 11

R3 9 4 2.5 10

2. R1 9 4.5 2 10

R2 12 6 3 14

R3 8 4 2.5 11

3. R1 10 6 1 15

R2 9 3.9 2 12

R3 9 4 2 13

4. R1 13 6 3 17

R2 10 5 2 14

R3 6 2 1 8

5. R1 13 6 2 19

R2 15 8 2.5 20

R3 13 9 2.5 19


Plant per

cavity



(cm)

Length of shoot

(cm)

Length of root

(cm)

No. of leaves

per plant

1. R1 10 5.8 2 12

R2 11 5.5 1 17

R3 9 5 2 12

2. R1 13 6 3 19

R2 6.5 3 2 8

R3 6 3 2 7

3. R1 16 8 3 20

R2 13 6 3 14

R3 9 4 2 10

4. R1 14 8 3 14

R2 12 8 2.5 16

R3 10 6 3.5 15

5. R1 8 3 1.5 12

R2 9 4 2 10

R3 9 4 2 10


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Plant per

cavity



(cm)

Length of shoot

(cm)

Length of root

(cm)

No. of leaves

per plant

1. R1 8 3.5 1 9

R2 5 2 1 8

R3 8.5 3 2 10

2. R1 5 2 1 3

R2 12 6 2 18

R3 15 8 3 19

3. R1 13 7 2.5 24

R2 10 5 2 13

R3 9.5 4.8 2 10

4. R1 14 6.5 3 18

R2 9 4.5 1 12

R3 10 5 2 15

5. R1 13 6 2.5 15

R2 10 5.8 1.5 12

R3 11 5 2 13

Table 11: Control.

Plant per

cavity


Total length of

tomato (cm)

Length of shoot

(cm)

Length of root

(cm)

No. of leaves

per plant

1. R1 10 6 2 16

R2 11 5.5 2 16

R3 11 4 3 15

2. R1 11 5 2 12

R2 8 3 1 15

R3 9 4 1 12

3. R1 12 5 3 12

R2 11 5 2 15

R3 12 4 1 12

4. R1 11 5 2 15

R2 12 5 2 12

R3 12 3 14

5. R1 13 5 2 16

R2 12 5 2 15

R3 12 4 3 14


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In vivo in pro trays

CONCLUSION

The phytotoxicity of the E.globulus leaf extracts with different concentration was studied.

Phytotoxicity tests were performed to evaluate the tomato seeds germination using standard

blotter method in petri plates and in In vivo using pro trays.

It is found that the phytotoxicity of the studied plant extracts, measured through seeds

germination rate and root length are, as follows:

5 mg/ml> 10mg/ml > 15mg/ml > 20mg/ml>25mg/ml

Taking into account the concentration of the leaf extract,5mg/ml and 10 mg/ml has no direct

impact on seed germination and seedling growth that could be found and 20 mg/ml and 25

mg/ml has highly negative impact on tomato seed physiology, however it is a clear

conclusion that it is a direct correlation of phytotoxicity with the content of the crude extract

and petroleum ether extract, higher concentration is being more toxics than the control.


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ACKNOWLEDGMENT

One of the authors (Surbhi Mehta) is thankful to University Grant Commission (BSR), New

Delhi for providing financial assistance in the form of SRF.

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