THE EFFECTIVENESS OF TRICHODERMA REESEI IN THE BIOCONTROL OF FUSARIUM OXYSPORUM AT DIFFERENT TEMPERATURES
A project report submitted for examination in fulfillment of the requirements for the course Unit SBT 414: RESEARCH PROJECT in the Bachelor of Science (Microbiology and Biotechnology) Degree.

AMWAYI ANGELA LUKALE
I23/3384/2008.
Signature…………………… Date………………………..
SUPERVISOR: DR. P.M WACHIRA
Signature …………………… Date……………………….

SCHOOL OF BIOLOGICAL SCIENCES.
UNIVERSITY OF NAIROBI.
2011-2012

DECLARATION iii
DEDICATION iv
ACKNOWLEDGEMENT v
ABSTRACT vi
CHAPTER ONE 1
INTRODUCTION 1
1.1 JUSTIFICATION 2
1.2 OBJECTIVES 3
1.2.1 Broad objective 3
1.2.2 Specific objectives 4
CHAPTER TWO 4
LITERATURE REVIEW 4
2.1 Trichoderma Spp. 4
2.2 Fusarium species 8
CHAPTER THREE 11
MATERIALS AND METHODOLOGY. 11
3.1 Media Preparation 11
3.2 Isolation of pathogenic fungi (Fusarium spp). 12
3.4 Dual culture inoculation 13
CHAPTER FOUR 13
RESULTS 13
CHAPTER FIVE 18
DISCUSSION 18
CHAPTER SIX 20
CONCLUSION AND RECOMMENDATION 20
REFERENCES 21
8. Dudley, N. S. (2007). Pathogenicity of four Fusarium species on Acacia koa seedlings. Missoula, MT: U.S. Dept. of Agriculture, Forest Service, Northern Region, Forest Health Protection. 21
12. James, R. L. (2002). Biological control of Fusarium oxysporum and Fusarium proliferatum on young Douglas-fir seedlings by a nonpathogenic strain of Fusarium oxysporum. Missoula, MT: U.S. Dept. of Agriculture, Forest Service, Northern Region. 21
21. Ozbay, N. (2003). Biocontrol of Fusarium crown and root rot of fresh market tomato with Trichoderma harzianum strains under greenhouse conditions. New York: Biological Association. 22

DECLARATION

I hereby declare that I am the sole author of this research paper. Every finding arrived at in this research paper is as a result of my own research and in instances where I have adapted from other sources I have clearly acknowledged.

Name: AMWAYI ANGELA LUKALE I23/3384/2008.
Sign……………………………………….. Date……………….

This work has been presented with approval from
Supervising Lecturer,
DR. P.M WACHIRA
Sign………………………………………… Date……………………

DEDICATION
I dedicate my findings to the farmers of Kenya both large scale and small scale as they go about their everyday lives trying to find a solution to the disease that destroys their tomatoes every year.

ACKNOWLEDGEMENT
First off, I thank God for giving me the strength and the capacity to conduct this research to completion. I would also like to thank my family and friends for their unending support during the time I was doing my project. Finally, I extend my gratitude to the Mycology laboratory technicians for making sure my project ran smoothly.

ABSTRACT
Physical properties such as temperature, pH and moisture are known to have a very significant impact on the biological activities of organisms. They are either slow down or speed up their activities. In this study, temperature was tested to see whether it had any impact on the bio-control of Fusarium by Trichoderma spp. The objective of this study is to effectively undertake isolation of the fungal phytopathogen, Fusarium and to successfully culture them together on one Petri dish to find out the how well Trichoderma will control the pathogens at different temperatures. The pathogen will be isolated from the diseased plant parts and incubated to grow separately. The fungus will then be incubated with Trichoderma and their growth measured. At the end of the experiment, it is expected that the phytopathogen will not grow in the region on the Petri dish where Trichoderma is growing. It is also expected that the different temperatures will have an effect on the growth of the two fungi. In conclusion, this study is expected to echo the fact that Trichoderma can indeed be used as a biocontrol agent but act differently at the different temperatures. The recommendation for the study will be to sensitize farmers on the best temperatures to use Trichoderma when using it to control Fusarium so as to get the best results. More research should be undertaken to ensure the best results under field conditions. Only then will this be beneficial to the farmers.

