Botanicals to Control Post-Harvest Decay of Colocasia

(Colocasia esculenta L.) Tubers in Odisha, India

 

Akhtari Khatoon¹, Ashirbad Mohapatra², Kunja Bihari Satapathy^1*

¹P. G. Department of Botany, Utkal University, Vani Vihar, Bhubaneswar-751004, Odisha, India

²Sri Jayadev College of Education and Technology, Naharkanta, Bhubaneswar-752101, Odisha, India

 

ABSTRACT:

The objective of the present work is to evaluate the in-vitro antifungal activity of petroleum ether and methonolic leaf extracts from eight different plant species against Colocasia esculenta L. post-harvest decay fungi including Aspergillus flavus, Aspergillus niger, Geotrichum candidum, Rhizopus oryzae and Penicillium sp. Previously, these fungi were isolated from the rotten colocasia tubers collected from different market places of some districts of Odisha, India. In-vitro antifungal efficacy of petroleum ether and methonolic leaf extracts of Abutilon indicum (L.) Sweet, Ageratum conyzoides L., Alstonia scholaris (L.) R.Br., Artocarpus heterophyllus Lam., Averrhoa carambola L., Cassia fistula L., Centella asiatica Urban. and Dillenia indica L. were tested against five test fungi by poison food technique. The efficacy of plant extracts was compared with four commercial fungicides such as Blitox-50, Dhanustin, Indofil and Macozeb. The result revealed that the plant extracts were more effective against the five pathogenic test fungi as compared to the four commercial fungicides. The results on the efficacy of test botanicals to control the growth of test fungi indicated that the petroleum ether extract of A. heterophyllus was most effective against A. flavus, petroleum ether extract of A. conyzoides against A. niger, petroleum ether extract of Ageratum conyzoides against G. candidum, methanolic extract of Artocarpus heterophyllus against R. oryzae, petroleum ether extract of Averrhoa carambola against Penicillium sp.

 

 

 

INTRODUCTION:

Colocasia esculenta L. is used as a staple food or subsistence food by millions of people in Asia, Africa and Central America like developing countries. It has much importance in ensuring food security. Nutritionally, C. esculenta contains 11% protein, 85-87% starch and other nutrients such as minerals, Vitamin C, thiamin, riboflavin and niacin better than other cereals¹. Nowadays, C. esculenta is considered as the fifth most consumed root vegetable throughout the world. Colocasia esculenta L. is an emergent, perennial, herbaceous species belonging to the family Araceae. As a root vegetable, colocasia plant is grown primarily for its edible corms².

 

The post-harvest loss of root and tubers has been of serious problem to farmers and it is a warning against food security³. Colonization of the tubers by fungi will lead to reduction in consumption materials and reduces market value⁴.

 

To control various pests and pathogenic microorganisms of crop plants, chemical pesticides are used in indiscriminate way that cause health hazard in both terrestrial and aquatic lives through their residual toxicity^5,6. To avoid use of synthetic fungicides, it is imperative to find alternative sources from naturally occurring compounds that are easily biodegradable and of low mammalian toxicity for a safe control of fungal pathogens⁷. Green plants are the best alternative of it because there is huge reservoir of green plants and it has various effective chemotherapeutics so it could serve as an environmentally friendly natural alternative to these toxic chemical pesticides⁸. Plants are the source of natural pesticides that make excellent leads for new pesticide developments^9,10. During the recent decades, many herbal extracts collected from plant extracts have been extensively tested to control the animal and plant diseases^11,12,13. Many medicinal plants have been reported to have antimicrobial properties to control plant disease¹³.

 

Considering the adverse effect of synthetic pesticides on environment and natural habitats, the present study was undertaken to evaluate the in-vitro antifungal activity of alternative and nontoxic medicinal plants against the fungi responsible for post-harvest storage rots of colocasia tubers in Odisha, India.

