SCREENING OF SOME COMMON WEEDS FOR NEMATICIDAL ACTIVITIES AGAINST THE ROOT-KNOT NEMATODE INFESTATION ON TOMATO (LYCOPERSICUM ESCULENTUM L) IN PLATEAU STATE, NIGERIA
The screening of some common weeds for nematicidal activities on Meloidogyne incognita infestation on tomato (Lycopersicum esculentum L.) was carried out at the Botanical nursery and Biology Laboratory, Plateau State Polytechnic, Barkin ladi, Nigeria, with a view to establishing their potential for organic amendment or as trap weeds for the control of Meloidogyne incognita. The common weeds were surveyed and identified by a taxonomist based on botanical nomenclature and voucher number to include Ageratum conyzoides, Crotalaria breviflora, Crotalaria juncea, Crotalaria retusa, Crotalaria spectabilis, Euphorbia hirta, Portulaca oleracea and Tridax procumbens. The common weeds were uprooted and taken to the laboratory for extraction and Phytochemical screening using standard procedure. Phytochemical screening of the test plants revealed the presence of Alkaloids, Carbohydrates, Flavonoids, Glycosides, Saponins, Sterols, Tannins, and Terpenes, which suggest that they are the active ingredients against Meloidogyne incognita, acting separately or in combinations. The root exudates of test plants were also prepared and collected by growing young actively growing seedlings of the test plants in test tubes wrapped with black carbon paper for five days under lighted florescent bulbs. Egg masses from root galls of tomato plants was teased out and identified. The eggs were placed in the exudates of susceptible tomato test plants for 24 hours to stimulate hatching of eggs. The resulting juveniles from hatched eggs were transferred in various concentrations of 1g in 5mls, 1g in 10 mls and 1g in 15 mls of extracts extracted with ethanol using soxhlet extractor and exudates of the test weeds. The set up were observed after an interval of 6 hours, 12 hours and 24 hours for live and dead juveniles in an in vitro study. The results of the in vitro studies reveal that at exposure time of twenty four (24) hours at concentration of 1g in 5 mls all the seventeen (17) juveniles were dead. The effects varied with concentration of the extracts and exudates (P< 0.05) using analysis
of variance (ANOVA). The effects of the test plants in terms of reduction in nematode population of soil increase in growth and yield of susceptible tomato plants grown in soil amended with decomposed test plants at concentration of 5 kg, 10 kg and 15 kg to beds measuring 120 x 240 cm was determined. Decomposed test plants in soil amendments also significantly reduced nematode populations at the varying application rates. The effects of decomposed plant parts (DPP) had a minimum mean number of thirty six (36) galls per three (3) tomato plants and with highest mean number of fruit yield of two hundred and fifty six (256) per three tomato plants. The results thus confirmed that all the test plants are potentially viable trap weeds, and can be used in organic amendments of soils for the control of Meloidogyne incognita infecting tomato variety UC 82 B and should be employed as such. The test plants, most of which are legumes have the added advantage of enriching the soil through nitrogen fixation. These test weeds can be composted and incorporated in soil organic amendment as well as grown on fallow fields as trap or catch plants to reduce nematodes populations and increase growth and yield of crops susceptible to Meloidogyne incognita. Farmers can now substitute the use of synthetic chemical of high cost for these obnoxious weeds.
