POTENTIAL OF SOME BACTERIA ISOLATED FROM COMPOST ORGANIC MANURE IN THE CONTROL OF COCOYAM (Colocasia spp) LEAF BLIGHT DISEASE

Jesica, O. Asagwara 1 , Martin, O. Onuh 1 , Ebenezer, O. Emeribe 1 , and Obinna, S. U. Nwosu 2 . 1. Department of Crop Science and Biotechnology ImoState University Owerri, Nigeria (PMB 2000) 2. Imo State Agricultural Development Progamme Owerri, Nigeria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

An investigation of bacteria isolates from composted manure and their potential in the control of Phytophtora colocasiae was carried out in the Teaching and Research Farm of Imo State University, Nigeria. Bacteria organisms isolated from composted organic materials (manure) were tested against the Phytophtora colocasiae which is the pathogen that causes leaf blight disease in cocoyam. Results revealed four bacterial isolates-Pseudomonas spp., Bacillus spp., Escherichia coli, and Staphylococcus aureus from the composted manure. The results also showed that Pseudomonas spp. Bacillus spp., Escherichia coli and Staphylococcus aureus gave mean diameter of growth inhibition of 43.167mm, 40.353mm, 19.667mm and 17.333mm, respectively. There was no significant difference (P≤0.05) in mean diameter of growth inhibition between Pseudmonas spp. and Bacillus spp. but the two were significantly different from that of Escherichia coli and Staphylococcus aureus. Observations showed that the bacterial isolates from the composted manure were more effective in their antagonism activity when the disease pathogen concentration was lower. Based on the antagonism properties of Pseudomonas spp. and Bacillus spp., they serve as potential agents in the program involving biological control of cocoyam leaf blight diseases.

…………………………………………………………………………………………………….... Introduction:-
The production and yield of Taro is faced with a lot of problems as a result of damage by Taro leaf blight disease caused by Phytophtora colocasiae. According to Helen and Graham, (2000), taro leaf blight reduces the life of leaves, thus instead of lasting for 40 days, they last for only 20 days resulting in plants with few leaves and small corms. The disease reduces yield by about 40%-70% (Jugumauth et al., 2002). Taro leaf blight disease also causes a firm, brown corm rot after harvest and it takes 7-10 days before the corm is completely decayed, (Sid et al., 2003). The disease is mainly foliar oriented, although post-harvest storage rots occur. Initially symptoms of the disease are small brown water-soaked lesions, often with a yellow margin (Wilson, 2007). Secondary infections lead to rapid destruction of the leaf, which may occur in 10-20 days after infection or less in susceptible varieties. The normal longevity of a healthy leaf is 40 days but the disease can significantly reduce the number of functional leaves which can lead to yield reduction in the magnitude of 50% (Gadre and Joshi, 2003). Leaf blight disease causes yield losses of 30-50%, including post-harvest decay of taro corms, shortages of planting materials and high production costs.

