Strategies for protecting plants against B. cinerea

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Botrytis cinerea: causal agent of grey mould:

B. cinerea is one of the most important plant pathogenic fungi, being the responsible of major crop losses on more than 200 plant species in temperate and subtropical regions worldwide. Based on its scientific and economic importance B. cinerea the causal agent of grey mould has been recently ranked as number 2 among the top 10 fungal pathogens in molecular plant pathology (Dean et al., 2012). It is a ubiquitous fungus which causes grey mould on many economically important crops including vegetables, ornamentals and fruits (Jarvis, 1992; Williamson et al., 2007). It can attack many organs including leaves, stems and fruits as a nectrotroph, often with heavy losses after harvest (Elad, 1997b). B. cinerea infects stem wounds of greenhouse tomatoes and can cause serious economic losses (Eden et al., 1996),infections of the pruning wounds result in stem cankers that can rapidly kill the plants (Decognet et al., 2010). In a study of 15 greenhouses in the south of France, (Nicot and Baille, 1996) showed that the incidence of B. cinerea between May and June 1991 ranged from 32-100% and plant mortality reached 46% in some greenhouses. In a larger survey carried out in the same area in late April 1993 (Terrentroy, 1994) showed that B. cinerea attacks were found in 58 of 73 tomato greenhouses studied and partial mortality of plants by the fungus was recorded in 31 of these greenhouses.
This pathogen can cause the partial or total destruction of the host plant and in some cases the whole crop. Economically, this fungus is considered such a major pest problem in viticulture worldwide (Martinez et al., 2005). Estimated global losses due to B. cinerea on alone grapevine to $ 2 billion per year (Elmer and Michailides, 2004a). In addition, the rapid and insidious B. cinerea produces the annual destruction of crops on hundreds of hectares of vines (Bolay and Pezet, 1987). Estimated losses for vineyards in France amount to 15-40% of crops depending on weather conditions. In Champagne, infection rates can reach 15 to 25% depending on the year (Cilindre et al., 2007). In protected corps, for example, tomatoes, peppers, lettuce and strawberries the risk of attack by this fungus is always standing on (Jarvis, 1992).

Taxonomic position:

Botrytis has been recognized as a genra by Micheli in 1729 where he was listed in his book  » Nova Plantarum Genera. » At the beginning it was sometimes confused with Sclerotinia spp. but details were made by (Smith, 1900) and the confusion was resolved by (Whetzel, 1945) in 1945. The genra, redefined by (Hennebert, 1973) and includes 22 species, most of which have a restricted host range, such as B. tulipae attacking on tulips, B. fabae on beans or B. squamosa on onion (Hennebert, 1973). In contrast, B. cinerea is ubiquitous and there are many species of plants on which it can cause serious damage before and after harvest. Botrytis cinerea name was given in 1801 by Persoon a pathogen of the vine. This fungus like many others experiencing double classification:
• Perfect form (teleomorph) Botryotinia fuckeliana (Barry) Wetzel. This is an Ascomycete, class of Discomycetes, the order of Leotiales and family of Sclerotiniaceae.
• An imperfect (anamorph), Botrytis cinerea Pers. This is a Deuteromycete the class of Hyphomycetes, order of Moniliales and family of Moniliaceae.
It is de Bary (1866) which established a genetic relationship between Botrytis cinerea Pers., asexual organism and Botryotinia fuckeliana originally called Peziza fuckeliana, sexual organism. Drayton and Groves (1939) observed for the first time, in vitro, the formation of apothecia by B. cinerea confirming the systematic relationship between the two forms of the fungus. However, it is the name of B. cinerea, widely known by mycologists and plant pathologists was retained while generally the scientific name of a fungus is given by its sexual form.

Life cycle of the pathogen:

During its life cycle, B. cinerea can produce mycelium, asexual spores (conidia), sexual spores and sclerotia. The mycelium of B. cinerea includes articulated filaments, grayish or olive-colored, cylindrical, sometimes vesicular at the central partition, whose diameter varies considerably depending on the conditions for the development of hyphae (Faretra and Antonacci, 1987; Faretra et al., 1988). When the mycelium is at the stage of fruit, it produces clusters of gray conidiophores. Sometimes this method of multiplication may disappear and give way to a white mycelial growth which corresponds to the elongation of hyphae slender, hyaline that spread in the form of web (Beever and Weeds, 2004).. The mycelium can be stored in plant debris from the previous crop. When conditions become favorable B. cinerea grows to give conidia. The development of conidia is characterized by the production of conidiophores erect in tufts that often extended, forming an intense gray chain. Their release is favored by a humid climate, and then they are transported by wind, rain and insects (Holz et al., 2004). When conditions become unfavorable to the development of mycelium and conidia sclerotia are formed. They consist of aggregated white mycelium (Coley-Smith et al., 1980).. As young get older, they become hard and black. They are composed of a thick cortex of cells forming thin barrier pseudo-parenchymal cells and a large central medulla composed of filamentous hyphae. In spring, the sclerotia germinate and produce mycelium or conidia (Beever and Weeds, 2004; Delcan and Melgarejo, 2002; Faretra et al., 1988)
The fungus overwinters as sclerotia or as mycelium in plant debris and may be seedborne as spores or mycelium in a few crops. Other crops may also serve as sources of the pathogen and are likely to cross-infect. Conidia are airborne and may also be carried on the surface of splashing rain drops (Williamson et al., 2007). In the field, spores landing on tomato plants germinate and produce an infection when free water from rain, dew, fog, or irrigation occurs on the plant surface (Fig.1). Dying flowers are a favorable site for infection, but infections can also result from direct contact with moist infested soil or plant debris. In the greenhouse, stem lesions develop either by direct colonization of wounds or through infected leaves. The presence of external nutrients, such as pollen grains in the infection droplet, can markedly increase infection (Elad et al., 2004).

Pathogenicity and host range:

B. cinerea produces a range of cell-wall-degrading enzymes, toxins and other low-molecular-weight compounds such as oxalic acid. This fungus secrete a variety of compounds among which oxalic acid, peptidases, and a pool of toxic metabolites(Williamson et al., 2007). These compounds allow the pathogen to modify the host redox status, perturb the host defence, alter the cell integrity and macerate the plant tissues (Alghisi and Favaron, 1995; Godoy et al., 1990; Riou et al., 1991).
B. cinerea has the vide host range of host plants, over 200 mainly dicotyledonous plant species and can cause grey mould on different plant organs, including flowers, fruits, leaves, shoots and soil storage organs (i.e. carrot, sweet potato), Vegetables (i.e. cabbage, lettuce, broccoli, beans) and small fruit crops (grape, strawberry, raspberry, blackberry) are most severely affected (Jarvis, 1992; Williamson et al., 2007). Culture of plants out-of-season in heated or unheated greenhouses and under plastic tunnels used increasingly to supply fruits, vegetables, herbs and flowers in northern latitudes greatly increases the risk of infection, especially in tomato, lettuce, cucumber and sweet pepper (Jarvis, 1989).

Symptoms

B. cinerea is responsible for a very wide range of symptoms. The most characteristic symptom is a grey-brown furry mould, which are masses of spores of the grey mould fungus, covering the infected area. When shaken, clouds of spores are released from these infected areas. On tomato, the infected areas can expand rapidly covering whole stems, leaves or petals. Stem infections can girdle the whole stem and cause wilting of the plants above the infected area (Fig. 2). On lettuce a quick-spreading grey mould appears on lettuce leaves, which renders them inedible. If the fungus strikes at the base of the plant, it turns yellowish-brown and becomes a slimy rot (Williamson et al., 2007).

Factors affect the development of the pathogen:

There are so many factors that greatly influence the development of the B. cinerea during the early stages of infection, the disease development and sporulation of the fungus (Elad and Yunis, 1993; Yunis et al., 1990) For example, nutrient requirements, light, climatic factors etc.

Nutrient requirements:

Among other factors, nutrient requirement of the pathogen play a key role in the development of the pathogen. Because of the lack of endogenous energy reserves, the conidia of B. cinerea, require an exogenous source of nutrients to germinate (Yoder and Whalen, 1975). According to (Blakeman, 1975), the presence of both carbon and nitrogen is necessary. The presence of nutrients such as glucose and fructose promotes germination and elongation of the germ filament and allow the elderly to regain their conidia germination (Clarck and Lorbeer, 1977). Thus the addition of sucrose, maltose, lactose, mannose, galactose or xylose stimulates the germination of conidia of B. cinerea (Shiraishi et al., 1970). Spraying onion leaves with conidia of B. cinerea suspended in the water does not result in injuries but the addition of a source of nutrients (sugars and minerals) leads to the germination of spores and leads to the formation of lesions (Clarck and Lorbeer, 1977). The molecular mechanisms of induction of germination by carbon sources were studied in detail by (Doehlemann et al., 2005; Doehlemann et al., 2006)
Nutrients are also required for the infection process of the pathogen and can be provided in the form of glucose, leaf extract of cabbage, PDA medium) or injury to the point of inoculation. The concentration of nutrients influences the ability of B. cinerea to cause rotting of tissues (Yoder and Whalen, 1975). Nutrients also play a key role in the sporulation of the Botrytis. For example, (Maas and Powelson, 1972) demonstrated that B. convoluta sporulate abundantly on glucose, sucrose, and fructose media as compared to maltose, galactose and starch media, sporulation was also increased by the casein hydrolysate, asparagine, ammonium tartrate, and ammonium sulfate, less profuse with glutamine and potassium nitrate, and absent with glycine, urea and no nitrogen.

