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PROYECTO FIN DE MÁSTER EVALUATION OF FOUR BOTANICAL FUNGICIDES AND SITUATIONAL REVIEW ON VIGNA UNGUICULATA (L. WALP.) ANTHRACNOSE PATHOGEN, COLLETOTRICHUM SPP. SACC & MANG. AUTOR: ENSEÑANZA: DIRECTOR/ES: PONENTE: FECHA: UNIVERSIDAD DE ZARAGOZA VITUS IKECHUKWU OBI Masters Degree On “Introduction To Research In Agricultural Science And Natural Environment Juan José Barriuso Vargas Joaquín Aibar Lete 29/09/2011 i DEDICATION This work is dedicated to my lovely spouse, Theresa and children, Chike and Chinenye, for their wonderful companion and co-operation in the course of the work. ii ACKNOWLEDGEMENT To God almighty be the glory for making this work the success it is today. To my project supervisor Professor (Dr.) Juan Jose Barriuso I thank so much for accepting to act in this capacity. The great role played in both the course of the project and the masters’ programme as whole by Dr. Jesus Carlos Romea of the MECOHISA cannot go undocumented. He was like an engineer as far as the programme was concerned and was always available to offer his wealth of experience in the successful completion of the project. Dr. Jesus Carlos accept my wholesome gratitude and may God almighty continue to grant you with all your heart desires according to His will, Amen. To my family members I owe a lot as regards this work. I recognize the special effort of my spouse, Theresa Chinwe Obi, for her spirited care and encouragement in the course of the study. Chike and Chinenye, my kids also aided in their own way during the study. I appreciate all their understanding during the period. iii ABSTRACT The completion of this research involved the revision of about seventy one referred subject related journals and academic materials. The parasitic organism Colletotrichum destructivum O`Gara was associated as the actual causal pathogen of cowpea anthracnose. Of all the twenty different pathogens linked with the various cowpea fungal diseases, in this work, only Colletotrichum (C. destructivum) was found to have the virulence and propensity of afflicting a 100% infection on a single susceptible cowpea crop each at a given pathogenic situation. Twenty Colletotrichum species along with their specific primary hosts were identified in this work. The study also provided eighteen plant families under the pathogenic affliction of Colletotrichum identified alongside eighteen different plant families representing the entire plants and plant materials screened for biofungicidal properties within a span of eleven years. The screened botanicals of four plants of Azadiractha indica, Cymbopogon citratus, Ocimum gratissimum, and Xylopia aethiopica were effective in reducing the spore germination and radial growth of Colletotrichum destructivum O`Gara in vitro and the growth of the pathogen in vivo. Cold water botanicals of C. citratus were the best in reducing the growth of the pathogen in vitro and in checking the spread of anthracnose disease of cowpea in vivo. An evaluation with cowpea (Vigna unguiculata) indicated the extracts applied before or after pathogen inoculation to be significantly effective in reducing the size of pathogen induced lesion. iv TABLE OF CONTENTS Page Dedication. ----------------------------------------------------------------------- i Acknowledgement. ------------------------------------------------------------- ii Abstract. -------------------------------------------------------------------------- iii Table of content. ---------------------------------------------------------------- iv List of table. ---------------------------------------------------------------------- v i i List of figures. ------------------------------------------------------------------- ix CHAPTER ONE . --- --- ------------------- ----------------------------- ---- ---- - 1 1.0 Introduction. ----------------------------------------------------------------- 1 CHAPTER TWO . --------------------------------------- ------ ---------- ---- -- -- 2 2.0: Literature review. --------------------------------------------------------- 2 2.1: Cowpea crop. -------------------------------------------------------------- 2 2.2: Colletotrichum and other phytopathogens of cowpea. ------ 7 2.3: Colletotrichum lindemuthianum. ------------------------------------- 9 2.4: Colletotrichum destructivum. ------------------------------------------ 11 2.5: One disease two pathogens. ----------------------------------------- 12 2.6: Anthracnose and the acervuli. ---------------------------------------- 17 2.7: The genus Colletotrichum. --------------------------------------------- 21 v 2.8: Systematic on Colletotrichum species. --------------------------- 23 2.9: Management of anthracnose problems of Vigna unguiculata 26 2.10: Chemical plant disease management. -------------------------- 28 2.11: The botanicals in plant disease management. --------------- 30 CHAPTER THRE E . --- ------------ ------------------------------ ----- --- - -- --- 37 3.0: Materials and methods. ------------------------------------------------ 37 3.1: Consulted material. ----------------------------------------------------- 37 3.2: Botanical materials. ----------------------------------------------------- 37 3.3: Botanical extractions. -------------------------------------------------- 43 3.4: Isolation and identification of pathogen. -------------------------- 43 3.5: Effect of extracts on spore germination. -------------------------- 44 3.6: Effect of extracts on colony growth of pathogen. -------------- 45 3.7: Field evaluation of extracts and Benomyl. ------------------------ 45 3.8: Experimental design. ---------------------------------------------------- 46 3.9.1: In vitro experiment. ---------------------------------------------------- 47 3.9.1.1: Botanical evaluation on pathogen spore germination. ---- 47 3.9.1.2: Botanical evaluation on pathogen colony growth. ---------- 47 3.9.2: In vivo experiment for botanical effectivity on disease development. ------------------------------------------------------------------- 49 vi CHAPTER FOUR . ------------------------------------- ---- ------------------ - -- - --- 50 4.0: Results. -------------------------------------------------------------------------- 50 4.1: Effect of Azadiractha Treatment on Spore Germination, Colony Growth and Sporulation Density. ----------------------------------------------- 51 4.2: Effect of Xylopia Treatment on Spore Germination, Colony Growth and Sporulation Density. ----------------------------------------------- 52 4.3: Effect of Cymbopogon Treatment on Spore Germination, Colony Growth and Sporulation Density. ----------------------------------------------- 54 4.4: Effect of Ocimum Treatment on Spore Germination, Colony Growth and Sporulation Density. ----------------------------------------------- 55 4.5: Effect of Plant Extracts and Benomyl Treatments on Vigna unguiculata Disease Development by Colletotrichum spp. ------------- 55 4.6: Effect of plant Extracts and Benomyl on in vivo Sporulation Density of Colletotrichum Spp. -------------------------------------------------- 56 4.7: Effect of treatments on pathological activities of Colletotrichum spp. ------------------------------------------------------------------------------------- 59 4.7.1: Treatments effect in vitro on three pathological activities of Colletotrichum spp. ----------------------------------------------------------------- 60 4.7.2: Effect of four botanical extracts and Benomyl fungicide treatment on Vigna unguiculata diseases development. ----------------- 61 CHAPTER FIVE . --- --------------------------------------------------------- --- - - -- 70 5.1: Discussion. --------------------------------------------------------------------- 70 5.2: Conclusion. --------------------------------------------------------------------- 76 BIBLIOGRAPHY. ------------------------------------------------------------------- 78 vii LIST OF TABLES Table 1: Relative contributions by geographical area to scientific publications on cowpea diseases. -------------------------------------------- 4 Table 2: Publications on cowpea disease according to pathogen groups. ------------------------------------------------------------------------------- 5 Table 3: Publication on cowpea disease control according to types- 6 Table 4: Colletotrichum and other fungal pathogen of cowpea. ------ 8 Table 5: Morphological and growth characteristics of Colletotrichum destructivum and Colletotrichum lindemuthianum. ----------------------- 14 Table 6: Morphological and growth characteristics of LAR 056, three isolates of Colletotrichum destructivum and an isolate of lindemuthianum. ------------------------------------------------------------------ 16 Table 7: Some species of Colletotrichum and their specific hosts.--- 20 Table 8: Plant families under the affliction of Colletotrichum. --------- 22 Table 9: Plant families screened for biofungicidal properties. -------- 33 Table10: Biological classification on Neem plant. ------------------------ 39 Table 11: Biological classification on Bush pepper plant.---------------- 40 Table 12: Biological classification on Lemon grass plant.--------------- 41 Table 13: Biological classification on Scent leaf plant. ------------------ 42 Table 14: In vitro experimental design. -------------------------------------- 48 Table 15: In vivo experimental design. -------------------------------------- 48 Table 16: Effects of extracts (HWE) in vitro on C. lindemuthianum. - 51 Table 17: Effect of extracts (HWE&OE) in vitro on C. lindemuthianum. ------------------------------------------------------------------ 52 viii Table 18: Effect of extracts on cowpea anthracnose disease. ------ 53 Table 19: Effect of Ocimum Treatment on Spore Germination, Colony Growth and Sporulation Density. -------------------------------- 54 Table 20: Effect of Plant Extracts and Benomyl Treatments on Vigna ungiculata Disease Development by Colletotrichum spp.---- 58 Table 21: Sporulation Density at Indicated Treatment Time.