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Antifungal activity of herbal extracts

against plant pathogenic fungi
a a
Manasi K. Bhagwat & Ajit G. Datar
a
Guru Nanak Institute for Research and Development
(G.N.I.R.D.) , G. N. Khalsa College , Mumbai , India
Published online: 16 Aug 2013.

To cite this article: Manasi K. Bhagwat & Ajit G. Datar , Archives Of Phytopathology And Plant
Protection (2013): Antifungal activity of herbal extracts against plant pathogenic fungi, Archives Of
Phytopathology And Plant Protection, DOI: 10.1080/03235408.2013.826857

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 Archives of Phytopathology and Plant Protection, 2013
http://dx.doi.org/10.1080/03235408.2013.826857

RESEARCH ARTICLE
Antifungal activity of herbal extracts against plant pathogenic fungi
Manasi K. Bhagwat* and Ajit G. Datar

Guru Nanak Institute for Research and Development (G.N.I.R.D.), G. N. Khalsa College,
Mumbai, India
(Received 15 July 2013; final version received 16 July 2013)
Downloaded by [University of Central Florida] at 09:21 24 September 2013

The present study was designed to evaluate in vitro antifungal activity of herbal
extracts against three plant pathogenic fungi (viz. Rhizopus stolonifer, Botrytis
cinerea and Colletotrichum coccodes). Extracts of leaves and rinds of Garcinia
indica, rhizomes of Curcuma aromatica, roots of Glycyrrhiza gahliae, leaves of Nyc-
tanthes arbour-tristis and seeds of Vernonia anthelmintica were used for screening.
Screening was done using poisoned food technique. Relatively potent extracts were
shortlisted from this study and were further assayed to find out their minimum fungi-
cidal concentration (MFC). From the above studies, it was observed that ethyl ace-
tate extract of rhizomes of C. aromatica and unripe fruit rinds of G. indica have
shown the lowest MFC values amongst the other tested plant extracts. This study
indicates that the potential of these plant extracts in the management of diseases
caused by plant pathogenic fungi.
Keywords: plant pathogenic fungi; poisoned food technique; MFC

1. Introduction
Globally, enormous losses of the crops are caused by the plant diseases. Plant diseases
are caused by pathogens (Singh 2007). Plant pathogens are considered as plant pests.
Pests are one of the serious problems faced by agricultural sector. Insect pests, plant
pathogens and weeds can be collectively termed as Pests. They represent a major con-
strain to crop production (Juroszek & Tiedemann 2013). Different kinds of organisms
can cause plant diseases, including, bacteria, fungi, viruses, nematodes, parasitic plants,
etc. Agricultural production incurs substantial yearly losses because of plant diseases.
Pathogenic fungi are the main infectious agents in plants. In the case of fruits and
vegetables, there is a wide variety of fungal genera causing quality problems related to
nutritional value, organoleptic characteristics, etc. (Dellavalle et al. 2011). Infection by
fungi and bacteria may occur, during growing season, at harvest time, during handling,
storage, transport and marketing or even after purchase by the consumer. Fungi are
more commonly found attacking fruits. In this study, the focus is on the problems
caused by three fungi, Rhizopus stolonifer, Colletotrichum coccodes and Botrytis
cinerea. The soft rot on the vegetables, fruits and ornamentals caused by Rhizopus spp.
occurs throughout the world (Kwon et al. 2001). R. stolonifer has a large host range.
Many fruits and vegetables are susceptible to this pathogen. Head rotting disease in
sunflower due to Rhizopus causes dramatic yield reductions in the number of seeds and
yield in sunflower (Yildirim et al. 2010). Rhizopus soft rot is one of the most costly