 

MATERIALS AND METHODOLOGY:

Collection and identification of plant material:

The leaves of Abutilon indicum (L.) Sweet (Malvaceae), Ageratum conyzoides L. (Asteraceae), Alstonia scholaris (L.) R.Br. (Apocynaceae), Artocarpus heterophyllus Lam. (Moraceae), Averrhoa carambola L. (Averrhoaceae), Cassia fistula L. (Caealpiniaceae), Centella asiatica Urban. (Apiaceae) and Dillenia indica L. (Dilleniaceae) were collected from the “Chandaka reserve forest” area near Bhubaneswar, Odisha in the month of March, 2015. Identification of the voucher specimen was done by available literature^14,15. The voucher specimens were deposited in the herbarium of Post Graduate Department of Botany, Utkal University, Vani Vihar, Bhubaneswar. The leaves were collected in bulk amount, washed in running tap water, dried under shade and made to coarse powder form.

 

Processing of plant material and preparation of extract:

The collected leaves were shade dried and ground to form coarse powder and had been successively extracted with the solvent petroleum ether and methanol by Soxhlet apparatus¹⁶ and the extract was recovered under reduced pressure in a rotatory evaporator. The extracts were kept in desiccators for further use.

 

In-vitro evaluation of antifungal efficacy of plant extracts:

For the evaluation of antifungal effect of petroleum ether and methanolic leaf extracts, the leaf extracts were diluted with dimethyl sulfoxide (DMSO) @ 20mg/ml. Then antifungal activities of those extracts were carried out based on the method of Satish et al. (2007) with modification. For sample treatment, 1 ml of diluted plant extracts plus 19 ml of Potato Dextrode Agar (PDA) were poured into each petri plate, mixed thoroughly and allowed to solidify. The concentration of plant extract on PDA medium was 1mg/ml. PDA medium without the plant extracts was served as control. Disc of 0.5 cm culture of the test fungi was placed at the center of the petri dish based on poison food technique (both sample and control) and incubated at 28 ± 1 ºC inside incubator for respective days of their growth of 8 cm diameter on petriplates such as Rhizopus oryzae was incubated for 1 day, A. flavus for 7 days,  A. niger for 8 days, G. candidum for 11 days, and Penicillium sp. for 15 days. The efficacy of each plant extract was evaluated by measuring fungal radial growth (cm) using ruler. The antifungal activity in terms of percentage inhibition was calculated by using formula as below:

 

% Inhibition = (X-Y)/X x 100

 

Where

X= average increase in mycelial growth in control,

Y= average increase in mycelial growth in treatment¹⁷.

 

Comparision of the efficacy of selected fungicides and plant extracts:

A separate experiment was conducted in order to compare the relative efficacy of four fungicides, viz., Dhanustin, Macozeb, Blitox-50, Indofil and 32 extracts (petroleum ether and methanolic extracts) of sixteen selected plant. The concentration of fungicides was 0.05 mg/ml. The required quantity of chemicals (0.5 %) and plant extracts (5%) were incorporated after sterilization of  PDA medium, and were inoculated by six test fungal isolates in four replicates and incubated for respective days of their growth as described earlier. At the end of incubation period, the colony diameter of each fungi chemical/ plant extract combination was measured, transformed to percentage of mycelia inhibition as per the method described earlier.

 

RESULTS:

In-vitro antifungal activity of medicinal plants:

In-vitro antifungal activity of sixteen medicinal plants against six plant pathogenic fungi revealed that almost all the test plants were effective to inhibit the mycelial growth of the isolated pathogenic fungi. The percentage of mycelial growth inhibition ranged from 24.08±2.25% (petroleum ether extract of Dillenia indica against Aspergillus niger) to 92.57±1.95 % (methanolic extract of Artocarpus heterophyllus). The petroleum ether leaf extract of Artocarpus heterophyllus was most effective (70.55±1.73%) to inhibit the mycelial growth of A. flavus followed by petroleum ether extract of Ageratum conyzoides (62.90± 0.29 %). The petroleum ether extract of Ageratum conyzoides was found to be more effective against A. niger than other test extracts. It controlled the mycelial growth of A. niger by 62.54±2.04 %. All the test plant’s extract were remarkly effective (60.66±2.49 % to 89.03±0.98 %) against the mycelial growth of G. candidum except petroleum ether extract of Dillenia indica (55.25±3.43 %) and petroleum ether and methanolic extracts of Abutilon indicum (56.08 ± 0.82 % and 55.19±1.38 % respectively). All the test botanicals were found to be severely efficient against Rhizopus oryzae. The inhibition of Rhizopus oryzae ranged from 72.53±2.74 %  to  92.57±1.95 %. The mycelial growth of Penicillium sp. was severely inhibited by both petroleum ether and methanolic extracts of Averrhoa carambola (75.20±1.28% to 73.08±1.27% respectively). (Table 1 and Figure 1-5).