--------- 59 Table 22: Treatment effect in vitro on the three pathological activities of Colletotrichum spp. --------------------------------------------- 61 Table 23: Effect of four botanical extracts and Benomyl treatments on Vigna unguiculata anthracnose disease development. ----------- 64 5 One important fungus induced pathogenic problems of cowpea is the anthracnose. Anthracnose disease of cowpea has long been associated with the species of Colletotrichum lindemuthianum (Sacc. & Magn.) Bri. & Cav. (Adebitan and Ikotun, 1996; Amadioha and Obi, 1998; Amadioha, 2003), and relatively of recent with the Colletotrichum destructivum O’Gara (Latunde- Dada et al., 1996; Allen et al., 1998; Akinbode and Ikotun, 2008). Whichever way the anthracnose of Colletotrichum species affects the above ground parts with the products of water soaked lesion in all tissues of the cowpea crop (Akinbode and Ikotun, 2008). According to Adebitan and Ikotun (1996) cropping pattern affects significantly the incidence of anthracnose on cowpea at various stages of the crop growth in a season. Cowpea anthracnose pathogen is a seed borne fungus, usually found on soil surface or plant debris (Amusa et al., 1994; Fokung et al., 1997; Akinbode and Ikotun, 2008). It thrives for at least 2 years on diseased stem tissue either on the soil surface or beneath the soil (Akinbode and Ikotun, 2008). Lush cowpea growth resulting from closer spacing, as indicated by Adebitan and Ikotun (1996), favored reduced air circulation, promoted higher humidity, prolonged dew periods and allowed cooler soil surface temperatures. All these Table 2: Publications on cowpea diseases according to pathogen groups (1995-2000). Group of pathogen Number of papers Percentag e of papers (%) Bacteria 21 6.10 Fungi 122 35.6 Nematodes 69 20.10 Parasitic plants 19 5.50 Viruses 112 32.7 Source: Emechebe & Lagoke (2002) 6 microclimatic changes as influenced by the exuberant cowpea vegetation they inferred are considered favorable for the development and progress of anthracnose disease on cowpea. Cowpea exhibits anthracnose systems within 35 to 40 days after planting susceptible varieties in the form of lesions as small angular brown spots on the leaf petiole, the lower surface of leaves and leaf veins (Adebitan and Ikotun (1996). These spots according to these scientists later coalesced to produce a brick-red to brown discoloration of the leaf. Table 3: Publication on Cowpea Diseases Control According to Types (1995-2000) Control type Number of papers Percentage of papers (%) Biocontrol . 17 11.49 Botanicals . 11 7.43 Cultural 22 14.87 Host Pest Resistance (HPR). 72 48.65 Pesticidal . 26 17.57 Source modified from: Emechebe & Lagoke (2002). 7 2.2 COLLETOTRICHUM AND OTHER PHYTOPATHOGENS OF COWPEA Cowpea crop has some other old and new fungal pathogens that influence its existence, reproduction and survival. Each of these pathogens has regions of interest on a whole cowpea plant (table 4.). Some fungal pathogens attack and infect the roots of the crop (Fernando and Linderman, 1997), stem (Bankole and Adebanjo, 1998, Smith et al., 1999a) leaves (Amadi, 1995, Santos et al., 1997 and Emechebe and Lagoke, 2002), pods and fruits (Munoz and Tamayo, 1994, Roy and Ratnayake, 1997), seeds/seedlings (Smith et al., 1999b, Aveling and Adandonon, 2002), whole plant parts (Akinbode and Ikotun, 2008). Interestingly of about twenty different pathogens associated with various cowpea crop fungal diseases, as indicated in this work, it is worthy to note that the Colletotrichum is the only one that has the potential to affect every part of its host (fig 2). From table 4, it is also obvious that about 30% of the fungal pathogens attack the foliar part of the crop, 25% on the stems, 15% on the roots, 10% on the pods /fruits, 25% on the seeds/seedlings and mere 10% on the whole parts of the plant. Incidentally, while the other fungal pathogens have the possibility each of attacking only about 20% of a stand, the Colletotrichum spp. (C.destructivum and C.truncatum) have the propensity of affecting 100% of a single crop each at a given situation. Fig 2: Various organs of Colletotrichum infected cowpea crop: A = cowpea seedling, B =cowpea leaf, C =cowpea pods, D= cowpea grains; E = Colletotrichum conidiophores and conidia. Adapted Source: Http://www.infonet biovision.org/default/ct/120/crops. B E A C D 8 Table: 4.Colletotrichum and Other Fungal Pathogens of Cowpea ( Vigna unguiculata). Pathogen Disease Organs affected Cited Ref. Alteneria cassiae Juria &Khan Alternaria leaf spot Foliar Grange and Aveling(1998) Cercospora canescens Ellis & Martin Cercospora leaf spots Foliar Amadi (1995) Choanephora cucurbitarum (Berk & Rav.) Choanephora pod rot Pods /fruits Munoz and Tamayo(1994) Colletotrichum dematium (Pers. ex Fr.) Colletotrichum stem disease Stem Smith et al., (1999a) Colletotrichum destructivum O`Gara Anthracnose Every part Akinbode &Ikotun(2008) Colletotrichum capsici (Syd.)Butl. & Bisb. (=Colletotrichum truncatum (Schw.) Brown blotch All parts Latunde-Dada et al ., (1999); Latunde-Dada and Lucas (2007) Fusarium oxysporum f.sp tracheiphilum Fusarium wilt Seedlings Smith et al., (1999b) Fusarium oxysporum f.sp vasinfectum (E.F.Smith)Synd & Hans Fusar ium wilt Seedlings Ushamlini et al., (1997a) Macrophomina phaseolina Macrophomina blight Seedlings(severe mortality) Ratnoo e t al., (1997) Mycospharella cruenta Latham.(Anormoph of Pseudocercospora) Pseudocercospra leaf spots foliar Emechebe &Lagoke(2002) Phomopsis longicola Phomopsis pod spot Pods/fruits Roy and Ratnayake(1997) Protomycopsis phaseoli Ramak&Subram.(=Entyl oma vinae Batista) Leaf smut Foliar Santos et al., (1997) Pythium aphanidermatum (Edison) Fitz Pythium soft rot Stem Bankole and Adebanjo(1998) Pythium ultimum Damping of(pre/post) Seed/seedling Aveling and Adandonon(2000) Phytophthora cactorum (Leb. &Chon.) Schroet. Red stem canker Stem/root Fernando and Linderman(1997) Phytophthora vignae Puress. Phytophtora stem rot Stem/root Fernando and Linderman(1997) Sphaceloma sp.(Anamorph of Elsinoe phaseoli Jenkin) Sphaceloma scab. Hypocotyls & epicotyls Emechebe &Lagoke(2002) Sclerotium rolfsii Sacc. (Teliomorph: Corticum rolfsii Curzi). Basal stem rot/wilt Stem Muqit et al., (1996) Uromyces appendiculatus (pers.) Unger (=U.vinae Barclay). Brown rust Foliar Heath (1997) Thanatephorus cumeris (Frank)Donk(=Rhizocto nia solani Kuhn) Web blight Root (root rot /seedling disease complex Root/seedling Emechebe &Lagoke(2002) 9 2.3 COLLETOTRICHUM LINDEMUTHIANUM Colletotrichum lindemuthianum (Sacc. and Magn.) Bri. And Cav. is one of the species of Colletotrichum being linked with the anthracnose pathogenic problem of cowpea. Anthracnose of cowpea has been described as the major disease of cowpea (Vigna unguiculata (L.)Walp), causing severe damage and lose under low temperature, high humidity and free moisture (Adebitan and Ikotun, 1996; Emechebe and Lagoke, 2002). In an infected cowpea crop the anthracnose symptoms first appears at thirty five to forty days after planting in the form of lesions as small angular brown spots on the leaf petiole, the lower surface of leaves and leaf veins of cowpea grown under different cropping patterns (Adebitan and Ikotun, 1996). These various spots created later coalesced to produce a brick red to brown discoloration of the entire leaf. Symptoms are usually delayed until production of flowering buds. Cropping pattern affects significantly, according to Adebitan and Ikotun, (1996) the incidence of anthracnose on cowpea at various stages of the growth. For instance anthracnose incidence of severity have been reported to be lower in the intercrop relative to the sole crop while reduction in both inter and intra row spacing resulted in an increase in the incidence and severity of anthracnose(O`Connell et al., 1993; Adebitan and Ikotun, 1996; Emechebe and Lagoke, 2002). Some scientists attributed the higher diseases incidence and severity values obtained on monocropped cowpea farming to lack of physical barriers provided in the intercrop. Adebitan and Ikotun, (1996) reported that up to forty to forty nine percent reduction in disease reduction can be obtained in cowpea intercropped system. Crop treatment with some phosphorus (P2O5) fertilizer has also been reported to reduce the severity of anthracnose problem when compared with crops without any fertilizer application (O`Connell et al., 1993; Emechebe and Lagoke, 2002). These same researchers reported that weed free plots of cowpea crops 10 can decrease incidence of anthracnose by about 56% and severity reduction by about 43% when compared to weed infested cowpea plots. The reduction in the possibility of infection has been reported in an intercropped cowpea by Adebitan and Ikotun (1996) who attributed the no host crop as being responsible due to their ability to slow down the dispersion and movement of spores and pathogen propagules within the cropping system. Adebitan and Ikotun, (1996) were of the view that the conidia which fall on a no host are eventually lost, resulting in a slower increase of disease in an intercropping system. Other observed mechanisms for disease reduction on intercropped stands as deduced by Adebitan and Ikotun, (1996) are: (a) that in a pure stand of cowpea crops with uniform susceptibility to species of Colletotrichum, for example, the replacement of a portion of these crops by resistant ones, like maize for example, reduces the amount of inoculums available for subsequent dispersal within the stands. (b) That the resistant crop may interfere with the passage of inoculums between crops. This is so, especially in the case of air borne diseases where the foliage of the resistant crop could act as a trap for the spore or potential propagules. This would subsequently reduce the number of propagules available for infecting the susceptible crop, and (c) that the resistant host provides an unsuitable environment for the development of the disease within the immediate or extended cropping system. As part of the observation on the Colletotrichum species effect on cowpea within a cropping systems, Adebitan and Ikotun (1996) reported that the closer the rows were together the greater the possibility of infection by the pathogen and that the disease was more severe on cowpea crops grown in rows which were more closely spaced than those which were grown in wider rows. Narrower plant spacing between and within rows led to increased incidence and disease severity. This according to Adebitan and Ikotun (1996) indicates that as a cowpea crops become more crowded together per given area, especially in the monocropped, the tendency for crop to contact increase. As a result the micro environment within the crop becomes more moist and thus more 11 conducive for disease development (Adebitan and Ikotun, 1996).Grain yield loss by 35-50% could be observed in Colletotrichum lindemuthianum susceptible cowpea cultivars (Amadioha and Obi, 1998; Amadioha, 2003). 