CONTENT TABLE OF CONTENTS
TITLE PAGE - - - - - - - - - i
DECLARATION - - - - - - - - ii
CERTIFICATION - - - - - - - - iii
ACKNOWLEDGEMENTS - - - - - - - iv
TABLE OF CONTENTS
LIST OF TABLES
LIST OF PLATES - - - - - - - - xi
LIST OF FIGURES - -
CHAPTER ONE INTRODUCTION
1.1 BACKGROUND TO THE STUDY - - - - - 1
1.2 STATEMENT OF THE PROBLEM - - - - 6
1.3 JUSTIFICATION OF THE STUDY - - - - - 7
1.4 AIM AND OBJECTIVES - - - - - - 8
1.5 HYPOTHESES - - - - - - - 9
1.6 SCOPE OF THE STUDY- - - - - - - 9
CHAPTER TWO LITERATURE REVIEW
SYMPTOMS AND DAMAGES OF ROOT-KNOT NEMATODES13
DISTRIBUTION OF ROOT-KNOT NEMATODES--18
SUSCEPTIBLE HOST RANGE OF ROOT-KNOT NEMATODES21
ECONOMIC IMPORTANCE OF ROOT-KNOT NEMATODES-25
2.6 CONTROL OF ROOT-KNOT NEMATODES - - - 27
2.6.1 Crop Rotation to Control Root-knot Nematodes - - - 28
2.6.2 Soil Steaming to Control Root-knot Nematodes - - - 29
2.6.3 Chemical Control of Root-knot Nematodes - - - - 29
2.7 ECOLOGICAL MANAGEMENT OF ROOT-KNOT NEMATODES 32
2.8 BIOLOGICAL CONTROL OF ROOT-KNOT NEMATODES - 36
NEMATODE SUPPRESSIVE CROPS
CHAPTER THREE MATERIALS AND METHODS
SURVEY AND COLLECTION OF WEEDS FOR IDENTIFICATION50
PREPARATION FOR PHYTOCHEMICAL SCREENING OF WEEDS52
Detection of Alkaloids------52
Detection of Carbohydrates------52
Detection of Flavonoids------52
Detection of Glycosides------53
Detection of Phenols -------53
Detection of Saponins------53
Detection of Sterols-------53
Detection of Tanins-------53
Detection of Terpenes------54
COLLECTION OF INFECTED TOMATO ROOT FOR ISOLATION
AND IDENTIFICATION OF ROOT-KNOT NEMATODES - 54
Isolation of Root-knot Nematodes-----54
Identification of Root-knot Nematodes----55
Used of Perineal Pattern Characteristic for Identification of
Root-knot Nematodes - - - - - - - 55
PREPARATION OF CRUDE AND ETHANOLIC EXTRACT OF
WEEDS - - - - - - - - 56
PREPARATION OF EXTRACT CONCENTRATION FOR IN
VITRO STUDIES - - - - - - 57
PREPARATION OF EXUDATE FROM THE WEEDS TO TEST
FOR NEMATICIDAL PROPERTIES - - - - 57
THE NEMATICIDAL EFFECT OF EXUDATES ON
MELOIDOGYNE INCOGNITA JUVENILES - - - 58
FIELD EXPERIMENTS TO CONTROL MELOIDOGYNE
INCOGNITA POPULATIONS- - - - - - 59
FIELD EXPERIMENT TO CONTROL MELOIDOGYNE INCOGNITA
BY VARYING THE CONCENTRATIONS OF THE DECOMPOSED
PLANT PARTS - - - - - - - 62
TEST WEEDS (PLANTS) GROWN ON INFESTED FIELDS TO MANAGE MELOIDOGYNE INCOGNITA POPULATIONS
CHAPTER FOUR RESULTS
SURVEY AND COLLECTION OF WEEDS FOR IDENTIFICATION66
THE PHYTOCHEMICAL ANALYSES OF THE TEST WEEDS
(PLANTS) - - - - - - - - 75
ISOLATION AND IDENTIFICATION OF ROOT-KNOT NEMATODES- 77
PREPARATION OF EXTRACT CONCENTRATION FOR IN
VITRO STUDIES - - - - - - - 79
EFFECT OF DIFFFERENT CONCENTRATIONS OF WEEDS
EXTRACT ON JUVENILES ORGANISMS - - - 82
Effects of C. breviflora Concentration on Juveniles Organisms --82
Effects of C. juncea Concentration on Juveniles Organisms ---85
Effects of C. retusa Concentration on Juveniles Organisms ---87
Effects of C. spectabilis Concentration on Juveniles Organisms --89
PREPARATION OF EXUDATES FROM THE WEEDS TO TEST
FOR NEMATICIDAL PROPERTIES - - - - 91
FIELD EXPERIMENT TO TEST EFFECT OF DECOMPOSED PLANT PARTS (DPP) ON MELOIDOGYNE INCOGNITA
POPULATIONS- - - - - - - - - 93
POT EXPERIMENT TO TEST EFFECT OF DECOMPOSED PLANT PARTS (DPP) ON MELIDOGYNE INCOGNITA POPULATIONS
ON L. ESCULENTUM PLANT - - - - - - 102
EFFECTS OF VARYING CONCENTRATIONS OF DECOMPOSED PLANT PARTS ON MELOIDOGYNE INCOGNITA POPULATIONS
ON L. ESCULENTUM PLANT- - - - - - 111
EFFECTS OF TEST WEEDS GROWN ON INFESTED FIELD
ON MELOIDOGYNE INCOGNITA POPULATIONS - - - 121
CHAPTER FIVE DISCUSSION
5.1 DISCUSSION- - - - - - - - 134
SUMMARY OF FINDINGS, CONCLUSION AND RECOMMENDATIONS
6.1 SUMMARY OF FINDINGS - - - - - - 141
6.2 CONCLUSION - - - - - - - 141
LIMITATIONS OF THE STUDY-----142
