ABSTRACT
A pot experiment was conducted in 2018 cropping season in the Research farm of Michael Okpara University of Agriculture, Umudike to assess the efficacy of some leaf extracts of Vernonia amygdalina, Gongronema latifolium, Ocimum gratissimum and the control (sterile water) in the control of bacterial leaf spot disease of vegetable cowpea. Also their effects on disease severity and incidence, sowing date; growth and yield performance were monitored. Four varieties of vegetable cowpea: Black vegetable cowpea, Brown vegetable cowpea, Ife brown and Ife 143, were subjected to infection by bacterial leaf spot pathogen with a view to identifying the most resistant variety that can be used to reduce the disease. The experiment was laid out in a completely randomized design (CRD) with three replicates, the pot size was (15cm), plant materials were washed, air dried, exactly 250g of each milled paste of the leaves were added in 200ml of sterile water for 10minutes in a 500ml beaker to obtain suspension and strained through two layers of cheese-cloth using Whatman No.1 Filter Paper, plant extracts were applied two weeks after planting (2WAP). Isolation was done by collecting diseased leaf part from field to the laboratory, pathogenicity test was also conducted both in vivo and in vitro to confirm the identity of the casual organism in the laboratory. Similarly some biochemical tests were conducted in which Gram, catalase, oxidase, indole, maltose, xylose and glucose all tested positive. Data were collected four weeks after planting (4WAP), for growth characteristics such as plant height (cm), number of branches, number of leaves per plant, stem girth, disease incidence and severity while data on yield performance were taken at maturity for grain yield, number of pods per plant, number of seed per pod, weight of seeds. Data generated from the study were subjected to Statistical analysis. Results obtained from the data analysis showed that significant difference (P≤0.05) existed among the leaf extracts used as treatment. The highest (80 and 74%) and lowest (10%) incidence of bacterial disease on vegetable cowpea were recorded in Vernonia amygdalina and control pot, respectively. Bacterial diseases severity indices of 5.16 and 6.14 were recorded for the Ocimum gratissimum and control pots, respectively. Results of the effect of plant extracts on the various growth component, yield and yield component per plant were significant (P≤0.05). Pots sprayed with plant extract significantly enhanced growth and yield when compared with the control pots. Based on the pathogenicity test conducted result obtained showed that pathogen caused symptoms on the leaves and was identified in the laboratory through some biochemical tests as Xanthomonas axonopodis pv vignicola
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables ix
List of Figures x
List of Plates xi
Abstract xii
CHAPTER 1: INTRODUCTION 1
1.2 Economic
Importance of Vegetable Cowpea 4
1.3
Objectives of the Study 5
CHAPTER 2: LITERATURE
REVIEW 7
2.1 Diseases of Vegetable Cowpea (Vigna unguiculata L. Walp) 7
2.1.1 Bacterial diseases 8
2.1.2 Fungal diseases 9
2.1.3 Viral diseases 10
2.1.4 Nematodes diseases 10
2.1.5 Insect pest of V. unguiculata 11
2.2 Management of Diseases of Vegetable
Cowpea (V. unguiculata L. Walp) 11
2.2.1 Cultural control 11
2.2.2 Physical control 13
2.2.3 Chemical control 13
2.2.4 Biological control 15
CHAPTER 3:
MATERIALS AND METHODS 21
3.1 Pot Trial 21
3.1.1. Experimental site 21
3.1.2 Experimental design 21
3.1.3 Soil pasteurization 21
3.1.4 Soil analysis 22
3.1.5 Source of plant materials 22
3.1.6 Preparation of plant extracts 22
3.1.7 Application of plant extracts 23
3.1.8 Assessment of disease incidence and disease
severity 23
3.1.9 Assessment of growth parameters 24
3.2 Laboratory Experiments 24
3.2.1 Sterilization of glass wares and inoculation
chamber 24
3.2.2 Preparation of culture medium 24
3.2.3 Isolation of pathogens 25
3.2.4 Preparation of inoculum 25
3.2.5 Identification of pathogens 25
3.2.6 Pathogenicity test 26
3.2.7 Morphological and biochemical tests and
identification of the pathogens 26
3.2.8 Characterization of bacterial isolates 27
3.2.9 Colony features 27
3.2.10 Microscopic features 28
3.2.11 Biochemical reaction tests 28
3.2.12 Carbohydrate utilization tests 29
3.2.13 Identification of bacterial isolates 29
3.2.14 Statistical
analysis 30
CHAPTER
4: RESULTS AND DISCUSSION 31
4.1 Pot
Trial 31
4.2 Effect
of Plant Extracts on Black Vegetable Cowpea at 21st June 34