 

Table 1: In-vitro antifungal activity of botanicals

Sl. No.	TEST PLANTS	PERCENTAGE OF INHIBITION OF MYCELIAL GROWTH
			Aspergillus flavus	Aspergillus niger	Geotrichum candidum	Rhizopus oryzae	Penicillium sp.
1	Abutilon indicum	A	46.08 ± 0.82	30.11 ± 0.4	56.08 ± 0.82	77.16 ± 0.23	33.46 ± 0.7
		B	56.45 ± 0.48	35.07 ± 0.28	55.19 ± 1.38	75.25 ± 0.69	43.58 ± 0.42
2	Ageratum conyzoides	A	62.90 ± 0.29	62.54 ± 2.04	89.03 ± 0.98	88.97 ± 1.74	70.29 ±1.39
		B	56.95 ± 1.18	46.51 ±  1.09	79.08 ±1.12	88.88 ± 1.03	58.72 ± 1.8
3	Alstonia scholaris	A	54.26 ± 1.42	34.32 ± 0.63	73.41 ± 3.24	86.29 ± 1.18	51.66 ± 1.69
		B	45.66 ± 2.49	54.72 ± 1.71	80.14 ± 1.79	89.14 ± 1.13	59.19 ± 0.92
4	Artocarpus heterophyllus	A	70.55 ± 1.73	36.38 ± 1.23	77.16 ± 1.64	89.13 ± 1.39	51.26 ± 0.89
		B	48.05 ± 1.53	42.57 ± 1.94	82.5 ± 2.04	92.57 ± 1.95	56.75 ± 0.88
5	Averrhoa carambola	A	57.39 ± 0.81	27.8 ± 0.21	77.75 ± 1.13	87.94 ± 1.08	75.20 ± 1.28
		B	54.57 ± 1.5	45.47 ± 1.31	70.46 ± 1.45	89.7 ± 2.70	73.08 ± 1.27
6	Cassia fistula	A	51.29 ± 3.44	47.24 ± 4.82	76.4 ± 2.72	85.47 ± 1.63	40.31 ± 3.21
		B	45.75 ± 2.87	38.22 ± 2.32	72.57 ± 2.8	86.05 ± 1.59	53.14 ± 2.85
7	Centella asiatica	A	56.25 ± 2.7	39.75 ± 2.76	60.66 ± 2.49	80.33 ± 2.05	25 ± 2.44
		B	45.76 ± 2.66	33.4 ± 2.27	63.5 ± 3.34	72.53 ± 2.74	43.33 ± 3.39
8	Dillenia indica	A	53.66 ± 3.29	24.08 ± 2.25	55.25 ± 3.43	75.08 ± 3.76	65.6 ± 2.21
		B	43.5 ± 3.08	51.93 ± 3.61	81.33 ± 4.98	83.13 ± 2.84	54.1 ± 2.88

Results expressed as mean ± S.D. of three determinations

A = Petroleum ether extract, B = Methanol extract

 

 

Fig 1: In-vitro antifungal activity of sixteen plants against Aspergillus flavus

 

 

Fig 2: In-vitro antifungal activity of sixteen plants against Aspergillus niger

 

Fig 3: In-vitro antifungal activity of sixteen plants against Geotrichum candidum

 

 

Fig 4: In-vitro antifungal activity of sixteen plants against Rhizopus oryzae

 

 

Fig 5: In-vitro antifungal activity of sixteen plants against Penicillium sp.

