Arabica coffee (Coffea arabica L.) is the single most important cash
crop that has been contributing a lions share to the Ethiopian economy
(Girma et al., 2007). Although, it plays significant
role in the economy of the country, the crop suffers from many production constraints.
Coffee Berry Disease (CBD) is the major threat to Arabica coffee production
since its outbreak in 1971 (Arega, 2006). It is an anthracnose
of green coffee berry, caused by the infection of a fungal pathogen Colletrichum
kahawae. Arega et al. (2008) pointed out the
concomitant spread of CBD to Southwest and Southeast of Ethiopia in the early
1970s. Furthermore, the disease was extended to Shewa and Gamugofa and Harraghe
areas of the country in the year 1971 and 1978, respectively (Hindorf
and Arega, 2006). Latter on it was also disseminated to other coffee growing
areas of the country.
CBD causes significant yield losses. The average national yield losses were
about 28% between 1974 and 1978 (Arega, 2006). Similarly
in Harraghe, the losses were estimated to be as high as 100% (Eshetu
et al., 2000). The severity of CBD and the losses caused are often
under estimated annually since young coffee berries drop off the tree at an
early stage of the disease. Largely, CBD causes 30% national average crop losses
to total harvestable coffee yield every year in Ethiopia (Eshetu
et al., 2000). At present, CBD has rapidly spread to all coffee growing
areas of Ethiopia and still inflicting significant crop loss (up to 100% on
susceptible land races) although the magnitudes vary from place to place and
from time to time (Arega, 2006).
Fungicides such as Daconil and Delan were considered as promising chemicals
against C. kahawae. However, later these products including Dyrene and
Octave were banned for a number of side effects (Eshetu et
al., 2000). The use of fungicides against CBD has been shown to induce
negative effects on beneficial microorganisms that can antagonize the CBD pathogen
(Masaba, 1991). The high cost of pesticides, the appearance
of fungicide resistant pathogen biotype and other social and health related
problems of the conventional agriculture on the environment have increased interest
in sustainable agriculture and biodiversity conservation. Additionally, millions
of coffee farmers are facing problem not only with low coffee prices but also
a growing interest in organically grown coffee across the globe. These problems
make it essential to look for alternative strategies that can ensure competitive
Many workers have reported antimicrobial activities of plant extract (phyotoproducts)
and gaining due attention because of their proven attributes such as specificity,
biodegradability and low toxicity (Okemo et al.,
2003; Saha et al., 2005). In an approach
toward the development of eco-friendly antifungal compounds for controlling
major foliar fungal diseases of tea, ethanol and aqueous extracts of Allium
sativum were tested against the pathogen C. camelliae. Ethanol and
aqueous extracts of A. sativum show 100% inhibitory to spore germination
of pathogen (Saha et al., 2005). In vitro
tests were carried out using extracts of Maesa lanceolata var. goulungensis
weir against a broad range of fungal plant pathogens such as Phytophthora
cryptogea, Trichoderma virens, Aspergillus niger, Phoma
sp., Fusarium oxysporium, Cochliobolus heterostrophus, Sclerotium
rolfsii and Pyrenophora teres. The extracts were very active against
all the pathogens tested (Okemo et al., 2003).
For instance, extract of some medicinal plants of Ethiopia for control of late
blight (Phytophtora infestans) on tomatoes and potatoes have been investigated
in vitro and in vivo (Mekuria et al.,
2005) and Hagenia abyssinica, Lepidum sativum and Lippia adoensis
showed the strongest suppression of mycelium growth. In Ethiopia, information
on antimicrobial activity of medicinal plants against fungal phytopathogens
is very scanty. However, the use of medicinal plant extracts (e.g., Allium
sativum) has a great potential in suppressing various plant pathogenic fungi
(Okemo et al., 2003; Satya
et al., 2005; Amin et al., 2009) that
may serve as better biological alternative in substituting the employment of
chemical fungicides. Therefore, the objectives of this study were to evaluate
the antifungal activities of the extracts of Hagenia abyssinica, Allium
sativum, Phytolacca dodcandera, Croton macrostachyus, Maesa lanceolata,
Eucalyptus globules, Eucalyptus citriodera and Lippia adoensis
on the inhibition of the growth of the fungus C. kahawae and disease
development on detached green coffee fruits and seedlings.