Int. J. Adv. Res. 4(8), 1388-1398
Phytophtora colocasiae belongs to the kingdom chromista and phylum Oomycota (Ooka 1994). It is known to infect taro (Colocasia esculenta) while Xanthosoma is resistant to it (Brooks, 2000). It has been found in all taro growing areas of Hawaii, and it is also present in Papua New Guinea, Solomon Islands, Philippines, Palua, Gaum, the Northern Marianas Island, West Samoa, American Samoa, Ponape, Yap, Truk, India, Taiwan and Trinidad (Trujillo, et al., 1993). The disease was first reported in Nigeria in 2009, and by 2010 it became an epidemic in Cameroon (Mbong, et al., 2013). It is believed to have originated in South East Asia and is widely distributed throughout the tropical regions of the world (CMI, 1997). Investigation carried out in Nsukka, Eastern Nigeria in 2011 revealed that the taro leaf blight diseases has become epidemic and has drastically reduced cocoyam production by 60% (Chiejina and Ugwuja, 2011) Other fungal organisms associated in the storage rotting of cocoyam include, Aspergillus niger, Fusaruim solani, Botryodiplodia theobroma, Fusarium oxysporum, Cortium rolfsii; Geotrichum candida and Sclerotium rolfsii (Ugwuanyi andObeta, 1996, Ohazurike et al., 2003). The early stages of leaf blight disease are characterized by the formation of small, frequently circular brown to olive green spots (Ooka, 1994). Helen et al., (1996) reported that the fungus is active in wet weather; spores produced on the leaves and spread in winds and rain to nearby plants or longer distance to new gardens. If they fall on a wet leaf they germinate and continue attack (Ugwuanyi and Obeta, 1996;Trujillo, 1996).The spores can germinate like a seed and infect leaves or they produce smaller spores, which burst out and swim short distances over the leaf before they germinate and cause infection, (Helen and Graham, 2000).In both cases, the fungus kills the cell of the leaf and brown spots occur.
The spots expand very fast and produce yellow margins with red-brown droplets developing on the under surface where the droplets dry as dark pellets, (Ngoka, 1997). Infection can occur anywhere on the leaf surface, but often starts at the edges where rain dew collects. After few days of infection, a white ring can be seen near the margin of the spot; this is the area where spores are produced. However, the spores dry out quickly in the sun and by midmorning they will shrivel and die, (Chiejina and Ugwuja, 2013). They only stay alive if it is cloudy or raining. Apart from wind, spread of the disease can occur in other ways, such as suckers planted with infected leaves attached or stalks of planting material probably on the cut ends in wet weather when stalks are trimmed for planting. Jackson, (1996) reported good control of the disease by spraying copper oxychloride applied at the rate of 4.5kg per 100 litres of water per hectare. Maheswari et al., (2001) again reported the use of copper fungicide (especially copper oxychloride), to be effective in the control of the disease but have to be used often and are only recommended where taro are grown at commercial scale.
Chukwu, (2012) also reported the effective control of phytophotora leaf blight disease of taro by spraying of Ridomil, Nordox and Kocide forthnightly on the leaves of affected plants; (One sachet of 100grams in 15 litres of water). Similar result was also reported by Scot et al., (2011) in Samoa when sprays of Ridomil; Manzate; and phosphorous acid product were used in the control of taro leaf blight disease. Misra, (1999)reported that Metalaxyland Mancozeb based fungicides have proved effective in controlling taro leaf blight disease but the waxy leaf surface and the occurrence of the disease during rainy season make fungicidal sprays ineffective.
Over the years, the protection of Agricultural crops and products was achieved almost entirely through the use of synthetic chemicals (Adams, 1991;Jacobson, 2001;Alabouvette et al., 2006). These chemicals though valued for their effectiveness are costly and constitute health hazard to farm households and the environment. This observation was reported by Ohazurike and Obi, (2000) who emphasized that, the ubiquitous use of these chemical pesticides pose numerous problems which include; development of resistance to the chemical pesticides by the target pest or pathogen species, outbreak of secondary pests or pathogen, extermination of beneficial organisms in the environment, environmental pollution and accidental poisoning of farmers and handlers. In view of the associated problems of synthetic chemicals, the U.S. Department of Agriculture specially recommended a general reorientation of research and extension to develop new technologies to reduce cost, increase efficiency and facilitate the economic viability of small to medium sized farms.
In the light of the above, biological control using microbial agents in diseases control cannot be over-emphasized. Alavi et al., (2013) described biological control as the purposeful utilization of introduced or resident living organisms, other than disease-resistant host plant, to suppress the activities and populations of one or more plant pathogens. Several works have been carried out using biological agents extracted from animal farm refuses. Compost tea, an extract from compost mixed with water was found to suppress certain plant diseases, such as Botrytis on green beans, straw berries, grapes and geraniums, powdery mildew on apples and others (Dianez et al., 2006). According to EL. Jahil and Zinedine, (2008), some microbial and chemical properties of poultry wastes manure after lactic acid fermentation at 30 0 C for 7-10 days using 10% molasses, humidified and inoculated with Lactobacillus planarum and Pediococcus acidilactici removed hazardous microorganisms like Enterobacteria, Enterococci, Clostridum and Salmonella.
Microbial fertilization from poultry manure were processed into Omug, Ecud and Pudret using hot air (60 0 C); fermentation at 40 0 C for 7-8 days and treatment with infra radiation at 48 0 C, respectively. Omug which has 55% dry matter and 15% water contained heterotrophic aerobic microorganisms (microflora) that improved the quality of fertilizer, promoted bacterial growth and suppressed phytopathogenic micro-organisms (Fisinin and Arkhipchenko, 2001). Jager et al., (1991), also confirmed that biological control of Rhizoctonia solani in potato by Verticillum biguttatum increased the percentage of harvests to 56%, compared to 24% in non-inoculated. Also gram-positive Bacillius spp. and Gram negative Pseudomonas spp were effectively used in the control of soil borne pathogens that affected Brassicas vegetables (Wulff et al., 2003).
Pseudomonas fluorescens and Bacillus pumulis significantly reduced the incidence, severity and vascular discolouration of Venticillium wilt in tested potato cultivars in growth room trials and field testing (Uppal et al., 2008). Okamoto et al., (2000) recorded that, three bacterial strains isolated from rhizosphere of angelica trees strongly inhibited mycelia growth of Cactorum. This is in conformity with work of the Rajkuma et al., (2005) in which 12 isolated Pseudomonas spp. from rhizosphere of pepper significantly inhibited Phytophtora blight disease. Hoopen et al. (2003) reported that mycoparasitic bacteria collected from aerial parts of the cocoa plant have shown great effect in the control of black pod disease, caused by Phytophtora palmivora.
Foliar application of biological control agents has some potential to protect taro plants from infection of taro leaf blight disease. Considering the importance and enormous potential of cocoyam in the human food chain and livestock feed in Nigeria, it is necessary that any factor militating against continued and increased production of the crop should be avoided or checkmated. Efforts have been made in the control of this disease especially, in the use of chemicals. But some of these chemicals are froth with challenges.
Therefore, efforts should be made in fashioning control measures that will guarantee the safety of animals, man and the environment. In view of this, the study investigated potential of some bacteria isolated from compost organic manure in the control of cocoyam (Colocasia spp) leaf blight disease.

