Effects of Selected Plants Extracts on in vitro Growth of Ralstonia slanacearum (Smith), the Causal Agent of Bacterial Wilt of Irish Potatoes
The antibacterial effect of crude medicinal plant extracts of Ocimum gratissimum, Brassica oleracea var. botrytis and Ipomoea batatas on Ralstonia solanacearum (Smith) extracted from infected potato tubers was determined by in vitro study using ethyl acetate and methanol solvents. The extracts were used at concentrations of 0.4, 0.2, 0.1, 0.05 and 0.025 mg mL-1. It was found that all the plant extracts used at their different concentrations except methanol extracts of Ipomea batatas at 0.025 mg mL-1 were effective to varying degrees in controlling the growth of bacterial colonies. The best results were observed with ethyl acetate extracts of Ipomoea batatas at concentration of 0.4 mg mL-1 giving mean inhibition zone of 4.2 mm followed by ethyl acetate extract of Brassica oleracea at concentration of 0.05 mg mL-1 that was 4.12 mm.
Received: March 07, 2011;
Accepted: April 26, 2011;
Published: July 20, 2011
Irish potato (Solanum tuberosum) is the second most important staple
food crop in Kenya after maize (Felix et al., 2010).
It is ranked fourth most important food crop in the world after wheat, maize
and rice with annual production approaching 300 million tons (Otipa
et al., 2003). In Kenya the crop ranks second after maize and plays
a major role in feeding the ever increasing population and providing source
of income to the local people (FAO, 2006). However, production
of potatoes has stagnated over the last 10 years with total yields ranging between
600 and 1200 thousand tones though acreage under the crop has been on the increase
(Ministry of Agriculture, 2007). The low yields have
been attributed to poor agronomic practices, low use of inputs especially fertilizers,
low soil fertility, limited access to good quality seeds, diseases (especially
bacterial wilt, late blight and viruses) and insect pests (Muthoni
and Nyamongo, 2009; Lemaga et al., 2001a).
Among the diseases that infect Irish potato is bacterial wilt (also known as
brown rot of potatoes) caused by race 3 biovar 2 of Ralstonia solanacearum
and ranks the second most important potato disease after late blight (Felix
et al., 2010; Lemaga et al., 2001b).
The pathogen invades the roots of the host plant and aggressively colonizes
the xylem vessels causing a lethal wilting (Tahat et
al., 2008). In Kenya, the disease has been reported to cause losses
ranging between 30-70% at altitudes ranging between 1800-2800 m above sea level.
It is considered more problematic than late blight since it has no known chemical
control procedures and many farmers do not know how to control it (Muthoni
and Nyamongo, 2009). Besides, available cultural control methods such as
crop rotation, use of clean seeds, planting in non-infested soils and growing
tolerant varieties (Tahat and Sijam, 2010) have individual
practical, technological and economic limitations (Lemaga,
2001; Muthoni and Nyamongo, 2009). Crop rotation
for example is not feasible because the disease has been noted even in first
planting in newly cleared land (Jinnah et al., 2002;
Khalequzzaman et al., 2002). In addition, R.
Solanacearum can survive in the soil for long periods in the absence of
host plants (Tahat et al., 2008) and small farm
sizes hinder effective crop rotation programmes (Muthoni
and Nyamongo, 2009).
Green plants have been shown to represent a reservoir of effective chemotherapeutants
and can provide valuable sources of natural pesticides (Dorman
and Deans, 2000). Among the plants that have been shown to contain some
antimicrobial components include Brassica oleracea var. botrytis (Kirkegaard,
2005), Ipomoea batatas (Graham et al.,
2000; Kirkegaard, 2005) and Ocimum gratissimum
(Mbata and Saikia, 2005; Adebolu
and Salau, 2005; Amadi et al., 2010). Use
of agrochemicals is becoming less favorable because of environmental pollution
and detrimental effects on a variety of non-target organisms (Bonjar
et al., 2006). Biological methods of control including use of natural
plant products have therefore been preferred because most of them are locally
available, environment friendly, have no side effects and development of resistance
is rare (Okigbo and Ogbonna, 2006; Soytong
et al., 2001; Khalequzzaman et al., 2002).