SUGGESTIONS FOR FURTHER STUDY ----143
CONTRIBUTION TO KNOWLEDGE----143
REFERENCES - - - - - - - 144
APPENDIX - - - - - - - - 160
CHAPTER ONE INTRODUCTION
BACKGROUND TO THE STUDY
The root-knot nematodes due to their frequency of occurrence and high levels of infestation with other pathogens have been recognized among plant nematodes as the major limiting factor in food production especially vegetables (Mohammed and Mashkor, 1990). Root-knot nematodes are plant parasitic nematodes from the genus Meloidogyne. They exist in soil and areas with hot climates or short winters; they cause approximately 5% of global crop loss (Adesiyan, Caveness, Adeniyi and Fawole, 1990).
Root-knot nematode larvae infect plant roots causing the development of root- knots or galls that drain the plant photosynthate and nutrients (Adesiyan et al., 1990). Infection of young plant may be lethal, while infection of matured plants causes decreased yield (Adesiyan et al., 1990). They injure plants by feeding on root cells with their needle-like mouthparts (stylets).The root system can become damaged to the point where the plant cannot properly absorb water and nutrients (Adesiyan et al., 1990). Root-knot nematodes first attracted attention in 1855 when it was found in a green house in England. Since then it has been a cause for concern over much of the world. Root-knot nematodes are cosmopolitan in distribution (Adesiyan et al., 1990).
The Meloidogyne spp infect more than 2,000 plant species and significantly reduce agricultural productions (Sasser and Freckman, 1987). After hatching from eggs, second stage juveniles invade roots of host plant, and migrate intercellularly to differentiating vascular regions. The nematodes then become sedentary and induce the formation of giant cells which act as the nutrient source for their development and reproduction (Jones, 1981; Williamson and Hussey, 1996). Between 2-12 giant cells are induced from pro-vascular cells in the differentiating vascular cylinder (Jones, 1981). They become multinucleate by repeated mitosis without cytokinesis and are filled with
metabolically active cytoplasm (Jones and Payne, 1978). There is an increase in the activities of many enzymes in giant cells, as well as in the rate of synthesis of Deoxyribose Nucleic Acid (DNA) and ribosomal RNA (Ribose Nucleic Acid) and protein, compared to that in the surrounding cells, which reflect the increased metabolic activity inside giant cells (Jones, 1981; Favery, Bedtold, Bouchez, Dalmasso and Abad, 1998). Formation and maintenance of giant cells requires continuous stimulus from nematodes oesophageal gland secretion (Williamson and Hussey, 1996),
The Meloidogyne incognita is endoparasite which must penetrate plant roots in order to establish a successful host-parasite relationship. Its juveniles readily penetrate host root near the apical meristem following attraction by root exudates (Wonang and Akueshi, 1997). Over the past 20 years plant and soil samples submitted to the University of Maryland Nematology laboratory routinely contained economically significant root-knot nematode populations. The most prevalent species observed was
M. incognita, and vegetables were the host plant most frequently encountered. In Maryland, M. incognita has been associated with damage to Zea mays L. (Corn), Cucumis melo L. (Muskmelo), Glycine max (L) Merr. (Soyabean), Ipomoea batatas L. Lam. (Sweet potato), Nicotiana tabacum L. (Tobacco), Lycopersicon esculentum Mill. (Tomato), Vicia villosa Roth. (Vetch), and Triticum aestivum L.(Wheat) (Jenkins, Taylor, Rahde and Coursen, 1957). Solanum tuberosum L. (Potato) and other vegetables grown throughout the region have experienced significant losses due to root- knot nematodes.