MATERIALS AND METHODS
Study area: The research was conducted at Jimma University College of Agriculture and Veterinary Medicine (JUCAVM), Jimma, Ethiopia, in Plant Pathology Laboratory and greenhouse from September 2009 to July 2010. JUCAVM is located at 7°42 N latitude and 36°50 E longitude and at altitude 1710 m a.s.l. The maximum and minimum temperatures of the area are 26.8 and 11.8°C, respectively, with relative humidity of 91% and the mean rainfall of 1500 mm per annum.
Preparation of fungal pathogen: Culture of Colletotrichum kahawae
was obtained from Jimma Agricultural Research Center (JARC), Jimma, Ethiopia
and sub-cultured on fresh PDA medium to obtain pure pathogen cultures for maintenance.
Blocks of fungal agar were cut out with a sterile surgical blade from the leading
edge of the actively growing portion and transferred to fresh agar medium and
incubated at 25°C for 3-5 days. The fungal pathogen was maintained in two
||By storing the pure culture of the pathogen in sterile distilled
||Keeping the pathogen on susceptible coffee host plant (selection 370)
on detached green berries (Van der Graaf, 1981)
Plant materials preparation: Ethiopian traditional medicinal plant sp.
H. abyssinica, P. dodcandera, C. macrostachyus, E. globules M. lanceolata
and E. citriodera were collected from natural habitats around Jimma,
Ethiopia, while A. sativum and L. adoensis were purchased from
the local market. The plant parts collected for extraction were leaf (H.
abyssinica, E. citriodera, E. globules and L. adoensis), fruit (R.
dodcandera and M. lanceolata), bark (C. macrostachyus) and
bulb (A. sativum). The collected samples of each plant species was washed
under tap water and surface sterilized with 5% sodium hypochlorite solution
followed by thorough rinsing with sterile water. The plant samples were air
dried at room temperature, ground and kept in bottles for subsequent activities.
Plant materials extraction: Plant samples were extracted using maceration
techniques following standard procedures of Amadioha (2002)
by some modification. Aqueous extracts were prepared 100 g/500 mL (w/v) of sterilized
distilled water and shaken on orbital shaker (130 rpm) for one hour. The mixture
was allowed to stand for 48 h and filtered using cheese cloth followed by filter
paper (Whatmann No. 1). A 70% ethanolic extract was prepared by the method of
Alade and Irobi (1993) by some modification, 100 g of
each dried powder plant was soaked in 500 mL of 70% ethanol for 48 h. The extracts
were filtered through cheesecloth followed by Whatmann filter paper No. 1. The
organic solvents were evaporated under oven 30-40°C at room temperature.
The remaining extract (2 mL) was diluted by adding appropriate quantity of sterilized
distilled water to make 20% extract. The stock extracts were transferred to
labeled sterile screw capped bottles and stored at 4°C for further use.
In vitro antifungal assays: The effect of the plant extracts
on the radial growth of C. kahawae was determined using the method described
by Amadioha (2002). One milliliter of the respective
extract was separately spread on the surface of the pre-solidified PDA contained
in the petri dishes. The control was not inoculated. Five-millimeter fungal
agar block was cut with a sterile scalpel from the 7 to 10 days old culture
at the actively growing portion on PDA and placed at the center of 9 cm diameter
petri dish. The plates were incubated at room temperature (20-22°C). The
radial growth of fungus for each treatment was measured at right angles for
each colony every 48 h after 5 days of inoculation for 21 days. The experiments
were carried out with three replications for each treatment.
Percentage radial growth inhibition by a given concentration of plant extracts was calculated as:
Based on the initial results obtained in in vitro, A. sativum bulb aqueous extract that showed the most suppressive effect on fungal radial growth was further tested against C. kahawae at different concentrations to determine the Minimum Inhibitory Concentrations (MIC). Accordingly, four concentrations [200, 150, 100 and 50 mg mL-1 (w/v)] of A. sativum bulb extracts were evaluated.
