INTERACTION OF Meloidogyne incognita WITH Botryodiplodia theobromae ON Manihot esculenta (CASSAVA) AND ITS BIOCONTROL

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INTERACTION OF Meloidogyne incognita WITH Botryodiplodia theobromae ON Manihot esculenta (CASSAVA) AND ITS BIOCONTROL

ABSTRACT

The Root-Knot Nematode (RKN), Meloidogyne incognita and fungus Botryodiplodiatheobromae, are important pests that cause yield losses in cassava and other crops. Chemicals have been used to manage these pests but with undesirable side effects. Information on pathogenicity of M. incognita, its interaction with B. theobromae and its biocontrol in Nigeria is very little. Therefore, pathogenicity of M. incognita on cassava, its interaction with B. theobromae and management with biocontrol agents were investigated.

A split–plot experiment was conducted in two cropping seasons with nematode-infested and denematised treatments (main-plot) on five cassava cultivars (sub-plots) to study pathogenicity in both field and pots. Two-week old potted sproutings were inoculated with 0, 1000, 10000 eggs of M. incognita per pot in four replicates in a 5 x 3 factorial experiment. Vegetative Growth (VG), galling index and yield-related traits were assessed using standard procedure. Interaction between M. incognita and B. theobromae on cassava was also investigated in pot and microplot field experiments. Two-week old sproutings of TMS 30572, TME 1 and Ofege cassava cultivars were each inoculated with 0, 1000 and 10000 M. incognita eggs, 5×105 spores mL-1 of B. theobromae and combined inoculation of M. incognita+B. theobromae per pot (r=4). Similar treatments were applied to sproutings in the microplots. Plants were assessed for VG, yield, percentage tuber rot and nematode reproduction. The assessment of Glomus mosseae and Paecilomyces lilacinus solely and in combination in the management of M. incognita was evaluated in pot and field studies following standard procedures. Plants were assessed for VG, yield, galling index and nematode reproduction. Data were analysed using descriptive statistics and ANOVA at p=0.05.

Meloidogyne incognita reduced by 35.0%, 30%, 18.8%, 54.3% and 53.0% for plant height, shoot weight, stem diameter, fresh tuber weight and number of tubers, respectively. Galling index increased with increase in inoculum density. In the interaction studies, sole inoculation with M. incognita reduced plant height (15.0%), fresh shoot weight (34.9%), number of tubers (35.6%) and tuber weight (32.0%). Inoculation with B. theobromae alone significantly reduced plant height (9.0%), fresh shoot weight (15.7%), number of tubers (22.7%) and tuber weight (25.0%). Combined effects of M. incognita and B. theobromae reduced plant height (25.6%), fresh shoot weight (44.6%), number of tubers (43.2%), tuber weight (72.2%) and increased tuber rot by 48.1% across cultivars. Paecilomyces lilacinus and G. mosseae reduced M. incognita population by 85.0% and 86.7% respectively; and, when added together, by 60.0%. Galling index was reduced by 66.6% and 66.5% respectively when P. lilacinus and G. mosseae were solely applied and when applied together by 35.7%. The use of P. lilacinus and G. mosseae increased VG by 30.4% and 26.7% and tuber weight by 55.9% and 58.3% respectively.

Meloidogyne incognita and Botryodiplodia theobromae reduced the growth, yield and quality of cassava. Applications of Paecilomyces lilacinus and Glomus mosseae have great potential in the management of Meloidogyne incognita in cassava production.

