Vegetables are soft textured edible plant materials characterized by high moisture contents and are Generally referred as perishable crops in their fresh state. They are essential for nutritionally balanced diet in man (Samir and Amnon, 2007).They are important sources of ascorbic acid (vitamin C), Carotene (Provitamin A), various vitamins, especially folic acid, as well as minerals such as calcium and iron. Some vegetables are source of carbohydrates, proteins and dietary fibres. Vegetables also provide variety, taste and aesthetic appeal to food (Opadokun, 2000). In UK as in many other countries vegetables provide the necessary variety and savoury flavor to make enjoyment of an otherwise predominantly starch diet possible.
Post harvest losses
Despite remarkable effort made in increasing food production at the global level, approximately half of the population in the third world does not have access to adequate food supplies(FAO, 1989). One major reason for this, is loss during post-harvest handling and marketing. Evidence suggests that these losses tend to be highest in those countries where the need for food is greatest. In the developing world about one quarter of what is produce never reached the consumer for whom it was grown, and the effort and money required to produced it is lost forever (FAO 1989).
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Causes of post - harvest losses.
Factors responsible for post harvest losses vary widely from place to place and become more complex as systems of marketing become more complex. However, in 1989, the Food and Agricultural organization named three causes of post-harvest losses of
vegetables. These are mechanical injury, physiological damage and pathological attack. Mechanical injury - includes cuts, bruises, punctures, insect scars, crushing and cracking. On account of their soft texture, vegetables are highly susceptible to mechanical damage.
Physiological damaged- includes various responses of the fresh vegetables to the post - harvest
Environment, eg. Respiration, loss of water, ripening, etc. Respiration is the important physiological Process in the post-harvest life of the vegetables and largely determines their shelf life.
Pathological damage is caused by fungi, bacteria, viruses and nematodes, insects can provide wounds that serve as portals of entry for micro-organisms as well as disseminating some plant pathogens into the crop. The pathological damage is generally enhanced by mechanical damage,
1.1.2 Classification of post-harvest losses.
Opadokun, (2000)classified post-harvest losses in vegetables into four broad categories, namely quantitative, nutritional and economic losses.
I.Quantitative losses are losses of quantity of weight of the crop. They may be the result of respiration or transpiration, losses due to consumption of the crop by micro-organisms, insects and rodents.
iI. Qualitative losses include deterioration in quality of the crop. These may be due to loss of flavor,
deterioration in texture and appearance to the extent of being rejected by the consumers.
iIi. Nutritional losses are caused by internal metabolic reactions, which results in the breakdown of
Nutrients normally associated with the crops. These losses are not readily evident to the consumers.
IV. Economic losses result from combined effect of quantitative, qualitative and nutritional losses resulting in reduction in monetary value of the crop.
1.2 The Fungal Botrytis cinerea
Botrytis cinerea is a member of the class Ascomycetes , it is a filamentous fungi with two stages in its life cycle consisting of asexual(anarmoph) and sexual (teleomorph). The sexual stage consist of vegetative hypae, sclerotia, macroconidia and microconidia while the sexual stage consist of a reproductive body, the apothecum, which contains ascospores in linear asci (Faretra and Grindle 1992). B. cinerea is regarded as the most important post harvest fungal pathogen that causes Significant losses in fresh fruits,vegetables and ornamentals(Theo et. al, 2000). Harvested crop are particularly vulnerable to infection of B. cinerea because unlike vegetative tissue harvested commodities a senescing rather than developing. It ability to attack a wide range of crops in a variety of modes of infection and its ability to develop under conditions prevailing during storage, shipment and marketing make its control a challenge(Samir and Amnon, 2007). In susceptible plants , new infections may begin in the spring as soon as weather conditions are favourable for disease development. Wet or very humid weather may be highly favourable for the spread of the disease. In some Botrytis species sclerotia develop on dead plant tissue and form the overwintering stage of the fungus. Fungal mycelium may also overwinter in woody stem debris. Sclerotia then germinate in the spring, or mycelium grows out of infected debris and conidia develops. Conidia may be windborne or rain splash to cause new infections on susceptible host tissue.