They, however, need to be applied in an integrated manner as little can be achieved
when they are solely applied. Fortunately, race 3 biovar 2 of R. solanacearum
has a narrow host range and can be successfully controlled by Integrated Disease
Management (Felix et al., 2010).
Very little work has been done to investigate the use of natural plant products
to control of brown rot diseases of potatoes. Muthoni and
Nyamongo (2009) reported that increased bacterial wilt occurrences can be
explained by the lack of appropriate management practices as research on this
subject has been at very low scales. This study was therefore carried out to
determine the effects of selected plant extracts and their concentrations on
the in vitro growth and development of R. solanacearum colonies.
MATERIALS AND METHODS
The study was conducted at Maseno University between September, 2006 and November,
2007. Fresh leaves of O. gratissimum, B. oleracea var.botrytis
and vines of I. batatas variety SPK 004 were collected from Maseno region
in Western Kenya. They were plucked, washed, shredded and dried as described
by Okigbo and Ogbonna (2006). They were then ground
into fine powder at Kenya Sugar Research Foundation in Kisumu, Kenya in readiness
for solvent extraction.
Sequential extraction: Cold extraction of the powdered plant materials
was done sequentially with organic solvents (ethyl acetate and methanol) of
varying polarities following the method described by Eaton
(1989). Known quantity of dry ground leaf material was soaked in extraction
solvents (Table 1) in Erlenmeyer flask and left for four days
with occasional shaking. The liquid portion was then filtered using Whatman
No.1 filter paper. The filtrate was then concentrated in vacuo in a round-bottomed
flask using rotary evaporator at 60 and 70°C for ethyl acetate and methanol
respectively (Junaid et al., 2006). The extracts
obtained were weighed and kept in vial bottles in readiness for bioassay tests
(Eaton, 1989; Llorach et al.,
2003). The amount of extracts obtained during the sequential extraction
of the test plants were weighed and recorded (Table 1).
Isolation of wilt bacteria from infected potato tubers: Infected tubers
were obtained from a test plot at the National Agricultural Research Laboratories
(NARL) fields, Nairobi. These were cleaned under running water to remove adhering
soil, air-dried, then cleaned using 97% ethanol to remove any microorganism
on its surface. The skin at the end of the stolon was removed using a disinfected
scalpel to make vascular tissues visible. A bacterial suspension was prepared
using the method described by Priou et al. (1999).
Approximately 0.5 mL of the bacterial suspension was spread on nutrient agar
in Petri dishes. The plates were incubated for 48 hours at 28°C and bacterial
colonies that were fluidal, flat, pearly white and irregular identified.
Pathogenicity test: The method of Kochs postulates was performed
with Solanum tuberosum var. Tigoni 381381 as the host. After a 24 h period
without water, one side of some potato roots were injured one centimetre from
the stem and approximately 20 mL of an aqueous suspension of R. solanacearum
of 1x107 cfu mL-1 was poured around the base of the stem.
Five days after inoculation (after the wilting symptoms were exhibited), vascular
flow test was run by cutting a piece of potato stem (5 cm long) and suspending
it in clear water in a glass container. The cut stem was held with a clip to
keep it in a vertical position until smoke like threads streamed downwards from
the cut stem (Priou et al., 1999).
Antibacterial assay: The antibacterial effects of the plant extracts
against R. solanacearum were evaluated using the method described by
Barry et al. (1979) and De
Souza et al. (2005). Inoculation was done by rubbing a sterile cotton
swab containing the pathogen on the surface of solidified agar as described
by Linnette et al. (1974).
|| Weight of raw material and yield of plant extracts
|EtOAc: Ethyl acetate, MeOH: Methanol
Experimental design, data recording and analysis: All tests were laid
down in Randomized Complete Block Design (RCBD) with four replications. The
antibacterial activity was recorded as the width (in mm) of clear zones of inhibition
surrounding the diffusion discs after 48 h (Reiner, 1982;
Baker et al., 1983; Deans
and Ritchie, 1987). The data were subjected to ANOVA using SAS version 9.1
(SAS, 2005) and effects declared significant at 5% level.