CHAPTER ONE
INTRODUCTION
Biological control is the use of specific microorganisms that act as antagonists and interfere with plant pathogens thus suppressing diseases. It is a nature-friendly, ecological approach to disease control and help in reducing the negative effects that are associated with standard chemical methods of plant protection (Kumar, 2008).
The efficacy of a biocontrol agent is biological in nature and this makes it dependent on many factors. The efficacy can be improved by adjusting these factors in favour of the biocontrol agents so that they can efficiently do their job (Spadaro & Gullino, 2005).Factors such as Temperature, soil moisture, pH and soil type are among the important factors that play a significant role in the efficacy of a biocontrol agent (Spadaro & Gullino, 2005).
Any biocontrol agent that shows some potential to be used in agriculture to control pests, diseases or weeds should be easy to prepare and apply, stable, have a long shelf life, have abundant viable propagules and have a low production cost (Boland, 1998). A good example of a fungal BCA that has these qualities is Trichoderma spp.
Trichoderma spp. is a free living fungus that is present in nearly all soils and other diverse habitats. It is favored by the presence of high levels of plant roots, which they colonize readily. It is highly rhizosphere competent, i.e., able to colonize and grow on roots as they develop. There are various species of the Trichoderma fungus. Up to 100 species have been identified through molecular characterization (Druzhinina et al., 2006). The most common species include; T. harzianum, T. viride, T. koningii, T. asperellum, T. reesei and others. Trichoderma spp have been widely studied for their capacity to produce antibiotics, parasitize other fungi and compete with deleterious plant microorganisms that are found in this rhizosphere (Elad, 2001). Trichoderma has been successful as a biocontrol agent because of the following reasons; ability to reproduce fast, ability to survive in adverse conditions effective use of nutrients, capacity to modify the rhizosphere, strong aggressiveness against phytopathogenic fungi and efficiency in promoting plant growth and defense mechanisms (Boland, 1998).
Fusarium spp is a phytopathogen that is as abundant in soil just like Trichoderma. This genus has various species that include; F. oxysporum, F. verticillioides, F. fumonisins, F. trichothecenes. Some Fusarium species are harmless and have no effect on the plants. They grow in the soil as saprophytes and help in the soil formation processes (Leslie & Summerell, 2006). Other Fusarium species are pathogenic causing diseases to plants growing in the soils in which they occur. A good example is Fusarium oxysporum that causes Fusarium wilt in a variety of plants. Fusarium oxysporum f. sp. Lycopersici is a species of Fusarium that causes vascular wilt in tomatoes. This is a destructive disease that causes losses in terms of millions to farmers around the world annually. There are several methods that are in place to control the vascular wilt but they are not very efficient (Amini, 2009). These methods include use of herbicides like methyl bromide and cultural methods that aim at reducing the effects of the disease. An alternative and workable method for the control of this disease had to be sought and the answer was found in biological control. Addition of the biocontrol agent directly to the roots of the plant is an efficient and cheap way to ensure that the disease causing pathogen is eradicated during transplanting.

1.1 JUSTIFICATION
Kenya is a land that is adorned with different weather patterns and climatic zones. This makes the country have different physical conditions such as varying temperature, rainfall, humidity levels and others. The climatic conditions dictate the type of crops to be planted. One crop that can grow in a vast array of climatic conditions is the tomato plant. Tomatoes are widely affected by Fusarium and this causes the disease Fusarium wilt in the plant. This is a destructive tomato disease worldwide (Jones et al, 2005). There are methods that have been put in place to control the spread of this disease but they are not that efficient and are difficult to apply. This has necessitated the use of alternative means to curb the disease and biological control using Trichoderma has proved to be a viable option. Even though the use of Trichoderma against Fusarium has been largely successful, it is still necessary to investigate whether the BCA is effective when employed under varying physical parameters specifically temperature. The aim of this study is to investigate whether farmers that are in regions that have temperatures that are higher or lower than the optimum for Trichoderma can still use it in their farms as a biocontrol agent against Fusarium or will its activity be curtailed by the changes in temperature.