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Mode of infection of B. cinerea after harvest:
Infection through blossom:
B. cinerea can infect fruit via the flower remaining in the very early stages of development and develop only after harvest when the fruit reaches full ripeness. Bloom blights often precede and lead to fruit and stem rots. Ageing flower petals of flower are particularly susceptible to colonization by B. cinerea, and under cool, humid condition abundant mycelium are produce. The fungus often grow from fading petals in to the rest of the inflorescence and develops on the fruits causing bloosom-end rot.From there it can spread and destroyed part or all the fruit(Phillip et. Al. 2007).
B. Infection through surface injuries and cracks:
Conidia can infect the fruit directly through grown cracks , cut stem scars, insect wounds or lesions made by other pathogens. Infected fruits may developed water saoked yellowish green or grayish brown irregular lesions which can be soft and spongy in texture(Elad et. al. 2004).
C. Infection through harvest cut and trimming:
Gray mould rot course by B. cinerea commonly occurs after harvest on the cut surface on leafy vegetables and fresh herbs (ie celery, and basil). Nutrients leaking from damaged tissues make the cut surfaces good infection site for B. cinerea. As soon as the infection is established decay can proceed to entire leaf tissue(Michailides and Elmer, 2000).
D. Insect mediated infection:
Activity of various invertebrate can play a role in contaminating the produce with inoculum. This was shown by infection of Kiwifruit through flowers with B. cinerea facilitated by thrips (Thrips obscuratus) and honey bees(Michailides and Elmer, 2000).High incidence of B. cinerea. Incidence was also reported on Kiwifruits with garden snail (Helix aspersa)damage; slime secreted by the snail stimulated the germination of the conidia. Honey bees and other types of insects visiting flowers potentially have the capability to disseminate Botrytis in Strawberry(MIchailides and Elmer, 2000).
Among the vegetable attacked by B. cinerea are Arabidopsis thaliana and Lettuce lectuca sativa, which are the subject of this research.
1.3 THE PLANT ARABIDOPSIS THALIANA
Arabidopsis thaliana is a member of the family Brassicaceae that is most often found in disturbed habitats. Although it is now naturalized in many parts of the world, its native range covers Eurasia, and Northern Africa (Mitchell -olds 2001; Okane jr. and Al-shebaz, 1997; Sharbel et al. 2000). Arabidopsis thaliana is an annual plant which has a life cycle of approximately 6 weeks . It is relatively Small in size, the diameter of the rosette rarely exceeding 12 cm, and is easy to grow under laboratory conditions. Arabidopsis thaliana is normally self pollinating and can produce thousands of seeds from one individual (Abbot and Gomes, 1989; Redei,1975). These are all advantageous features of the plant features of the plant for its use in experimental systems, as they allow relatively large number of plants to be grown in limited spaces, over progeny from single mutant or transgenic plants. The large number of research group focusing on Arabidopsis thaliana means that there is a wide range of tools that facilitate the ease of working with these species including efficient transformation methods(Clough and Bent, 1998).
A characteristics difference between Arabidopsis thaliana and other Arabidopsis species relates to its chromosome number, with Arabidopsis thaliana possessing the lowest diploid chromosome number at 2n = 2x = 10 (1e x = 5), and representing the smallest genome within the Brassicaceae family as a whole (-157 mbp) (Johnson et al, 2005). Chromosome base numbers of x = 8 are found in the other species which all are diploid except for Arabidopsis succia (2n = 4 x 26), an allopolyploid formed by the hybrization of Arabidopsis thaliana and Arabidopsis arenosa (O kane et al. 1996 ;AL- Shehbaz and O kane, 2002 ; O kane and Al-shebaz, 2003 ; Hoda, 2004).