TABLE OF CONTENTS

PAGE

Title Page i

Abstract ii

Acknowledgements iv

2.1.1 Origin 7

2.1.2 Adaptation 7

2.2 Importance of cassava 8

2.3 Some Uses of cassava 9

2.3.1 Food for man 9

2.3.2 Animal feed 10

2.3.3 Pharmaceutical 10

2.3.4 Ethanol 10

2.3.5 Biodegradable plastics 11

2.4 Morphology of Cassava 11

2.5 Agronomy 14

2.6 Land Preparation and Planting 14

2.7 Cassava Production in Nigeria 15

2.8 International trade in Cassava 16

2.9 Nematode Pests of Cassava 16

2.9.1 Root-knot nematode (Meloidogyne spp.) on cassava 17

2.10 Economic losses due to root knot nematodes 19

2.11 Nematode interactions with other plant pathogens 21

2.12 Importance of nematode-fungi interaction 22

2.13 Taxonomy and importance of Botyrodiplodia theobromae 24

2.14 Biological Control of Pests 26

2.15 Biological Control of Nematodes 30

2.15.1 Arbuscular Mycorrhizal Fungi 32

2.15.2 Paecilomyces spp. 34

2.16 Effects of Carbofuran on plant-parasitic nematodes 37

CHAPTER 3: MATERIALS AND METHODS 39

3.1 Experimental Site 39

3.2 Sources of cassava cultivars 39

3.3 Nematode inoculum extraction and population estimation

Procedures 39

3.4 Extraction of nematodes from soil samples using extraction

tray method 40

3.5 Soil sterilization 40

3.6 Counting of Nematodes 40

3.7 Preparation of culture media 41

3.8 Isolation of Botryodiplodia theobromae from cassava tubers 41

3.9 Estimation of Botryodiplodia theobromae spores 42

3.10 Isolation and multiplication of Glomus mosseae 42

3.11 Source of Paecilomyces lilacinus 44

3.12 Inoculation of plants 44

3.13 Soil sampling 45

3.14 Data analyses 45

3.15 Experiment One: Pathogenicity of root-knot nematode

(Meloidogyne incognita) on cassava (Manihot esculenta) 45

3.15.1 Pot Experiment 45

3.15.2 Field Experiment 46

3.16 Experiment Two: Effects of the interaction between root-knot nematode (M. incognita) and Botryodiplodia theobromae on

growth, yield and quality of cassava 49

3.16.1 Pot Experiment 49

3.16.2 Micro-plot Experiment

3.17 Experiment three: Evaluation of Glomus mosseae and Paecilomyces lilacinus in the management of M. incognita

on cassava 53

3.17.1 Pot Experiment 53

3.17.2 Field Experiment 54

CHAPTER 4: RESULTS 57

4.1 Pathogenicity of Root-Knot nematode in cassava in pots 57

4.1.1 Effects of various inoculum densities of Meloidogyneincognita on vegetative growth of five cassava cultivars from inoculation to the twelfth month 57

4.1.2 Effects of various inoculum densities of Meloidogyneincognita on root galling and yield parameters of cassava across cultivars 58

4.1. 3 Pathogenicity of Root-Knot Nematode on cassava in the field 74 4.1.3.1 Effects of M incognita infection on Plant Height, Stem Diameter and fresh shoot weight of five cassava cultivars 74

4.2. Interaction of Meloidogyne incognita and Botryodiplodia theobromae on growth, yield and nematode reproduction on

cassava cultivars (Pot) 81

4.2.1 Effects of M. incognita and B. theobromae interaction on plant

height of three cassava cultivars 81

4.2.2 Effects of M. incognita and B. theobromae interaction on fresh

shoot weight of three cassava cultivars 84

4.2.3 Effects of M. incognita and B. theobromae interaction on stem

diameter of three cassava cultivars 87

4.2.4 Effects of M. incognita and B. theobromae interaction on yield

of three cassava cultivars 94

4.2.5 Effects of M. incognita and B. theobromae interaction on tuber rot severity and percentage tuber rot of three cassava cultivars 100 4.2.6 Effects of M. incognita and Botryodiplodia theobromae

interaction on Galling Index and Nematode Reproductive Factor on

three cassava cultivars 101

4.2.7 Effects of the main treatment on growth, yield, galling index

and reproductive factor on three cassava 107

4.3.1 Effects of Glomus mosseae, Paecilomyces lilacinus and Carbofuran on plant heights and stem diameter of three

cassava cultivar 117

4.3.2 Effects of Glomus mosseae, Paecilomyces lilacinus and

Carbofuran on the fresh shoot weight of Cassava 125

4.3.3 Effects of Glomus mosseae, Paecilomyces lilacinus and

Carbofuran on galling index and nematode population

on three cassava cultivars. 127

4.3.4 Effects of Glomus mosseae, Paecilomyces lilacinus and

Carbofuran on the yield of three Cassava cultivars 128

4.3.5 Effects of Glomus mosseae , Paecilomyces lilacinus and carbofuran on the number of nematodes on three cassava

cultivars. 133

4.3.6 Effects of Glomus mosseae, Paecilomyces lilacinus and

Carbofuran on vegetative parameters on the field 137

4.3.7 Effects of Glomus mosseae, Paecilomyces lilacinus and Carbofuran on the yield of Control of Cassava 139

4.3.8 Effects of Glomus mosseae, Paecilomyces lilacinus and Carbofuran on Galling Index and Meloidogyne incognita

reproduction on Cassava in a field. 142

5.0 CHAPTER 5: DISCUSSION 144

6.0 CHAPTER 6: SUMMARY AND CONCLUSIONS 154

REFERENCES 157

APPENDICES 185

CHAPTER ONE

INTRODUCTION

Cassava (Manihot esculenta Crantz), a perennial woody shrub of the Euphorbiaceae family, grown principally for its tuber, originated from Brazil and Central America, the two centers of the Manihot species (Jones 1959; Leon 1986). Cassava is the principal source of carbohydrate for man as well as a food reserve during the period of drought. Out of the 123 Manihot species reported, cassava is the most economically important. It is the crop with the highest total production in Africa, with 140 million tonnes across the continent in 2012. It contributes significant energy input to the population with an average 196 k cal/capital/day (FAO, 2012; Moyib et al., 2012).

