ISOLATION AND IDENTIFICATION OF BACTERIAL PATHOGENS ASSOCIATED WITH THE GROWTH RETARDATION OF PLANTS

ISOLATION AND IDENTIFICATION OF BACTERIAL PATHOGENS ASSOCIATED WITH THE GROWTH RETARDATION OF PLANTS

CHAPTER ONE

1.0     INTRODUCTION

Bacteria are microscopic, single-called organisms that have a cell wall. Their genetic materials, a circular stand of DNA, floats inside the cell and is not surrounded by a nuclear membrane. Therefore, bacteria are prokaryotic (i.e do not have a true nucleus) as do plants, animals and fungi.

Bacteria have other small gene-carrying entities within them called plasmids. Some of the characteristics exhibited by bacteria, such as resistance to streptomycin, copper and other antibiotics are controlled by the plasmid genes. While most bacteria in the environment are beneficial, several are able to cause diseases such as leaf spots, stem rots, gall wilts, blights and cankers (Moorman, 2012).

Plant pathogenic bacteria generally survive in infected plants, in debris from inflected plants and in a few cases in infested soil. Most require a wound or natural warm, moist conditions in order to cause disease. Bacteria grow between plant cells on the nutrients that leak into that space or within the vascular tissue of the plant. Depending on the species of bacteria involved and the tissue inflected, they release enzymes that degrade cell walls, toxins that damage cell membranes, growth regulators that disrupt normal plant growth and complex sugars that plug water conducting vessels. In most bacterial diseases, photosynthesis and respiration are severely altered to the detriment of the plant (Moorman, 2012).

Bacteria reproduce very rapidly. They are splashed easily from the soil to the leaves and from leaf to leaf by overhead irrigation. They are also easily moved from soil or debris when a worker handles such material and the handles the live plant. The most important means of avoiding ornamental crop loses caused by bacteria is to purchase plants that have been shown to be free of such pathogens by the process of culture indexing. In this procedure pieces of plant tissue are incubated in a nutrient broth which will encourage the growth of plant pathogenic bacteria. If the test is repeated two to three times and no pathogenic bacteria are detected, the plant is said to have been indexed and free of bacterial pathogens.

Plants are usually indexed at the same time for fungi that grow within the vascular tissue of the plant. In different procedures, elite propagators also index plants for viruses. Plants found to be free of the organisms for which they are tested are said to be culture/ virus indexed (Arthur, 2002).

1.1            Aim and Objectives

To isolate bacterial pathogens affecting growth retardation of plants from three different locations in Kaduna metropolis or environment, Kawo Sabon – Tasha and Kaduna Polytechnic.

Objectives

1.                 To identify some sources of these bacterial pathogens associated with the growth retardation of plants

2.                 To find out possible ways or measures that can be adopted to reduce the prevalence of such pathogens causing the growth retardation of plants.

CHAPTER TWO

2.0     LITERATURE REVIEW

Many foliage plants are susceptible to bacterial diseases, especially during gloomy winter months. Common symptoms include leaf spots, blights and wilting. Bacterial diseases restricted to the leaves can often be controlled.

It has been researched over the years and found that bacteria are microscopic single-cell organisms that reproduce by dividing in half. This process may occur often as once every 20 minutes or it may take several hours. In some of the faster multiplying species, a single bacterium can produce over 47 million descendants in 12hours (Pfleger and Gould, 2012).

Approximately 170 species of bacteria can cause disease on foliage plants. Bacteria cannot penetrate direct into plant tissue, but must enter through wounds or natural opening such as stomata (pores for air exchange) in leaves.

Bacteria are normally present on plant surfaces and will only cause problems when conditions are favourable for their growth and multiplication. These conditions include high humidity, crowding, and poor air circulation around plants. Misting plants will provide a film of water on the leaves where bacteria can multiply.

Too much, too little or irregular watering can put plants under stress and may predispose them to bacterial infection. Other conditions that produce stress include low light intensity, fluctuating temperatures, poor soil drianage, too small or too large a pot and deficient or excess nutrients.