In vivo antifungal assays: In vivo antifungal potential
of the extracts was examined on detached green coffee berries and on seedlings
under greenhouse conditions. For the in vivo test on detached green berries,
coffee berries were inoculated following standard methods (Van
der Graaf, 1981). Berries having the same size and developmental stage were
collected from the same line of coffee selection 370. The berries were harvested
and surface sterilized with 5% sodium hypochlorite for 2 min and rinsed 3 times
for 3 min with sterile water. Thereafter, the berries were placed on clean plastic
petri dish and covered with sponge soaked in sterile distilled water to obtain
100% humidity. A total of 20 berries were used for each three replications.
For inoculation of the detached green berries, conidial suspension (2x106
conidial mL-1) was used. The concentration of conidia in the suspension
was determined using a haemocytometer. Twenty-five microliter conidia in the
suspension was dropped at the center of the berries at different times with
the medicinal plant extracts that showed high inhibitory effects in the in
vitro test. The selected plant extracts were sprayed on berries by using
an air-pressurized hand sprayer per the following schedule: (1) 48 h before
spore inoculation, (2) 48 h after spore inoculation and (3) at the same time.
The interaction between medicinal plant crude extracts and inoculated pathogen
were recorded starting from 15 day after inoculation every 2 day until 21 days
after inoculation. The disease assessments on green berries were performed using
0-5 scale (Table 1).
Disease Index (DI) on berries was calculated using the following equation:
where, b0, b1, b2, b3, b4, b5 are No. of berries in class 0, 1, 2, 3, 4 and 5, respectively.
For the in vivo seedling test in greenhouse, the plant extracts with
the highest inhibition percentage under in vitro test were further tested
in greenhouse. In this test was performed following standard procedures (Tegegne
et al., 2008) with some modifications on susceptible coffee seedlings
to evaluate their ability to control coffee berries disease two days before,
after and at the same time of inoculation.
|| Classification for Coffee Berry Disease (CBD) assessment
on detached green berries
Plant extracts (200 mg mL-1) were sprayed on the seedling plants.
Data were taken after 15 days 2 to 3 times with 2 day intervals until 21 days.
For inoculation of coffee seedlings, susceptible coffee seedlings were raised in greenhouse from freshly harvested seeds of 370 cultivar. To obtain seedlings, ripe cherries were picked from Jimma Agriculture Reseach Center and dried under shade after removing the pulp by hand. Seeds of 370 coffee cultivar were prepared by removing the parchment and soaked in sterile distilled water and kept for 48 h. Thereafter, seeds were sown (12 seeds pot-1) in heat sterilized and moistened forest soil,compost and sandy soil (2:1:1 ratio) in disinfected plastic pots arranged on benches and covered with mulch in greenhouse. Six weeks after sowing, the emerging seedlings were kept at 20-22°C. Two days before inoculation, the hypocotyls of the seedlings were sprayed with sterile distilled water and covered with plastic sheet for 48 h to obtain 100% relative humidity.
Plastic pots containing 10 seedlings pot-1 of 370 coffee cultivar
were inoculated with 1 mL fungal spore suspension in different time of applications
by stem brushing procedure with fine camel hairbrush (Van
der Graaf, 1981). Fourty eight hours before inoculation, the seedlings were
treated with medicinal plant extracts (aqeoues and 70% ethaol), respectively
at the rate of 200 mg mL-1 by using an air pressurized hand sparyer.
Mycelia colonies of fungal pathogen was carefully removed with a sterile scalpel from the PDA medium while washing with sterile distilled water to harvest conidia from 10 days old cultures. The suspension was stirred with magnetic stirrer for 10-15 min and filtered through double layers of cheese cloth. After repeating the procedure, the spore concentration in the suspension was adjusted to 2x106 conidia mL-1. Completely Randomized Design (CRD) was employed to see the interactions of medicinal plant extrats and the fungal pathogen on 370 coffee seeedlings cultivar. The seedlings were maintained wet with sponge soaked in sterile distilled water for additional 48 h under plastic cover. Temperature and relative humidity were adjusted by using digital sling psychrometr (Gm Nievw-vennep the Netherlands model 8706) to 80-85%, 20-22°C, respectively for 3 weeks. The reaction of each seedling of coffee cultivar against the fungal pathogen was assessed 15 and 21 days after inoculation using the symptom classifications (0-4 scale).