Separation of means was done only for those parameters where the ANOVA was significant,
using Least Significant Difference at 5% level of significance (LSD5%)
(Steel and Torrie, 1980).
Activity of plant extracts against R. solanacearum: The combined analysis of variance (Table 2) showed that the three plant extracts exhibited highly significant (p<0.0001) differences on their effects against growth of R. solanacearum at different concentrations (Fig. 1a). In addition, the two solvents (ethyl acetate and methanol) used in extraction of plant extracts portrayed highly significant (p<0.0001) differences in their suitability as extraction solvents (Fig. 1b). The interactions between plants extracts, their concentrations and the different extraction solvents were also highly significant (p<0.0001). This indicated that R. solanacearum responded differently to different plant extracts, their different concentrations and to different solvents used.
The antibacterial activity of both methanol and ethyl acetate extracts of I. batatas against wilt bacteria increased as their concentration increased. However best activity was observed with ethyl acetate extracts compared to methanol extracts. The best (p<0.05) results were observed with ethyl acetate extracts of I. batatas at concentration of 0.4 mg mL-1 giving a mean inhibition zone of 4.2 mm. Methanol extract of I. batatas at concentration of 0.025 mg mL-1 were inactive against R. solanacearum (Fig. 2a).
Activity of ethyl acetate extracts of O. gratissimum against the wilt bacteria increased as concentration increased up to 0.2 mg mL-1 beyond which it declined. Statistically, similar inhibitory activity was recorded at extract concentrations of 0.1 and 0.4 mg mL-1. Methanol extracts of O. gratissimum at concentration of 0.025 and 0.05 mg mL-1 exhibited similar activity where inhibition zone measured 2 mm but the activity then increased as the concentration was increased. At concentrations of 0.05 and 0.1mg mL-1, both the methanol and ethyl acetate extracts of O. gratissimum were not significantly (p>0.05) different in their inhibitory activity against the wilt bacteria (Fig. 2b).
|| Combined analysis of variance for the three plant extracts
||(a) Comparative suitability of Ethyl acetate and Methanol
as extraction solvents and (b) comparative combined antibacterial effects
of O. gratissimum, I. batatas and Brassica oleracea var. botrytis
extracts against R. solanacearum
Inhibitory effects of B. oleracea var. botrytis are illustrated in Fig. 3. The best results were realized with ethyl acetate extracts of B. oleracea at concentrations of 0.025 and 0.05 mg mL-1 which recorded an inhibition zone of 3.78 and 4.18 mm, respectively. The two treatments were, however, not significantly (p>0.05) different from each other. Inhibitory activity of methanol extracts of B. oleracea increased as concentration increased attaining an inhibition zone of 2.94 mm at the highest concentration of 0.4 mg mL-1. Unlike methanol, suitability of ethyl acetate as an extraction solvent appeared to decline at higher concentrations.
||Antibacterial effects of (a) I. batatas and (b)
O. gratissimum extracts extracted using ethyl acetate and methanol solvents
||Antibacterial effects of B. oleraceae var. botrytis
extracts extracted using ethyl acetate and methanol solvents
This study demonstrated that compounds extracted from the three plants using
the two organic solvents vary in their efficiency in inhibiting bacterial growth.
The difference observed in antibacterial activity of the extracts is likely
to be due to the solubility of the active compound(s) in ethyl acetate and methanol
or the presence of inhibitors to the antibacterial principle. This agrees with
the report of Okigbo and Ogbonna (2006). The two solvents
have different polar abilities with methanol having higher polarity and thus
they tend to dissolve different compounds from the plant materials dipped in
them. Polar solvents dissolve polar compounds best and non polar solvents dissolve
non polar compounds best (Siddhuraju and Becker, 2003).
The presence of antibacterial substances in the different extracts which caused
the inhibition of radial growth in vitro agree with reports of other
studies (Olayinka, 2009; Mbata and
Saikia, 2005). The active principles present in plants are influenced by
many factors which include the age of plant, extracting solvent, method of extraction
and time of harvesting plant materials (Ajalie and Okigbo,
2005; Okigbo et al., 2005; Okigbo
and Ogbonna, 2006).