2.4 COLLETOTRICHUM DESTRUCTIVUM There has been scientific suggestions that the cowpea anthracnose pathogen be regarded as species that is distinct from Colletotrichum lindemuthianum (Emechebe and Lagoke, 2002), a phytopathogens also associated with the Phaseolus bean anthracnose (Amadioha and Obi, 1998). Colletotrichum destructivum is the other pathogen of the species being associated with the anthracnose disease of cowpea. According to Akinbode and Ikotun, 2008; Allen et al., 1998, anthracnose disease of cowpea (Vigna unguiculata (L.) Walp) is initiated by Colletotrichum destructivum O`Gara. The pathogen is described as seed borne fungus which can be found on soil surface or plant debris (Amusa et al., 1994; Fokung et al., 1997), and can survive for at least two years on diseased stem tissues either on the soil surface or beneath (Akinbode and Ikotun, 2008). Colletotrichum destructivum is hemibiotrophic pathogen which is associated with a whitish fluffy mycelia colour (Akinbode and Ikotun, 2008). Ricinus communis plant extracts has been found to encourage the growth and survival of this pathogen instead of its inhibition for which it was bio-assayed. This pathogen C. destructivum sporulates readily on infected cowpea at localized infection foci and produce symptom within 96hrs of inoculation (Latunde-Dada et al., 1999). In the note of Latunde-Dada et al., (1999) the anthracnose disease of cowpea induced by Colletotrichum destructivum O’Gara was erroneously, in the past, ascribed to Colletotrichum lindemuthianum (Sacc. and Magn.) Bri. and Cav. The pathogen gains ingress into the host by elaborating from the melanized 12 aspersorium, an infection peg, which penetrates the cuticle directly to initiate infection (Latunde-Dada et al., 1999). According to Akinbode and Ikotun (2008) a weekly or biweekly application of Benomyl are effective against C. destructivum pathogen. They also observed in their investigation that an application of phosphorus fertilizer gave lowest severity of C. destructivum. Fungicide application and an integrated control system are some of the efforts geared towards the management of problems from C. destructivum in cowpea crop reported Akinbode and Ikotun (2008). Only of recent Colletotrichum destructivum has also been linked to Colletotrichum higginsianum as synonyms. This, according to Sun and Zhang (2009) has been supported in scientific investigations where the sequences of the rDNA ITSxs region of C. destructivum isolates from cowpea were identical with 100% similarity to that of isolates of C. higginsianum originating from cruciferous plants. 2.5 ONE DISEASE TWO PATHOGENS Two phytopathogens, Colletotrichum lindemuthianum (Saccardo & Mangnus) Briosi & Cavara and Colletotrichum destructivum (O`Gara) have at different times and space been allegedly linked with the anthracnose of cowpea (Vigna unguiculata (L.) Walp). But recently, based on many scientific investigations and findings on detailed, physiological and morphological characteristics of the different organisms and in comparism to the characteristics of the diseases inflicted, mycologist of the phytopathology are already on to the distinction and proper classification of these agents. In the works of Sheriff et al., 1994; Adebitan and Ikotun, 1996; Amadioha and Obi, 1998; Amadioha, 2003; Adebanjo and Bankole, 2004, the anthracnose of cowpea was linked with the Colletotrichum lindemuthianum as the sole causal organism and no mention was made of Colletotrichum destructivum. But anthracnose disease of cowpea according to Latunde-Dada et al., (1999) was erroneously ascribed to the Colletotrichum lindemuthianum pathogen. 13 There have been numerous scientific calls that the cowpea anthracnose pathogen be regarded as a species that is completely distinct from the Colletotrichum lindemuthianum, which instead should be restricted to as a causal agent responsible for the anthracnose of kudzu bean (Phaseolus vulgaris L.) (Emechebe and Lagoke, 2002). Incidentally researchers in the field of plant pathology have before now also associated the anthracnose of Kudzu bean with the same Colletotrichum lindemuthianum pathogen. In their work Amadioha and Obi (1998) documented Colletotrichum lindemuthianum as the causal organism responsible for the anthracnose disease in both cowpea (Vigna unguiculata (L.) Walp) and Kudzu bean (Phaseolus vulgaris L.). The anthracnose disease of cowpea according to Latunde-Dada et al., (1996) is caused by a species of Colletotrichum which produces ovoid shaped conidia that are not of Colletotrichum lindemuthianum but of the Colletotrichum destructivum. They indicated that the initial accreditation of the cowpea anthracnose to the Colletotrichum lindemuthianum was faulty since the pathogen is more characterized with the anthracnose of Kudzu bean (Phaseolus vulgaris L.). And considering that the data arising from scientific studies of Colletotrichum destructivum pathogen morphology, mode of infection and rDNA sequences are distinct from that of C. lindemuthianum (O’Connell et al., 1993; Sheriff et al., 1994; Latunde-Dada et al., 1996), an increasing body of evidence has questioned the designation of C. lindemuthianum as the ovoid spored cowpea anthracnose pathogen (Latunde-Dada et al., 1996). In the investigation of Latunde-Dada et al., (1996) though, both the cowpea anthracnose fungus and the bean anthracnose fungus infect their respective hosts through an initial intracellular biotrophic phase, it was however, observed that the infection structures produced by the cowpea anthracnose fungus differ markedly from those produced by C. lindemuthianum (table 4). Within a comparative evaluation of the hyphae lacerating structure, it was observed that during the biotrophic phase, C. lindemuthianum produces spherical vesicles and swollen primary hyphae which pass from initially-infected 14 epidermal cell to several other epidermal and cortical cells (Latunde-Dada et al., 1996), whilst the biotrophic phase of the hemibiotrophic cowpea anthracnose fungus was restricted to single epidermal cells (Table 5). According to Latunde-Dada et al., (1996 &1999) the causal organism of cowpea anthracnose produces large, multilobed infection vesicles during the biotrophic infection of the host. The pathogen gains ingress into the host by elaborating from melanized appressoria, an infection peg, which penetrates the cuticle directly to initiate infection and never through the stomata (Latunde-Dada et al., 1999). In addition Latunde-Dada et al.,(1996 &1999) inferred that anthracnose of cowpea has a straight conidia (14-18 µm) and penetrate host cells directly to establish transient intracellular biotrophic infections which are restricted to the initially infected epidermal cells (Table 5). Source: modified from Latunde-Dada et al., (1999). Latunde-Dada et al., (1999) have provided strong evidence in favour of considering the cowpea anthracnose pathogen as a form of C. destructivum (O`Gara). Recognizing C. destructivum pathogen as a real causal organism of anthracnose in cowpea crop has been accepted by some authors on recent review of cowpea diseases (Emechebe & Lagoke, 2002; Allen et al., 1998). Table 5: Morphological and growth characteristics of C. destructivum and C. lindemuthianum. Structures and Activities C. destructivum C. lindemuthianum Conidia shape Ovoid Ovoid Conidia size (µm) 16.3±2.0x4.0 12.1±1.0x4.0 Conidial Septation (upon germination) Yes No Appressorial shape Subglobose (with variable margins) Globose Stomatal penetration No No Intracellular biography Large,multilobed vesicles Small spherical vesicles+primary hyphae Intracellular One cell Many cells Ability to infect cowpea Yes No Prolonged symptomless infection No No Acervulus One seta Many setae 21 2.7 THE GENUS: COLLETOTRICHUM Corda The plant pathogenic genus, Colletotrichum, is an important genus within the parasitic microorganisms of crop production. Its damaging activities have been reported in the temperate, subtropical and tropical agricultural crops (Gomes et al., 2008). It is a genus associated with setose acervuli, relatively large cylindrical or falcate phialoconidia, and appressoria, (Thaung, 2008), attacking a very broad range of host plants (table 6). The genus embraces some forty species of plant parasites and provides anamorphs of Glomerella with a large reservoir of synonymy too cumbersome for accurate and conclusive systematic study stated Thaung, (2008). The present work has been able to associate about twenty seven Colletotrichum species to their specific primary plant hosts (table 7). Scientists such as Gomes et al., (2008) reported the genus to include many of the most damaging plant pathogens in the field of agriculture, whose anthracnose effects on a wide range of plants are well documented (Martin & Garcia- Figures, 1999). Colletotrichum species have also been reported as the casual agents of anthracnose and blight on cereal crops (Amusa & Ikotun, 1995) grasses (Gomes et al., 2009), grain crops (Adebitan & Ikotun, 1996, Amadioha & Obi, 1998, Latunde-Dada, 1999, and Amadioha, 2003), vegetable plants (Liu et al., 2007), perennial tree crops (Latunde-Dada et al.,1999 and Gomes et al., 2009) and even feeds (Mould et al., 1992 and Liu et al., 2007). Some postharvest disease problems have also been attributed to some species of the Colletotrichum genus of the plant pathogen (Sanders et al., 2000). The genus with its devastating prowess can infect all plant surfaces, but favours young leaves, branches and fruits of herbaceous species growing in humid climates reported Gomes, et al., (2008). Incidentally, this work has counted about eighteen total plant families under the pathogenic attack and disease affliction of Colletotrichum (table 8). It has been pretty documented that the ability of Colletotrichum to develop a series of specialized infection structure, including germ tubes, appressoria, intracellular hyphae, and secondary necrotrophic hyphae including the evolving of a hemibiotrophic strategies singled out the wide host range linked with the 22 species of this pathogenic genus (Sun and Zhang, 2008). Studies have also shown that Colletotrichum conidia will adhere rapidly to a wide range of plant and artificial surfaces, including cellophane, polystyrene, polycarbonate and glass. These strong and relatively unique pathogenic characteristics must have led to the acceptance of the genus as an excellent model for studying the molecular and cellular bases of fungal pathogenicity as indicated by Sun and Zhang (2009). Table 8: Plant Families Under The Affliction Of Colletotrichum Corda (1995 - 2009) Family Cited Authors Amaranthaceae Juss. Liu et al.,(2007) Anacardiaceae Lindl. Sanders et al.,(2000) Asteracea Bercht. & J. Presl. Amusa & Ikotun(1995), Liu et al.,(2007) Brassicaceae Juss. Sun & Zhang(2009) Carcaceae Dumort. Palhano et al.,(2004) Convovulaceae Juss. Amusa & Ikotun (1995) Cucurbitaceae Juss. Liu et al.,(2007) Cuscutaceae Dum. (=Convovulaceae) Amusa & Ikotun (1995) Fabaceae Lindl. Mould et al. , ( 1992) , Amadioha & Obi ( 1998) , Latunde-Dada et al., (1999), Emechebe & Lagoke (2002), Liu et al., (2007). Lauraceae Juss Sanders et al (2000) Leguminosae Juss., Non. con (=Fabaceae Lindl.) Adebitan & Ikotun (1996), Latunde - Dada et al., (1996),Emechebe & Lagoke (2002),Amadioha (2003) Linaceae.L Latunde-Dada and Lucas(2007) Malvaceae Juss Liu et al.,(2007) Musaceae Juss Liu et al., (2007) Oleaceae Hoffmgg.& Link Salazar et al., (2007) Gomes et al., (2009) Poaceae Barnhart (=Gramineae Juss, Non. Con.) Amusa & Ikotun (1995), Moore et al., (2008) Roasaceae Adans Liu et al ., (2007) Salazar et al., (2007) Garrido et al., (2008) Rubiaceae Linn Moriwaki et al ., (2003), & Nguyen et al ., (2010) 23 Colletotrichum species are haploid organisms which can be cultured axenically and transformed. This in extension, according to Sun and Zhang (2009) greatly facilitates mutational analysis and the critical assessment of gene function by targeted gene disruption. For example, isolates of Colletotrichum from cruciferous plants has recently been reported to lead to the production of a new Arabidopsis pathosystem for infecting Arabidopsis thaliana plant (Sun and Zhang, 2009). 