Prior to the molecular era, the vast majority of plant species used experimentally were those of either agricultural or horticultural relevance. In genetics studies members of those group which had unusual or prominent features were concentrated upon, such as pisum sativum, zea may, and Antirrhinum majus (Sturtevant, 1965). It was Fredrich Laibach who was initially responsible for an increased interest in the use of Arabidopsis thaliana as a model plant. In 1937, with the aim of studying natural variation and the effects of light quality on flowering time and seed dormancy, he began to collect different accessions of Arabidopsis thaliana having notice the large variation present in physiology traits between them (Somerville and Koornneef,2002). He subsequently suggested the suitability of Arabidopsis thaliana as a model plant for research on genomics and developmental biology ([ Laibach, 1943] cited in [Somerville and Koornneef, 2002]). At that time the suitability of Arabidopsis thaliana as a model plant was based mainly on its reproductive features, size and natural. It was also noted that Arabidopsis thaliana cells had just five chromosome pairs ([Laibach, 1907] cited in [Somerville and Koornneef,2002]), but it was not until the 1980 s and down of the molecular era that some other fundamental benefits of the use of Arabidopsis thaliana as a model plant became clear. It was found that Arabidopsis thaliana has a very low quantity of nuclear DNA (approximately 70 mb)with relatively few high repetitive sequence(Leutwiler et al .1984), and this allowed the use of recombinant DNA techniques with relatively ease compared to organisms with larger genomes. It was later shown that Arabidopsis thaliana could be transformed by incubation with Agrobacterium cultures to produce transgenic transfer DNA insertion mutants (Feldmann and Marks, 1987). This has since been improved upon so that modern techniques involved merely spraying the Agrobacterium onto the plants flowers for
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successful transformation to occur (Somerville and Koornneef. 2002). As molecular techniques boomed, so did the popularity of Arabidopsis thaliana as a model plant (Griffing and Scholl, 1991; Meyerowith, 1987 ; Meyerowith, 1989 ; Somerville, 1989) and this has resulted in the establishment of Arabidopsis thaliana newsgroups, stocks centers containing large numbers of accessions, mutant and transformed lines and ultimately the publication of the entire genome sequence of the plant (The Arabidopsis Genome Initiative, 2000). Arabidopsis thaliana has now been used to model a diverse range of systems including floral morphogenesis (Bowman et al, 1991; Weigel and Meyerowith, 1994), trichome development(Marks and Feldmann 1989 ; Marks, 1997) and plat - pathogen interactions (Davis and Hammer Schmidt, 1993). A more detailed review of the steps which led to the emergence of Arabidopsis thaliana as model plant can be found in Somerville and Koornneef (2002).
The production and description of large numbers of Arabidopsis thaliana lines by molecular community, has provided a huge infrastructure and resource to scientist examining the ecology of plants which has been aided by the placement of these lines in easily accessible stocks centers, such as Nottingham Arabidopsis Stock Center(NASC) . Loughborough, UK, studies using Arabidopsis thaliana as a model plant can potentially produce economically and socially important insights and ideas for research, particularly as extrapolation of results is likely to be most relevant to other members of the Brassicaceae(Price et al. 1994),which includes many crops of agricultural or horticultural importance across the world. Many Arabidopsis thaliana genes have been found to have homologues in cultivated Brassicaceae and in addition, many aspect of plant defence that are employed by Arabidopsis thaliana have also been widely observed in many other plants families (Mitchel -Olds, 2001). It has been concluded that because of the availability of a complete genomic sequence, Arabidopsis provides an efficient system for understanding plant defence against insect natural enemies in natural populations.
and in agriculture (Mitchell -Olds, 2001). Therefore, Arabidopsis thaliana is becoming increasingly important as an initial plant, particularly for works on members of the Brassicaceae.
