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Bio-Fungicides in Allium Sativum (L) Had Significant Inhibition on Phytophthora Megakarya (Brasier & Griffin) and Cocoa Blac

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Bio-Fungicides in Allium Sativum (L) Had Significant Inhibition on Phytophthora Megakarya (Brasier & Griffin) and Cocoa Blac International Journal of Multidisciplinary Research and Development International Journal of Multidisciplinary Research and Development Online ISSN: 2349-4182, Print ISSN: 2349-5979; Impact Factor: RJIF 5.72 Received: 26-05-2019; Accepted: 29-06-2019 www.allsubjectjournal.com Volume 6; Issue 8; August 2019; Page No. 162-169 Bio-fungicides in Allium sativum (L) had significant inhibition on Phytophthora megakarya (Brasier & Griffin) and cocoa black pod rot disease Ayodele M Ajayi Department of Crop. Soil and Pest Management. Federal University of Technology, Akure, Ondo State, Nigeria Abstract Aqueous extracts from three plants (Chromolaena odorata, Zingiber officinale and Allium sativum) were evaluated In vitro and In vivo for the management of cocoa black pod rot disease caused by Phytophthora megakarya. Four concentrations (20, 40, 60 and 80%) of hot (70o C) and cool (35o C) aqueous extracts were obtained from fresh and dry samples of each plant. Efficacy of extracts was determined by inhibitions of mycelial growth of P. megakarya (In vitro) and disease progression on cocoa pods (In vivo). Treatments were replicated four times, and incubated at 28o C ± 2o C and 75% relative humidity. Data were collected on mycelial growth of pathogen on artificial media and radial growth of infection on cocoa pods. Statistical analysis of data collected showed significant differences among the concentrations of aqueous extracts. Inhibition of mycelial growth increased with increasing concentration of extracts but decreased with increasing days after inoculation. A. sativum was the most effective In vitro. Mycelial growth inhibition of 100% (40% concentration and above) was recorded at 3 days after inoculation. C. odorata was the least effective, as its highest percentage mycelial growth inhibition was 54.12% (at the highest concentration of 80%). A. sativum was also the most effective In vivo. It inhibited infection by 66. 10% and 79.03% (60% and 80% concentrations respectively) at 11 days after infection. A. sativum can be recommended for use in the management of cocoa black pod rot disease. Keywords: aqueous extracts, poison food technique, antifungal properties, cocoa black pod rot, Phytophthora megakarya 1. Introduction are the most commonly used (Agbeniyi and Oni, 2014) [2]. Global annual yield loss of cocoa resulting from cocoa black Unfortunately, however, these chemicals are extremely pod rot disease (CBPRD) has been projected to be around toxic. In addition, the residue of synthetic chemicals, the 30% (Guest, 2007) [16]. In Africa, 100% yield loss has been organochlorines in particular, have been found in cocoa reported in farmers’ fields in Ghana (Opoku et al, 2008) [29] produce, processed products and by-products (Aikpokpodion and Nigeria (Oluyole and Lawal, 2008) [24] with devastating et al, 2013; Okoffo et al, 2016 and Ibigbami and social and economic implications (Tijani, 2005; Adebawore, 2017) [4, 22, 17]. To prevent these chemical Aikpokpodion and Adeogun, 2011) [30]. residues from attaining damaging levels with serious Phytophthora spp. are known to incite CBPRD. P. capsici implications on public health, there is an urgent need for and P. citrophthora are the causative organisms of the alternative management strategies for CBPRD. disease in South America, while P. palmivora, with the This study was designed to evaluate aqueous extracts from reputation of infecting cacao trees in addition to the pods, three plants (Chromolaena odorata, Zingiber officinale and was the predominant pathogen of CBPRD in West Africa Allium sativum) for antifungal properties against P. for a very long time, until 1979 when P. megakarya was megakarya. The test crop was CRIN TC-2 Cocoa cultivar described for the first time (Brasier and Griffin, 1979) [8]. developed by the cocoa research institute of Nigeria Today, P. Megakarya is the dominant cause of CBPRD in (CRIN). It has many desirable agronomic characteristics but West Africa (Guest, 2007; Akofi, 2015; Ali et al, 2017) [16, 5, is susceptible to the cocoa black pod rot pathogen (CBPRP). 6]. It differs from P. palmivora by being more virulent, having larger nuclei in its gamentagia, less number of 2. Materials and methods chromosomes and longer sporangia (Samson et al, 1975; 2.1 In vitro studies Froster et al, 1990) [14]. Heavy and continuous rain along 2.1.1 Collection of infected cocoa pods with high relative humidity and warm temperature are some Infected cocoa pods were collected in August, a period of the pre-disposing environmental conditions for infection characterised by high incidence and severity of CBPRD, due and pathogenesis. Man, rodents, birds, ants as well as to the high humidity and warm temperature associated with rainwater running down the stems of infected cocoa trees all this period. Five pods, at various stages of infection, were acts as agents dispersing pathogen’s inoculum. obtained