2.8 SYSTEMATICS ON COLLETOTRICHUM The genus Colletotrichum Corda was first described by Tode in 1790 under the name Vermicularia, but was later established as Colletotrichum by Corda in 1837. According to Thaung, 2008, and Gomes et al., 2008, Colletotrichum parasite is the known single genus among the Coelomycetes that has garnered most attention probably because of the diversity, distribution and devastating activities of most of its members. Coelomycetes are Deuteromycetes referred to as fungi imperfecti associated with structural acervuli, pycnidia, or stromata as conidiomata (Dugan, 2006; Schumann & D`Arcy, 2006 and Thaung, 2008). And within the Deuteromycetes, fungi Coelomycetes are Mitosporic and microscopic (Thaung, 2008), ubiquititious (Gomes et al., 2008), parasitic, saprobic or facultative phytopathogens (Schumann & D`Arcy, 2006). The Coelomycetes generally exist as conidial, spermatial / microconidial states or anamorphs of ascomycetes (Thaung, 2008). This group might as well be called fungi mitospori or fungi Anamorphic. According to Thaung, (2008) some authors have referred to Coelomycetes as polyphyletic, artificial (non phyletic) (Schumann & D`Arcy, 2006) and untenable or redundant form of fungi (Fig. 7). The characterization and identification for species classification within the genus of the form Coelomycetes traditionally has been based on conidial shape & size, presence of sclerotia, appressoria production, and often on host range and pathogenicity (Liu et al., 2007). Though molecular technologies based on the analysis of DNA have come on stream for the examination in details of the relationship that exist within species of Colletotrichum (Liu et al., 2007), the 24 traditional methods based on both morphological characteristics and host specificity are still employed in the genus studies (Garrido & Carbu, 2008). Analysis of DNA sequences continues to be a valuable tool to help resolve relationships among and within species and species complexes of Colletotrichum. According to Liu et al.,(2007),DNA sequence along with morphology and host range were used to delineate species within the broader Colletotrichum graminicola complex on sorghum (Sorghum bicolor) , maize (Zea mays), wheat (Triticum aestivum),oat (Avena sativa), feeds, and amenity grasses. For lack of sexual (perfect meiospore) stages in their life cycles, Coelomycetes are usually consigned to a form – class/division of Deteromycota (also Dikaryyomycota) or fungi imperfecti (Thaung, 2008). This author (Thaung, 2008) in his studies pointed out that the form – taxa classificatory system in the Coelomycetes are exclusive, restrictive and of limited use and not indicative of clear relationships among taxa, because in his words “they serve nomenclature and identification purpose only”. 25 Fig. 7: Classification on Colletotrichum Source: Schumann and D`Arcy (2006). Deuteromycetes Mitosporic fungi. Nonphyletic (artificial). Assemblage of fungi that reproduce asexually. Anamorphic state of ascomycetes (in practice). Melanconiales (traditional división) forms Forms produce acervuli (with setae). Colletotrichum (Genus) Over 40 known species. Melanconiaceae (Family) Artificial (Nonphyletic). Coelomycetes Produces conidia in acervuli (practically en closed structure). Conidiogenous cells. 26 2.9 MANAGEMENT OF ANTHRACNOSE PROBLEM The great economic importance of the Colletotrichum phytopathogens in agriculture cannot be over emphasized (Thanug et al., 2008). On animal feeds, anthracnose by Colletotrichum trifolii of alfalfa (Medicago sativa L.), has been reported to lead to reduced forage yields, losses in plant vigour and stand depletion (Mould et al., 1992). Species of Colletotrichum parasites like C. acutatum have been creating some known food health problems in the European regions over some time. According to Garrido et al., (2008), this dreaded phytopathogens, Colletotrichum acutatum, thought to have been introduced in to European territory from California (USA) was first reported on strawberry plants in France in the year 1981. For the impact of this organism, it is considered a quarantine pathogen in the European union and, as revealed by Garrido et al., (2008), has subsequently been included in the list of regulated A2 pests in the European and Mediterranean Plant Protection Organization (EPPO) region since 1997 (EPPO/CABI,1997). An understanding of the mode of infection of individual Colletotrichum species is a prerequisite for developing effective control strategies, particularly those based on host plant resistance. Knowledge of the factors influencing infection processes also provides epidemiologists with information which can be developed into forecasting models, and aids agronomists developing appropriate agricultural practices, based on crop sanitation and removal of volunteer, reservoir or collateral hosts. Nevertheless, control of anthracnose problem of Colletotrichum species tends to focus mainly on inoculum reduction and prevention of latent infection (Sanders et al., 2000). Adequate preharvest spray programme, therefore, have been recommended to check the post harvest anthracnose problems of agricultural products such as Avocado and Mango fruits. 27 In some countries previously registered fungicides programmes included monthly pre-harvest application of Benomyl, followed by cupric hydroxide or copper oxychloride for fruits such as avocado (Persea americana) and cupric hydroxide alone or with alternate mancozeb or Benomyl sprays for fruits such as mango (Mangifera indica). According sanders et al., (2000) with the exception of Benomyl, the aforementioned compounds are all contact fungicides whose timing of application should, therefore, coincide with periods of high rainfall when inoculum is dispersed. The early 1960 introduction of benzimidazole agrochemicals such as Benomyl, carbendazin and thiophanates revolutionized fungicides disease control in crop production (Sanders et al., 2000). However, the extended use of such agrochemicals as revolutionized by the introduction of benzimidazole fungicides in early 1960s resulted in selection for resistant pathogen genotypes which has remained predominant for several years after discontinued use indicated Sanders et al., (2000). Some Colletotrichum species resistant to Benomyl, thiabendazole and prochloraz fungicides are documented (Sanders et al., 2000). The use of resistant cultivars implying recurrent phenotypic selection has been reported to increase anthracnose resistance from 1-20% to 59- 88% (Mould et al., 1992). In the study of the infection process of Colletotrichum destructivum ,a hemibiotrophic fungus, using a light microscope in two cowpea cultivars, (TVX3236 and IT82E-60) resistant and susceptible respectively, Latunde-Dada et al., (1999), observed that the production of appressoria and their melanisation were impaired in the resistant cultivar, resulting in reduced organ penetration. The scientists further observed that where penetration occurred in the course of their research, within the resistant varities, the initially infected epidermal cells underwent a hypersensitive response, restricting the destructive necrotrophic phase of the disease development. This action they attributed to the activities of the phytoalexins, Kieviton and Phaseollindin substances which they observed accumulated earlier and more rapidly in the stem tissues of the resistant cultivars, associated with the appearance of delimited necrotic spots on inoculated surfaces. 28 It is the presence and accumulation of the same substance, Kieviton, in times of problems, in the resistant cultivars that give Phaseolus vulgarus (Fabaceae) protection against ravaging pathogens such as Colletotrichum lindemuthianum (Diabate et al., 2010). In the susceptible cowpea cultivar IT82E-60, as against the resistant cultivars, Latunde-Dada (1999) revealed that there was delayed and slower accumulation of phytoalexins, Kieviton and Phaseollindin in the compatible interaction, together with the development of typical spreading water –soaked, anthracnose lesion. 2.10 CHEMICAL PLANT DISEASES MANAGEMENT The agricultural crop and forestry production is presently largely dependent on the use of chemicals to control various pests and diseases or to retard unwanted soil microbial processes. Though some of these agrochemicals are designed to affect only specific target organisms or processes, most of them, however, have general toxic effects and hence often cause strenuous interactions with the biological soil ecosystem. In general species compositions of the soil microflora and fauna is reconstituted by pesticide substances. The inhibitory effect of copper sulphate on microbial glucose degradation in red latosol soil is reported by Airoldi and Critter, 1996. They discovered that increasing masses of copper sulphate caused a decrease of the original thermal effect to reach a null value at 6.19mg of inhibitor. Soil life and fertility for crops, according to Pell et al., (1998) depends partly on the delicate “balance” that exists between the various types of microorganisms that determine the turnover of carbon, nitrogen, and other valuable plant nutrients. Thus, it is clear that the addition of any potentially toxic compound is a serious threat to this equilibrium and hence to the sustainable fertility of the soil. 29 The use of fungicides in agriculture to protect plants from both soils and non soil born pathogen are a common practice. According to a review of the ecological effects of the accumulation of copper in soil by Jansch et al., (2009) copper and copper based fungicides were introduce into European agriculture since 1885 and for its non degradable quality, and extended toxicity to the soil and its inhabitants, is under review whether they can be included in the recent positive list of active substance authorized for use in plant protection products in Europe (Annex 1) of the EU council Directive 91/4141. However, there exists a dearth of information on the side effects of fungicides (conventional) on key soil ecological processes (Chen et al., 2001; Sahin and Ugur, 2003; and Jansch et al., 2009). In their investigation, Perrin and Plenchette (1993) observed that “because of its harmful effect, Benomyl must be avoided in any management strategy that aims to preserve arbuscular mycorrhizal fungi”. According to Chen et al., (2001), Benomyl, Captan and Chlorothalonyl exhibited adverse effect on Soil microbial activity (substrate induced respiration and dehydrogenase activity) and nitrogen dynamics (NH4-N and NO3-N) in their laboratory batch incubation. All the three fungicides suppressed the peak soil respiration in unamended soil by 30-50%. However the