1.3.1 Morphology and life cycle of Arabidopsis thaliana
Arabidopsis thaliana usually grows to a height of about 20-25 cm tall. The leaves form a rosette at the base of the plant with a few leaves also on the flowering stem. The basal leaves are green to slightly purplish in colour, 5-15 cm long and 2-10 mm broad with an entire to coarsely serrated margin, the stem leaves are small, unstalked, usually with an entire margin. Leaves are covered with small unicellular hairs called trichomes. The flower are 3mm in diameter, arranged in a corymb, their structure is that of the typical Brassicaceae. The fruit is about 5-20 mm long, containing 20-30 seeds. Roots are simple in structure, with single primary root which grows vertically downwards, later producing smaller lateral roots. These roots form interaction with rhizosphere bacteria such as Bacillus megaterium.The life cycle of Arabidopsis thaliana is completed within 6 weeks. The central stem produce flower which grows after about 3 weeks, which self pollinate. In laboratory Arabidopsis thaliana is grown in petri dishes or pots under fluorescent light or in greenhouse.
1.4 The plant lettuce
Lettuce lactuca sativa is a member of the family Asteraceae (Compositae), Lettuce is an annual herbaceous plant grown in UK since 16th centuary, with chromosome number of 2n=18. Lettuce lack extensive root system, it has thick mass of root which are around the first 15 cm of the soil. It has main root with fibrous lateral branches. The stem is cylindrical containing latex which may be up to 1m in length. The leaves which are sessile arranged in rosette their colour vary from light or yellowish green to dark green The flower are in dense clusters, yellow in colour and pentamerous . The seeds are oval in shape 3-4 mm on length, ribbed, with a white, yellow, grey papppus of silky hairs (Tindal, 1983).
1.4.1 Types of Lettuce:
Sowey and Shaw (2006) classified Lettuce cultivars into five main types on the basis of predominant use and plant form. The crisphead (icebaerg)form closed heads resistant to mechanical damage. This is the type Predominantly grown in Europe and America. The butter head type forms open loose Head with soft leaves which are easily damaged by handling. Leaf lettuce share this fragile nature. Romaine lettuce has elongated erect leaves which forms a loose loaf-shape head. While stem lettuce is grown for its thick parenchymatous stem, which is harvested during vegetative stage of the plant.
1.4.2 Composition and Nutritional value of lettuce:
Lettuce contain high water content of about 94-95%.However, the nutritional contents varies in
different forms of lettuce. Romaine lettuce is more nutritious and provides 22mg of ascorbic acid, 1925 I.U. of vitamin A and 44mg of calcium in 100mg of edible product. Crisphead lettuce provide moderate Amounts of ascorbic acid(7mg 100mg), vitamin A (470 I.U./100g) and calcium (22mg/100g).Leaf lettuce Provide 18mg ascorbic acid,1900 I.U. of vitamin A, 68mg in 100g of edible product. Butterhead lettuce Supply 8mg of ascorbic acid, 1065 I.U. of vitamin A and 35mg of calcium per 100g of edible product. Irrespective of the type all lettuce supplies iron, sodium, phosphorus and potassium (Ryder, 1999).
2.0 LITERATURE REVIEW
2.1 Contributions of Botrytis cinerea on post-harvest losses:
Botrytis cinerea is probably the most commonly distributed diseases of fruits and vegetables throughout the world(Agrios, 1997). A survey carried out by ONeil et al. (1997) stated that B. cinerea is the common disease affecting both field and protected crops in UK. B. cinerea causes a disease called gray mould rot or botrytis blight . Botrytis cinerea is a necrotic fungi which infects a wide range of plant species annual and perennial plants. Botrytis infection is favoured by cool, rainy spring and summer weather usually around 15 0C (60F).Botrytis cinerea can infect many ornamental plants including anemone, begonia, calendula, chrysanthemum, dahlia, dogwood, fuchsia, geranium, hawthurm, heather, hydrangea, marigold, pansy, periwinkle, petunia, rose, snapdragon, sunflower, sweet pea, violet zinnia(Elad et. al, 2007a).