4.3 Effect
of Plant Extracts on Variety one at Late Planting at 19th July 36
4.4 Effect of Plant Extracts on Growth
Parameters at Early Planting
for
21st June 38
4.5 Effect
of Plant Extracts on Growth Parameters at Late Planting at 19th July 40
4.6
Effect of Plant Extracts on Brown Vegetable Cowpea at 20th July 43
4.7 Effect
of Plant Extracts on Brown Vegetable Cowpea at 17th August 45
4.8 Effect
of Plant Extracts on Growth Parameters at 20th July 47
4.9 Effect
of Plant Extracts on Growth Parameters at 17th August 49
4.10 Effect
of Plant Extracts on Variety Three at 7th August 51
4.11 Effect
of Plant Extracts on Ife Brown at 4th September 53
4.12 Effect
of Plant Extracts on Growth Parameters at 7th August 55
4.13 Effect
of Plant Extracts on Growth Parameters at 4th September 57
4.14 Effect
of Plant Extracts on Sowing Dates 59
4.15
Morphological and Biochemical
Characterization of Leaf Bacterial Spot
Pathogen
of V. unguiculata L. Walp 60
4.16 Pathogenicity
Test 61
4.17 Soil
Analysis of Experimental Site 64
4.18 Discussion 66
CHAPTER 5:
SUMMARY CONCLUSION AND RECOMMENDATIONS 71
5.1 Conclusion 71
5.2 Recommendations 72
References
Appendices
LIST OF TABLES
4.1: Effect of plant extracts on black vegetable
cowpea at 21st June 33
4.2: Effect
of plant extracts on variety one at late planting at 19th July 35
4.3: Effect of plant extracts on growth parameters
at early planting
for
21st June 37
4.4: Effect of plant extracts on growth parameters
at late planting at 19th July 39
4.5: Effect of plant extracts on
brown vegetable cowpea at 20th July 42
4.6: Effect
of plant extracts on brown vegetable cowpea at 17th August 44
4.7: Effect
of plant extracts on growth parameters at 20th July 46
4.8: Effect
of plant extracts on growth parameters at 17th August 48
4.9: Effect of plant extracts on variety three at 7th August 50
4.10: Effect of plant extracts on
Ife brown at 4th September 52
4.11: Effect
of plant extracts on growth parameters at 7th August 54
4.12: Effect of plant extracts on
growth parameters at 4th September 56
4.13: Effect
of plant extracts on sowing dates 58
4.14: Soil
analysis of experimental site 60
LIST OF FIGURES
1: Seeds of different varieties of Vigna ungiculata 62
2: Appearance
of bacteria leaf spot on the leaves 63
3: Pathogencity test on potted seedling. 63
LIST
OF PLATES
1.
Culture
of Xanthomonas
axonopodis on nutrient agar 62
2.
Confirmatory
pathogenicity test 62
CHAPTER 1
INTRODUCTION
Vigna
unguiculata L. Walp
also called vegetable cowpea is a dicotyledonous plant, which fits into the
family of Fabaceae. (Cronquist, 1988; Singh et
al., 2002; Ali et al., 2004). Vegetable
cowpea is a member of the Genus Vigna and belongs to the species unguiculata.
According to Ano and Ubochi (2008) it originated from West and Central Africa
but widely grown in Latin America and South-East Asia. It is an important
source of livelihood to Nigerians (Ali et
al., 2004). The immatured seeds, pods and leaves of legumes can be eaten fresh
(Dugje et al., 2009; Remison, 2012). V.
unguiculata is a staple food crop of significant economic importance in
Nigeria and worldwide (Magloire, 2005; Emeasor et al., 2007). Its cultivation provides social and economic
benefits; cash to small holder farmers due to its many uses (Langyintuo et al., 2003; Kristjanson et al., 2005; Langyintuo and
Lowenberg-DeBoer, 2006). It is also cultivated in South-Eastern, Nigeria
(Akpan, 2014; Ano and Ubochi, 2008; Martin et
al., 2009). This explains why Udealor (2002) and Ano, (2006) reported little
research has been carried out on this crop in Nigeria. Diouf et al., (2007), reported that vegetable
cowpeas are often neglected by developing communities. It is often cultivated by
poor farmers at the subsistence level, which does not meet up with the local
demands. It is mostly grown in the South-Eastern part of Nigeria such as; Enugu,
Abia, Anambra and Ebonyi States. FAO STAT, (2016) reports showed that V. unguiculata production in Nigeria
accounted for 58% of the total world’s output; making Nigeria the largest
producer with, an output of 2.9 million metric tons. In Nigeria, V. unguiculata are grown in large
quantities in Northern part of the country (Rachie, 1985). However, the
cultivation spread to the Western, Eastern and Southern parts of Nigeria (Emosiarue
et al., 2004). Areas in which production
are highest in Nigeria are the North East (703.13 metric tons from 641.03 ha),
followed by North West (519.51 metric tons from 1068,02 ha and North-Central
166.58 metric tons from 307,70 ha).