In-vitro antifungal activity of test fungicides:

Four fungicides were tested against the 5 isolated plant pathogenic fungi namely Blitox-50, Dhanustin, Indofil and Macozeb. The growth of A. flavus was completely inhibited by Dhanustin (100 %) followed by Macozeb (82.5 ± 2.04 %), A. niger by Dhanustin (81.41 ± 1.23 %), Geotrichum candium by Macozeb (61.05 ± 1.59 %), Penicillium sp. by Macozeb (72.57 ± 2.13 %) followed by Dhanustin (68.58 ± 1.31 %) while the growth of Rhizopus oryzae was moderately inhibited by Macozeb (37.16 ± 0.95 %), Indofil (37.16 ± 1.64 %) and Dhanustin (6.48 ± 1.23 %) but Blitox-50 had no effect

on it. (Table 2 and Figure 6)

 

Table 2. In-vitro antifungal activity of four commercial fungicides against five test fungi

TEST FUNGICIDES	PERCENTAGE OF INHIBITION OF MYCELIAL GROWTH
	Blitox-50	Dhanustin	Indofil	Macozeb
Aspergillus flavus	21.74 ± 1.33	100	19.63 ± 2.01	82.5 ± 2.04
Aspergillus niger	17.66 ± 1.05	81.41 ± 1.23	22.75 ± 1.96	18.11 ± 1.85
Geotrichum candidum	17.09 ± 2.37	31.41 ± 1.23	31.41 ± 1.23	61.05 ± 1.59
Rhizopus oryzae	0	6.48 ± 1.23	31.75 ± 1.67	37.16 ± 1.64
Penicillium sp.	12.5 ± 1.22	68.58 ± 1.31	19.25 ± 0.54	72.57 ± 2.13

Results expressed as mean ± S.D. of three determinations

 

 

Fig 6: In-vitro antifungal activity of four commercial fungicides against the five test fungi

 

Comparative efficacy of test plant extracts and fungicides:

The comparative result of plant extracts (Table 1) and test fungicides (Table 2) revealed that plant extracts are more efficient to inhibit the mycelial growth of all the isolated fungus. For inhibiting the mycelial growth of A. flavus and A. niger, Dhanustin is more efficient than the test plant extracts but rest of the test fungicides were found to be less effective as compared to all test plant extracts. For the inhibition of mycelial growth of G. candidum, Blitox-50, Dhanutin and Indofil were found to be less efficient than all the test plant extracts. Against Rhizopus oryzae, all the plant extracts were found to be much more efficient than the test fungicides. Antifungal activity of Averrhoa carambola and Macozeb was found to be more effective against the mycelial growth of Penicillium sp. than other plant extracts and Dhanustin but all those rest plant extracts were showed comparatively more activity than Indofil and Blitox-50.

 

DISCUSSION:

From the present investigation it was found that plant extracts were more efficient to inhibit the mycelial growth of test isolated fungi than the synthetic fungicides.  The use of herbal extracts to control plant diseases is an environment friendly approach and it is an effective alternative to toxic chemical pesticides. Plant extracts of many higher plants have been reported to exhibit antibacterial, antifungal and insecticidal properties under in-vitro study^{18,19,20,21,22,23}. The use of synthetic fungicides is not only providing potential danger to both the farmer and environment²⁴, but also it is unaffordable by most farmers. From a recent study it is clear that both crude extracts and purified compounds isolated from plants can effectively be used as natural fungicides for the management of plant diseases.

 

 

 

 

CONCLUION:

In this study, the effects of plant extracts and their efficiencies against fungal storage rot of colocasia were evaluated in vitro, and quite satisfactory results were obtained with leaf extracts of all plants. The inhibitory activity of plant extracts was most likely due to antimicrobial components present in plant extracts. However, the exact chemical compounds and their controlling mechanism to the fungal storage rots need to be elucidated. Since leaves of all those plants are readily available without any cost. Thus the use of plant leaf extracts should not have any phytotoxic effects on other plants.

 

ACKNOWLEDGEMENT:

The authors are thankful to the Head, Post Graduate Department of Botany, Utkal University, Bhubaneswar, Odisha for providing necessary laboratory facilities to conduct the study. The financial assistance received from the University Grants Commission, Government of India, New Delhi in the form of Maulana Azad National Fellowship to the first author is deeply acknowledged.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 20.12.2017           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(5):1804-1809.