Cassava is exclusively a tropical crop grown between latitudes 30oN and 30oS, between altitudes 0-2300 m and between temperatures 15oC and 40oC (Cock, 1985). However, Allem and Hahn (1991) and Omodamiro et al. (2007) have reported cassava growing in the semi-arid areas where cassava was not cultivated before. The crop is also potentially highly resilient to future climatic changes and could provide Africa with options for adaptation whilst other major food staples face challenges (Jarvis et al., 2012). It can also withstand drought and it yields on poor soils better than other crops (Dorosh, 1989).

Cassava is a major staple food crop in the tropics and is the most important food in Africa (Adekunle et al., 2005). In most of these countries, cassava is grown mainly for its starchy tuberous roots, which is a valuable source of cheap calories, particularly for the low-income earners (Allem, 2002; Adenle et al., 2012). Cassava is simply the most important staple food grown and consumed in the Western Region of Nigeria and it plays a major role in the effort to alleviate the country‘s food crisis (Fakoya et al., 2010). It accounts for approximately one-third of the staple produced in sub-Saharan Africa (FAO, 1999; Sewando, 2012). It is adapted to various climatic and edaphic conditions. It can also remain on the field up to two years from where it is harvested when needed; and thus, serves as food for household and food security (Cock, 1985; Dorosh, 1989). Both leaves and storage roots of cassava serve as feed for livestock (Sarma and Kunchai, 1989).

In some African countries, cassava is being more and more perceived, not only as a food security crop, but also as a raw material for various types of industries (Sewando, 2012). Cassava can be converted into a large number of products ranging from traditional and novel food products, to livestock feeds, ethanol and starch and its numerous derivatives. In such countries, there are concerted efforts on cassava development being initiated, sometimes with strong political support at the highest level (Kenyon et. al., 2006). For example, special presidential initiatives on cassava exist in Nigeria and Ghana to make cassava the engine for economic growth (Ukpongson et al., 2011). The Cassava Initiative of the government of Nigeria has projected to the limelight the multifarious uses of cassava; and as a consequence, it has become a viable export crop (Ukpongson et al., 2011).

The New Partnership for African Development (NEPAD) has recognized cassava as a crop which can reduce poverty in Africa (Sewando, 2012). In cassavagrowing households, approximately 26% of cash income from all food crops can be derived from sale of cassava. In such areas, cassava frequently forms the basis of cottage industries to produce cassava products for domestic consumption and local/export markets. Export of cassava, mainly through increased private sector awareness of international market opportunities for cassava chips and pellets as animal feed ingredients, is strengthening the value of the crop and provides another tool in the fight against poverty. Cassava production and processing provide employment and income for many rural people, especially women (Sarma and Kunchai, 1989).

The Food and Agricultural Organization‘s food outlook global market analysis (FAO, 2012) predicted that world cassava output will vigorously increase in 2013. In 2012, 282 million tonnes were estimated to have been produced worldwide (FAO 2012). The expected increased growth is expected to be more rapid in Africa where cassava remains a strategic crop for both food security and poverty alleviation Cassava is vegetatively cultivated and thus planting materials are derived from stems. Yields in farmers‘ fields range from 3 t/ha for local varieties (Moyo and Pelletier,

1989) to 40 t/ha for improved clones in Nigeria (Fermont et al., 2008; Njoku et al.,

2010). Despite the importance of cassava as a staple food in many developing countries, particularly in Africa, several production constraints such as poor soils, use of unimproved local varieties, land tenure and damage by pests and diseases, have kept the production of cassava below its full potential in Africa (FAO, 2000; Fakoya et al., 2010). Although a relatively hardy crop, the low average yield of cassava has been attributed to various factors. According to Campo et al. (2011), insect pests and plant diseases reduce cassava yields substantially, posing a threat to food security throughout the developing world.

Cassava is attacked by more than 50 bacterial, fungal, viral, mycoplasma and nematode agents (IITA, 1990, Coyne et al., 2004; Mohammed et al., 2012). These pathogens cause losses in crop establishment, lessen normal plant vigour, reduce photosynthetic efficiency or cause pre- and / or post-harvest stem and root rot (Lozano et al., 1981; IITA, 1990; Onyeka et al., 2005; Buensanteai and Athinuwat, 2012; Amodu and Akpa, 2012). The particular importance of pathological diseases in cassava lies in the fact that cassava is a long cycle crop (8-24 months), and it is vegetatively propagated. These favour easy multiplication and dissemination of the pathogens (Lozano et al., 1981).