V. unguiculata seeds
contain about 25% protein, making it extremely valuable in areas where many people
cannot afford proteinous foods such as meat and fish (Lephale et al., 2012). The crop is a legume
cultivated mainly in the savanna regions of the tropics and subtropics of
Africa, Asia and America (IITA, 2010). Those regions contribute to 70% of global
production of V. unguiculata (Boukar et al., 2012). All over the world, over
12 million ha are used to produce V.
unguiculata with a yearly production of less than 6.9 million tons. The
largest V. unguiculata producers in
the world are Nigeria, Niger, and Brazil (FAO, 2018). Nigeria produced 2.1
million tons of this, making it the world’s major producer, followed by Niger
(650,000 metrictons) and Mali (110,000 metric tons) (IITA, 2004). According to
FAO (2012), about 7.56 million tons of V.
unguiculata are produced worldwide annually on about 12.76 million hectare.
Statistically 3.3 million tonnes of V.
unguiculata were produced globally in year 2000. Nigeria, being the major producer
and consumer, recorded 61% of V.
unguiculata production in Africa and 58% worldwide (IITA, 2015). Between
2000 and 2013 the country produced an average of 2.7 million metric tons of V. unguiculata (FAOSTAT, 2015). Out of
the V. unguiculata total output in
Africa, 52% is used as food, 13% as animal feed, 10% for seeds, 9% for others
and 16% is wasted. This crop is a source of food for man and animals, also
provides cash for farmers, in addition to benefits to farmlands through the decay
of roots residues which serves as manure and ground cover from vegetable cowpea’s
spreading habits (Quin, 1997). In Nigeria, V.
unguiculata is grown for its grains, leaves, green pods, anti-erosion crop and
stover.
The
nutrient composition of V. unguiculata leaves
changes as they grow (Bubenheim et al., 1990; Ohler et al.,
1996). The rainfall ranges between 1,150 – 1,500mm yearly and occurs mainly
between April - October with heavy rainfall in June and September. The plant
can be grown in a wide range of soils but does best on well-drained soil. Relative
humidity of (80 – 95%) are recorded within the rainy season and (20–50%) in the
dry season. The lowest and highest temperatures are 19 and 35°C, respectively, optimum
soil temperature for rapid germination of V.
unguiculata seed is above 18.3℃, but the minimum temperature to initiate
germination can be (7℃ - 14℃). The vegetative period of V. unguiculata ranges between 90–240 days. Most diseases occurs
regularly under high relative humidity, which links with high rainfall and
atmospheric temperature that are seen in humid forest of Southern Nigeria. V. unguiculata strives well in a wide
range of soil texture. The crop grows well in slightly acidic to slightly
alkaline (pH 5.5–8.3), sandy loam soils. The crop cannot thrive in water logged
soil and must be grown in well drained soils. V. unguiculata production is faced with
several limitations such as
pests and diseases (Ajibade and
Amusa, 2001). The influence of diseases
on V. unguiculata has led to huge
yield loss in the humid forest of Nigeria.
Over
65% of vegetable cowpea is produced in Africa with Nigeria and Niger producing
about 50% of the world supply (Henshaw, 2008). Many V. unguiculata varieties such as Vita-5; IT84E; IT84E-124;
IT84E-108 and IT84S-2246-4 have been established in Nigeria to suit the
different part of the country and consumer’s taste (Udohet al., 2005; Remison, 2012). Reports on early maturing erect and
semi-erect V. unguiculata varieties
example IT8ID-1228-10; IT8ID-1228-12; IT81D-1228-14; IT81D-1228-15 grown as
mono crop which can yield up to 18 t/ha green pods with 2-4 harvest, at 45 days
after planting (Remison,
2012). Early erect vegetable cowpea cultivation which begins flowering at 30
days after planting in the tropics has shown great importance in some dry areas,
because of its ability to withstand drought (Okaka et al., 2002). Fresh pods yield of V. unguiculata includes number of pods per plant, weight of pods and
number of seeds is the most significant attribute (Xu et al., 2010; Reddy, 2012).