*Corresponding author. Email: khalsagnirdm@gmail.com

Ó 2013 Taylor & Francis

 2 M.K. Bhagwat and A.G. Datar

postharvest diseases of sweet potatoes. Integrated practices designed to minimise sweet

potato losses to farmers and marketers are recommended (Nelson 2009). Anthracnose
because of C. coccodes is common fungal disease on ripe tomato fruit in field and
during storage. The disease can cause significant losses in yield and marketability
(Yonghao 2013). Black dot is a tuber blemish and foliar disease of potato caused by the
fungus C. coccodes. It has also been demonstrated that potato early dying characterised
by stunting, wilting, premature senescence and reduced yields is associated with
complexes of soilborne parasites including Verticillium gahliae and C. coccodes (Lees
& Hilton 2003). Anthracnose of pepper (Capsicum annuum L.) caused by C. coccodes
is one of the most destructive diseases in pepper. C. coccodes has a wide host range
that includes at least 58 species in 17 families, primarily in Leguminosae, Solanaceae,
and Cucurbitaceae (Hong & Hwang 1998). B. cinerea is one of the world’s most impor-
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tant fungal pathogens, being reported on more than 230 host plants including economi-
cally important agricultural and horticultural crops. In grape, the disease frequently
occurs on ripe berries close to harvest. Grey mould due to B. cinerea lead to financial
losses for the growers, reducing not only yield but also grape quality (Viret et al.
2004). Grey mould is also a major cause of postharvest losses of strawberry fruits dur-
ing storage, transportation or shipment. The disease can cause important fruit losses on
strawberry plants before or after harvest worldwide and it is estimated that they can
cause yield losses up to 25% (Donmez et al. 2011). At present, growers often rely heav-
ily on chemical fertilisers and pesticides for improvement in crop productivity and qual-
ity (Pal & McSpadden Gardener 2006). In recent years, a large number of synthetic
pesticides have been banned in the western world because of their high and acute
toxicity, long degradation periods, accumulation in the food chain, etc. (Satish et al.
1999). However, the environmental pollution caused by excessive use and misuse of
agrochemicals has led to considerable changes in people’s attitudes towards the use of
pesticides in agriculture. The need for effective and safe alternative has increased. The
natural plant products derived from plants effectively meet this criterion (Guleria &
Tiku 2009). In recent years, a lot of interest has been developed in the antimicrobial
effects of medicinal plants for plant disease control. In view of above reports present
study has been undertaken to evaluate antifungal activity of herbal extracts against three
fungal plant pathogens viz. R. stolonifer, C. coccodes and B. cinerea.

2. Materials and methods

2.1. Collection and authentication of plants
Curcuma aromatica (Ranhalad/Wild turmeric) dried rhizomes (The wealth of India
2010, Vol. 2), Glycyrrhiza glabra (Jeshthmadh) dried roots (Bently & Trimen 1991; The
wealth of India 2006, Vol. 1; The wealth of India 2009a, Vol. 4; Quality standards of
Indian Medicinal plants 2011, Vol. 9) and Vernonia anthelmintica (Kalijiri) dried seeds
(Kapoor 1990; The wealth of India 2010, Vol. 5) were purchased from the local market
(Thane district in Maharashtra, India). The leaves and unripe fruit rinds of Garcinia
indica (Kokam) (Quality standards of Indian Medicinal plants 2008, 7) and the leaves of
Nyctanthes arbour-tristis (Parijat) (Database on Medicinal plants used in Ayurveda
2002; The wealth of India 2009b, Vol. 4) were collected from the organic farm (District
Thane, Maharashtra, India), and then shade dried. C. aromatica rhizomes, G. glabra
roots and V. anthelmintica seeds were authenticated from Agharkar research institute
Pune, India. G. indica and N. arbour-tristis herbariums were authenticated from Botanical
survey of India, Pune, India. The selected plants are easily and widely available.
 Archives of Phytopathology and Plant Protection 3

2.2. Extraction and reconstitution of herbal extracts

All the six plant parts mentioned above were powdered using mixer grinder and sieved
through 40 mesh. Fifteen grams of each powder were extracted separately in 150 cm3 of
Hexane, Dichloromethane (DCM), Ethyl acetate (EtOAc) and Ethanol by soxhlet extrac-
tion. Extraction was done for about 18–24 h. Organic solvents were evaporated on water
bath and all dried extracts were reconstituted using dimethylsulfoxide (DMSO) to achieve
the final extract concentration as 5% w/v. All the extracts were then used for screening
their antifungal activity against the three fungi. Glacial acetic acid (GAA) 5% v/v pre-
pared in distilled water was used as positive control. There are references on antifungal
activity of acetic acid. Short-chain organic acids, such as acetic and propionic acid, are
commonly used by food manufacturers as antimicrobial preservatives or acidulants in a
variety of food products. Acetic, formic and propionic acids significantly reduced
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decay in eight cherry cultivars inoculated with spores of M. fructicola, P. expansum and
R. stolonifer (Sholberg 1998).