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Effect of light:

Light is an important factor to consider for the control of B. cinerea in greenhouse crop protection. Germination of conidia of B. cinerea occurs better in light as compared to darkness, on the condition there is water and nutrients provided in sufficient quantity (Blakeman, 1980). (Nicot et al., 1996) showed that the germination of spores of B. cinerea on PDA medium has no difference when the spores were placed under a selective ultraviolet filter film (UV) or when placed in a film that does not absorb UV. Sporulation of B. cinerea is dependent on the quality of the light received and especially UV (Elad, 1997a; Nicot et al., 1996; West et al., 2000). Spore production in a selective filtering ultraviolet film in a petri dish represents 0.05% of the spores under a non-control film for several weeks after inoculation. This demonstrates that the absence of UV inhibits sporulation rather than delay it (Nicot et al., 1996). Finally, it appears that some isolates are able to sporulate in the dark and are not affected by the quality of light (Dik and Wubben, 2004b). (Nicot et al., 1996) estimate that more than 15 million spores are produced in 7 days on a tomato stem segment of 2 cm and that reduces sporulation in a radical way to wait only 6 to 18,000 spores in a selective UV-filtering film. In greenhouse crops, the light filtering using a polyethylene film of green or pink and can inhibit sporulation of B. cinerea from 35 to 75%, respectively (Elad, 1997a). The spread of symptoms associated with this pathogen could be effectively limited by the effective management of light in protected crops, due to the reduction of spore production (Sutton, 1983; Sutton and Peng, 1993). The survival of conidia in the air is also influenced by the quality of light.

Climatic factors:

Environmental conditions, especially relative humidity and temperature play a key role for plant infection by B. cinerea and development of the disease. B. cinerea can thrive under a range of temperatures between 2 and 30oC (Elad and Yunis, 1993). The optimum temperatures for the different growth phases are range from 12–30 oC. B. cinerea will therefore always be a potential threat in greenhouse crops. Other studies have also evaluated the temperature requirements for the spores of B. cinerea germination. (Jarvis, 1977) reported that conidia of B. cinerea germinate to 100% at 20 °C, 15 oC and 5 oC with 100% humidity.
A 95% relative humidity, only 80% of conidia of B. cinerea germinate at 15 oC and 5 oC, however100% of the spores germinate at 20 oC. At 90% relative humidity, 85% of conidia germinated at 20 oC, and germination is stopped when the relative humidity and temperature are lower (Jarvis, 1977). Under conditions of relative humidity of about 80 to 100%, the incidence of the disease is more severe on cucumber in dry condition (Yunis et al., 1990). The aerial mycelium and sporulation develop a more rapid at 21 oC, 94% relative humidity (Thomas and Marois, 1986). An area of open water or high relative humidity (95%) even seems necessary for conidial germination and penetration of germinal filaments, and the success of the infection (Williamson et al., 1995). According to (O’Neill et al., 1997), the sporulation phase is favored by high relative humidity and the interruption of these conditions causes a delay in sporulation.

Strategies for protecting plants against B. cinerea

Cultural control

Cultural methods that ensure ventilation and drying of plant canopy after rain, whilst maintaining adequate water supply to the roots, are the most effective means developed so far for prevention of Botrytis epidemics (Elad and Shtienberg, 1995). Increasing public awareness of some potential drawbacks of chemical fungicides was addressed by the development of alternative control measures making use of microbial antagonists that are capable of disease suppression (Dubos, 1992). Cultural measures can be a powerful means to suppress plant diseases in greenhouses where the value of crops is high and the farmers make considerable efforts during long cropping seasons. Such measures are usually aimed at altering the microclimate in the canopy and around susceptible plant organs, prevention of inoculums entrance into the greenhouse and its build up and rendering the host plants less susceptible to diseases. Calcium loading of plant tissues and alteration of nitrogen fertilization reduce susceptibility to Botrytis (Elad and Shtienberg, 1995a). Cultivars resistant to B. cinerea are not available. In straw berry only the combination of a one-row-system, leaf sanitation and fruit sanitation decreased almost half gray mould damage in the first crop year compared to a two-row-system without leaf and fruit sanitation. B. cinerea damage correlated significantly and positively with the biomass of plants (Schmid et al., 2005).

