ABSTRACT
This study was conducted to examine the In vitro antifungal activity of leaf ethanol extracts of Azadirachta indica, Senna alata, Eucalyptus camaldulensis and Tithonia diversifolia against Phytophthora colocasiae Racib. Extracts were tested against isolated pure culture of P. colocasiae at 5, 10 and 20mg/ml. 3mg/ml Ridomil and untreated culture served as the standard and absolute control respectively. Experiment was conducted in a completely Randomized Design (CRD) and all treatments were replicated thrice. Data were subjected to analysis of variance (ANOVA) using statistical package for social scientists (SPSS). Means were separated by Duncan Multiple Range Test (DMRT) and test of significance at 5%. Results indicated that 20mg/ml extracts of A. indica and 3mg/ml of Ridomil had the best percentage radial growth inhibition of P. colocasiae at 100%, followed by S. alata (78.19%) at 5mg/ml while extract of E. camaldulensis showed the lowest anti-fungal activity at 5mg/ml (19.33%). P. colocasiae growth in absolute control was un-inhibited (0.00%). Mycelia dry weight of P. colocasiae at 10mg/ml of E. camaldulensis was recorded at 0.10 +- 0.61g contrary to 0.00g recorded against other plant extracts. At 20mg/ml, A. indica supported a mycelia dry weight of 0.09 +- 0.06g but none was recorded against other plant extracts. Ridomil did not support mycelia growth in PDB while absolute control had a significant (p≤0.05) mycelia dry weight of 0.27+- 0.08g. Leaf extract of A. indica should be characterized to obtain the pure active component responsible for anti-fungal activity and used for further studies in the control of the pathogen in the field.
TABLE OF CONTENTS
Cover page
Title page i
Declaration ii
Certification iii Dedication iv
Acknowledgements v
Table of Contents vi
List of Figures viii
List of Plates ix
Abstract
x
CHAPTER ONE 1
1.1.
INTRODUCTION 1
1.2.
Statement of problem 6
1.3.
Justification of study 7
1.4.
Aims and
objectives 8
CHAPTER TWO 9
2.0. LITERATURE REVIEW 9
2.1. History of taro (Colocasiae
esculenta) 9
2.2. Taro
leaf blight (TLB) (Phytophthora
colocasiae Raciborski)
10
2.3. Symptoms of taro leaf blight
11
2.4. Temperature
requirement of P. colocasiae 12
2.5. Management of P. colocasiae
13
2.5.1. Cultural control 13
2.5.2. Biological
control 14
2.5.3. Chemical control 15
2.6. Efficacy of plant extracts on Phytophthora colocasiae 17
CHAPTER THREE 19
3.0. MATERIALS AND METHODS 19
3.1 Source of plant materials
19
3.2. Study
area 19
3.3. Experimental design 19
3.4. Preparation
of plant extracts 19
3.5. Preparation
of media 20
3.6 Isolation
of pathogen from diseased taro leaf tissues
20
3.7 Pathogenecity test 21
3.8. Experimental procedure 22
3.9. Inoculation and measurement of radial growth 22
3.10. Measurement of the mycelia dry weight
23
CHAPTER FOUR 24
4.0. RESULTS AND DISCUSSION 24
4.1 Results 24
4.2 Discussion 31
CHAPTER FIVE
5.0
CONCLUSION AND RECOMMENDATIONS 36
5.1 Conclusion 36
5.2 Recommendations 37
REFERENCES
38
APPENDIX 43
LIST
OF FIGURES
Fig. 1: Growth
inhibition of P. colocasiae at 5, 10
and 20mg/ml ethanol leaf extracts.
Fig. 2: Mycelia wet weight (mg) of P. colocasiae.
Fig. 3: Mycelia dry weight (mg) of P. colocasiae.
LIST OF PLATES
Plate 1: Micrograph
of P. colocasiae x 400.
Plates 2:
Micrograph of Aspergillus niger x100.
Plate 3: Artificially inoculated taro leaf showing symptoms
of TLB 3dai
Plate 4: Artificially inoculated taro leaf showing symptoms
of TLB 3dai
Plate 5: Micrograph
of P. colocasiae x100.
Plate 6: Micrograph
of Aspergillus niger x 400.
Plate 7: Picture of taro leaf tissue on agar medium.
CHAPTER ONE
1.0 INTRODUCTION
Plant
disease epidemics have influenced the course of history in countries where they
have had a devastating effect and continue to be of great importance especially
for those people whose day to day survival depends on their crops.