Materials and method:-Study Area:-
The study was carried out at the Teaching and Research Farm of Faculty of Agriculture and Veterinary Medicine, Imo State University, Owerri Nigeria. The samples used in this study were; organic manure and cocoyam leaves (Colocasia esculenta) (L) Schott) infected with Phytophtora colocasiae. Nutrient Agar incorporated with 1% Nystatin which inhibits fungi but promotes the growth of bacteria.
Making Compost:-Organic manure comprising of animal dungs saw dust, grasses, legumes and dry plantain leaves were collected from Alex farms, Ubomiri, Somachi cattle market Egbu road; Umuonyeali-Ugo timber market, Imo ADP zonal office and the Teaching and Research Farm of Faculty of Agriculture and Veterinary Medicine, Imo State University, Owerri. All the areas of material sources are located in Owerri. Two pits were dug to 60cm depth and the organic materials were piled in each of the pits. Water was sprinkled on the materials to facilitate decay. An iron peg was driven at the Centre of the organic materials in the pit to create space for aeration. The pit was then covered with perforated black polythene sheet. The compost materials were checked after one week for decomposition, by removing and feeling the hotness of the iron peg thereafter turning started after four weeks. Turning continued fortnightly till 16 weeks when the manure became fully decomposed Collection of Samples and Sterilization of Materials:-Five (5) grams of the composted manure was aseptically collected using sterile rubber container with stopper. The container was wrapped with foil and taken immediately to the Department of Microbiology, Imo State University, Owerri laboratory for microbiological analysis. Also, leaves of cocoyam infected with Phytophtora colocasiae were collected and cleaned with disposable alcohol swab pad and the alcohol allowed to evaporate. A sterile knife was used to cut off the infected leaves and packed in a sterile paper packet ensuring that there was no delay, to retain the viability of the organisms. The samples were taken to the same laboratory for microbiological analysis and the study was done under aseptic conditions. The materials used were sterilized by standard laboratory methods as described by Chesbrough, (2002); Obiajulu and Ozumba, (2009).