researchers observed that captan appeared to have more pronounced overall effects on soil microbial activity and nitrogen dynamics than either Benomyl or chlorothalomyl. Due to high side effect of copper based fungicides to soil microbial activities, Sonmiez et al., (2006) observed in their study that fruit number, total yield, dry root weight and plant height decreased with increasing copper application to soil. Combined applications of copper to soil and leaves could be more deleterious to the plant, soil and the entire ecology than when the product is applied only to soil or leaves, they inferred. They reported that copper toxicity, as expressed by reduced root length, appeared to be a direct result of the accumulation of excess copper in the soil. Important natural antimicrobials such as streptomycin and actinomycetes are soil inhabitants and should be protected and not dislodged using fungicides or other copper based pesticides, these researches further argued. In their investigation of the antimicrobial activity of some Streptomyces isolates, Sahin 30 and Ugur (2003) recovered from 46 soil samples, a total of 74 different Streptomyces isolates. The researchers listed rhizosphere of plants, agricultural soil, reserved areas and forest soils as the potential habitats of these microbial elements. It is worth the interest to note, however, that these aspects of the result would not have been possible if the soil regions were under the bombardment of synthetic fungicides. In recent time the active search for bioactive molecules in insect, micro organisms and plants for disease control in Agriculture has been on the increase (Amadioha and Obi, 1999; Enikuomehin and kehinde, 2007; Colpas et al., 2009). This is to give a commensurate check and balance to the rapid devastation our soils and ecology are being subjected to from conventional pesticide. 2.11 THE BOTANICALS IN PLANT DISEASES MANAGEMENT For the economic importance of this disease ,anthracnose on cowpea , several control methods have been adopted including the application of chemical (fungicides) and integrated pest and disease management (Amadioha and Obi, 1998;Amadioha,2003;Adebanjo and Bankole, 2004).But for the conscious environmental sustenance and ecological compatibility , there is the need and desirability to search for the alternative which employs natural agro biological(Biopesticides) balance to address this all important cowpea disease. The Environmental protection Agency (EPA-USA) defines a Biopesticides as a pesticide derived from natural materials such as animals, plants, bacteria and certain minerals (http://www.polyversumla.com). Biopesticides of plant origin are the botanicals (Fig: 8). 37 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 CONSULTED MATERIALS: Materials on literatures bordering principally on cowpea anthracnose by Colletotrichum lindemuthianum and Colletotrichum destructivum, and botanicals spanning through 1992 to 2010 were sourced and reviewed accordingly. A total of seventy one referred literatures and four on line materials provided direct information for the study. Crop families under the afflicting influence of Colletotrichum were grouped, and species of Colletotrichum with their specific hosts identified. Colletotrichum with other related pathogens of cowpea crop were assembled and percentage virulence comparism evaluated among them. The plant families so far screened (between 1998 and 2009) for potential source of biofungicidal substances, together with their botanical forms were also included in the work. 3.2 BOTANICAL MATERIALS: Seeds from mature dehisced fruits of Azadiractha indica A.Juss (neem) (fig 9; table 10) were oven dried for two days at 60oC and the seed coats then split to remove the cotyledons which were subsequently washed in sterile, distilled water and oven dried together with the sterile water washed fruits of Xylopia aethiopica (fig10; table 11) at 60oC for 24h. After drying, the fruits and the seeds were separately ground in a mortar to obtain 1000g of dry powder from each material. Harvested fresh leaves of Cymbopogon citratus (fig 11; table 12) and Ocimum gratissimum (fig12; table 13) were washed thoroughly in tap water and sterile distilled water, airdried at 27oC, weighed (100g) and ground separately in sterile mortar. 38 Fig 9 c : Azadiractha indica , young tree. Source: UPTH, Porthacourt Fig 9d: Azadiractha indica tree with immaturefruits.Source:Http://www.bhg.com/ gardening/plant dictionary/vegetable/cowpea/. Fig 9 a: Matured and ripped fruit of Azadiractha indica. Source:UPTH, Porthacourt Fig 9 b : Dehisced seeds of Azadiractha indica . Source: UPTH, Porthacourt 39 NA: Not available. Table10 :Biological Classification on Neem plant Kingdom Plantae Plants Subkingdom Tracheobionta Vascular Plants División Magnoliophyta Flowering Plants Superdivision Spermatophyta Seed Plants Clase Magnoliopsida Dicotyledons Subclase Rosidae N A Order: Sapindales( Rutales) NA Family: Meliaceae Mahogany family Subfamily: Melioideae NA Tribe: Melieae NA Genus Azadirachta A.Juss azadirachta Species: Azadirachta indica A.Juss neem 40 Fig10a: Xylopia aethiopica tree with unripe fruits. Source: Spiced Africa – Grains of Paradise and Grains of Selim (http://www.justfoodnow.com) Fig10b: Xylopia aethiopica harvested ripe fruits.source: ChAI EXOTICOS ZGZ. NA: Not available. Table 11: Biological Classification on bush pepper plant Kingdom Plantae Plants Subkingdom Tracheobionta NA División Magnoliophyta(Angiospermae) Seed producing Superdivision NA NA Clase Magnoliopsida Dicotyledoneae Subclase Magnoliidae NA Order: Magnoliales woody Plants Family: Annonaceae Juss Woody Plants Subfamily: NA NA Tribe: NA NA Genus Xylopia Pepper Species: Xylopia aethiopica (Dunal) A. Rich. Guinea pepper 41 NA: Not Available. Fig11 a: Cymbopog on citratus (Young Lemon grass Source:UPTH, Porthacourt Fig11b: Cymbopogon citratus plant (Matured). Source:UPTH, Porthacourt Table12: Biological Classification on Lemon grass plant Kingdom Plantae Plant División Magnoliophyta Angiospermas (flower plant) Clase Liliopsida Monocots Subclase Commelinidae Commelinids Order Poales Monocots flowering Plants Family Poaceae Herbácous plants,rarely woody Subfamily Panicoideae Racemos inflorescence Tribe Andropogoneae NA Genus Cymbopogon Tea grass Species Cymbopogon citratus Lemon grass 42 Fig12 a. Ocimum gratissimum (Young sce nt plant). Source: UPTH, Porthacourt Fig12 b .Ocimum gratissimum plants at fruiting stage. Source: UPTH, Porthacourt. NA: Not available. Table13: Biological Classification on scent leaf plant Kingdom Plantae Plants Subkingdom Tracheobionta Vascular Plants División: Angiosperms Flowering Plants Superdivision Spermatophyta Seed Plants Clase: Magnoliopsida Dicotyledons Subclase: Asteridae NA Order: Lamiales NA Family: Lamiaceae Mint family Subfamily: NA NA Tribe: NA NA Genus Ocimum L. basil Species Ocimum. gratissimum L. African basil/Scent leaf 43 3.3 BOTANICAL EXTRACTION: An oil soluble extract was made of each sample of A. indica (fig 9) and X. aethiopica (fig 10) by placing 80g of the dry powder in thimble and extracting with 500 ml diethyl ether for 6h using Soxhlet extractor (Amadioha & Obi 1998 and 1999). The ether was evaporated initially using water and then left overnight at a laboratory temperature for evaporation of the remaining ether. Hot water extracts (HWE) were obtained by infusing the four ground test materials separately with 100ml sterile distilled water using 250ml Erlenmeyer flasks in water bath at 80OC for 1.5h. Each paste from Cymbopogon citratus (fig 11) and Ocimum gratissimum was added to 100ml beaker, stirred vigorously and allowed to stand for 1h and then filtered to obtain cold water extracts (CWE). The crude hot and cold extracts were both obtained by several filtrations through 4 folds of sterile cheese cloth. Fig13. Azadiractha indica oil. Fig.14. Xylopia aethiopica oil. Fig15.Processed Cymbopogon citratus leaves. 3.4 ISOLATION AND IDENTIFICATION OF PATHOGEN. The test pathogen, Colletotrichum destructivum was isolated from an infected cowpea plant. An infected cowpea plant with anthracnose symptoms was collected from the farm site of the International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria. Lesions were observed under a stereobinocular microscope(X 12-60) for the presence of acervuli .Mounts of the fruiting bodies were examined under the microscope to ascertain the identity of the isolates with reference to 44 illustrated genera of imperfect fungi (Dugan, 2006). Pure cultures of the pathogen were prepared through aseptic transfer of acervuli to PDA in Petri dishes and Koch Pasteur `s postulates (fig 16) observed for further authenticity. 06/09/2011 BOTANICALS 20 Petridish microscope Inoculum transfer Laboratory assay Field evaluation Pathogen isolation and inoculation process (koch-pasteur postulates) Fig 16: Pathogen isolation and inoculation process according to Koch- Pasteur postulates 3.5 EFFECT OF EXTRACTS ON SPORE GERMINATION. Suspension of 10-day –old cultures of the pathogen were prepared using a disc (3mm diameter) in 1ml each of the undiluted (100% concentration) and diluted (50% concentration) extracts in test tubes. Similar spore suspensions were prepared in sterile distilled water as control. The contents of the tubes were subsequently centrifuged at 100 revolution/min for 10 minutes and then filtered through four folds of cheese cloth. With a Pasteur pipette , a drop (0.05ml) of each spore suspension (10x104 spores /ml) was placed on triplicate sterile slides inside Petri dish-moisture chambers and incubated at 27±1oC for 24h.Further spore germination was then stopped by adding a drop of lactophenol cotton blue (a biological stain) to each spore suspension on the 45 slide and 100spores were observed at random with microscope (X 10) and recording the number generated for each replicate treatment to determine the number that germinated which was used to determine the percentage inhibition of spore germination. 3.6 EFFECT OF EXTRACTS ON COLONY GROWTH. The effect of botanicals on fungal growth was determined by growing Colletotrichum lindemuthianum on a PDA (potato dextrose agar) media containing extract in a Petri plate. A 50 percent concentration of crude extract in PDA was prepared by adding 50ml of the oil or hot water extract to 50 ml molten PDA (prepared by dissolving 3.7g PDA in 50 ml sterile distilled water). One hundred percent botanicals in PDA was prepared by spreading 1ml of full strength of each botanical on the surface of the solidified PDA-botanical medium contained in Petri dish previously marked at the bottom with two perpendicular lines indicating the center of the Petri plates. The control contained PDA without added botanicals. A disc (3 mm in diameter) of 10-day old Colletotrichum lindemuthianum culture was aseptically transferred to the center of the solidified PDA-extract medium in the Petri plates (above the marked perpendicular lines). The Petri plates were subsequently incubated for 7 days at 27ºC. Radial growth of the Colletotrichum lindemuthianum was measured on each plant and compared with the growth of the organism in plates without botanicals. The experiment was replicated five times per treatment. 