O. gratissimum extracts: Antibacterial effects of O. gratissimum
extracts have also been reported by Ntezurubanza et al.
(1984), Nakamura et al. (1999), Iwalokun
et al. (2003), Lemos et al. (2005),
Akinyemi et al. (2004), Lemos
et al. (2005) and Lopez et al. (2005)
reported that Ocimum oil is active against several species of bacteria (Staphylococcus
aureus, Listeria monocytogenes, Escherichia coli, Shigella,
Salmonella and Proteus) and fungi (Trichophyton rubrum T. mentagrophytes,
Cryptococcus neoformans, Penicillium islandicum and Candida
albicans). Although there was a gradual increase in the inhibitory activity
of ethyl acetate extracts of O. gratissimum, the activity was less consistent
compared to the methanol extracts. The significantly higher activity of methanol
extracts compared to ethyl acetate extracts could be an indication that methanol
absorbed more antibacterial principles from O. gratissimum than ethyl
acetate. This could therefore mean that the anti microbial principles of African
basil against wilt bacteria are polar compounds. Similar findings were reported
in a study of Junaid et al. (2006) while examining
the methanol and hexane extracts of O. gratissimum against E. coli.
They found higher inhibition in methanol extracts and concluded that the active
components of the plant could be highly polar.
I. batatas extracts: The study showed that I. batatas
extracts contains antibaterial compounds against R. solanacearum. This
agrees with Cevallos-Casals and Cisneros-Zevallos (2002)
who reported that sweetpotato extracts caused growth inhibition of Salmonella
enteritidis. The significantly high activity of ethyl acetate extract for
all concentrations used compared to methanol extract is an indication that ethyl
acetate extract contained more anti microbial principles against R. solanacearum
than methanol extracts. This could probably mean that the active compounds
of this plant against the bacteria are less polar compounds. Ishiguro
et al. (2004) and Islam (2006) reported that
the leaves of I. batatas contains large amounts of polyphenolics such
as anthocyanins and phenolics acids and are rich in vitamins and minerals. When
ethanol and water were used in their extractions, higher polyphenols concentration
was found with ethanolic extracts. The compounds extracted using the solvent
may be similar to those released when the vines were used in a biofumigation
study (Kirkegaard, 2005) to control bacterial wilt where
the effectiveness of the vines in controlling bacterial wilt increased with
increase in the amount used.
B. oleracea var. botrytis extracts: There was significantly
high activity of ethyl acetate extracts of B. oleracea var. botrytis
at lower concentrations of 0.025, 0.05 and 0.1 mg mL-1 compared to
methanol extract. This indicates that most antimicrobial principles of ethyl
acetate extract of cauliflower are highly effective against R. solanacearum
in lower concentrations. This could be interpreted to mean that the less polar
active compounds of this plant interact best with the bacteria at low concentrations.
The activity of methanol extract increased gradually with increase in its concentration
surpassing the effectiveness of ethyl acetate indicating that the polar compounds
were more effective at higher concentrations. Walters (2009)
reported the release of isothiocyanates (ITCs) from Brassica species like cauliflower
that suppressed the bacterial wilt of potatoes when used as green manure and
that the effectiveness increased as materials incorporated increased. Larkin
and Griffins (2007) also reported the release of surfur compounds from Brassica
species that suppressed soil borne potato diseases when chopped leaves were
incorporated in potato fields.
The study demonstrated that O. gratissimum, I. batatas and B.
oleracea var. botrytis contain antibacterial compounds which can
be utilized in preparation of a potential phyto-bactericide to control the pathogenic
bacteria that causes brown rot of potatoes. It was apparent that the use of
raw plant extracts of the three plants has a potential to substitute the chemical
approach of controlling the disease. This kind of biological approach would
be economically viable and environmental friendly. The plants are also available
to all farmers including those that do not have ready access to other chemicals.
The study did not ascertain the specific chemical compounds that were responsible
for antibacterial activity. Further research is therefore recommended to identify
the active compounds against the wilt bacteria.
The authors wish to acknowledge Maseno University for providing laboratory space, equipments and technical assistance. Thanks are due to Prof. George Odhiambo of Department of Botany and Horticulture, Maseno University, for his valuable assistance in data analysis.
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