Bacterial diseases tend to be prevalent on foliage plants during the winter months when light intensity and duration are reduced. During this time, plants are not growing actively and are easily stressed (Pfleger and Gould 2012).

2.1     Bacteria features (features of bacteria cells)

Bacteria are single –celled micro organisms, generally ranging from 1 – 2um in size that cannot be seen with the unaided eye. Plant associated bacteria may be beneficial or detrimental. All plants surfaces have microbes on them (termed epiphytes), and some microbes live inside plants (termed endophytes). Some are residents and some are transient. Bacteria are among the microbes that successively colonize plants as they mature. Individual bacterial cells cannot be seen without the use of a microscope, however, large populations of bacteria become visible as aggregates in liquid as biofilms on plants as viscous suspensions plugging plant, vessels or colonies on petri-dishes in the laboratory. For beneficial purposes or as pathogens populations of 106cfu (colony forming units/milliliter) or higher are normally required for bacteria to function as biological control agents or cause infectious disease.

Plant pathogenic bacteria cause many serious diseases of plants throughout the world (Vidhyasekaran, 2002), but fewer than fungi or viruses and they cause relatively less damage and economic cost (Kennedy et al., 1980). Most plants, both economic and wild have innate immunity or resistance to many pathogens. However, many plants can harbor plant pathogens without symptoms development (asymptomatic) (Arthur, 2002).

2.2     History of bacterial pathogens

Individual bacteria were first seen by humans about 325 years ago when they were magnified by the first microscope. Its only been a little over 100years since a bacterium was first implicated as a causal agent in a plant disease. Bacteria were shown in 1878 to be associated with fire blight of apples and pears in Illinois and New York, USA (fire blight disease lesson) (Burill, 2000).

The disease caused by Erwinia amylovora, now wide – spread throughout much of the temperate world remains a limiting factor in growth of healthy apple and pear trees. In 2002 Arthur was able to isolate a bacterium from diseased plants, culture it, and then inoculate the same host to reproduce a naturally occurring disease. He recovered it subsequently from diseased tissue fulfilling what is known as Koch’s postulates (Arthur, 2002). And its only been about 120years since the development of sterilized semi-solid media, first gelatin and then agar with various nutrients added that enabled the isolation of purified cultures, a technique taken for granted today (Koch, 1983).

Bacteria as plant pathogens can cause severe economically damaging diseases, ranging from spots, mosaic patterns or pustules on leaves and fruits or smelly tuber rots to plant death. Some cause hormone – based distortion of leaves and shoot called fasciation or crown gall, a proliferation of plant cells producing a swelling at the intersection of stem and soil and on roots (Arthur, 2002).

2.3     Basic biology of bacterial pathogens

Bacteria associated with plants have several morphological shapes such as can be seen with conventional microscopes at 400x to 1000x magnification. These shapes initially provided simple ways to differentiate them. There are bacilli (rods), cocci (spherical), pleomorphic rods (tendency toward irregular shapes) and spiral shapes. The majority of plant associated bacteria are rods. However, modern science has shown by biochemical, genetic and molecular biological analyses that these bacteria are quite heterogeneous. Some are related to and grouped with animal and human pathogens. Stains are often useful in the differentiation of structures.

On laboratory media, plant pathogens usually grow more slowly than non – pathogenic bacteria isolated from plants with optimal temperature of 20 – 30⁰C (68 – 86⁰F). This makes isolation sometimes very challenging. A few grow at 37⁰C (99⁰F) (or higher), the temperature at which human pathogens e.g Burkholderia cepacia are able to grow. Some can grow slowly at 10 – 12⁰C (50 – 54⁰F). Most are aerobic, some are facilitative anaerobes (i.e they can grow with or without oxygen) and a few are anaerobes (Coplin et al., 1981).