1.2 OBJECTIVES
1.2.1 Broad objective
The broad objective of the study was to investigate the efficiency of Trichoderma as a biological control agent against Fusarium at different temperatures.
1.2.2 Specific objectives
The specific objectives of the study were to;
• Establish the mechanism of action of Trichoderma against the fungal phytopathogen
• Establish the rate of inhibition of Fusarium by Trichoderma at the different temperatures.

CHAPTER TWO
LITERATURE REVIEW
2.1 Trichoderma Spp.
Trichoderma species are effective biological control agents (BCAs). They grow really fast when cultured in vitro at their optimum growth temperature 23-28oC. When growing in field conditions, they rapidly colonize areas with an abundance of roots. They use this as a vantage point to attack and parasitize the pathogenic fungi. Most biocontrol agents from the genus Trichoderma are T. viride, T. harzianum, T. reesei and T. hamatum.
It has been observed that there are Trichoderma strains that are more effective at controlling phytopathogens than others. Several experiments have been carried out to test the efficiency of one strain against another (Miyauchi, 2010). Their biocontrol abilities are based on the ability to turn on the antagonistic mechanisms that they possess. Some strains will induce plants to increase their native defense mechanisms so that they are better placed to fight off pathogens. These strains will be less effective at controlling fungal pathogens. Several new general methods for both biocontrol and for causing enhancement of plant growth have recently been demonstrated and it is now clear that there must be hundreds of separate genes and gene products involved in these processes. A recent list of mechanisms follows. mycoparasitism, antibiosis, competition for nutrients and space, tolerance to stress through enhanced root and plant development, solubilization and sequestration of inorganic nutrients, induced resistance and inactivation of pathogen’s enzymes(Vincent et al., 2007).
2.1.1 Life cycle and biology of Trichoderma species
Most Trichoderma strains have no sexual stage but instead produce only asexual spores. However, for a few strains the sexual stage is known, but not among strains that have usually been considered for biocontrol purposes. The sexual stage, when found, is within the Ascomycetes in the genus Hypocrea.
During the asexual reproduction cycle, mitotic spores develop on a haploid mycelium. Conidiophores, bearing these mitotic, and in many cases, green spores (conidia), can usually be found associated with immature stromata and develop skywards to get the conidia distributed by wind, water or other vectors (e.g., animals).

Most strains are highly adapted to an asexual life cycle. In the absence of meiosis, chromosome plasticity is the norm, and different strains have different numbers and sizes of chromosomes. Most cells have numerous nuclei, with some vegetative cells possessing more than one hundred (Vidhyasekaran, 2004). Various asexual genetic factors, such as parasexual recombination, mutation and other processes contribute to variation between nuclei in a single organism (thallus). Thus, the fungi are highly adaptable and evolve rapidly.
Wild strains of Trichoderma are heterokaryotic and this makes them highly variable. Strains used for biocontrol in commercial agriculture should be homokaryotic. They are genetically distinct and non variable. This is an extremely important quality control item for any company wishing to commercialize these organisms.
2.1.2 Susceptibility to chemicals
Trichoderma spp. possesses innate resistance to most agricultural chemicals, including fungicides, although individual strains differ in their resistance. Some lines have been selected or modified to be resistant to specific agricultural chemicals. Most manufacturers of Trichoderma strains for biological control have extensive lists of susceptibilities or resistance to a range of pesticides.