3.7 FIELD EVALUATION OF EXTRACTS AND BENOMYL The effects of botanicals on disease development in vivo were determined using cowpea crops. From the international Institute of Tropical Agriculture (IITA), seeds of cowpea, cv.ITE 2246-4, susceptible to Colletotrichum lindemuthianum were surface sterilized in 0.5 percent sodium hypochlorite solution for 1 min and rinsed in sterile distilled water. The sterile seeds, at three seeds per pot (fig18) were sown in 4 kg of top -soil contained in 22.5cm diameter earthen pots, previously sterilized with metham sodium or Vapam (a soil fumigant). 46 Potted plants were randomly arranged in three groups (fig 17) in a glass-house and watered twice daily through a tap water source. Crops in the first group were spray-inoculated with a spore suspension (10x105 spores /ml distilled water) of the pathogen two days before spraying with either botanicals (undiluted) or Benomyl (3.0 g/l). Crops in the second were inoculated two days after application of botanicals or Benomyl .Plants in the third group were sprayed with the botanicals or Benomyl after symptoms of anthracnose were evident (21 days after inoculation).Control crops within each group were similarly inoculated with spore suspension ,but sprayed with sterile water instead of with neither botanicals nor Benomyl. The size of individual leaf lesions (fig 18) were measured with a metric ruler 25 days after spraying the leaves with botanicals or Benomyl. Each test consisted of five replicates pots per treatment in a completely randomized block design. Twenty 3.8: Effect of tissue extracts of Azadiractha, Cymbopogon Ocimum, and Xylopia on sporulation density of Colletotrichum spp The culture plate used for the study on colony growth were used In the study to study the effect of the tissue extracts on sporulation density of Colletotrichum spp. Five mllimometres of sterile distilled water was added to each of four replicate plates per treatment .Spores from each plate were washed into suspension with the aid of a flame–sterile scalpel and filtered through 3 layers of muslin cloth into a test tube. Spores count in each of 5 small squares per replicate spore suspension were made using hemacytometer slide for each treatment. Number of spores per treatment was calculated using the formular: (A+B+C+D+E) x50 equal numbers per cubic millimeter ,multiplied by 1000 to get value in milimetre.,where letters A,B,C,D and E represent spore counts in 5 one square millimeter rulings of the hemacytometer. The ruled surface is 0.100mm below the cover glass on the hemacytometer grid and volume of liquid over a square millimeter is 0.1mm3. To compensate for differences in colony 53 Pathogen spore germination of 100% was recorded in sterile distilled water treatment as against low 21.3% and 41% spore germination recorded in 100% and 50% hot water extract concentration respectively. Twenty eight percent spore germination were associated with 50% oil as against zero spore germination with 100% oil concentration. (Table 17) X = Mean of 3replicates each of 100spores /extract, 24hrs after incubation. Y = means of 5 replicates 7 days after incubation. Z = Means of 4 replicates 7 days after incubation. * = Means in the same column followed by the same letter are not significantly different at 0.05 level of significance (Duncan’s Multiple Range Test, DMRT). ** = Numbers in bracket are percentage reduction or increase in parameter indicated. The Xylopia extracts also had some significant inhibitory effect on colony growth of Colletotrichum. There was no concentration dependence in hot water case, hence there was no significant difference within the treatment unlike with the oil treatment where there was significant difference within mean colony values. One hundred per cent and fifty per cent water extracts caused 86% and 52% reductions in sporulation density, respectively, whereas 100% and 50% oil extracts were associated with 18% and 58% reduction in sporulation density. There was no significant difference between oil extract at full strength and the control mean sporulation density. Table 18: Effect of Cymbopogon Treatment on spore germination ,colony growth and sporulation density Treatment Mean percentage spore germination X Mean colony growth (mm) Y Mean sporulation density Z (x103). 100% Hot water - Extract (Chwe) 79.7b * (20.3)** 24.7a* (53.9)** 9.3b* (-86.0)** 50% Chwe 90.7 d (9.3) 25.0a (53.4) 16.6 c ( - 232.0) 100% Oil (Coe) 68.7 a (31.3) 24.9a (55.2) 8.6 b ( - 72.0) 50% Oil (Coe) 85 . 0 c (15.0) 24.4a (54.5) 16.1 c ( - 222 .0) Water ( C) 100.0 c (0) 53.6b 5.0 a 54 4.3: Effect of Cymbopogon Treatment on Spore Germination, Colony Growth and Sporulation Density. The expression of Cymbopogon on in vitro spore germination, colony growth and sporulation density of Colletotrichum spp. is presented in table18. All extract treatment had a significant inhibitory effect on spore germination 24 hours after incubation. The highest percentage reduction of 31.3% in spore germination was recorded in 100% cold water extract. X = Mean of 3replicates each of 100spores /extract, 24hrs after incubation. Y = means of 5 replicates 7 days after incubation. Z = Means of 4 replicates 7 days after incubation. * = Means in the same column followed by the same letter are not significantly different at 0.05 level of significance (Duncan’s Multiple Range Test, DMRT). ** = Numbers in bracket are percentage reduction or increase in parameter indicated. This was followed by 100% hot water extract (20.3%), 50% cold water extract (15%), and 50% hot water extract (9.3%). Though the extracts showed over 50% inhibitory effect on colony growth of the pathogen at all levels, the leaf extract was not concentration dependent and statistically, therefore, were no significant difference within the treatment values under mean colony growth. The leaf extracts instead significantly stimulated sporulation of Colletotrichum spp. One hundred per cent and fifty percent hot water leaf extracts impacted 86% and 232% increase in sporulation density respectively, while 100% and 50% cold water extracts were associated with 72% and 222% increase in sporulation density respectively. Table19 :Effect of Ocimum Treatment on spore germination ,colony growth and sporulation density Treatment. Mean percentage spore germination X Mean colony growth (mm).Y Mean sporulation density Z(x103). 100% Hot water- Extract (Ohwe) 60.7c* (39.7)** 25.9a* (51.7)** 1.8b* (64.0)** 50% Ohwe 80.3d (19.7) 27.1a (49.4) 4.4c (12.0) 100% Oil (Ooe) 51.3a (48.7) 25.5a (52.4) 1.2a (76.0) 50% Oil(Ooe) 59.0b (41.0) 26.8a (50.0) 4.1c (18.0) Water (c) 100c 53.6b 5.0d 55 4.4. Effect of Ocimum Treatment on Spore Germination, Colony Growth and Sporulation Density The record on the effect of Ocimum is shown on Table19 Also all extract treatments (four levels) had a significant inhibitory effect on spore germination 24 hours after incubation. The inhibitory effect was all concentration dependent .As against the 100% germination recorded in sterile distilled water , only 80.3 and 60.7 germination per cent were recorded in 50% and 100% hot water leaf extract respectively. The extracts showed significant reduction in sporulation density with all treatments. One hundred per cent cold water extract was the most potent causing 76%reduction in sporulation density .This was followed by 100% hot water extract associated with 64% reduction in sporulation density. At 50% concentration, the potency of both hot and cold water extracts caused 12% and 18% reduction in sporulation density respectively. Again mean colony growth, though, were reduced close to, or above 50 %,( table 19) the values on analysis did not show the existence of any significant difference within values at all treatment levels. 4.5 Effect of Plant Extracts and Benomyl Treatments on Vigna ungiculata Disease Development by Colletotrichum spp The effect of botanical extracts and Benomyl on Vigna ungiculata is presented in table 20. Oil plant extracts of Azadiractha and Xylopia reduced lesion spread by the same margin of 37.8% on plants sprayed 2 days before inoculation. This value was significantly different from the 25.7% reduction recorded for Benomyl treatment two days before pathogen inoculation (2dbi). Lesion sizes in leaves sprayed with Ocimum and Cymbopogon cold water extracts were similar to those on Benomyl sprayed leaves but significantly smaller than those on watersprayed control leaves. Similar, but significantly smaller lesion spreads were observed on leaves sprayed with Azadiractha oil, Xylopia oil and Xylopia hot water extract than those observed on control leaves. Least control of lesion spread was associated with Ocimum and Cymbopogon hot water extracts with only 8.9% and 10.4% reduction in lesion spread respectively. 56 Lesion development was significantly inhibited in all leaves sprayed with either plant extract or Benomyl two days after artificial inoculation with Colletotrichum spp. spore suspension. Treatments with Ocimum, hot and cold, and Cymbopogon hot and cold water extracts were as effective as foliar spray with Benomyl .Foliar spray with Azadiractha and Xylopia oil and hot water extract were more effective than foliar spray with Benomyl. Lesion spread was also significantly reduced in leaves sprayed with all plant extracts (except Cymbopogon cold water extracts) and Benomyl twenty one days after inoculation with test pathogen: Foliar spray with Benomyl, Cymbopogon hot water extract and Ocimum cold and hot water extracts resulted in similar but significantly lower percentage reduction in lesion spread than percentage lesion spread reduction associated with Azadiractha-oil, Xylopia-oil and Xylopia-hot-water extracts. Fig. 17 Shows Vigna crops treated at three different levels including the control Fig 17A Shows crops treated two days before inoculation (2dbi) .Fig 17B were crops treated two days after inoculation (2dai) .Crops treated twenty one days after inoculation (21dai) are shown in fig 17C. Fig 17D indicates crop samples from the control lots .Health conditions of the test crops tend to decrease in the order of D >C >B >A as indicated by the general appearance .Fig 18 shows heavily infected control crops with stem anthracnose (K) and almost completely blighted leaf with excised lesion(J). 4.6 Effect of plant Extracts and Benomyl on in vivo Sporulation Density of Colletotrichum Spp. The effect of plant extracts and Benomyl on in vivo sporulation density of Colletotrichum is shown on table21 .Generally, sporulation density control on Vigna crops treated two days before inoculation (2dbi) was higher than that on crops treated two days after pathogen inoculation (2dai) but lower than sporulation density on crops treated twenty ones days after inoculation (21dai). 