2.4     Reproduction in bacteria pathogens

In general, bacteria reproduce by binary fission (one cell splitting into two), but the process is complex. If present the extra diromosomal DNA elements or plasmids are usually reproduced in synchrony with the bacterial chromosome, but under some conditions can be lost naturally or by chemical manipulation (cured). Many plant pathogens harbor plasmids e.g strains of pantoea (syn. Erwinia) stewartii sub sp. Stewarti (stewart’s wilt of corn disease lesson) may have up to 13 plasmids of unknown function (Coplin et al., 2003).

Genetic variation in natural settings e.g fields, is probably underestimated due to lack of sampling and characterization. For example at least seven pathogenic variants of the gross wilt and blight bacterium of corn, clavibacter michiganensis sub sp. Nebraskensis have been detected in a single field (Smith and vidaver,1987).

2.5     Survival of bacterial pathogens

Survival of pathogenic bacteria in nature occurs most commonly in plants debris left on the soil surface in and on seeds, in soil and in association with perennial hosts. But some bacteria can also survive in water and some do well on inanimate objects or on or inside insects. Clavibacter michiganensis, sub sp. Sepedonicus, causative agent of potato ring rot, is notoriously known for surviving on machinery and packaging material. Knowledge of survival is usually essential to intervene in dissemination and for disease management (Gelvin, 2003).

2.6     Dissemination of plant pathogenic bacteria

Dissemination of plant pathogenic bacteria is easy but fortunately does not always result in disease. Dissemination commonly occurs by windblown soil and sand particles that cause plant wounding, particularly during or after rain or storms. Wounding is essential for entry by many plant pathogens.

Aerosols generated by diurnal temperature fluctuations enable dissemination, if temperature and humidity are aligned (Hirano et al., 1989). Some plants diseases require certain temperature conditions e.g Pseudomonas syringae (Synonym: Pseudomonas savastanoi) Pv. Phaseolicola causes disease below 22⁰C (72⁰F) and Xanthomonas campestris (syn: Xanthomonas axonopodis) Pv. Phaseoli, above 22⁰C on dry bean (Phascolus vulgaris). Both diseases can occur simultaneously under growth conditions in which day and night temperature differentials enables diseases progression in susceptible plants. Infested (surface contamination) or infected seed or any plant part can be sources of bacterial innoculum. Machinery, clothing, packing material and water can also disseminate pathogens, as can insects and birds. Continual monoculture in an area will usually enable increases in innoculum, making it easier for pathogens to be disseminated (Cao et al., 2001).

2.7     Host – pathogen interactions

Infection of plants by bacteria can occur in multiple ways. Infection is generally considered to be passive i.e accidental, although a few cases of plant chemo attractants have been reported. Bacteria can be sucked into a plant through natural plant openings such as stomata, hydathodes or lenticels. They can enter through abrasions or wounds on leaves, stems or roots or through placement by specific feeding insects. The nutrient conditions in plants may be such as to favour multiplication in different plant parts e.g flowers or root. Wind–driven rain carrying inoculums can be highly effective. Artificially, bacteria are most commonly introduced into plants by wounding by pressure – driven aerosols mimicking wind – driven rains, vacuum infiltration or by seed immersion into innoculums (Jones et al., 2001).

2.8     Symptomatology of bacterial diseases

Symptomatology of bacterial diseases is extremely varied, but usually characteristic for a particular pathogen symptoms can range from mosaics, resembling viral infections to large plant abnormalities, such as galls or distorted plant parts. Hormone disruption can produce characteristic abnormal growths on roots, stems and floral structures (phyllody) and sometimes abnormal flowers colours (virescence). The most common symptoms are spots on leaves or fruit, blights or deadening of tissue on leaves, stems or tree trunks and rots of any part of the plant, usually roots or tubers. Wilts can also occur due to plugging of vascular tissue. Symptoms may vary with photoperiod, plant variety, temperature and humidity and infective dose.

In some cases symptoms may disappear or become inconsequential with further growth of the plant. For example, Holcus spot of corn cause by pseudomonas syringae pv. Syringae is arrested at the onset of hot dry weather (Opel et al., 1993).