Media Preparation and Dilution of Composted Manure:-
Twenty-eight (28) grams of the powder was dissolved in 1 litre of de-ionized water and allowed to stand for 10 minutes for complete dissolution. It was swirled to homogenize and sterilized by autoclaving at 121 0 C for 15 minutes. It was allowed to cool to 40 0 C and 1% Nystatin suspension was added to it to inhibit fungal growth. It was then poured; 20ml into each sterile disposable petridishes and allowed to solidify at room temperature. The petridishes were then stored in the refrigerator until required for use.
One (1) gram of the composted manure was weighed out and mixed with 10ml of distilled water and swirled to make a good solution. Five test tubes were arranged in the test tube rack, each was filled with 9ml of distilled water, and 1ml of the composted manure solution was collected with pipette and added to the first test tube containing 9ml of distilled water, thereby making it 10ml. From the first test tube, 1ml was also collected and added to the second test tube. This process was carried out in successive manner up to the fifth test tube where 1ml was discarded, leaving it as 9ml. With the five-fold serial dilution, different concentrations of the manure were achieved in each of the test tube. From each test tube, 0.1ml of the diluted sample was cultured directly on duplicate plates (petridishes) of nutrient agar with Nystatin (Uwaezuoke et al., 2008). The inoculated plates (petridishes) were labeled properly with different concentrations and dilutions of the corresponding test tubes. Plates containing nutrient agar were incubated at 37 0 C for 24 hours and were further examined for bacterial growth.

Isolation and Identification of Bacteria from Composted Manure:-
The bacterial isolates were sub-cultured on nutrient agar (NA) to obtain pure cultures and their characteristics and morphologies were identified. They were further subjected to series of test, which include:-gram staining reaction and biochemical tests such as catalase, coagulase, citrate, indole methyl red, oxidase and motility.

Isolation and Identification of Cocoyam Leaf Blight Pathogen (Phytophtora colocasiae):-
One (1) gram of sections of the leaves which was infected with cocoyam leaf blight disease was cut off with a sterile razor blade. This was mashed to make a paste, and 10ml normal saline was added to it. Five test tubes were arranged in a test tube rack, and each was filled with 9ml distilled water. 1ml of the infected cocoyam leaf solution was taken with pipette and added to the first test tube containing 9ml of distilled water, making it 10ml.
From the first test tube, 1ml was also collected and added to the second test tube. This process was carried out in successive manner up to the fifth test tube. From the fifth test tube, 1ml was discarded, leaving it as 9ml; with the five-fold serial dilution, different concentrations of the cocoyam leaf blight was achieved in each of the test tubes.

Antagonism of Composted Manure Microbial Isolates to Cocoyam Leaf Blight Pathogen Using Well In Agar
Method:-From each test tube containing different concentrations of the diseased cocoyam leaf solution, 0.1ml was collected and inoculated on NA agar plate and spread evenly on the surface of the media with a glass rod spreader. For each concentration four agar plates were prepared to test the four microbial isolates of composted manure.
Control plate of only cocoyam leaf solution on agar was also prepared for the different concentrations. Hole of 8.0mm in diameter was created on each of the agar plates using a sterile core borer by removing circular plugs of agar. For each of the four agar plates, the four microbial isolates were introduced into the hole using sterile wire loop.
Plates containing the isolates were incubated at 37 0 C for 24-48 hours. At the end of incubation, plates were checked for areas of antagonism or diameter of clearance. The arrangement was laid out in a completely randomized design, while the experiment was replicated three times.

Data Collection:-
Data were collected on the following parameters: Morphological appearance of bacterial culture, gram staining and biochemical qualities of bacteria isolates. Types and number of bacterial isolates from the composted culture, level of antagonism of cocoyam leaf blight pathogen by isolates of bacteria from composted cultures. The level of antagonism was expressed as the diameter of zone of growth inhibition in the culture, measured in millimeters (mm).

Data Analysis:-
Data on the diameter of zone of clearance (antagonism) was subjected to analysis of variance of the completely Randomized Design procedure, under the following model; X ij = µ+t i +∑ j .However, the above analysis was achieved with the use of Statistical Analytical System (SAS) package 10.1 editions (2007). Means were separated with Least Significance Difference (LSD) test and interpreted according to methods of Onuh and Igwemma (2007).

