ANTAGONISTIC EFFECTS OF BACILLUS MEGATERIUM ISOLATED FROM RHIZOSPHERE OF TUMERIC PLANTS ON COCOYAM FUNGAL PATHOGENS

ABSTRACT

Cocoyam and other related tuber crops are susceptible to infection by various species of fungi, at all stages of growth and also during storage of tubers. Tuber rot is a major factor limiting the post-harvest life of cocoyam. Sixteen (16) spoilt cocoyam samples were purchased from Ahiaeke market, Abia State. Fresh and healthy cocoyam cormels and cormels showing no symptoms of rot were also obtained for pathogenicity testing. The points of advancement of rot were inoculated on a solidified Sabouraud Dextrose Agar (SDA) agar. Four (4) different fungal isolates were obtained from rotten cocoyams. The isolates included Aspergillus niger, Sclerotium rolfsii, Fusarium solcmi, and Penicillium which were implicated in the rots. Sclerotium rolfsii and Fusarium solani had the highest frequency of occurrence 56.3% and 37.5% respectively followed by Aspergillus niger and Penicillium species with 31.2% and 18.7% frequency of occurrence. In the pathogenicity testing, species of Sclerotium rolfsii and Fusarium solani were very pathogenic while, Aspergillus niger and Penicillium was mildly pathogenic, Sclerotia rolfsii caused a rot severity of 69.34% on the sample with Fusarium solani following it closely with a rot severity of 43.56% Aspergillus niger and Penicillium recorded 27.2% and 14.8% rot respectively. The inhibitory zones produced by the Bacillus species were ranged from 12mm to 16mm in diameter. Four isolates showed inhibitory effect on the pathogens. Bacillus megaterium exhibited the highest inhibition of 16mm and 12mm against Sclerotium rolfsii and Aspergillus niger. Bacillus subtilis and Bacillus licheniformis recorded inhibition zone of 13mm and 15mm respectively against Penicillium species and Aspergillus niger. Fusarium solani was however only susceptible to Bacillus subtilis with a clear zone of 12mm. From the results obtained in this study, it was revealed that Bacillus species possess potential inhibitory activity against rot-inducing fungi of cocoyam.

TABLE OF CONTENTS

LIST OF TABLES

Table 4.1:  Morphological and Biochemical Characteristics of the isolates

Table 4.2: Cultural and Microscopic Features of the fungal Isolates

LISTS OF FIGURES

Figure 1: Percentage occurrence of mold isolated from diseased corms of cocoyam

Figure 2: Percentage severity of rots obtained from test corms during pathogenicity test

CHAPTER ONE

1.0       INTRODUCTION

Root and tubers comprise of staple food crops, being the source of daily carbohydrate intake for a large population of the world. The tuber refers to any growing plant store edible materials in subterranean root, corm or tuber from which cocoyam is a member of this important class of food (Tilak et al., 2005).

There are several limiting factors for the production, processing and quality of cocoyam in the world. Yam and other related tuber crops cocoyam inclusive are susceptible to infection by fungi, bacteria and viruses at all stages of growth and also during storage of tubers. Tuber rot is a major factor limiting the post-harvest life of cocoyam and losses can be very high which has been estimated to be about 26% in the world (Amusa et al., 2003). Rot is the process of decomposition or decaying of tubers by the action of fungi and bacteria. Most rots of yam tubers in the storage are caused by pathogenic fungi such as Aspergillus flavus, Aspergillus niger, Botryodiplodia theobromae, Fusarium oxysporum, Fusarium solani, Penicillium chrysogenum, Rhizoctonia spp, Penicillium oxalicum and Rhizopus nodosus (Okigbo and Ikediugwu, 2002; Okigbo and Emoghene, 2004). The quality of yam tubers are affected by rots, which makes them unpleasant to consumers. In Nigeria, over 60% of white yam varieties get rotten when stored for less than six months (Okigbo and Emoghene, 2004). It has been observed that some farmers lose as high as 70% of their stored cocoyams to rot causing fungal pathogens (Aidoo, 2007).

Postharvest deterioration has been a major problem associated with cocoyam storage for both farmers and traders and it is caused mostly by micro-organisms especially fungi.