In agriculturally developing countries like Nigeria, Ghana, Kenya, it has been noted that among root-knot nematodes, Meloidogyne incognita causes immense damage to vegetable crops. Meloidogyne spp (root-knot nematodes), are important pests of Lycopersicon esculentum Mill. (Tomato) worldwide (Sasser, 1980; Jones, Jones, Stall and Zitta, 1991; Wonang and Akueshi, 1996). Four major species, namely
M. arenaria, M. hapla, M. javanica and M. incognita have been reported to infect tomatoes in the tropics (Sasser, 1979). These species cause gall or root-knot on infected plants. Other symptoms include stunted growth, wilting, and poor fruit yield. Infection by Meloidogyne incognita can increase root weight and decrease shoot weight (Fortnum, Kasperbauer, Hunt and Bridges, 1991). In the tropics estimated production losses of tomatoes due to Meloidogyne spp reach as high as 50 %. However, the overall impact on tomato is highly variable, as disease intensity is influenced by many biotic and abiotic factors (Sasser, 1980).
Diseases such as Meloidogyne root-knot affect cowpea adversely. Ehlers and Hall (1997) reported that these diseases could cause up to 64-80 % yield losses in cowpea production under severe conditions. Meloidogyne, the causal organism of cowpea root-knot disease is the most pathogenic species of nematodes to the crop (Khan, Khan and Khan, 1996). They are very numerous and adaptable to many soil ecology where they feed on roots, live and reproduce entirely within the roots of the crop.
The Meloidogyne spp are major pathogens of vegetables throughout the United States and the world, impacting both on the quantity and quality of marketable yield. In addition, root-knot nematodes interact with other plant pathogens, resulting in increased damages caused by other diseases. In susceptible plants, the population of the nematodes build up to a maximum, usually, as the crop reaches maturity (Shurtleff and Averre, 2000) and in some cases the plants die even before reaching maturity (Singh and khurma, 2007).
Three species of root-knot nematodes were known to be most prevalent in Nigeria. These are Meloidogyne incognita (Kofoid and White) Chitwood, M. javanica (Treub) and Meloidogyne arenaria (Chitwood) (Caveness, 1976, Chindo and Khan.1986), Meloidogyne arenaria, Meloidogyne incognita are generally known to be
the most common species in tropical and sub-tropical regions of the world. While root- knot nematodes alone are capable of causing severe plant injury and reduction in crop production, they are also often involved with pathogenic fungi and bacteria.These combinations often result in more than additive effects such as the breaking down of resistance or production of symptoms differing from those usually produced by other organisms alone. Such associations are sometimes referred to as disease complexes (Chindo and Khan, 1986).
Several control strategies such as host plant resistance, rotation with non-hosts, sanitation and avoidance, destruction of residual crop root, and judicious use of nematicides have been reported to effectively control root-knot nematodes (Becker and Koenning, 1998). However, the use of resistant varieties remains the most viable option, particularly for small-scale farmers with limited resources (Williamson, 1998). Even with these control strategies root-knot nematodes are often cited as major limiting factor of crop production. Despite their relative importance in the biology and growth and yield of crops, root-knot nematodes have not been fully addressed in Nigeria. Taking into account the world-wide distribution of root-knot nematodes, it is necessary to find out the most effective and feasible control measure. The use of chemical for nematode control on large scale is an expensive and impracticable operation (Akhtar and Malik, 2000).