Raciborski
(1900) was the first person to study the leaf blight disease of taro in Java
and was also responsible for naming the causal pathogen. Taro leaf Blight (TLB) is the most commonly
observed and destructive Oomycete disease of taro (Fullerton and Tyson, 2003)
capable of causing corm yield losses up to 30% (Miyasaka et al., 2001), leaf yield losses 95% (Brooks 2005) and causing
devastating epidemic.
Mbon
et al. (2013) reported that Phytophthora
colocasiae was first reported in Nigeria in November 2009. The pathogen is
soil borne and favoured by flooding conditions in fields (Grade and Joshi,
2003). It is believed to have originated in South East Asia and is widely distributed
throughout the tropical regions of the world (CMI, 1997). Symptoms typical of
TLB begin as small, brown, water-soaked lesions that rapidly become enlarged to
form large, dark brown lesions, zonate spots on the leaves, often coalescing to
destroy large parts of the leaf, sometimes with numerous droplets of orange or
reddish exudates. The pathogen can cause rapid and complete defoliation and
crop destruction within 14 days which under normal conditions lasts for 40 days.
Under certain circumstances the disease can attack harvested corms and cause
heavy losses during storage. The disease usually appears during the rainy
season and spreads throughout the period by means of zoospores and sporangia (Misra
et al., 2007). A comprehensive
history of the pathogen or the disease is not yet available Mpong et al. (2013). However, there has been
reports of taro blight disease attributed to P.
colocasiae in Nigeria, Ethiopia,
Equatorial Guinea and this has occurred more recently in Cameroon which led to
a huge economic lose (Mpong et al.,
2013).
Taro
(Colocasia
esculenta ( L.) Schott) is grown in many parts of the world due to its wide
adaptability, large scale acceptability and high return per unit area. The
leaf, petiole and corm of Taro are edible. Most people in the Southern region
of Nigeria depend on taro (C. esculenta)
as their primary source of food. The crop provides the indigenous population
with food in periods of food scarcity. Echebiri (2004) reported that taro is an
important tuber crop after yam and cassava in Nigeria, with diverse forms of
production. It can be produced on wet or dry land system with regular supply of
water throughout the growing season either by rainfall or supplement irrigation.
Taro can be planted at any time of year between April – July after rains have
been well established; and also after early season cropping of maize, yam,
cassava, eggplant and yellow pepper have been done in most parts of Nigeria. During
the dry season it is grown in waterlogged areas and in the forest under canopy
cover (Mpong et al., 2013). In
Nigeria, the corms are, boiled, pounded and used as soup thickener. Savory (2007) reported that the corms are
boiled and eaten for their high starch contents. Taro is easily digestible (97%)
and has very small starch grains (0.05-0.08µm) which are rich in amylases
(28%), amylopectin (72%) (Combie and Ferguson, 2003; Sefa-Dede and
Agviv-sackey, 2002).Taro corms are also rich in vitamins A, B1, B2 and good
source of protein and calcium phosphorus (Savory, 2007).
The Oomycota are
now placed by most authorities in the kingdom Chromista, though, Perterson (1989)
put them in the kingdom Straminipila (Stramenopila). The division Oomycota
contains many important plant pathogens. Two of its most destructive orders are
the downy mildews and the water molds, the latter including Phytophthora and Pythium. Though traditionally taught along with fungi in plant
pathology courses, studies indicate that these organisms are more closely
related to algae, such as the giant brown kelps.
Phytophthora colocasiae is
the most serious disease which infects leaves, petioles, corms and cormels
leading to heavy reduction in yield which may exceed 60% in severe cases
(Gurung, 2001). The pathogen is soil borne and favoured by flooding conditions
in fields (Grade and Joshi, 2003). In Nigeria, the disease usually appears during
the rainy season and spreads throughout the period by means of zoospores and
sporangia (Misra and Chowdhury, 1997; Misra et
al., 2007). Gurung (2001) noted that
the disease assumes severe dimensions in areas having high relative humidity
with frequent rainfall and causes 25-50 per.
According
to the recent report by Onyeka, (2011); the National Root Crops Research
Institute (NRCRI) (2012), cocoyam production is seriously declining due to poor
agronomic practice, climatic stress, socio- economic perceptions and above all,
fungal disease due to taro leaf blight disease.
Mpong
et al. (2013) reported that the
epidemic of taro leaf blight may occur throughout the year during continuous
rainy season over vast weather where night temperatures are 20 – 22oC
and daily temperatures of 25 - 28oC with little seasonal variation,
resulting in rapid taro defoliation, death and heavy to total yield losses
under favourable weather conditions. In addition to corm yield losses that
arise as a result of the reduced leaf area in diseased plant, Danny et al. (2008) maintained that there is
also a corm rot caused by P. colocasiae. This is mainly a problem when taro corms are
stored for longer than 7 days but not in subsistence economies where corms are
harvested and consumed within days.