Experimental design and statistical analysis: The experiment was run
using complete randomized design with three replications and repeated. Data
were subjected to analysis of variance using SAS version 9.2. Single and interaction
effect of factors were determined using the GLM procedure of SAS. Whenever,
significant interactions were observed between factors, the level of one factor
was compared at each level of other factors. Mean values among treatments were
compared by the Tukeys test (Montgomery, 2008) at
α = 0.05% level of significance.
In vitro antifungal activity: The in vitro antifungal activity of aqueous and 70% ethanol extracts of 8 medicinal plants at 20% (w/v) against Colletotrichum kahawae was studied through measurement of radial growth of the fungus. There was significant (p<0.0001) interaction effect between type of medicinal plants used and methods of extraction in inhibiting radial growth of C. kahawae. As a result, the effect of medicinal plants on radial growth of the pathogen was presented for each method of extraction. Generally, extracts of all the tested medicinal plants except aqueous extracts of R. dodcandera significantly inhibited the mycelia growth of C. kahawae compared to the untreated control (Table 2). The inhibitory effect of aqueous extracts of the 8 medicinal plants ranged from 13 to 100% with the highest inhibition by A. sativum (100%) followed by C. macrostachyus (88%). With the ethanol extract, inhibition of the mycelia growth of C. kahawae ranged from 41 to 83%. The highest inhibition showed by A. sativum (83%) followed by R. dodcandera (76%), E. citriodera (70%) and C. macrostachyus (68%). Overall, A. sativum and C. macrostachyus had significantly higher inhibitory effect on C kahawae with both aqueous and ethanol extracts (Fig. 1). While, R. dodcandera had significantly higher inhibitory effect only with ethanol extract (Table 2).
Results in the present study revealed that extract of A. sativum gives
significantly higher inhibition zone compared to all the tested medicinal plants.
Thus, the antifungal activity of A. sativum aqueous and ethanol extracts
at different concentrations was tested against C. kahawae to determine
Minimum Inhibitory Concentration (MIC). The results showed that there was an
interaction effect between methods of extraction and concentration of extracts
used indicating that the antifungal effect of A. sativum depends on the
method of extraction used and the concentration level. Generally, both aqueous
and ethanol extracts at 10, 15 and 20% significantly reduced the radial growth
of C. kahawae compared to the control, while there was no significant
difference between 5% concentration and the control in radial growth (Table
||Effect of medicinal plant extracts and methods of extraction
(aqueous and ethanol) on radial growth of Colletotrichum kahawae in vitro
|*Means with the same letter are not significantly different
( α = 0.05)
||In vitro antifungal activity of the most effective
medicinal plant extracts against Colletotrichum kahawae pathogen.
(a) Allium sativum aqueous extract; (b) and (e) untreated plates;
(c) Croton macrostachyus aqueous extracts; (d) Allium sativum
70% ethanol extract and (f) Croton macrostachyus 70% ethanol extract)
|| Effect of extraction method and concentration of Allium
sativum on radial growth of Colletotrichum kahawae in vitro test
|*Means with the same letter are not significantly different
(α = 0.05)
The minimum inhibitory concentration for radial growth of C. kahawae
under in vitro condition was 10% for both aqueous and ethanol extracts.
In vivo antifungal activity: The aqueous and ethanol extracts
of A. sativum and C. macrostachyus were tested on detached coffee
berries to evaluate their in vivo antifungal activities against C.
kahawae because of their high antifungal effect observed under in vitro
conditions. There was significant (p<0.0001) interaction effect between
type of medicinal plants used, extraction method and time of application of
the extracts. Application of aqueous extracts on green berries 48 h before and
after inoculations of the test organism significantly (p<0.001) reduced severity
of coffee berry disease on the berries (Table 4).