2.1.3 Mode of Action
Trichoderma spp uses various antagonistic mechanisms during the biocontrol of fungal pathogens. These mechanisms are activated once the pathogen is present and they depend on the type of Trichoderma strain, the target fungus, and the environmental conditions such as the pH, temperature and nutrient availability (Elad, 2001)
Some of the antagonistic mechanisms that Trichoderma uses to control the phytopathogens include:
• Mycoparasitism – in this mode of action, Trichoderma grows towards the pathogen and parasitizes on its hyphae. It coils around this pathogen and dissolves the cell wall of the hyphae through the production of lytic enzymes (Djonovic, 2007). This process leads to dissolution of the pathogen’s cytoplasm and this limits the growth of the plant pathogenic fungus.
• Cell wall degrading enzymes – Many Trichoderma species are involved in the production of enzymes that can break down the cell walls of phytopathogenic fungi. The enzymes secreted include chitinases, gluconases and proteases. They dissolve chitin, glucans, proteins and polysaccharides that are responsible for the rigidity of the cell wall and this interferes with the integrity if the cell wall. The fungus cannot function with a faulty cell wall this then leads to its death.
• Antibiotic and toxin production – The Trichoderma strain simply releases antibiotics and toxins in the environment and this prevents the growth of any fungal pathogen in the surrounding regions. This way, the plants are able to grow without any disease development. T. virens produces a toxin glovirin that prevents the growth of some Pythium and Phythophthora species.
• Competition and rhizosphere competence – For any microorganism to be an efficient biocontrol agent, it must be able to compete for nutrients and space with its pathogenic counterpart. Trichoderma is both competitive and rhizosphere competent. It is able to readily colonize the roots of plants and in this way gain an advantage when competing for space and nutrients. As it grows, it uses up the readily available nutrients and most probably controls the pathogens through starvation because it makes sure that the pathogen doesn’t get any nutrients or space to multiply.
• Induction of defense responses in plants – some Trichoderma species are known to have an effect on the defense responses of plants. When applied to soils where these plants are growing, they bring about a change in aspects of the defense responses such as; increase in peroxidase activity, increase in chitinase activity and re-enforcement of the cell walls (Djonovic, 2007). This makes it hard for the pathogens to invade the cells of these plants.

2.1.4 Uses of Trichoderma
Trichoderma is a fungus that can be used in numerous other ways other than biocontrol. The characteristics it has and the products that it produces such as enzymes can be applied in other sectors. It can be used in:
• Food and textiles – the extracellular enzymes that are produced by Trichoderma can be useful in the food and textile industries. Cellulases derived from these fungi are produced commercially in these industries and used to degrade polysaccharides (Kubicek & Harman 1998). This helps to make the textiles produced from cellulose rich materials softer. The enzymes are also used in poultry feed to increase the digestibility of hemicelluloses from barley or other crops.
• Enhancing plant growth – Trichoderma is known to induce defense responses in plants when it is applied where they are growing. As a result, these plants are able to resist disease establishment and they are able to accelerate their growth and development. Trichoderma is also known to induce the production of strong roots as they colonize the roots.
• Source of transgenes – Trichoderma contains a large number of genes that code for the different products that are involved in the mechanisms employed during biocontrol. Several genes have been cloned from Trichoderma spp. that offer great promise as transgenes to produce crops that are resistant to plant diseases. No such genes are yet commercially available, but a number are in development. These genes, which are contained in Trichoderma spp. and many other beneficial microbes, are the basis for much of "natural" organic crop protection and production (Arora, 2005)