Among vegetables and fruits, B. cinerea can infect Arabidopsis thaliana, lettuce, asparagus, beans, beef, carrot, celery, chicory, crucifers, cucubits, rutabaga, shallot, strawberry onion, pepper, potato, raspberry, cucubits, eggplant, endive, grape, lettuce, tomato, turnip, and others. The two other damaging Botrytis blight have strict host preference : B. paeonie infect peony, and B. tulipae infect tulip causing the disease known as tulip fire(Elad et. al. 2007a ).
2.2 Epidemiology of Botrytis cinerea
In 1977 Agrios defined epidemiology as the spread of diseases in a population, and gave three main components of epidemiology as parasite, environment and the host. The level of the virulence, type and quantity of inoculums and the mode of spread are factors of the pathogen that affect a diseases. The environmental factors include temperature, moisture, relative humidity and natural enemies; and host factors include genetics composition, age and nutrients status of the host. Botrytis cinerea causes many problems to the greenhouse and field crops. However, the epidemiology of B. cinerea and it control possibilities differ between greenhouse crops with that of field crops. The structure of the greenhouse, the materials used and the cropping method have great influence on the host plant and diseases. The infection of B. cinerea of potted plants in the greenhouse can occur during production or after harvest(Kohl et. al. 2000). In(2004) Aleid et.al also stated that the type of greenhouse covering influences sporulation by absorbing near- UV light, but may enhance disease by influencing the greenhouse climate.
Aleid et. al (2004) stated that B. cinerea causes problems in many greenhouse crops such as tomato, cucumber, pepper, strawberry, sweet basil, rose, gerbera and most potted plants. In vegetables it may infects fruits, leaves and stems. Stem infection resulting from growth through petiole or from direct infection of wounds may causes plant death. In cut flowers , symptoms mostly occur during the post harvest phase.
Van Denber and Lentz (1968) working on the effect of temperature and relative humidity on growth survival of B. cinerea on potato dextrose agar, their work shows that mycelium survived longer than conidia and survival decrease with decreasing relative humidity and was significantly less at 20 0c than at 0 0c. They also shows that germination of B. cinerea conidia occurred only in solutions in which the mycelium could grow and survive. Similar effect is shown by Temperature on the growth rate of B. cinerea. The rate of growth increase with temperature to a maximum of about 20 0c , but goes down very fast to above 25 0 c.
ONeill et al. (1997) reported that the optimum temperature for the growth of tomato flowers and l leaves by B. cinerea was between 10 and 20 0c . They also reported sporulation to be maximum at 15 0c. They also shows that susceptibility of tomato stem pieces to infection decreases with increasing wound age.
Elad et al. (1992) observed that the nutritional status of host plant is affected by their
susceptibility to the development of grey mould epidermics in vegetables greenhouses. They also reported that high humidity reduces the movement of calcium to the upper part of the plant and calcium enrichment of host tissue reduces the susceptibility of tomato, pepper and eggplant to grey mould. Greenhouse epidemics are also enhance by hormone treatments. Hormone auxin which is applied to enhance fruit setting and development in tomato , greenhouse seem to affect the susceptibility of young fruits to grey mould. The hormone results in early maturing of the plants which make them more susceptible to infection.
Coertz et al. (2001) found that surface colonization of the pathogen was enhance by both wetness and host phenology. The study shows that free water on grape berry had a negative effect on colonization, but the detrimental effect of free water was differentially influence by the mature berries.
Apart from the environmental factors pathogen and host interaction may depend on the availability of nutrients to the infecting pathogen, these nutrients may either originate from the host tissue or from external sources (Elad 1997). Plant exudat secondary metabolites which may affect the behavior of B. cinerea in the phyllosphere. Nutrient supplements may not only influence the pre penetration activities, but, may also influence subsequent symptoms development (Elad 1997).
Anuja and Shaw (2008)stated conidial density as an important factor which affect infection and symptom development in B. cinerea. Visible penetrations produced by inoculum of high conidial concentration was found to be between 5-10% of the conidial germlings. The percentage was higher 20-80% when the concentration of conidia was low.