||Effect of type of extraction methods, plant species used and
time of application on severity of coffee berry disease on detached green
|*Means with the same letter are not significantly different
(α = 0.05)
||Effect of methods of extraction, plant species used and time
of application on coffee berry disease severity on seedlings in greenhouse
|*Means with the same letter are not significantly different
( α = 0.05)
For C. macrostachyus, its aqueous extract significantly reduced severity
of the disease when applied at the time of inoculation of the test organism
and 48 h after inoculation but not when applied 48 h before inoculation of the
pathogen. However, its ethanol extracts reduced severity of the disease significantly
only when the extract was applied at the time of inoculation of the pathogen
In order to evaluate the antifungal activity of A. sativum and C. macrostachyus against coffee berry disease, their aqueous and ethanol extracts were tested on coffee seedlings by applying the extracts at the time of inoculation of the test pathogen and 48 h before and after inoculation and measuring severity of the disease on the seedlings. The results showed that there was significant (p<0.0001) interaction effect among methods of extraction, plant species used and time of applications of the extracts on the seedlings. Aqueous and ethanol extracts of A. sativum significantly reduced CBD severity on coffee seedlings when applied at the time of inoculation of the pathogen and 48 h after and before inoculation (Table 5). However, in C. macrostachyus the aqueous and ethanol extracts consistently reduced the disease severity only when applied 48 h before inoculation of the pathogen.
Incidence of CBD on coffee seedlings varied depending on type of medicinal
plant used methods of extraction and time of application of extracts. The study
showed that A. sativum extracts significantly reduced CBD incidence on
the seedlings except when its ethanol extract was applied 48 h after inoculation
of the seedlings with the pathogen inoculums (Fig. 2).
||The effect of method of extraction (aqueous and ethanol extracts)
and plant species used on coffee berry disease incidence on seedlings 21
days after inoculation with pathogen in greenhouse. (ASHA) Allium sativum
aqueous extract applied 48 h before pathogen inoculation; (ASHB) A. sativum
aqueous extract applied 48 h after pathogen inoculation; (ASHC) A. sativum
aqueous extract and the pathogen spore treated at the same time; (CRBHA)
Croton macrostachyus bark aqueous extract applied 48 h before pathogen
inoculation; (CRBHB) C. macrostachyus bark aqueous extract applied
48 h after pathogen inoculation; (CRBHC) C. macrostachyus bark aqueous
extract and the spore treated at the same time; (ASEA) A. sativum
ethanol extract applied 48 h before pathogen inoculation; (ASEB) A.sativum
ethanol extract applied 48 h after pathogen inoculation; (ASEC) A.sativum
ethanol extract and pathogen spore treated at the same time; (CRBEA)
C.macrostachyus bark ethanol extract applied 48 h before pathogen inoculation;
(CRBEB) C. macrostachyus bark ethanol extract applied 48 h after
pathogen inoculation; (CRBEC) C. macrostachyus bark aqueous extract
and pathogen spore treated at the same time and (Control) Seedlings inoculated
only by the pathogen spore. Means followed by different letters are significantly
different (α = 0.05)
Whereas, in C. macrostachyus its aqueous extract significantly reduced
CBD incidence at the three times of applications but its ethanol extract did
not show significant difference at all the three times of applications.
Plants are rich source of potentially useful antimicrobial products for the
development of new chemotherapeutic agents (Mousavi et
al., 2009; Musyimi et al., 2008; Ferreira
et al., 2009; Safary et al., 2009).
Many reports are available on the antifungal, antibacterial, antiviral, anthelmintic,
antimolluscal and anti-inflamatory properties of plants (Dey
and De, 2010; Mahesh and Satish, 2008; Samy
and Ignacimuthu, 2000; Palombo and Semple, 2001).
Some of these observations have helped in identifying the active compounds responsible
for such activities and in the developing drugs for the therapeutic use in human
beings. However, not many reports are available on the exploitation of antifungal
or antibacterial property of plants for developing commercial formulations for
applications in crop protection.
In this study the antifungal effect of the aqueous and ethanol (70%) extracts
of 8 medicinal plants were evaluated against Colletotrichum kahawae under
both in vitro and in vivo conditions. The findings showed that
the effect of plant extracts on C. kahawae radial growth and disease
development vary depending on the type of plant species used, method of extraction
and concentration of the extracts applied. A. sativum and C. macrostachyus
were found to be the most effective plants extracts in inhibiting the radial
growth of the pathogen in vitro and reducing disease development on detached
green berries and seedlings in vivo condition. Overall, however, A.
sativum is the most effective plant extract in reducing the radial growth
of the pathogen in vitro and in reducing severity and incidence of the
disease in vivo with both aqueous and ethanol extracts consistently.