2.3. Test fungi

All the three plant pathogenic fungi namely, R. stolonifer Microbial Type Culture
Collection and Gene Bank (MTCC 7370), B. cinerea MTCC 2104 and C. coccodes
MTCC 2076 were obtained from The MTCC, Chandigarh, India.

2.4. Poisoned food technique

The antifungal activity of plant extracts was evaluated against three plant pathogenic
fungi using poisoned food technique (Amadioha 2000). Five hundred microliters of
each herbal extract reconstituted in DMSO was spread evenly onto solidified potato
dextrose agar (PDA) plates using a sterile glass spreader. A mycelial disc of 8 mm
diameter, cut out from 5- to 7-day old fungal culture was aseptically positioned onto
the centre of the PDA plates containing plant extracts. The PDA plates spread with
500 μL DMSO were used as negative control and plates spread with 500 μL 5% v/v
GAA were considered as positive control. The negative control and positive control
plates were prepared with respect to each fungus. A plate only with PDA and fungal
disc was considered as control, and the diameter of growth of fungi in this plate was
used as a control for the calculation of percent inhibition of test fungi. The inoculated
plates were incubated at 25 °C and colony diameter was measured and recorded after
five days. The percentage of mycelial growth inhibition was calculated as given below
(Das et al. 2010):

% Mycelial growth inhibition

Mean dia. of fungal colony for control  Mean dia. of fungal colony for plant extract
¼  100
Mean diameter of fungal colony for control

Mean diameter of fungal colony for control is a fully grown fungus in petri plate which
is equal to 9 cm.
The plant extracts showing percent inhibition > 50% were considered as potent and
were further evaluated for their MFC values.
 4 M.K. Bhagwat and A.G. Datar

2.5. MFC for test fungi

MFC is defined as the lowest concentration of antimicrobial substance resulting in the
death of 99.9% of the microbial cells. MFC of shortlisted potent extracts namely EtOAc
extract of rhizomes of C. aromatica, unripe fruit rinds of G. indica and DCM extract of
roots of G. glabra were determined by macrodilution broth method (CLSI 2002;
M38A). The concentration range studied for plant extract was 2–0.25% and 5–0.625%
w/v. For positive control GAA, concentration range of 1–0.0625% v/v was studied.

2.6. Assessment of safety of potent herbal extracts

EtOAc extract of rhizomes of C. aromatica and unripe fruit rinds of G. indica along
with DCM extract of roots of G. glabra were studied for their safety. Acute oral
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toxicity study was performed as per OECD guidelines test No. 420. The study was
approved by Institutional Animal Ethics Committee. It was performed on Albino swiss
mice at dose concentration of 2000 mg/kg body weight. This study was carried out in
Animal testing unit, Ramnarain Ruia College (Mumbai, India), CPCSEA/315.

3. Results and discussion

The fungal rots are very common in occurrence and have been reported almost in all
parts of the world (Nisa et al. 2010). Some of the examples of losses can be noted as
diseases caused by Colletotrichum spp. that occurs on a wide range of plant spp. and
have been recorded worldwide as both pre and postharvest causes of crop loss
(Phongpaichit & Liamthong 2001). Amongst the postharvest fungal pathogens, infection
caused by B. cinerea lowers the shelf life and adversely affects the market value of the
fruits (Tripathi et al. 2008). R. stolonifer caused the most rapid rotting on stored tomato
fruits in Nigeria (Abdel-Mallek et al. 1995). The growing concern about food safety
has recently led to the development of natural antimicrobials to control pathogens
(Pundir & Jain 2010). The approach of this study is the same. Different herbal extracts
were studied for their antifungal activity. From the result of the poisoned food tech-
nique, it was observed that EtOAc extract of unripe fruit rinds of G. indica, rhizomes
of C. aromatica and DCM extract of roots of G. glabra have shown inhibition more
than 50%, hence were considered as potent amongst other tested extracts (refer to
Table 1). These extracts were further studied to find out their MFC values. EtOAc
extract of rhizomes of C. aromatica and unripe fruit rinds of G. indica have shown the
lowest MFC values that is, 1% w/v (refer to Table 2). Similar findings on the fungicidal

Table 1. Percent inhibition given by plant extracts against plant pathogenic fungi, using
poisoned food technique.