2.2 Fusarium species
Fusarium spp are classified under the Hyphomycetidae subclass of the Deuteromycetes. It is a large genus of filamentous fungi that are found in abundance in the soil. Most species of this genus are saprobes and are involved in the decomposition of dead decaying matter, contributing to the soil formation processes. Some Fusarium species parasitize on plants causing diseases while others produce mycotoxins in cereal crops causing contamination. When these cereals are consumed by humans or animals they cause serious diseases. This calls for care when handling the cereals so as to ensure that these mycotoxins producing Fusarium species do not get access to the grains. The main toxins produced by these Fusarium species are fumonisins and trichothecenes (Earl, 2004).
Some of the species that cause economically important diseases include; Fusarium graminearum which causes head blight in barley. This has an effect on the brewing industry because the quality of barley influences the quality of the beer. This disease develops when there is rain in the late season. This Fusarium species also causes root rot and seedlings blight (Walsh, 1996).
Fusarium oxysporum f. sp. cubense causes Fusarium wilt of banana. This disease is also known as Panama disease of banana. The disease easily affects a wide range of banana seedlings because of their little genetic variation due to vegetative propagation. This is a serious disease that can wipe out an entire population of bananas (Carlier & Waele, 2003).
Fusarium oxysporum f. sp. Lycopersici causes vascular wilt in tomatoes. The disease affects seedlings while they still on the seed bed and even after transplanting. It causes wilting of the plant even when it is watered. This is because the fungus infects the vascular vessels of the plant and causes them to rot. This is demonstrated by the brown discolouration observed when the plant is cut open. This disease when not managed can result in many losses. One of the control methods for this condition is the use of resistant varieties of tomatoes. It has however been reported that there are some pathogenic strains of F. oxysporum f. sp. Lycopersici that are still effective against some resistant tomato strains (Amini, 2009).

2.2.1 Life cycle and biology of Fusarium species
Fusarium oxysporum produces three types of asexual spores; micronidia, macronidia and chlamydospores (Nelson et al. 1983). Micronidia are uninucleate and germinate poorly. Macronidia are multinucleate and are produced in large numbers. They germinate rapidly as compared to the micronidia. Chlamydospores are accessory spores resulting from the structural modification of vegetative hyphal segments or conidial cell having a thick wall. Its main function is primary survival in the soil.
The life cycle begins when the chlamydospores that survive in the soil germinate and produce a thallus from which conidia form. The conidia then penetrate the epidermal cells of a host plant and subsequent establishment of disease in the vascular system.
2.2.2 Susceptibility to chemicals
Fusarium is susceptible to a number of chemicals mainly in the form of herbicides and formulations used in its control. Fusarium spp is particularly affected by chemicals that contain highly-concentrated organic acids specifically designed to penetrate soil and eradicate the soil-based disease pathogens,
2.2.3 Mode of action
The mode of action of Fusarium or the way it infects its host plants involves a variety of processes that are clearly defined. These processes are as follows;
• Adhesion: hyphae adhere to the root surface. The fungus uses site specific binding to anchor the propagules at the root surface. This step is important because it sets the pace for the other processes to occur.
• Penetration: This step is controlled by many factors that include; fungal compounds, plant surface structures, activators or inhibitors of fungal spore germination and others (Mengden et al. 1996). The pathogen can penetrate the root directly or through the wounds on the roots of the plant. The common sites that are fungus uses for direct penetration are located at or near the root tip of both tap and lateral roots (Lucas 1998).
• Colonization: The mycelium advances intercellularly through the cortex until it reaches the xylem vessels. The fungus uses this means to colonize the whole plant. Microconidia are formed within the xylem vessels and they are then detached and carried upwards in the sap. This enables rapid spread of the fungus. The fungal mycelium may accumulate in the vessels leading to the manifestation of the disease symptoms such as wilting (Dudley, 2007).

2.2.4 Uses of Fusarium spp
Some Fusarium species have been use as bio control agents. It has been used as a bio control agent against weeds e.g. the knapsack weed in the United States of America. A good example is F. avenaceum. This fungus is used because of its wide host range and its high virulence. It parasitizes on the weed causing diseases to it and then ends up destroying it.
The downside of this method is the very valuable aspect that makes it a biocontrol agent - its virulence (James, 2002). It can easily attack the non target plants causing diseases and subsequently huge losses. This type of biocontrol involves a very delicate balance.