Colocasia esculenta (Cocoyam) is next to yam in importance in oriental economies. It serves as both a vegetable crop and a root tuber. The corm is an important component of the diet with a very high starch content which is nutritious, containing dietary fibre and easily digested (Amusa et al., 2003). The corm is eaten fried, boiled, baked, or converted into breadstuffs. Colocasia esculenta has more carbohydrate and protein than potato, and has a pleasant nutty flavour. The fried corm is a major delicacy in many areas in Southern Eastern Nigeria. C. esculenta leaves serve as a vegetable and is rich in vitamins and minerals. They are also good sources of thiamine, riboflavin, niacin, iron, phosphorus, zinc, potassium, copper, and manganese.

Urbanization resulted in decreased production of C. esculenta but recent decline in growth and development of this crop has resulted from pests and diseases (Hao, 2006). Diseases of Colocasia significantly reduce the number of functional leaves and have led to yield reduction of about 50% worldwide (Kuklinsky-Sobral et al., 2004). C. esculenta is affected by a number of infectious diseases caused by fungi, bacteria, nematodes, and viruses as well as noninfectious or abiotic factors. According to Taiga (2012), among these diseases, fungal diseases of C. esculenta are the most significant. Diseases caused by fungi are the most prominent, aided by climatic conditions which favour the growth of C. esculenta.

Storage losses in cocoyam have been identified as a major factor limiting the quantity and quality of the crop available for human consumption. Storage losses in cocoyam are known to result from weight loss, sprouting and microbial decay. Ogaraku and Usman (2008) isolated Aspergillus sp., Botryodiplodia theobromae, Fusarium sp., Rhizopus sp. and Penicillium sp. as pathogens causing rots in cocoyam.

Amusa et al. (2003) inferred that cocoyam cormels mature for harvesting 9-12 months after planting. They, however, reported that no serious deterioration occurs if the crop is left in the ground for a few weeks after maturity and harvested as needed. Even though this practice provides field storage for the crop it, no doubt, discourages the commercial production o f the crop as it places undue restriction on the availability of land.

There is therefore the need for an in-depth study into factors that influence storage rots of cocoyam cormels and how these could be controlled.

Post-harvest spoilage of cocoyam arises from improper handling of the cocoyam either during storage or harvest. The greatest cause of root rot and tuber loss in storage is the highest disease in cocoyam (Zhang et al., 2011). The post-harvest loss of root and tuber crops has been a very serious problem to farmers, as more than 40% of their harvest maybe lost because of decay. It is estimated that in the tropics each year between 25% and 40% of stored agricultural products are lost because of inadequate farm and village-level storage. The principal species of microorganism associated with cocoyam rot in Nigeria are; Aspergillus flavus, Penicillium digitatum, Botryodiplodia theobromae and Erwinia carotovora (Zhang et al., 2011). These fungi are believed to be pathogenic to various cultivars of cocoyam, causing rot of several parts of Southern Nigeria (Amusa et al., 2003). Fungi spoil the cocoyam by colonizing it by depolymerizing certain specific cell wall polymers such as proto-protein, the cementing substance of the produce (Arora et al., 2001).

Disease management practices can contribute to sustainability by protecting crop yields, maintaining and improving profitability for crop producers, reducing losses along the distribution chain, and reducing the negative environmental impacts of diseases and their management. Crop disease management supports sustainability goals through contributions to food security, food safety, and food sovereignty for producers and consumers alike.

While pesticides have done much to contribute to food security and food sovereignty for many millions of people worldwide, pest and disease control through the regular use of pesticides is neither desirable nor sustainable over the long term. Pesticide use raises significant concerns over impacts on health and the environment (Hameeda et al., 2008). Furthermore, we cannot address the challenges to sustainability posed by synthetic pesticides by simply switching to the application of natural pesticides, because the same concerns apply to them (Hameeda et al., 2008).