2.9     Diagnosis of bacterial pathogens

Diagnosis of non-fastidious bacterial diseases depends on characteristic symptomatology, isolation of the presumed infectious agent and physiological and or molecular test (plant disease diagnosis). In heavily infected plants, bacterial populations in leaves or lesions may reach 10⁸ or 10⁹ cfu/gram of plant tissue and actually visibly ooze from leaves or stems. A simple way to determine if a disease is caused by a bacterium is to cut a typical lesion or discoloured area near its boundary with healthy tissue and suspend it in a droplet of water on a microscope slide. If a mass of moving small rods or ‘dots is seen at 400 – 100x magnification flowing from the cut tissue under a microscope, you are observing bacterial streaming which is an indicator of a bacterial disease. However, not all bacterial infections show streaming, or it may not be visualized without special microscope attachments. Serological test, usually enzyme-linked and physiological assays are available commercially for a few common economically important bacteria (Schaad et al., 2001).

2.10   Epidemiology and management

Bacterial diseases, in principle can occur in any plant. Minimizing plant disease requires understanding the mechanisms of survival and spread. A competitive exclusion mechanism by beneficial bacteria can be effective in protection against disease. Notably, in crown gall of  roses. Agrobacterium radiobacter strain K84 and its genetically engineered, transfer – minus derivative, strain K1026, provide excellent protection against Agrobacterium tumefaciens (Ryder and Jones, 1991).

2.11   Useful plant pathogens and relatives

A few bacterial plant pathogens or their relatives have been widely used in agriculture and food production. The thickening agent, Xanthan gum, is an extra – cellular polysaccharide derived from the plant pathogens Xanthomonas campestris pv. campestris and is found in an enormous variety of products (Sutherland, 1993).

Transformation or genetic engineering of plants is best carried out by disarmed vectors (plasmids) of Agrobacterium tumefaciens. The elimination of a gene from a non-pathogenic Pseudomonas syringae – that codes for ice formation at relatively high temperatures made history in an ice – minus derivative that prevents frost damage when applied to plants other properties awaits discovery and exploitation (Sutherland, 2000).

2.12   Control of Bacterial Diseases

The strict sanitation practices required to control bacterial diseases include the destruction of infected plants as well as cleaning and disinfesting tools, benches, flats and pots that are used repeatedly.

Soil used in potting should be treated to kill all pathogens. Soil in which infected plants were grow or rooted should be discarded or thoroughly treated.

Workers should be trained to not handle soil or debris and then the living plant tissue unless they stop work immediately and wash their hands. Do plants handling procedures and debris/ soil handling operations completely separately (Moorman, 2012).

The most important cultural practice used against bacteria is irrigating in a manner that keeps foliage surfaces dry and which avoids splashing.

Overhead irrigation should not be used in crops particularly susceptible to bacterial disease. When overhead watering is employed, watering should be done early in the day so that free moisture evaporates quickly provide good air circulation within the crop canopy. It is best to force air under benches and up through the canopy. Horizontal air flow with rows of plants oriented parallel to the air movement, can greatly reduce relative humidity within  the canopy. Various  types of  trickle irrigation  and capillary mat   watering  are  techniques  that avoid providing  the  condition  required for bacteria   spread  and infection.

  

Some bacteria have been shown to spread in ebb and flow systems steps should be taken to filter crop debris out of the water and chemically treat the water.

Once disease begins on the plants chemical control is not effective. Although research reports may indicate 80 to 90% control with chemicals under experimental conditions, often less than 50% control is achieved under commercial conditions with chemicals (Moorman, 2012).

2.13   Specific classification of bacterial plant pathogens: Erwinia chrysanthemi and Erwinia carotovora survive in plants debris that is not completely decomposed, on or in infected plants, on other green house plants without causing disease, and under some conditions in soil. Both species infect a wide range of plants in the green house.  Erwinia chrysanthemi has been shown to survive on plants that it does not actually inflect. They can cause a mushy, brown, smelly, soft rot or leaf spots.