CHAPTER THREE
MATERIALS AND METHODOLOGY.
3.1 Media Preparation

Potato Dextrose Agar (PDA)
To prepare the media, 39g of PDA was measured and dissolved in 1000ml of sterile distilled water that was in a clean glass bottle. The mixture was then placed in a steam autoclave so as to sterilize the media. The autoclave was first filled with a small amount of water up to a certain level. This water is essential as it is the source of the steam that provides the high temperature and pressure necessary for the sterilization. The autoclave was then switched on and the media sterilized for about 40minutes. It was then switched off and left for a while to allow the temperature and pressure to fall. When it was safe to open the autoclave, the media was removed and left to cool in the biological safety cabinet (BSC) until it could be poured out on Petri dishes.
The Petri dishes were then placed in the refrigerator for storage. They were removed 24hrs before use to allow thawing.

3.2 Isolation of pathogenic fungi (Fusarium spp).

Fusarium oxysporum f.sp. lycopersici was isolated from diseased tomato plants obtained from Kabete farm that had shown signs of wilting and had a brown discolouration of vascular vessels. The roots were cleaned, and cut into small pieces using a sterile blade. The pieces were sterilized using jik for 30 seconds to remove surface contaminants, then rinsed in sterile distilled water and dried using sterile filter paper.
A clean Petri dish was obtained and lined with a sufficient amount of cotton wool. A small amount of distilled water was poured on the cotton wool just to wet it. Two slides crossing each other at the middle were then placed at the centre of the Petri dish. A portion of the cut tomato roots were washed with tap water and then placed on top of the two slides. The Petri dish was then covered and left at room temperature to sporulate.
After about six days, the fungi had sporulated and the mycelia that had grown were transferred to plates of potato dextrose agar and incubated to allow for growth at 250C. Upon growth Fusarium was identified as the fungus with fuzzy growth and a pink colouration.
A portion of agar where the fusarium had grown near the edge of the colony was cut out using an inoculating needle and inoculated on a fresh agar plate and allowed to grow at room temperature so as to obtain a pure culture.

3.3 Isolation of Trichoderma reesei
Some old cultures of Trichoderma reesei were provided by the University of Nairobi Mycology laboratory. It was therefore necessary to make fresh pure cultures of this fungus. The old culture was obtained and using an inoculating needle that was sterilized using 70% alcohol and flaming using the spirit lamp, the active part of the colony near the edge was cut out with a piece of agar and inoculated on a fresh agar plate. The agar plates were incubated at room temperature (250 c). After about 5 days, colonies of the pure cultures had formed and this was to be used during the rest of the experiment.

3.4 Dual culture inoculation

The basis of this experiment was to demonstrate the inhibition of Fusarium spp by Trichoderma reesei so they must be cultured together to demonstrate the confrontational assay. From the pure cultures small discs of the colonies of about 5mm were cut out and each of them placed about 1.5cm from the edge of the Petri dish. This means that a disc of T. reesei was on one side and a disc of Fusarium on another.
The paired cultures were then incubated at three different temperatures 200 C, 250C and 300C for about six days. After this time, the fungal growth was observed and recorded and their percentage growth in terms of area coverage on the Petri dish calculated.

CHAPTER FOUR
RESULTS
In all cases Fusarium was inhibited by T. reesei but the percentage of inhibition varied at the different temperatures. Single cultures of Fusarium grew and colonize almost the whole surface of the agar plate in the six days of incubation but when incubated with T. reesei the Fusarium growth was limited to only a few milimetres on one side of the Petri dish. This was at 25oC The graph below shows the growth of the two fungi when incubated together at 250C. Trichoderma was able to multiply quickly and overwhelm Fusarium. Fusarium was inhibited on the 3rd day.

At 200C, the Fusarium growth was slow during the first few days but finally picked up and colonized the agar surface. Trichoderma also exhibited slow growth at this temperature but showed better nutrient utilization than Fusarium because it colonized the surface of the agar better. When incubated together, Trichoderma was able to inhibit Fusarium but at a lesser level than at the optimum temperature.

The graph below shows the growth of the two species at 200C. From the graph, we can conclude that Fusarium was inhibited on the 5th day.