Sr. R. stolonifer C. coccodes B. cinerea

No. Name of extract (%) (%) (%)

1. C. aromatica EtOAc extract. 100 71.33 67.44

2. G. indica EtOAc extract. 39.22 64.78 79.0
3. G. glabra DCM extract. 31.89 82.56 0
4. GAA in water 5% w/v (positive 100 100 100
control)
5. DMSO (negative control) – – –
Note: – indicates no inhibition.
 Archives of Phytopathology and Plant Protection 5

Table 2. Data orientation of MFC of potent plant extracts using macro dilution broth method.

Sr. No. Name of extract R. stolonifer (%) C. coccodes (%) B. cinerea (%)
⁄ ⁄
1. C. aromatica EtOAc extract 1 1 1⁄
2. G. indica EtOAc extract 1⁄ 1⁄ 1⁄
3. G. glabra DCM extract 2 2 2.5
4. GAA in water 5% v/v 0.25 0.5 0.5
Note: The concentration range studied for plant extracts was 2–0.25% and 5–0.625% w/v.
The concentration range studied for positive control was 1–0.0625% v/v.
⁄
indicates lower MFC values given by herbal extracts, for respective fungus.
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activity of some plant extracts in controlling different plant pathogens have been
reported by several researchers (Okigbo & Ogbonnaya 2006). Most of the essential oils
have been reported to inhibit postharvest fungi, tested under in vitro conditions (Feng &
Zheng 2007). Azadirachta indica, Hyptis suaveolens and Carica papaya have been
used to control fungal pathogens. The fungitoxic properties of ethanol extracts of 10
plants were reported against five pathogenic fungi (Mogle 2011). The antifungal activity
of thyme essential oils has been well proven against fungi such as Botrytis and
Rhizopus (Amini & Safaie 2012). The GAA used as a positive control in this study was
found to be effective. However, it may cause irritation to eyes, skin and throat, if comes
into contact with human. Being an acid it, can act non-specifically. Sometimes it can
show adverse effects on the usage e.g. there is a report which says that stem browning
of sweet cherries was seen when acetic acid was used to control Botrytis (Chu et al.
1999). Although these chemicals seem to give the desired results at a lower concentra-
tion than herbals, the above-listed issues with respect to them cannot be eliminated. On
the contrary many times herbals give a target-specific action and being complex
structures, it is difficult for them to develop resistance to microorganisms. It was
observed from acute oral toxicity study that the extracts namely EtOAc extract of
unripe fruit rinds of G. indica, and rhizomes of C. aromatica were safe too. These
findings show that plant extracts can serve as a good alternative to synthetic fungicides
considering their safety.

4. Conclusion

• In this work, the in vitro efficacy of herbal extracts of five plants namely,
C. aromatica, G. indica, G. glabra, V. anthelmintica and N. arbour-tristis was
studied against three plant pathogenic fungi namely R. stolonifer, C. coccodes
and B. cinerea.
• The EtOAc extracts of C. aromatica and G. indica were more potent as well as
safe.
• It can be concluded that these potent herbal extracts can be used as potential
source of herbal fungicides for green farming, and can be a safe alternative to
synthetic chemicals.
• Moreover these herbal fungicides can be subjected to in vivo efficacy study, both
in pre and postharvest stages.
• Besides, isolation and identification of bioactive molecules can also be performed.
It will serve to get purified active compounds and can be manufactured in bulk.
 6 M.K. Bhagwat and A.G. Datar

Acknowledgements
We are grateful to Dr. R. T. Sane, Dr. B. V. Vakil and Ms. Komal D. Barbade for providing
technical support and writing assistance. We are also thankful to Guru Nanak Institute for
Research and Development, Guru Nanak Khalsa College, for providing the well-equipped
research facility where the work has been conducted.

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