2.3 Gernination of B. cinerea in a susceptible host:
B. cinerea can produce conidia on every host plant. They are unbiquitous in the air and can be transported by wind, rain and insects over long distance before infecting the next host(Theo et. al 2000).After attachment on the host, conidia germinates under favourable condition and produce a germ tube that penetrates the host surface. After penetration the underlaying cells are killed and the fungus established a primary lesion, in which the necrosis and defence responses may occur. At certain stage the defence barriers are reached and the fungus start a vigorous outgrowth, resulting in rapid maceration of plant tissue, on which the B. cinerea finally sporulates to produce inoculum for the next infection(Theo et. al, 2000).
Hawker and Hendy(1963)reported that germination of conidia took place after soaking water for 6h at 20 0C. Studies with light microscope showed that the first visible stage in germination was the swelling of the spore through intake of water and germ -tube emerged.
Cotoras et. al (2009) showed that B. cinerea present high variability in several biological traits, which can be explain by the degree of genotypic diversity among isolates. The results obtained from investigation on the requirements for conidial germination in 3 natural isolates of grapes G1, G5and GII showed that contact with solid surface is a common requirements for conidial germination of the isolates but differ in their nutritional requirements to germinate. Isolate GII was able to germinate in the absence of carbon or nitrogen source. While isolate G1 and G5 required the presence of carbon source such as glucose, fructose or sucrose. The germinating conidium of B. cinerea produce enzymes capable of hydrolyzing the wax cuticle and cell wall of infected plant(Elad and Evenson, 2000). Enzymes secreted into the liquid culture by B. cinerea are thought to be in infection, particularly the cell wall degrading enzymes, such as endo and exo polygalacturonases ,pectin lyse pectin methyl esterase, and cellulose have been reported(Robert, 1999) Polygalactoronase was readily detectable in extracellular matrix preparation and was eliminated by boiling. Pectin methyl esterase, pectin lyse and cellular activities were observed with both boiled and unheated preparations. In another study it was reported that cellulose from B. cinerea retained activity after boiling and that pectin methyl esterase exhibit activity at 80 oC. The response of pectinlyse to heating has been reported.
Another enzyme though to be important in the infection process of B. cinerea is cutinase. Culture filterate from B. cinerea posses cutinase activity, and modifications of the cuticle in the vicinity of the infection hypha at the site of penetration have been noted(Robert, 1999).In contrast to cell wall degrading enzyme,laccase, is thought to play a role in protecting B. cinerea from plant defence compounds. Suppression of the synthesis of laccase could increase the virulence of the pathogen toward the host(Bar-Nur et. al. 1998).
2. 4 Symptoms of Botrytis cinerea
Van kan(2005) stated that Botrytis cinerea is a fungal pathogen of wide range of species, with different diseases cycles. However, not all the process occur in every infection. The fungus is able to infect all aerial part of its host plant to a certain extent. Infection may results in a serious damage both during plant growth and in the post harvest phase during cold storage or during transport. B. cinerea is a pathogen which invade plant tissue directly or through wounds and senescent or dead organs(Elad,2007a). The pathogens infect many species and causes many diseases in many crops including those grown in the open and those grown in the greenhouse,(Elad, 1997).Infection causes electrolyte leakage from host cell, reduction of chrolophyll contents and photosynthetic rate which results in visible diseases symptom9Zhang et. al. 1996).
2.5 Mechanism of infection of Botrytis cinerea
Kamoen,(1992) reported that infection of the tissue of the host by fungal fungi occurs through three main stages which include germination of the conidia on the surface of the plant, penetration of the epidermis and expansion of fungal hyphae within host tissue. It is not certain weather mechanism of penetration of the plant tissue by B. cinerea is mechanical or enzymatic. Method of penetrations may differ based with the conditions and host type.