It
contains protein, vitamin A, thiamin, riboflavin, iron, phosphorus and
potassium and a very good source for vitamin C folate, magnesium and manganese
and its nutritional value is per 100g (3.5) (a) Energy 197kj (47 keal) (b)
Carbohydrates 8.35g (c) Fat 0.4g (d) Protein 2.8g (e) Vitamins Vitamin A
equivalent (5%) 43g (f) Thiamine (B1) (9%) 0.107mg (g) Riboflavin (B2) 9%,0.11mg (h) Niacin (B3) 3%, 0.41mg (i)
Pantotheric acid (B5) 11%, 0.55mg (j) Vitamin (B6) 20%, 0.024mg (k) Folate (B9)
16%, 62 g (1) Vitamin C (23%), 18.8mg (m) Minerals, calcium (5%), 50mg (n) Iron
(4%), 0.47mg (o) Magnesium (12%), 44mg (p) Manganese (10%), 0.205mg (q)
Phosphorus (8%), 59mg ® Potassium (5%), 240mg (s) Sodium (0%), 4mg (t) Zinc
(40%), 0.37mg. Leaves and seeds of V.
unguiculata are cherished for their nutritive content for humans and
animals. Seeds contain 25% protein, macro and micronutrients, while the leaves also
contains cherished nutrients used mostly as fodder for livestock (Singh, 2006).
1.2 ECONOMIC IMPORTANCE OF VEGETABLE COWPEA
It
is an essential crop in the agriculture of African countries in particular for
the following reasons:
1.2.1 Provision of
nutritious food: V.
unguiculata can be used for food which includes its
leaves, unripe pods are nutritious. In some countries in Africa, some varieties
of V. unguiculata have been grown for
food purposes (Cook et al.,
2005). The seed is said to contain 24%
crude protein, 53% carbohydrates and 2% fat. (FAO, 2012). V. unguiculata leaves are important sourcesof vitamins, it is a
dual purpose crop grown for both the green pods and vegetable. V. unguiculata grain is eaten directly
after cooking, or as a part of meals made from other crops (Latunde-Dada,
1993). The fresh pods are snapped into small pieces to produce good dietary
balance (Uguru, 1996). V. unguiculata tender
pods are eaten both fresh and cooked. Food legumes particularly V. unguiculata is a source of protein,
carbohydrate and vitamins in the diet of many populations especially in
developing countries.
1.2.2 Used as feed for
animals: V.
unguiculata can be grown above ground plant parts,
processed into feed for animals examples are cattle, sheep and goats. (FAO,
2012).
1.2.3 V. unguiculata as cover crop:
The spreading indeterminate and semi-determinate bushy growth of V. unguiculata provides ground cover, which
destroys the development of weeds and guides against soil erosion by running
water during heavy rains, through theallelopathic compounds in the plant (Clark,
2007). It serves as ground cover in orchards and can be intercropped with cash
crops such as cotton. A whole ground cover helps to reduce hotness in the soil.
(Clark, 2007). Its long taproot, and wide vegetative spread plays a vital role for
erosion prevention and weed suppression.
1.2.4 Provision of cash: Trading
of fresh V. unguiculata leaves,
harvest provides opportunities for earning money. (Langyntuo, et al., 2003).
1.2.5 Used as forage: V. unguiculata can
be used as forage, hay and also silage. When used as forage, it should be grazed
after flowering (FAO, 2018). The plant will renew, if there are buds left after
defoliation. When used as silage, it shoud be mixed with sorghum, and maize
which provides sugar for fermentation (FAO, 2018). V. unguiculata is eaten as forage by deer. Birds which includes
pigeons, wild turkey, eat the seed. Some varieties of V. unguiculata are used specifically for wildlife purpose (Ball et al., 2007).
1.3
OBJECTIVES OF THE STUDY
The objectives of this study included
to:
1. isolate,
characterize and identify the casual organism for cowpea bacterial leaf spot.
2. compare
the efficacy of the plant extracts with the control in reducing the bacterial
spot disease.
3. conduct
pathogenicity tests both invivo and invitro.
4. determine
the efficacy of plant extracts on disease incidence, severity and yield.
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