Practices for managing crop diseases fall into four general categories: host plant resistance, cultural practices, biological control, and chemical control. If pesticide use is to be reduced, it will be necessary to depend more on the remaining three approaches. Cultural practices (examples include crop rotation, polyculture, manipulation of planting date, etc.) certainly play a central role in disease management (Zhang et al., 2011). However, control achieved via cultural practices is sometimes inadequate, impractical, or economically nonviable. Natural biological control of plant pathogens is a fact of life, as it undoubtedly occurs at some level in all agricultural soils.

Soil is replete with microscopic life forms including bacteria, fungi, nematodes, and algae. Over 95% of the bacteria exist in the plant roots and those plants obtain many nutrients through the soil bacteria. Nitrogen is used to synthesize plant proteins and nucleic acids, including DNA. Although, it is found naturally in the atmosphere, it cannot be used by the plants in the available form (N₂).

These bacteria significantly affect plant growth by increasing nutrient uptake, producing biologically active phytohormones and suppressing pathogens by producing antibiotics, siderophores, and fungal cell wall-lysing enzymes (Arora et al., 2001; Kuklinsky-Sobral et al., 2004). PGPB could also promote plant growth by suppressing plant pathogens indirectly. This enhanced state of resistance is effective against a broad range of pathogens and parasites, including fungi, bacteria, viruses, nematodes, parasitic plants, and insects (Ryu et al., 2004). In the last few years, the number of PGPB that have been identified has seen a great increase. Species of bacteria like Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus and Serratia have been reported to enhance the plant growth (Kuklinsky-Sobral et al., 2004). In a previous report, a nitrogen fixing bacteria, Bacillus megaterium was isolated from maize rhizosphere which did not show any antifungal activity (Liu et al., 2006). In another recent report, Bacillus subtilis was iso-lated from roots of banana plant and it was concluded that although, B. subtilis is not a nitrogen fixing bacterium, it can be efficiently used in a bio-organic fertilizer against Fusarium wilt (Zhang et al., 2011).

Antagonistic bacteria produce antimicrobial substances as important compound for self-defense function towards other organisms e.g., Bacillus sp. producing antimicrobial compounds have been used as biocontrol agents against plant pathogenic fungi (Yilmaz et al., 2005).

Many studies exploring of beneficial organisms have been carried out, such as P. fluorescens, which was one of the examples used for the control of Fusarium wilt of tomato. Similarly, P. fluorescens were found to be effective biocontrol agents against the Phytophthora disease in black pepper (Diby et al., 2005). In addition, most of the species from the genus Bacillus are considered as safe microorganisms and they possess remarkable abilities to synthesize many substances that have been successfully used in agriculture and for industrial purposes. The secondary metabolites produced by several species and strains of the genus Bacillus have been found to show antibacterial or antifungal activity against different phytopathogens (Ongena and Jacques, 2008).

 The control of this disease through fungicide application however has adverse effects on the environment and negatively affects the soil microbiota. These chemicals either induce the generation of mutant varieties or reduce or suppress other microbial populations. The increased reflection on environmental concern over pesticide use has been instrumental in a large upsurge of biological disease control. Development of fungicide resistance among the pathogens, ground water and foodstuff pollution and the development of oncogenic risks have further encouraged the exploitation of potential antagonistic microflora in disease management. Among the various antagonists used for the management of plant diseases, plant growth promoting rhizobacteria play a vital role.

Studies have shown that various types of microorganisms are a potential substitute for inorganic chemical compounds (fertilizer and pesticides) that can be applied in the field in a wide scale. A number of microbes have been reported to be effective as biological control agents of plant diseases i.e. Bacillus, Bdellovibrio, Dactylella, Gliocladium, Penicillium, Pseudomonas, and Trichoderma (Diby et al., 2005). Soil microorganisms associated with the rhizospheres of plants have been known to contribute in many processes in the soil which in turn may influence the plants growth and progression (Shimoi et al., 2010)

1.1       Aims and Objectives

The specific objectives of this research work were:

1.     To isolate, identify and characterize Bacillus species from the rhizospheric soil of turmeric plants

2.     To isolate, identify and characterize fungal pathogens of cocoyam cormss.

3.     To study the pathogenicity of the fungal isolates on healthy cocoyam corms and re-isolation of the fungal pathogens.

4.     In vitro evaluation of antifungal ability of Bacillus megaterium isolates against the fungal phytopathogens.

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