Javis ,(1977) reported that penetration of an intact host cuticle is purely mechanical. A conidium which lands on the cuticle becomes attached to the substratum by means of an adhesive mucilaginous sheath which surrounds the germ tubes. Penetration often occurs directly from the distal end of the germ tube. Apart from direct penetration of the cuticle in germ tubes of B.cinerea are capable of penetrating stomata. Mycelium which always has a nutrients providing saprophyte base infects in the same manner as germ tubes . The mycelium has s greater inoculums potential because it is less dependent on the external environment.
Infection area may arise from conidia, mycelium,, and ascospores (Tenberge, 2004).Fungal species have develop various means of penetration strategies in to plant cell walls. The encounter between B. cinerea and the host plant start when conidia firmly attach to the surface of the host and go on to germinate culminating in the infection after penetration into the host plant. The first stage in the penetration occurs upon hydration normally characterized by weak adhesion forces. The second stage , delayed adhesion occurs after conidia have been incubated under favourable conditions. The delayed adhesion is normally associated with secretion of extracellular matrix (Cotoras and Silva, 2005).
Attachment to the leaf surface by conidia is enhanced by the environmental conditions. Temperature changes , can alter the adhesion properties of conidia. Constant changes in temperature has an influence on respiration and rate of metabolic activities which could thus effect adhesion of the spores. The correct penetration mechanism of the host by the spore may involved a specific interactions, orhydrophobic contact with the plant cuticle. It is believed that the attachment of spore to the substrata is often related with production of extracellular matrix by the fungal spore. In B. cinerea , additional matrix polymers are released following germ pore Lysis and concurrent with germ tube growth. Fungal extracellular matrices have been shown to consist of two classes of adhesive substances like polysaccharides and proteins glycoproteins(Tucker and Talbot,2001).
Attachment of germinated conidia to the host is enhanced by the extracellular matrix. The secretion of extracellular matrix is by the germ tubes and appresoria not by the conidia. The extracellular matrix secreted by B. cinerea play a role in tropism along the infection site, preventing dessication of germlings, and providing a matrix in which enzymes and fungal toxins required for the infection could be sequestered. Some the enzymes used by B. cinerea during infection are cell-wall degrading enzymes including endo and exo- polygalacturonases, pectin lyse, pectinmethyl esterase and cellulose and cutinase. In contrast to the cell wall degrading enzymes laccase, which is thought to play a role in protecting B. cinerea from plant defence compounds (Viterbo et al, 1992). Majority of the enzymes believed to be in the infection process are in the extracellular matrix (Doss, 1999).
Reino et al (2004) working on the relationship between toxic production and virulence on B. cinerea on bean leaf disc reported that isolates which produce less toxin were less virulent. Virulent refers to the extend to which a pathogen can cause disease to it host. Delon et al. (1977) found that B. cinerea Causes fast spreading rot in lettuce when conditions are favourable. The tissue of the host is completely destroyed within 72 hours. Also degeneration of host protoplasm is completed when the fungus invade the host cells. For sometimes cell walls are able to resist lysis for sometime but they are eventually resulting in the soft rot and collapse of the tissue. The fungus seems to be very active when it moves through the tissue of the dead host. This is supported by the hypae of many ribosomes, polyribosomes, mitochondria, and multivesicular bodies. The fungus it self degenerates during host lysis and this is indicated by a decease in cytoplasm and organelle density and the appearance of vesicular structures.
2.5.1 Latent infection of B. cinerea
One of the interesting phenomena with B. cinerea is ability of the pathogen to be quiescent in the host tissue for a varying periods either during the crop growing seasons or post harvest(Aleid et. al. 2004).
Verhoeff(1974) stated that Latent infection is a dormant or quiescent period of parasitic relationship were the pathogen takes long period during the host life cycle in quiescent until, under certain specific circumstances, when it become active. Elad et. al.(2004)described latent infection as quiescent infection in which the fungus germinates on the surface of the plant but remains dormant under the cell epidemic confine to few cells or as mycelial fragments which progress in to the plant tissues. Many researchers believed that latent infection act as a source of transport for inoculum from infected to diseases-free areas. The results obtained by Sowley and Shaw(2006)shows that latent infection is seed borne in lettuce, with seed acting as the transporting medium for the inoculum.