Pseudomonas cichoni can cause leaf spots and blights on Chrysanthemum, geramin inpatiens and many other ornamental plants. The spots are generally water soaked (wet-looking) and dark brown to black. Depending upon the plant infected, the leaf spots may have a yellow halo.

Xanthomonas is another genus of bacteria containing plant pathogenic species. Xanthomonas campestris PV pelargoni causes bacterial blight or wilt of geranium. Other species of Xanthomonas attack dieffenbachia, philodendron, Syngonium, Aglaonema and other foliage plants.

(Pfleger and Gould, 2009).

Rhodococcus fascians (formerly Corynebacterium) causes abnormal branching and stem development near the base of infected plants such as geranium.

The bacterium is carried on infected cuttings and may enter the propagation medium.

Ralsotonia solanacearum (formerly Pseudomonas) causes vascular wilting of many herbaceous ornamentals, including geraniums. Cross symptoms in geraniums mimic those of bacterial blight caused by Xanthomonas compestris PV. pelargoni. Unlike most other bacteria, Ralstonia, Solanacearum, survives well in the soil.

Once a greenhouse is contaminated with this organism, it is difficult to eliminate and poses a threat to many different crops. Symptoms include leaf wilting, discoloration of the vascular tissue, leaf yellowing and death of the plant (Sutherland, 2000).

2.14   Management  

a.                 Purchase culture-indexed plants known to be free of the most important bacterial pathogens.

b.                 Discard infected plants.

c.                  Do not used over head irrigation

d.                 Pasteurize the propagation bed and medium between crops

e.                  Do not handle soil or debris on the potting soil surface and then the plant

Under some conditions in soil both species infect a wide range of plants in the green house. Erwinia dryeantheni has been shown to survive on plant that it does not actually infect. They can cause a mushy, brown, smelly, soft rot or leaf spots (Sutherland, 2000).

2.15   Classification and identification of bacterial plant pathogens

The following is a general classification of phytopathogenic prokaryotes with the exception of the division, tenericutes, class mollicutes which will be addressed in a later section. Genera in bold type are common plant pathogens. According to Agrios (2005), bacteria are classified dose:

Kingdom, procaryotae

Bacteria – have cell membrane and cellwall and no nuclear membrane

Division – bacteria – gram – positive

Class – proteabacteria – mostly single celled bacteria

Family: Enterobacteriaceae

Genus : Erwinia causing fire blight of pear and apple, stewarts wilt in corn and soft rot of fleshy vegetables. Pantoea, causing wilt of corn

Serratia marcescens, a phloem inhibiting bacterium causing yellow vine disease of cucurbits

Sphingomonas, causing brown spot of yellow Spanish melon fruit

Family: Pseudomonadaceae

Genus: Acidovorax causing leaf spots in corn, orchids and water melon

Pseudomonas, causing numerous leaf spots, blights, vascular wilts, soft rots, cankers and galls.

Ralstonia, causing numerous leaf spots, blights, vascular wilts, soft rots, cankers and galls

Ralstonia, causing wilts of solanaceous crops

Rhizobacter, causing the bacterial gall of carrots

Rhizomonas, causing the corky root rot of lettuce

Xanthomonas, causing numerous leaf spots, fruit spots, blights of annual and perennial plants, vascular wilts and citrus canker

Xylophilus causing the bacterial necrosis and canker of grape vines

Family: Rhizobiaceae

Genus: Agrobacterium, the cause of crown gall disease

Rhizobium, the cause of nitrogen fixing root modules in legumes

Family: Still unnamed

Genus: Xylella, xylem – inhibiting, causing leaf scorch and dieback disease on trees and vines

Candidatus liberobacter, phloem inhabiting causing citrus greening disease

Unnamed, laciticifer – inhibiting, causing bunchy top disease of papaya

Division firmicutes – Gram – positive bacteria

Class: Firmbacteria – mostly single celled bacteria

Genus: bacillus causing rot of tubers, seeds and seedlings and white stripe of wheat