Due to the phase out of methyl bromide and constraints of use of other fumigant nematicides, such as the length of the period following fumigant application when crops cannot be planted, growers developed an increase interest in non-chemical control options (Ferguson and Padula, 1994, Noling and Becker, 1994). Maryland potato growers met in fall in 1999 and expressed interest in cover crops, non-host rotation crops, and use of poultry manure in the potato cropping system.The growers
were especially interested in practices that had been effective, even experimentally, in other regions.
The use of chemicals (nematicides) which are the most effective method of controlling nematodes is, however, not economical, because these chemicals are very expensive particularly on large scale farming and most farmers cannot afford them.They are currenly being reappraised with respect to the environmental hazards and human health (Wachira, Kimenju, Okah and Mibey, 2009).
Indiscreminate use of synthetic pesticides for controlling nematodes is likely to give rise to phytoxicity, environmental pollution and nematode resistance. Unsafe use of pesticides may result in poisoning of humans and is a problem especially in developing countries (Conway, 1995; Yudelman, Ratta, and Nygaard, 1998). There is a need to develop naturally occurring nematicides, which may be less toxic to man and animals but as effective against nematodes of various crops as synthetic ones. Toxicity of root extracts of different plants against nematodes has been reported by many researchers (Onifade and Egunjobi, 1994). Control and management are two important concepts for dealing with nematodes problems. Nematode control refers to specific tactics applied to reduce or eliminate nematode population, while management describes efforts to reduce nematode numbers to non-damaging level through the application of severe control procedures in combination or in sequence (Thomason and Caswell, 1987).
Identification of plants with nematicidal or nematostatic properties facilitates safer, cheaper, practical and profitable control of nematodes through organic amendments with such plant residues or biological control by cultivation of such plants with nematicidal properties on heavily infected agricultural soils to bring the population of root-knot nematodes down to safe levels. Generally, in pest control, the method used must be of economic value, that is the increase in monetary value of the crop must be
more than enough to offset the cost of control measures. According to Taylor and Sasser (1978) the expected benefits should exceed the expenses by a ratio of three to one (3:1).
STATEMENT OF THE PROBLEM
Root-knot disease of tomato caused by Meloidogyne incognita is common throughout Nigeria. It is destructive in the savannah regions including Plateau State. Severe losses in tomato yields occur in fields infested with root-knot nematodes (Wonang and Akueshi, 1997). Tomato cultivars UC 82 B, Mermande Vfn, Mamade VF, Ronita, Ife l, Rossol, Jos local, Marzinino, Newyorker, Pusa early dwarf, Enterpriser and Nematex are commonly cultivated in Jos Plateau State and other parts of Nigeria. They have exhibited varying degrees of susceptibility/resistance to Meloidogyne incognita and M.javanica (Wonang and Akueshi, 1997).
Root-knot nematode Meloidogyne incognita is known to attack tomato in many different parts of the world including Nigeria; tomatoes have been regarded as most favourable host of root-knot nematodes causing significant yield losses (Dropkin, 1980). Yet, tomato is one of the most important vegetable crops grown throughout the world for consumption in various forms. All the four major species of Meloidogyne viz., M. incognita, M. javanica, M. arenaria, M. hapla and their known races attack tomato crops in outdoor as well as indoor cultivation. Studies have shown that root- knot nematodes can cause suppression in yield of tomato as high as 85 % (Taylor and Sasser, 1978; Sasser, 1979). Tomatoes being a cash crop in many parts of the world, control measures to safe the crop from root-knot disease are usually taken seriously especially as it is known to be highly susceptible to this disease. Several methods have been used for the control of root-knot nematodes on various crops including tomato, but the most spectacular of all is the use of chemicals nematicides. However, they are not much used by peasant farmers because they are expensive, toxic and persistent, and
require skill for their application (Umar, Muhammad and Okusanya, 2010; Umar and Chubado, 2009).
Root-knot nematodes impact heavily on beans production in Kenya (Kimenju, Muiru, Karonja, Nyongesa and Miona, 2004). Besides causing significant yield reduction, the nematodes interact synergistically with other plant pathogens resulting in disease complexes that impact more heavily on crop yield. Continuous cropping on the same piece of land further aggravates the nematodes menace as the population increases above the economic threshold level.