from a cocoa farm in Idanre, a popular town The process of developing cultivars of cocoa that are known for cocoa production in Ondo State, Nigeria. The resistant to P. megakarya and making such available to pods were transported to the pathology lab of The cocoa farmers is still at its infancy (Thevinin et al, 2004; Department of Crop, Soil and Pest management (CSP), Paulin et al, 2008) [29, 26], hence the management of CBPRD Federal University of Technology, Akure (FUTA), (a in Africa and most tropical countries, where cocoa is distance of about 30 km) in sterile glass jars covered with cultivated, has mainly been through the use of synthetic aluminium foil. chemicals. Copper/Copper and Metalaxy based fungicides 162 International Journal of Multidisciplinary Research and Development 2.1.2 Preparation of artificial growth medium 2.1.6 Preparation of PTJA/Aqueous extract complex and Phytophthora megakarya is an Oomycete, requiring assessment of its anti-fungal properties against P. specialised artificial medium like V8 agar for rapid growth. megakarya An improvised medium was prepared as a result of the A PTJA/Aqueous extract complex was achieved by adding scarcity of V8 agar. It consisted of 100 ml of tomato juice, 3 ml of extract to 12 ml of hot (70o C) PTJA in a Peri-dish prepared following the method described by Oluyemi et al, with gentle swerving to allow for proper mixing of the two. (2014) [23] and the same volume of potato infusion (obtained This procedure was repeated for the 48 concentrations. The from 20 g Irish potato in 100 ml of distilled water). Four (4) standard check had aqueous extract replaced with 3 ml of g each of dextrose sugar and agar were added to obtain Copper Hydroxide (Kocide 2000) at the manufacturer’s potato/tomato juice agar (PTJA). Sterilisation was done at recommended rate, while the control had 3 ml of sterile O 121 C for 15 minutes in a Surgifriend (2010 model) autoclave. distilled water. The PTJA/Aqueous extract complexes were Prepared PTJA was amended with 0.4 g of Streptomycin allowed to cool and gel. Thereafter, 5 mm agar disc each antibiotic to prevent bacterial growth. Pour plating, 20 ml from one-week-old pure culture of P. megakarya were O PTJA/sterile Petri-dish, was done when PTJA cooled to 45 C. punched out with the aid of sterile cork borers and transferred, upside down, to the centre of each 2.1.3 Isolation and identification of P. megakarya from PTJA/Aqueous complexes with the aid of a sterile wire infected cocoa pods loop. A total of 50 treatments, standard check and control Isolation of P. megakarya was done about 1 hr after inclusive, were evaluated. Each treatment was replicated collection of infected pods. Small segments, about 2 mm four times and was laid out in a completely randomized wide, from the active zones of infections were obtained with design (CRD). Incubation was done for 9 days at 28oC±2oC. the aid of sterile scalpels. The segments were surface sterilised in 70% ethanol for 20 seconds, rinsed in 4 changes 2.2 In vivo studies of sterile distilled water and pat-dried with sterile blotting 2.2.1 Collection/selection of healthy cocoa pods paper. Inoculation of the segments was done on prepared Ripe and unripe cocoa pods (Plate 1a) were obtained from a PTJA after gelling. A total of 10 Petri-dishes were cocoa field beside the new crop type museum of CSP inoculated and each had two pod segments placed at Department, FUTA and transported in sterile polythene bags opposite ends, about 8 cm apart. Incubation was done at 28O to the pathology lab of the same department immediately C ± 2O C. Sub-culturing was done at 48 hours after after collection. The exocarp of each pod was examined inoculation and re-incubated as described above. carefully with a 2 MP, 1000 magnification digital Identification was through visual observation of colony in microscope/endoscope camera and only healthy pods were culture, microscopy, aided by standard text, (Dugan, 2006) selected for the In vivo studies. and pathogenicity test. 2.2.2 Evaluation of aqueous extracts for inhibition of 2.1.4 Collection of plant materials infection on detached cocoa pods Fresh Allium sativum, garlic (bulb), Zingiber officinale, 2.2.2.1 Preparation of inoculum/ aqueous extract ginger (rhizome) and dry (pulverized) of both plants were complex purchased from Isinkan market in Akure, Ondo state, Mycelium and spores were harvested from the one-week-old Nigeria. Chromolaene odorata, (Siam weed) leaves were culture of P. megakarya, using a sterile spatula and distilled obtained from an abandoned farmland beside the old crop water (20 ml/Petri-dish). Filtration was done to remove type museum of CSP Department, FUTA. The dry pieces of agar discs and tangled mass of mycelium. A small (pulverized) leaves of C. odorata was obtained after air quantity of the filtrate (0.2 ml), which consisted mostly of drying for 8 days. chlamydospores and fragments of mycelia, was mixed with an equal volume of the most effective extract concentration 2.1.5 Preparation of aqueous extracts from ginger and garlic, in the In vitro experiment, to form Fresh samples of the three plants were ground separately inoculum/aqueous extract complex.
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