57 In all cases, sporulation density control on lesions of non–treated leaves (C) was significantly lower than sporulation density control associated with lesions on leaves sprayed with plant extracts and Benomyl. A highly significant reduction in sporulation density of 88.1% was associated with Benomyl on crops treated two days before inoculation.(2dbi) This was closely followed by Azadiractha oil and Xylopia oil which caused 78% and 69.6% reduction in sporulationdensity respectively .The least fungitoxic effect was expressed by Ocimum cold water extract (Ocwe) with only 22% reduction in sporulation density. There was also no significant difference within the values of Ocimum and Cymbopogon extracts at the whole treatment levels ,two days before crop inoculation(2dbi) On Vigna crops treated two days after artificial inoculation (2dai), the highest fungitoxic effect was shown by Azadiractha oil with 63.2% reduction in sporulation density. Benomyl was the second in effectiveness with 57.9% reduction in sporulation density two days after inoculation (2dai). This, however was not significantly different (P<0.05) from the 56.1% inhibition from Xylopia oil Extract from Cymbopogon (cwe) was the least effective with only 13.8% inhibition on sporulation density. two days after inoculation (2dai) Fig 17: Potted Vigna unguiculata crop treated at three different levels ,including the control. 58 * Data are means of 5 replicates each of 4 lesions at random. ** Columns means followed by the same letters are not significantly different at 5% level (DMRT) *** Numbers in brackets are percentage reduction in lesion spread T able 20 :Effect of Plant Extracts and Benomyl t reatments on Vigna ung u iculata d ise ase development by Colletotrichum spp Treatment. Lesion diameter (mm)* at indicated treatment time. 2 days before inoculation (2dbi). 2days after inoculation (2dai). 21days after inoculation (21dai). Azadiractha Hot Water (Ahwe). 9.5c** (29.6) *** 10.4d** (23.0)*** 11.7dc** (13.3)*** Azadiractha Oil (Aoe). 8.4f (37.8) 9.4c ( 30.4) 10.8f (20.0) Xylopia Hot Water (Xhwe). 9.1ef (32.6) 11.3c (16.3) 11.4c (15.6) Xylopia Oil (Xoe) . 8.4f (37.8) 10.1de (25.2) 10.5f (22.2) Ocimum Hot Water (Ohwe). 12.3b (8.9) 12.2b (70.4) 12.3bc (8.9) Ocimum Cold Water (Ocwe). 10.6cd (21.5) 11.9bc (11.9) 11.9cd (11.9) Cymbopogon Hot Water (Chwe). 12.1b (10.4) 12.1b (10.4) 12.3bc (8.9) Cymbopogon Cold Water (Ccwe). 10.0de ( 25.9) 11.9bc (11.9) 12.7ab (8.0) Fig 18: Infected Vigna unguiculata crop (control) with stem anthracnose (K), and leaf lesion ((J). 59 *Data are means of 5 replication each of two hemacytometer readings **Columns mean followed by the letters are not significantly different at 5% level (DMRT). ***Numbers in brackets are percentage reduction in sporulation density. Observation on crops treated twenty one days after inoculation(2dai) showed Xylopia oil to be more fungitoxic with 56.9% inhibition on sporulation density compared to the significantly lower percentage inhibition of 49.2 associated with the Benomyl fungicide .Ocimum hot water extract (Ohwe) was the least effective causing only 6.2% reduction in sporulation density,. twenty one days after artificial crop inoculation (21dai 4.7 Effect of treatments on pathological activities of Colletotrichum spp. This heading tends to present in total but concise the various treatments evaluated on the pathological activities of Colletotrichum spp in the course of this study.These include the major in vitro and in vivo fungicidal determination of four botanical extracts from Azadiractha indica, Cymbopogon citratus, Ocimum gratissimum and Xylopia aethiopica and in comparation with Benomyl, a conventional fungicide. Table 21. Sporulation Density at Indicated Treatment Time Treatment Lesion Diameter (mm)* at indicated treatment time. 2daysbefore (2dbi) inoculation (x106). 2daysafter (2dai) inoculation (x106). 21daysafter (21dai) inoculation (x106). Azadiractha Hot Water (Ahwe) 2.1d** (64.4)*** 3.1c (45.6)*** 5.1dc (21.5)*** Azadiractha Oil (Aoe). 1.3c (78.0) 2.1c (63.2) 2.8g (56.9) Xylopia Hot Water (Xhwe). 2.7cd (54.2) 3.0 ef (47.4) 4.9e (24.6) Xylopia Oil (Xoe) 1.8dc (69.5) 2.5 fg (56.1) 3.2fg (50.8) Ocimum Hot Water (Ohwe) 4.5 b (23.7) 4.3cd (24.6) 6.1 b (6.2) Ocimum Cold Water (Ocwe). 4.6b (22.0) 3.9d (31.6) 5.5c (15.2) Cymbopogon Hot water (Chwe). 4.5b (23.7) 4.7bc (17.5) 6.0bc (7.7) Cymbopogon Cold Water (Ccwe). 4.3 b (27.1) 4.8b (13.8) 5.3cde (18.5) Benomyl (B). 0.7 f (88.1) 2.4fg (57.9) 3.3 f (49.2) Water (C). 5.9a 5.7a 6.5a 60 4.7.1 Treatments effect of extract in vitro on three pathological activities of Colletotrichum spp. Table 22 displays the inhibitory potential s of the extracts in vitro of Azadirachta indica, Cymbopogon citratus, Ocimum gratissimum and Xylopia aethiopica on three pathological activities (spore germination. colony growth and sporulation density). The comparative effect of treatment as indicated in fig 19 showed the oil extract of Azadiractha and Xylopia to inhibit spore germination of Colletotrichum up to 100% at full strength extract concentration. Least extracts effectiveness contra spore germination was obtained with Cymbopogon water extract at half dose concentration In all spore germination of Colletotrichum was controlled between 10 % and 100%., with the average from the activity of Ocimum at full strength water extract. Fig 20 indicates a comparative effect of extract treatments on colony growth of test pathogen. Extract activities of test plants existed on 48 to 58%. Again the Xylopia treatment at full strength oil concentration showed the highest potential against colony growth while the least was obtained with Ocimum hot water at half dose concentration. A rather fascinating result was obtained of extracts` activities on sporulation density. (fig 21).Though there was inhibitory extract effect of over 60%from both Azadiractha (100% oil), Ocimum (100%cold water) and Xylopia (100%hot water), Azadiractha and Cymbopogon extracts supported sporulation in the test pathogen While Azadiractha extracts (hot water and oil at half dose) aided pathogen sporulation with well over 180% intensity, Cymbopogon extracts at all levels of treatment concentration assisted heavily pathogen sporulation with more than 230% (fig 21) 61 Table22: Treatments effect (WE&OE)1 in vitro on three pathological activities of Colletotrichum spp. Treatment Percentage inhibition 2 Spore germination Colony growth Sporulation density Water Extract Oil Extract Water Extract Oil Extract Water Extract Oil Extract A 3 B 4 C 3 D 4 E 3 F 4 G 3 H 4 I 3 J 4 K 3 L 4 Azadiractha indica 56,0 45,7 100 69 51,5 50,8 56,2 56,0 -26 -140 80 -184 Cymbopogon.cit ratus 20,3 9,3 31,3 15 53,9 53,4 55,2 54,5 -86 -232 -72 -222 Ocimum.gratissi mum 39,3 19,7 48,7 41 51,7 49,4 52,4 50,0 64 12 76 18 Xylopia.aethiopi ca 78,7 59 100 72 53,9 53,4 57,3 52,2 86 52 18 58 1: Extracts were water (WE) or oil (OE) with data means of 5replicates 2: Inhibition measured as a reduction on number of spore germination, extent of colony spread and quantity of inocula available. 3: Treatment at full extract concentration. 4: Treatments at diluted (50%) extracts concentration. 4.7.2 Effect of four botanical extracts and Benomyl fungicide treatment on Vigna unguiculata diseases development The information on table 23 is on the effect the four evaluated botanical extracts and Benomyl fungicidal expressed on the test pathogen, Colletotrichum spp In this in vivo treatment evaluation lesion development was inhibited at various degrees in all leaves sprayed with either botanicals or Benomyl through all periods tested. Comparatively the highest value of 70.4% lesion reduction (fig 22) was associated with hot water extract of Ocimum at 2days after crop inoculation (2dai) The two subsequent values of relatively high percentages associated with reduction in lesion development after the 70.4% were 37.8% each from extracts of Azadiractha and Xylopia oil but from crop plants treated 2days before pathogen inoculation (2dbi). 62 0 20 40 60 80 100 120 100% Hot water- Extract 50%hot awter extract 100% Oil/Cold water 50% Oil/Cold water Fig 19: Comparative effect of Azadiractha, Cymbopogon, Ocimum, and Xylopia extract base on Colletotrichum spore germination Azadractha Cymbopogon Ocimum Xylopia Con → ↑ % 69 0 10 20 30 40 50 60 Biocontrol. Botanicals Cultural HPR. Pesticidal. % Fig 25: Information on Vigna unguiculata disease control tecniques utilized for a media decade 70 CHAPTER FIVE 5.1 DISCUSSION Cowpea crop (Vigna unguiculata L) is an important and indispensable crop whose livestock and human nutritive values cannot be underestimated at least in the world regions with limited source of protein supply. This opinion has been corroborated by many scientific works in this field including Emechebe & Lagoke (2002), Adebanjo & Bankole (2004), Owolade et al., (2006). This precious crop has always been exposed to the affliction of several pathogens ranging from Bacteria, to viruses, fungi to nematodes and of course recently the ravaging parasitic plants. However, the crops problem appears to originate most from the fungi group of pathogens. In addition to the information in support of this assertion (Emechebe & Lagoke, 2002), this study also identified about twenty different fungi pathogens associated with various cowpea diseases (table 4). In this work it was observed that in the consideration of single cowpea crop into six parts(fig 24), 30% of the fungal infections occur on the foliar part of the crop, 25% on the stems, 15% on the roots, 10% on the pods/fruits, 25% on the seeds/seedlings and 10% on the whole parts of the plant (table 4). Incidentally, while the other fungal pathogens posses the ability each of attacking only about a meager 20% of a stand, the Colletotrichum species (C.destructivum & C. truncatum) have in stock 100% virulence on a single crop each at a given pathogenic situation(table 4). This is in line with the findings of some scientists such as Latunde-Dada et al., (1999), Latunde-Dada &Lucas (2007), and Akinbode & Ikotun (2008). This degree of virulence on cowpea often leads to grain yield loss between 35 % and 50% as indicated by Amadioha & Obi (1998) and Amadioha (2003). After due scientific documents collation and an in-depth review of same, the author also affirm the pathogen Colletotrichum destructivum O`Gara the answerable causal organism of the anthracnose disease of cowpea (tables 5&6).This is also in support of the earlier assertion by Latunde-Dada et al., (1999) and Emechebe & Lagoke (2002). In the work of Akinbode & Ikotun (2008), the causal organism 71 of cowpea anthracnose was also recognized as the Colletotrichum destructivum O`Gara. Attempts have severally been made to adequately arrest the pathogenic problems of cowpea arising from Colletotrichum affliction among other pathogens.