JUSTIFICATION OF THE STUDY
Nematodes are recognized as important agricultural pests and have been implicated in crop failure world wide especially in the tropical regions. They usually attack the roots, stems, leaves, flowers and even bulbs causing galling, lesion, stunting, poor development of the leaves and fruits, yellowing of the leaves, decrease in yield and increased susceptibility to pathogens and sometimes plant death. The use of chemicals (nematicides) which is the most effective method of controlling nematodes is, however, not economical; most farmers cannot afford them or lack the experience to handle them. These chemicals are hazardous if not properly handled. Dropkin (1989) gave some control methods to include sanitation, crop rotation/fallowing, soil amendments, solar pasteurization, but he equally stated that certain factors such as expenses and type of crops may limit their application in some cases. Taylor and Sasser (1978) stated that the major problem with crop rotation is that the rotation of resistant or immuned crops may not always be profitable as susceptible crops and also if weeds are not adequately controlled, the success of crop rotation is jeopardized. It is also land demanding.
The use of chemical agents is highly effective in controlling root-knot nematodes and other plant-parasitic nematodes. However, the majorities of the
fumigant-type nematicides are no longer available and are costly and difficult to apply properly by most farmers (Widmer, 2007). The non-availability of synthetic pesticides when needed by the farmers and adulteration and sale of expired products also compound the problem (Salako, 2002). Cases of side effects such as phytotoxicity, accidental poisoning of the farmers leading to ill-health and residual effect such as soil toxicity and water pollution have been reported too (Pimental and Porking, 1980).
Organic amendments have been suggested and used in the control of root-knot nematodes in crop fields. Amendments such as Cow dung, Poultry manure and neem leaves have been used in different crop fields to control Meloidogyne species (Egunjobi, 1992, Umar and Jada, 2000). Under this context, the use of plants with nematicidal properties in land fallowing or organic amendment of soils can be effective, cheaper and safer. It is against this background that the present investigation has been designed.
AIM AND OBJECTIVES
The aim of this study is to survey some coomon weeds for nematicidal properties against root-knot nematodes (Meloidogyne incognita) with a view to integrating them in the control of the nematodes, through organic amendment or as trap or catch weeds during fallowing.
The specific Objectives are:
1. Collection and identification of common weeds
2. To determine their active ingredients (Phytochemical screening)
3. Isolation and identification of Nematodes from diseases plants
4. In vitro control of the Plant Parasitic nematodes using extracts from weeds
5. To determine the efficacy of decomposed weeds as organic amendment of soil in the control of infecting susceptible tomatoes.
6. To determine the efficacy of the identified weeds in the control of nematodes through their incoporation as trap or catch weeds during fallowing of field infested by root-knot nematodes
1. There is no significant difference between the mortality of Meloidogyne incognita treated with common weeds extract and exudates and those not treated with common weed extract and exudates.
2. There is no significant difference between the phytochemical compositions of common weeds extracts with nematicidal properties and those without nematicidal properties.
3. There is no significant difference between the mortality of Meloidogyne incognita treated with decomposed common weeds with nematicidal properties and those not treated with the decomposed common weeds with nematicidal properties.
4. There is no significant difference between Meloidogyne incognita mortality in infested field fallowed with common weeds with nematicidal properties and those without common weeds with nematicidal properties.
5. There is no significant difference between the growth and yield of susceptible tomato plants grown in soil treated for the control of root-knot nematodes and those on untreated control fields or soil.
SCOPE OF THE STUDY
The scope of this study was limited to screening of common weeds found on the plateau for their nematicidal potential using the root-knot nematodes as test organisms in an in vitro trial. The study also encompassed in vivo trials involving the utilization of varying quantities of decomposed test plants in organic amendment of soil infested with root-knot nematodes. A susceptible variety of tomato to root-knot nematode was grown in the soil as experimental plant. Also the study covered phytochemical screening of extracts of common weeds utilized for the trial to determine their bioactive constituents..