Clostridium causing rot of stored tubers and leaves and wet wood of elm and poplar

Class: Thallobacteria – branching bacteria

Genus: Arthrobacter, causing bacterial blight of holly, thought to be the cause of Douglas – fir bacterial gall. Clavibacter, causing bacterial wilts in alfalfa potato and tomato

Curtobacterium, causing wilt in beans and other plants leifsonia, causing ratoon stunting of sugarcane

Rhodococcus, causing fasciation of sweet, pea

Streptomyces, causing common potato scab (Agrios, 2005)

CHAPTER THREE

3.0     METHODOLOGY

Samples of Tomato seeds were collected from Kawo, Sabon-Tasha and Kaduna Polytechnic. The culture medium was prepared using the nutrient broth or nutrient agar (Sani, 2013) then it was used to culture the samples collected. After culturing, it was incubated for 48-72 hours at a temperature of 45-60⁰C and allowed in the incubator for growth to occur.

3.1     Nursery preparation

A proper preparation of the nursery bed was carried out, after clearing or removing the stumps of trees and also weeds or grasses, the soil was tilled using a small hoe in making the soil into a fine state so as to make it suitable for planting by spraying.

After that has been done, the tomato seeds were now spread (planted) on the nursery bed and then dry grasses was used to cover the nursery bed. These dry grasses or leaves serves as materials for mulching to prevent the heat from the sun and help to trap moisture back to the soil which aids the seed germination. Watering was done whenever necessary to speed up the germination of the seeds. Then it was allowed to certain height as seedling for three weeks before transplanting to the field was carried out.

3.2     Field preparation

After clearing the field by removing plant debris, farm manure was then applied on the soil before making of the ridges for the transplanting of the tomato seedlings. The ridges were now made or constructed using a big hoe to make the ridges not be to high and also not to be to wide.

A spacing of about 10 – 12cm was given in between each seedling transplanted and a depth of about 5 – 6cm was given for each plant. Then watering, manuring and weeding were all done whenever necessary.

3.3     Collection of isolates

An already prepared isolate of both Staphylococcus and Escherichia coli aureus was collected from one of the laboratories in KASU (Kaduna State University), and then sub-cultured in the laboratory where the work was carried out.

 After sub-culturing it was left in the incubator for 48 – 72hours at a temperature of 45-60⁰C for it to grow. A light microscope was used to view the colonies that where formed on each plate.

3.4     Preparation of culture media

After a proper sterilization of the apparatus or equipments to be used has been carried out using – the autoclave to rid them of micro organisms or microbes the media was now prepared through the following procedures.

About 20 – 30 grams of nutrient broth was measured and then poured into a beaker. Then 100ml or 250ml of water was then poured into the beaker depending on the required amount needed for the culturing. Then the mixture was stirred properly to mix up properly. It was then heated to a temperature of about 45 – 60⁰C on a hot plate. After about 10 – 15minutes of heating it was brought down and allowed to cool for sometimes.

The culture medium was poured into plates or petri-dishes and allowed to solidify before the bacteria Staphylococcus aureus and Escherichria coli were sub-cultured into the plates by streaking the plates using sterilized wire loop that was used to pick from the existing isolates that were collected earlier.

3.5     Isolation of pathogens

The pathogens Staphyloccus aureus and Escherichia coli were isolated using nutrient broth to sub-culture them again, using the same method as in 3.4 (preparation of culture media) but this time using slant nutrient broth in tubes. The cooled nutrient broth was poured into sterile tubes, five (5) for Staphylococcus aureus and five (5) also for Escherichia coli and allowed to solidify for 5-10minutes then the isolates sub-cultured in the plates were picked and cultured in the tubes using sterile wire loop from the colonies formed on the plate of both Staphylococcus aureus and Escherichia coli. Then they were placed in a beaker and incubated at a temperature of 35⁰C and allowed to incubate for a period of 48-72hours for growth to occur.