Several
attempts have been made in the past to control the devastating and destructive
effect of Phytophthora colocasiae. Chemical
control using Fungicide sprays with co-formulation: Ridomil plus, Ridomil Gold
has been used in managing the disease. Fullerton and Tyson (2004) reported that
successful control of taro leaf blight is technically possible with fungicides
like Mancozeb (Dithane M45), copper (Copperoxychloride), Metalaxy (Ridomil
MZ-containing Mancozeb) and phosphorus acid (Phoschek). Mancozeb and copper
have protectant activity only. Metalaxyl and phosphorus acids have systematic
effect and are specific for Phytophthora
and Pythium diseases.
Cultural
methods such as exclusion are important. Where the organism is likely to be
dispersed over long distances by fungi propagules, or infected plant materials,
constant vigilance is required to ensure that the disease is not imported.
Suppression of diseased leaves (Sanitation) which is an effective control
measure in subsistence gardens particularly when plots are relatively separated
from one another seems not to be effective for taro disease. This strategy according
to Fullerton and Tyson (2004) would be most effective when the disease is in an
endemic phase with a relatively low and restricted disease incidence. When the
disease is in an epidemic phase, the removal of all the leaves with lesions
would quickly lead to almost complete defoliation of the crop with consequent
effects on yield. Plant spacing was also ineffective.
Several
experiments have been done on antifungal activity of aqueous and ethanol extracts
of different plants. Shakywar et al. (2012) reported the use of Azadirachta indica leaf
extract, Allium sativum and Allium cepa bulb extracts to inhibit the growth
of P.
colocasiae in vitro. Inhibition of mycelia growth of P. colocasiae was also recorded in neem, garlic; Lantana camera extracts (Anandraj and
Leela, 1996; Guha et al. 2004 and
Choudhury et al. 2006). Integration
of more efficient eco-friendly treatments like plant extracts with lesser use
of fungicides may provide a better management of the disease.
The
need to protect taro against disease is therefore, a crucial aspect of
enhancing the crops production (Onyeka et
al., 2008).
1.1 STATEMENT
OF PROBLEM
Taro
leaf Blight (TLB) is the most destructive disease of taro (Fullerton and Tyson,
2003) capable of causing corm yield losses up to 30% (Miyasaka et al., 2001,) leaf yield losses 95%
(Brooks, 2005) and causing devastating epidemic. There were over 300 different
cultivars of taro, today there are less than 70 of those cultivars in
cultivation due to effect of taro leave blight and unfavourable climatic
condition (Savory, 2007). Gurung (2001) maintained that leaf blight of taro
caused heavy reduction in yield which exceeds 60% in severe cases in most
countries. In 2009, taro farmers in Nigeria encountered heavy corms and leaf
losses of up to 100% due to outbreak which made farmers become sceptical of the
aetiology and health consequences. This made them to abandon the crop in the
field which led to widespread poverty. The
serious threat posed by this disease has adversely affected production and
biodiversity of this important food crop as many synthetic fungicides used
against it are observed to be environmentally unfriendly. Hence, the urgent
need for alternative and more environmentally friendly means of controlling
taro leaf blight using plants leaf extracts.
1.2 JUSTIFICATION
OF STUDY
The use of natural remedies such as
plant extracts has been advocated by many experts as a good alternative to
synthetic chemicals. This is because of possible health risk associated with
the use of synthetic fungicides in the eradication of plant diseases. Senna alata, Eucalyptus camaldulensis, Azadirachta
indica and Tithonia diversifolia are
common plants often found around homes and farm lands in the south-eastern part
of Nigeria. According to Bipasha (2011), plant extracts have also been used in
the treatment of bacterial and fungal diseases of plants and animals. The
adoption of these botanicals in the treatment against P. colocasiae would save farmers from the high cost of synthetic
fungicide and hence, reduce the cost in cocoyam (taro) production, minimize the
possible risk associated with the consumption of synthetic fungicide treated-cocoyam
and encourage massive interest in cocoyam production in the current
agricultural value chain policy of the Federal Government of Nigeria.
1.3 AIMS
AND OBJECTIVES
The main aim of this study is to determine the effect
of four plant extracts in the control of Phytophthora
colocasiae. Other specific objectives include to:
i.
Determine the effect of plant extracts on
the radial growth of Phytophthora
colocasiae.
ii.
Evaluate the influence of plant extracts
on mycelia wet and dry weights of Phytophthora
colocasiae.
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