(table 4).These management efforts includes the use of Biocontrol systems (bioagents), Pesticides (conventional/synthetic chemicals), cultural observations (clean seeds/hygienic fields and practices), HPR (host plant resistance) and botanicals (Biopesticides/no synthetic chemicals) (table 3). Regrettably the Biopesticides (Botanicals) which tends to confirm to the global yearning for a natural agro biological balance in the fight against agricultural pests and disease is associated with about a meager 7% of the total cowpea disease management options (Fig 25). This was also the observation in the work of Emechebe & Lagoke (2002). These findings appear to confirm to the indication on table 9 of this work, where it was recorded that of all the entire plant families in existence only about eighteen (18) were screened for their biofungicides characteristics between 1998 and 2009. The study also reported that the products screened during this eleven years span were of various forms or state, such as (i) Aqueous: botanicals extracted using water as the solvent. The water also forms the extract solution (Akinbode & Ikotun, 2008; Nduagu et al., 2008); (ii) Syrup: botanicals of a higher measure of viscosity having been extracted with a solvent other than water , and also containing some of the extracting liquid in its solution (Win et al.,2007; Colpas et al., 2009);(iii) Oil: botanicals in the form of essential oil of the test plants, usually extracted through a condensation system (Amadioha & Obi, 1998); and (iv) Ash: botanicals produced in the form of residues powder left after the combustion of a test plant material (Enikumehin & Kehhinde, 2007; Obi & Ugwunze, 2009). Nevertheless, the botanical forms enumerated in this study could be extended to produce additional form by the application of further processing treatment on the original form. For example the Syrup produced in the work of Win et al., 72 (2003) was utilized in its dry crude botanical extract state after subjecting the initial extract syrup to an evapoconcentration system. However, this study observed that most of the botanicals screened for this span of eleven years (1998 to 2009) were produced and also utilized in their aqueous form. And considering the total 33 frequency occurrence of botanical forms (table 9), the aqueous botanicals was 51.52%, followed by the syrup (15.15%), ash (18.18%) and the oil form of 15.15%. The relative easy and economy of production could be responsible for the high percentage value obtained with aqueous botanical evaluation. Close to 28% family interaction existed between plant families under the affliction of Colletotrichum and the plant families screened for antifungal properties as expressed in tables 8 & 9. These occurrences were observed within the five plant families of Asteraceas, Caricaceae, Fabaceae, Lauraceae and poaceae. The rest of about 72% were unique in occurrence as there were no family interactions among them (tables 8 & 9). The reason for this pattern of existence leaves for further investigation. Tissue extracts of Azadiractha indica, Cymbopogon citratus, Ocimum gratissimum and Xylopia aethiopica in the present study demonstrated, both in vitro and in vivo, to contain some degree of antimicrobial substances. These antimicrobial activities could be attributed to the presence of some toxic substances in these plants that were fungitoxic to Colletotrichum destructivum O`Gara, both in vitro and in vivo. (Tables 16 to 21) It was observed that the tissue extracts of Xylopia aethiopica and Azadiractha indica were generally more effective than the Ocimum gratissimum and Cymbopogon citratus leaf extracts in the in vitro experiments. (Tables 16 to 19) However, the same Azadiractha and Cymbopogon extracts (at different concentrations) were found to rather stimulate, greatly, sporulation in the Colletotrichum. Test pathogen.(Fig21) All levels of extract concentration evaluated in Cymbopogon positively influenced sporulation in Colletotrichum destructivum while Azadiractha tissue extract (water and oil) could archive the 73 same feet only at product dilution levels with 140% sporulation density in water and 184% in oil and a relatively small sporulation density of 26% from the full strength of water extract The sporulation potentiality conversely exhibited by these two materials could suggest the suitability of extracts from Azadiractha indica and Cymbopogon citratus for substrate base/components in the culture of phytopathogens such as Colletotrichum destructivum. The Azadiractha seed extracts is known to contain some phytoalkaloids such as azadirachtin as one of its active ingredients (Amadioha &Obi, 1998). The high antifungal activity exhibited by the oil extract in the present study may be attributed to such a substance (Nduagu et al, 2008). In the Cymbopogon treatment one hundred per cent and 50% hot water leaf extracts caused 86% and 232% increase in sporulation density respectively, while 100% and 50% cold water leaf extracts were associated with 72% and 222% increase in sporulation density respectively The inhibitory effect of the cold water leaf extract of Cymbopogon on colony growth of Colletotrichum was concentration dependent unlike with the hot water leaf extracts Cymbopogon citratus contains between 75-85% aldehydes consisting largely of the organic substance called citral, a preservative and food flavorings especially in India and Java. It is possible that the antifungal property exhibited in the study was due to the citral content. This seems to support the work of Palhano et al, 2004, who successfully inactivated spores of Colletotrichum gloeosporiodes using high hydrostatic pressure separate and combined with citral essential oil. That also needs a further study to isolate the pure oil and determine the exact chemical content(s) that is (are) responsible for such antifungal activity. in conjunction with its converse sporulative potentials on Colletotrichum destructivum.of Vigna unguiculata 74 The oil of Xylopia aethiopica was found to possess a chocking and irritating odour. The antimicrobial property of X.aethiopica exhibited on Colletotrichum destructivum test pathogen could perhaps be due to the Xylopia acid among other interesting diterpenes contained in the fruit extract (Amadioha and Obi 1998) Further work, however is needed to ascertain the specific chemical responsible for the fungitoxicity of this oil. All treatment with Ocimum leaf extracts had a significant inhibitory effect on spore germination. The inhibitory was concentration dependent. There was also significant reduction in sporulation density with all treatments. In colony growth however, its effectiveness fell below that of A. indica, C. citratus and X. aethiopica at half dose concentration of both hot and cold water extract. (Fig 20) In the in vivo experiment , the tissue extracts of Xylopia and Azadiractha also exhibited a more antifungal effect on Colletotrichum destructivum anthracnose than the leaf extracts of Cymbopogon and Ocimum (Tables 20 & 21) .Plant tissue extracts and the standard fungicide exhibited more fungitoxic effect as protective or prophylactic than as eradicative or chemotherapeutic fungicides against Vigna unguiculata disease in vivo This suggestion is based on the fact that the percentage reduction in lesion spread on plant treated 2 days before artificial inoculation(2dbi) was more than those treated after the expression of disease symptoms. This experimental assertion was further supported by the graphic run of line of tendency (Fig 23) where the lineal distribution located more points from treatments administered two days before the artificial crop inoculation. (2dbi). The more prophylactic tendency of the test materials was further expressed in the physiological condition of the crop shown in fig 17, where plants treated 2 days before artificial inoculation (17A) looked healthier than the ones treated two days after pathogen inoculation (17B), which in turn were healthier than those treated after macroscopic symptom expression (17C) The oil extract of Azadiractha seeds used in the present study caused some physiological wilting of the sprayed crops. 75 The plants, however recovered at a later stage. The synthetic fungicides could not, however reduce the spread of the disease as readily as the test plant extracts in vivo This relatively unimpressive control from the synthetic Benomyl fungicide (tables 20 & 21) could be due to the reported tolerance of the pathogen to the standard formulation (Amadioha, 2003 and Akinbode et al, 2008). In general the fungitoxic activities of the plant tissue extracts, especially in vitro, tend to be adversely affected by heat, as the cold water tissue extracts showed more fungitoxic effect than the hot water tissue extracts on the test pathogen, colony growth and spore germination (Figs 19 &20). .The considerable disease control potentials expressed in this study, however suggest that the extracts of the plants are suitable for exploitation as potent fungicides for enhanced crop production practices and safer and ecological compatibility. The four test plants could be possible source of substitutes for synthetic chemicals in controlling anthracnose development in Vigna unguiculata, at least in the tropical regions of the globe. Interestingly the advantage and potential use of the higher plants in the controlling crop diseases have been emphasized (Amadioha, 2003; Hernandez-Albiter et al, 2007; Akinbode & Ikotun, 2008; Ogwulumba et al 2008; and Colpas et al, 2009) 76 5.2CONCLUSION 1. Anthracnose disease remains a devastating health problem to cowpea crop and an equated hindrance to its economic cultivation. The afflicting pathogen Colletotrichum destructivum O`Gara has the virulence of 100% infection on a single crop stand (that is every part of the crop is subject to attack and infection by Colletotrichum destructivum O`Gara at a given pathogenic situation).The use of botanicals remain suitable contest to adequate disease management options, at least for its characteristics ease of production economy and ecological amiability. 2. Results from the present study, established the fact that the four test plants: Azadiractha indica, Xylopia aethiopica, Cymbopogon citratus, and Ocimum gratissimum possess antifungal substances significantly toxic to Colletotrichum destructivum O`Gara. It is however pertinent to state here that further studies are needed to isolate and characterize these antifungal substances from the four test plants. 3. The potential of these botanicals as source of fungicides is enormous including the following: (i) The plants can be locally grown particularly in the global tropics. (ii) The extracts can also be obtained with crude or relatively refined cheap methods. (iii) The extracts have no mammalian toxicity, judged from the fact that they are already in use by man as food and flavorings or drugs and associated medicaments. (iv) They have little or no phytotoxic effect on sprayed crops. 4. 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