Results:-
Tables 1 and 2 below showed the appearance of the bacterial culture for 24 and 48 hours in terms of colour, elevation, number of colonies, capacity, surface, shape and type of colony. It was observed that higher concentrations of the manure produced higher colonies of bacteria.   Table 3 summarized gram staining reactions and biochemical tests of samples from pure culture of manure. The tests were used to identify the particular species of the bacteria organism.
NA is nutrient Agar while T is the treatment. In the course of the study the treatments were repeated to get pure cultures in order to avoid doubt.  Four bacteria species were isolated from composted manure cultured in nutrient Agar medium. Out of the 4 bacteria organisms isolated, Pseudomonas spp occurred highest (14 in number) followed by Bacillus spp (8), Escherichia coli (6), and Staphylococcus aureus (3) giving a total of 31 (Table 4) The result presented in the Table 5 showed that Pseudomonas spp had the highest antagonism to cocoyam leaf blight pathogen with a clearance diameter of 43.167mm. This was followed by Bacillus spp that produced diameter of clearance of 40.333mm, but it was not significantly different (P≤0.05) from the diameter of clearance produced by Pseudomonas spp (Table 5). However, there was no significant difference (P≤0.05) in the diameter of clearance produced by (Eucherichia coli (19.667mm) and that of Staphylococcus aureus (17.333mm) as presented in the Table  6. But the performance E. coli and S. aureus was significantly different from that of Pseudomonas spp and Bacillus spp. (Table 5).
There was no significant difference between pseudomonas spp and Bacillus spp while significant difference exists between them and Escherichia coli and staphylococcus. Also there was no significant difference between Escherichia coli and staphylococcus aureus.   It was observed that number of bacteria colonies decreased as the concentration of the composted manure samples decreased. Thus, the 10 -1 manure concentration recorded the highest number of bacteria colonies while 10 -10 manure concentration recorded the lowest colonies. This observation may have been as a result of concentration of more manure samples in the 10 -1 dilution, which gradually decreased in manure concentration as the dilution factor increased; given rise to lower bacterial colonies in the higher diluted manure samples.

Discussion:-
Gram staining reaction and some biochemical tests such as catalase, coagulase, citrate indole, methyl red, oxidase, and motility were done based on the characteristics of the bacterial organisms. Four species of bacteria were isolated and they are: Pseudomonas spp, Bacillus spp, Escherichia coli and Staphilococcus aureus. It was observed that Pseudomonas spp was the most frequent organism isolated from the composted manure, followed by Bacillus spp, Escherichia coli and staphylococcus aureus, respectively.
Studies made on the mean diameter of growth inhibition (antagonism) of leaf-blight disease pathogen (Phytophtora colocasiae) by bacteria isolates from composted manure recorded Pseudomonas spp as having the highest antagonistic properties followed by Bacillus spp, even though there was no significant difference between them. This efficacy of Pseudomonas spp was also reported by Maryem et al., (2013) in biological control of root-knot nematode. Also Alavi, et al., (2013) confirmed this in his work; biological control of take-all disease by isolates of Pseudomonas and biosynthesis of silver nanoparticles by the culture supernatant of Pseudomonas. Pal and Gardener, (2006) in their study, reported the efficacy of Bacillus spp in the biological control of plant fungal pathogens such as Alternaria, Pythium, Aspergillus, Fusarium, Rhizoctonia, Phytophtora etc. Escherichia Coli and Staphylococcus aureus also showed some level of antagonistic properties to cocoyam leaf blight disease but they were not as effective as Pseudomonas and Bacillius spp.
It was observed from the results that the level of antagonism of the bacteria isolates from the composted manure increased with decrease in the concentration of the leaf blight disease pathogen samples. Thus, the antagonistic bacteria were effective in antagonizing the pathogen in the lower concentrations, perhaps as a result of reduction in the density of the pathogens in the lower concentrations to be attacked. Hence, it was easier for the level of antagonist bacteria used to attack the pathogen under scanty density. The results generally indicated that composted manure can yield bacterial isolates such as Pseudomonas spp and that can be harnessed in the biological control of cocoyam leaf blight disease.

Conclusion:-
The potential of bacteria in the control of cocoyam leaf blight disease was evaluated. Responses from the different group of micro-organisms, viz-bacteria showed antibiotic and parasitic properties to the disease pathogen .It was also observed that the microbial isolates from the composted manure were more effective in the antagonism activity when the disease pathogen concentration is lower. It is therefore, recommended that Pseudomonas spp and Bacillus spp. should be explored further in their possible use as biological agents in the control of leaf blight disease of cocoyam.