At 300C both fungal species had their growth affected especially Trichoderma. The colonies of Fusarium formed were a little bit larger that those formed at the other two temperatures. When incubated together, Trichoderma was still able to overwhelm Fusarium and inhibit it but the rate of inhibition was at its lowest. This is evidenced by the size of Fusarium colonies formed. Both fungal colonies had also lost their pigmentation. The graph below illustrates the growth of the two fungal species at 300C. The growth was generally low in both of them. Fusarium was inhibited by Trichoderma on day 5.

At 35oC none of the fungi was able to grow. They all recorded zero growth.

The graph below shows the area covered by the fungi in mm2 against the different temperatures. They all attain their maximum growth at 250C.

CHAPTER FIVE
DISCUSSION
From the above results, it is clear that temperature has an effect on the growth of these fungi. The most effect was felt at temperatures 30oC and 35oC. At these temperatures, the fungi struggled to grow. Their enzymes might have been inhibited or denatured thus reducing their metabolism and eventually having an effect on their overall growth and activity (Chakraborty, 2005). The slow growth might also be attributed to stress. The fungi might have been having trouble adjusting to these temperatures.

Trichoderma spp was able to inhibit the growth of F. oxysporum because it possesses some characteristics that make it an effective biocontrol agent. Some of these features include ability to grow much faster than the phytopathogenic fungi. Trichoderma spp was able to colonize almost the entire area of the Petri dish in two days as compared to Fusarium spp that took almost 6 days to have established sufficient growth. This advantage enabled Trichoderma spp to compete effectively for space and nutrients leaving little or none for Fusarium spp and this made it hard for it to colonize more areas (Ozbay, 2003). This finally contributed to its biological control.
Trichoderma spp is known to produce antibiotics that inhibit the growth of the phytopathogen that it is incubated with (Dubey and Suresh, 2006).

Another mechanism of pathogen control in Trichoderma is mycoparasitism. Microscopic observation of interaction region between T. reesei and Fusarium spp showed that the mycelia of Trichoderma grew on the surface of the pathogen always coiling round the mycelia and later penetrating the cell wall directly (Djonovic, 2007).The pathogen mycelia then disintegrated suggesting enzyme action. (Metcalf et al., 2001). These enzymes function by breaking down the polysaccharides, chitin and glucans that are responsible for the rigidity of the fungal cell walls, thereby destroying the cell wall integrity limiting the growth of the pathogen.

The slide above represents the region between the two fungi when they are grown together during a confrontational assay. The mycelia at the top of the slide (Trichoderma spp) are thicker than those at the middle of the slide (Fusarium spp). This could be a case of Trichoderma spp depleting the nutrients or rapidly colonizing them and this made it hard for Fusarium spp to obtain the nutrients hence the thin mycelia. The thick mycelia strands then go on to invade the region that has the thin strands.

CHAPTER SIX
CONCLUSION AND RECOMMENDATION
From the experiment conducted, it can be concluded that Trichoderma viride is an effective control agent at varied temperatures. At the three temperatures that the experiment was based on, it was observed that T. viride quickly established itself and colonized the surface of the agar plate faster than F. oxysporum at all temperatures. It is not majorly affected by the stress associated with growing at temperatures above or below the optimum growth requirements. Fusarium on the other hand takes time to adjust to these temperatures and thus, its growth is slowed down. Trichoderma can be described as hardy due to its persistence and this feature contributes to its success.
Another conclusion that can be drawn from the results is that the fungi found it easy to adjust to the low temperatures that they did with the high temperatures. The low temperature did not have a great effect on their enzyme activity, metabolism and activity like the high temperatures.
In conclusion, Trichoderma spp can be a success when used in areas that have temperatures ranging from 20-280C. Above that, its growth is slowed down and chances are it will not be effective at controlling the pathogens.
Recommendation
This experiment focused on only one physical condition that is associated with the effectiveness of a biocontrol agent. As a recommendation, more research should be carried out on this topic under field conditions so as to capture the real scenario. Other conditions that affect the activity of this biocontrol agent include moisture, pH, sunlight etc. When more research is carried out while factoring in all the other conditions then this report will be useful to farmers because they will have the big picture of what affects the effectiveness of Trichoderma spp other than just temperature.

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