Yermiyahu et. al.(2006)stated that a high humidity level, greater than 95% for a period of more than 3 hours , is critical threshold for the germination of spores of B. cinerea. Once this period is exceeded then spore germination will continue even if the humidity is reduced to 80% or less. However, where high humidity periods are kept at 3 hours or less there is little germination or disease development, even after a period of fluctuating high and low humidity periods.
Once infection has occurred, leaf wetness is important for symptom expression, with disease severity increasing with leaf wetness duration. Symptoms may develop within hours, or the infection may remain dormant and symptomless, not becoming active until days or even weeks later. This is known as latent infection. Due to the potential for latent infection by B. cinerea, cuttings taken from apparently healthy but infected plants can subsequently develop grey mould symptoms. Similarly, produce that appears healthy at the time of harvesting and packaging can later develop symptoms of grey mould during transit or storage (Elad et. al. 2007 b).
2.5.2 Systemic infection
2.6 Role of bacteria in the aggressiveness of B. cinerea infection
Dewey et. al (2004) reported that like all other fungi bacteria, infection of B. cinerea is aggressiveness is enhanced by bacteria.s The bacteria assit B. cinerea to invade the host either through wound or senescent tissue.
2.7 Plant defence against infection of B. cinerea:
Verhoff(2000)reported an increased in aggressiveness of B. cinerea with plant age, that lead to the loss of defence. The loss of defence may arise when most of the resources are channel for flowering or may be the result of disesea that multiply over time. Elad, (1997)noted that susceptibility to grey mould of plant organs, particularly fruits and flowers increases with ageing or ripening. Factors which accelerate senescence such as ethylene, tend to increased susceptibility, while treatment which delay senescence such as calcium, cytokinin and gibberellins tend to increase resistance. Senescence is a combination of many processes however, it is unknown which of these are important for B. cinerea susceptibility.
Odjakova(2001),Dangl and Jones92003), and Ferrari et. al. 92007) agrees that plants are the major target of microbes seeking nutrition. A complex array of infection between plants microbes evolved that reflect both the nutrients acquisition strategies of microbes and defence strategies of plants. Part of the strategy includes an active offence against microbes using an array of antimiccrobes gene products.The majority of of plant microbes encounter do not results in diseases. Preformed components, antimicrobial gene peptides, proteins, proteins and non proteinecious secondary metabolites that prevents invasion have been proposed to contribute significantly to the range of pathogens(Heath, 2000). The importance preformed defence has been inferred from observation that plants can be rendered susceptile by a deficiency in the synthesis of secondary metabolites(Morris and Osbourn, 2000).
Infection by B. cinerea is largely dependent on the host and part of the plant involved.
Infected plant during wet or humid condition develop masses of silvery grey spores on
he dead or dying tissue. These spores are readily liberated, and may appear as dust
coming off of infected plant material. Some species of Botrytis form tiny black resting
structures called sclerotia that may be evident on dead plant tissue in late summer.
However, not all species of Botrytis readily form these, so they may not be observed on
all plants(Anonymous, 2000). Botrytis blight can affect leaves, stems, crowns, flowerbuds, seeds seedling bulbs with exception of roots. Depending on the location of the crop, symptoms may initially appear either on the flower and pods or flower in the crop canopy. The most damaging symptoms became apparent after the canopy. The disease appear first as discrete cream coloured lassions on lower leaves . These enlarge and coalesce to infect whole leaflets which later senesce and fall to the ground. If conditions remain conducive for disease, that is warm and wet under the crop canopy for atleast 4 days, infection can spread to the stems. These lesions will girdle the stem and become covered with a furry layer of grey mould, eventually causing stem death and whole plant death, occur before the onset of flowering and pod fill. Infection will continue to spread resulting in patches of dead plants with crops(Anonymous, 2000).