Plant-parasitic nematode infection and damage on agricultural crops in Africa are widespread (Bridge and Muller, 1984; Bridge, 1988). The most important and widespread plant-parasitic nematodes with high damaging potential are, perhaps, the root-knot nematodes, Meloidogyne spp. which have been shown to cause 31- 87% reduction in top and root yield of cassava (IITA, 1985; Nwauzor, 1990), with 98% loss in extreme cases (Theberge, 1985, Coyne, 1994). Loss on a global basis due to nematodes in general has been put at 10% annually (Whitehead, 1998); a loss the hungry masses of the developing world cannot afford.

Plant-parasitic nematodes are among the pests that have been implicated in yield losses of cassava (McSorley et al., 1983; Jatala and Bridge, 1990). Many nematode species have been reported associated with cassava but few are reported to have caused economic damage to the crop (Coyne, 1994). Meloidogyne spp. has been identified as the main nematode species affecting cassava (McSorley et al., 1983, Jatala and Bridge, 1990). They are cosmopolitan and their worldwide distribution, extensive host ranges and involvement with fungi, bacteria and viruses in disease complexes rank them among the major plant pathogens affecting the world‘s food supply (McSorley et al., 1983). Of the various species, M. incognita (Kofoid and White) Chitwood and M. javanica (Treub) Chitwood are the most important on cassava (Jatala and Bridge, 1990) although, M. arenaria (Neal) Chitwood and M. hapla (Chitwood) have been recorded (Coyne and Talwana 2000) and are of major concern in Uganda

Among the diseases of cassava, root and stem rots are important in different ecozones of West Africa (Banito et al., 2010) and various pathogens have been implicated in cassava root rot in different environments and under different conditions. Among the commonly reported organisms are Phytophthora drechsleri Tuker (Booth, 1978; Theberge, 1985); Armillaria mellea (Vahl : Fr) Kummer and Rigidoporus lignosus (Booth, 1978; IITA, 1990); Rosellinia necatrix Prill. (Booth, 1978); Botryodiplodia theobromae (Akinyele and Ikotun, 1989; Onyeka etal., 2005, Banito et al., 2010); Sclerotium rolfsii (IITA, 1990; Banito et al., 2010); Fusarium (Bandyopadhyay et al., 2006) and Pythium (Poltroneiri et al., 1997, Banito et al., 2010). Studies under controlled conditions and in the field showed that root rot disease incidence was related to susceptibility of varieties and also by the presence of nematode in the soil (Okechukwu et al., 2005).

Root-knot nematodes have been the subject of many of the studies on biocontrol combinations (Meyer and Roberts, 2002; Hashem and Abo-Elyousa, 2011). Numerous microbes are antagonistic to plant–parasitic nematodes, and some of the organisms reduce pathogen populations and disease (Siddiqui and Mahmood, 1999). Development of natural resistance to nematicides by nematodes and the tendency to withdraw nematicides from the market led to the search for new methods of control (Brand et al., 2010). Biological control of nematodes with fungi is being investigated thoroughly (Brand et al., 2010). Of the microorganisms that parasitize or prey on nematodes or reduce nematode populations by their antagonistic behavior, fungi had important positions and some of them have shown great potential as biocontrol agents. Fungi continuously destroy nematodes in virtually all soils because of their constant association with nematodes in the rhizosphere. A large number of fungi are known to trap or prey on nematodes but the most important genera include Paecilomyces, Verticillium, Hirsutella, Nematophtora, Arthrobotrys, Drechmeria, Fusarium and Monacrosporium (Hashem and Abo-Elyousar, 2011). Arbuscular mycorrhizal fungi (AMF) are ubiquitous in the soil and are commonly symbiotic with the root systems of many crops, supporting shoot growth and phosphorus nutrition (Bethlenfalvay and Liderman, 1992; Barea and Jefferies, 1995). Glomus mosseae has been confirmed as a potential bio-control agent for M. incognita. The fungus significantly suppressed nematode reproduction and damage on cowpea (Odeyemi et al., 2010).

Effective control of plant pests including plant-parasitic nematodes, especially root-knot nematodes, will be enhanced if some of these biocontrol-fungal agents are used in integrated nematode management programmes. Despite huge losses in cassava production due to root-knot nematodes and tuber rots, very little attempt has been made to evaluate the interaction of root-knot nematodes and B. theobromae on cassava fungal rot disease complex. Though G. mosseae and P. lilacinus have been found to be able to suppress nematode populations in some other crops (Inbar et al., 1994; Elsen, 2002), work on cassava is lacking. The objectives of this work were, therefore, to:

growth and yield of cassava fungus-Botryodiplodia theobromae on growth and yield of cassava. agents in the protection of cassava against M. incognita

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