This indicates that A. sativum possess antifungal activity against C.
kahawae. The result of this study corresponds with work done by William
(2000) who reported that sprays made from aqueous garlic extracts have antibiotic
and antifungal properties and will suppress a number of plant diseases, including
powdery mildew on cucumbers and to some extent black spot on roses. Similar
results were reported by Slusarenko et al. (2008)
who tested the effectiveness of garlic juice against a range of plant pathogenic
bacteria, fungi and oomycetes in vitro. Another investigation by Charimbu
et al. (2009) on the antifungal activity of garlic extracts against
Phaeoisariopsis griseola in common bean also showed that the aqueous
and methanolic crude extracts of A. sativum showed highest antifungal
activity compared to commercial fungicide Ridoml.
In an approach towards the development of eco-friendly antifungal compounds
for controlling major foliar fungal diseases of tea, (Saha
et al., 2005) tested ethanol and aqueous extracts of A. sativum
against the pathogen C. camellia for evaluation of antifungal properties.
Results showed that aqueous extracts of A. sativum was 100% inhibitory
to spore germination (Saha et al., 2005). Garlic
extracts were tested against three pathogenic fungi namely A. flavus, C.
lunata and F. moniliforme. The results of phytochemical screening
showed that water and ethanol extracted more components from garlic extract.
In most traditions, decoctions or infusions of herbs are usually made with either
alcohol or water as the solvent (Olusanmi and Amadi, 2010).
This may be related to their efficiency in extracting most of the active principles
in plants. At times, marked differences exist between the phytochemical profile
of alcoholic and aqueous extracts of plants. For A. sativum, the aqueous
extract is recommended because no vital phytochemical constituent seemed to
be left out and also because of probable unwanted effects that alcohol which
is another drug on its own may produce (Olusanmi and Amadi,
Aqueous extracts of A. sativum, has been reported to inhibit the growth
of Alternaria alternata, A. brassicola and Myrothecium roridum
(Khan et al., 1998). Su
and Cheng (2009) also reported total inhibition of mycelia growth of Phytophthora
capsici at different concentrations. In general, voluminous number of literature
reported the effectiveness of A. sativum against most pathogenic
microorganisms. A crude extract of A. sativum, however, showed the lowest
inhibitory effect (<16%) at 10% concentration against Botryodiplodia theobromae
Pat and Macrophomina phaseolina (Tassi) Goidanich. among six tested
fungal pathogens isolated from rotted cassava roots (Okigbo
et al., 2009).
The effects of the antifungal compounds may be on spore germination leading
to its inhibition or may be due to effect of these compounds on the cell wall
altering its permeability (William, 2000). The antifungal
compounds may also suppress the early stages of mycelia growth leading to inhibition
of the fungus.
The antifungal activity of A. sativum may be due to sulfur containing
compounds such as ajoene allicin found in it. Allicin is produced in garlic
when the tissues are damaged (Slusarenko et al.,
2008) and it has been found to effectively control seed-borne Alternaria
sp. in carrot, Phytophtora leaf blight of tomato, tuber blight of
potato and downy mildew of Arabidopsis.
The present study suggested that aqueous and ethanol extracts of A. sativum bulb parts have the potential to be applied as a control measure against infection of coffee berry disease caused by C. kahawae. The application of aqueous extract of A. sativum bulb seems promising as it is easy, effective and cheap alternative means of C. kahawae management for the majority of Ethiopian subsistence farmers, who cannot afford synthetic chemicals. Moreover, the risk associated with synthetic chemicals as well as consumers resistance, towards its application in agriculture make the product more attractive natural product for organic agriculture. This study provides new scientific information on antifungal activity of A. sativum against C. kahawae. The extracts should be tested against the disease under field conditions.
The authors are grateful to Jimma University, Ethiopia, for providing finance for the research and to the Jimma Agricultural Research Centre, EIAR, Jimma, Ethiopia, for the provision of fungal isolates for the study.