3.6     Infections of healthy plants

The tubes incubated were brought out of the incubator after the time stipulated for it, then a thin smear of the innoculum was made on a sterile glass slide and staining procedure was carried on it staining it with methylene blue to get a clear view using the light microscope to view.

The innoculum was then diluted in water of about 100-250ml which was then used to infect the healthy plants by sprinkling it on the plants.

Measurements of the heights of the plants was carried out using a rope and a ruler to take their measurements. The heights of the infected plants were measured on weekly basis and recorded.              

CHAPTER FOUR

4.0     RESULTS

The result of the re-infested tomato plants with Staphylococcus aureus and Escherichia. coli are shown in Table 4.1

Table 4.1    Heights (cm) of tomato plants re-infected with Staphylococcus aureus and Escherichia coli

Treatment	Time (Weeks)
	Re-infected plants	1	2	3	4	5	6
Control	A	25.30	27.40	29.50	37.70	59.00	59.00
	B	24.20	25.20	25.20	29.00	34.20	34.20
	Average	24.75	26.30	27.30	33.35	46.60	46.60
Staphylococcus aureus	A	23.40	25.30	27.80	28.60	30.90	32.00
	B	22.50	24.20	28.80	29.43	31.80	33.00
	C	23.30	24.20	28.40	28.64	30.20	34.10
	D	20.40	24.08	27.83	28.83	31.85	32.60
	Average	20.40	24.08	27.83	28.88	31.20	32.93
Escherichia coli	A	21.20	23.50	22.40	25.50	27.00	32.80
	B	21.40	24.30	22.20	26.50	27.00	33.00
	C	23.10	24.30	22.80	25.59	26.10	32.10
	D	20.40	22.30	22.90	26.20	28.20	31.40
	Average	21.53	23.60	22.58	25.93	27.10	32.33

Table 4.2 The average heights (cm) of plants re-infected with Staphylococcus aureus and  Escherichia coli 

	Time (Weeks)
Treatment	1	2	3	4	5	6
Control	24.75	26.30	27.35	33.35	46.60	46.60
Staphylococcus aureus	20.40	24.08	27.83	28.88	31.20	32.93
Escherichia coli	21.53	23.60	22.58	25.93	27.10	32.33

A plot of treatment, Staphylococcus aureus and Escherichia coli from Average height of plants re-infected.

Figure 4.1: Average height of tomato plants re-infected with S. aureus and E. coli compared to the control.

CHAPTER FIVE

5.0     DISCUSSION, RECOMMENDATION CONCLUSION,

5.1     Discussion

Staphylococcus aureus and Escherichia coli from the result gotten in chapter four i.e from (table 4.1), after re-infection the plants heights were stable but after two to three weeks the plant heights increased. From research work carried out, bacteria are normally present on plant surfaces and will only cause problems when conditions are favourable for their growth and multiplication. These conditions include high humidity crowding and poor air circulation around plants. Misting plants will provide a film of water on the leaves where bacteria can multiply. Too much, too little or irregular watering can put plants under stress and may predispose them to bacterial infection. Other conditions that produce stress include low light intensity fluctuating temperatures, poor soil drainage, too small or too large a pot and deficient or excess nutrients.

Bacterial diseases tend to be prevalent on foliage plants during the winter months when light intensity and duration are reduced. During this time, plants are not growing actively and are easily stressed. 

5.2     Recommendations

          Based on the conclusion drawn above the following can be recommended;

-                     Plants that are susceptible to bacteria pathogens should not be grow on a soil that has bacterial pathogens living in it (the soil).

-                     Plants resistant to bacterial organisms should always be cultivated

-                     Spray any soil or plant using appropriate pesticides before and after planting of the seeds or plants on the soil

-                     Proper irrigation method should be used to water the farm land where plants are grown.

-                     And then finally further study and research should be carried out to check for more pathogens that affects the growth of plants such as fungi and viruses.      

5.3     Conclusion

In conclusion it can be concluded that the higher or the more the concentration of the bacterial organisms the higher their effectiveness on the plant growth retardation.

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