Chemical control

The most common means for disease management is by application of chemical fungicides. Both spraying of fungicides and application of fungicides directly to sporulating wounds is practiced. However, high activity of several fungicides is being lost, at least in part, due to the development of resistance. As fungicides still remain an important tool for control of epidemics caused by B. cinerea, it is important to monitor populations of the pathogen for their resistance towards potential fungicides (Elad and Shtienberg, 1995a) Chemical control remains the main way to reduce the incidence of grey mould and other Botrytis diseases on major crops. The use of botryticides is an efficient way to protect crops against Botrytis spp. The most common interventions consist of spraying aerial parts of plants with fungicides. The applied doses vary from 2000-3000 g/ha (e.g. maneb, thiram, dichlofluanid) to 400-500 g/ha (e.g. carbendazim, fludioxonil, pyrimethanil). The number of treatments during a season ranges from one or two, to more than twenty.

Biological control

Biocontrol of Botrytis-incited diseases has been extensively investigated over the last 50 years. Biocontrol of Botrytis-incited diseases with filamentous fungi, bacteria and yeasts has been intensively studied over the last two decades (Blakeman, 1993; Elad and Shtienberg, 1995b; Tronsmo, 1991). Biocontrol offers an attractive alternative or supplement to the use of conventional methods for disease control since microbial biocontrol agents (BCAs) are perceived to be less demanding to the environment and their generally complex mode of action reduces the risk of resistance development. For example Ulocladium atrum showed a real potential to protect stem wounds from B. cinerea. The level of protection provided by the BCA was as good as that provided by the fungicide both in heated glasshouse and in unheated plastic tunnels (Fruit and Nicot, 1999). In 16 greenhouse trials conducted in southern France, an antagonistic strain of Fusarium sp. significantly reduced the incidence of stem lesions caused by B. cinerea on tomatoes (Decognet et al., 1999).
The efficacy of combined biological control against B. cinerea, it is concluded that M. dimerum, L. lecanii and R. sachalinensis extract are compatible for application on tomatoes (Bardin et al., 2004). Yeast isolates consistently reduced incidence of disease and sporulation of B. cinerea in tomato. Several isolates reduced disease by more than 75% in all experiments (Dik et al., 1999) U. atrum was most effective in suppressing Botrytis spp. Colonization fruit rot of strawberries was significantly reduced by weekly applications of U. atrum in 3 out of 4 field experiments. The antagonist was as effective as fungicide treatments (Kohl and Fokkema, 1998). U. atrum is a strong competitor on necrotic above-ground plant tissues. In onion leaf spot the potential of the antagonist to reduce colonisation of necrotic leaf tissue by Botrytis spp. and subsequent sporulation was studied in two field experiments. U. atrum colonised necrotic tissues and consistently reduced the sporulation of fungal competitors.(Kohl et al., 2003)

Table of contents :

GENERAL INTRODUCTION
Objectives of the thesis work:
Chapter I: Review of literature
1. Botrytis cinerea: causal agent of grey mould:
1.1 Taxonomic position:
1.2. Life cycle of the pathogen:
1.3. Pathogenicity and host range:
1.4. Symptoms:
1.5. Factors affect the development of the pathogen:
1.5.1. Nutrient requirements:
1.5.2. Effect of light:
1.5.3 Climatic factors:
2. Strategies for protecting plants against B. cinerea
2.1. Chemical control
2.2. Curtural control
2.3. Biological control
3. Effect of nutrition on the susceptibility of plants to Pathogens
3.1 Effect of nitrogen nutrition on the host pathogen relationship
3.2 Relationship between the form of nitrogen and host susceptibility
3.3 Biological interactions between nutrition and soil microorganisms.
3.4 Trophic host-pathogen interactions
3.5 Effect of nitrogen nutrition on the capacity of host defence
3.6 Effect of nitrogen on the development of plant disease
4. Relationship between nutritional status of the host plant and Botrytis cinerea
4.1 Nitrogen nutrition of the host and Botrytis
4.2 Effect of form of nitrogen on severity of B. cinerea
4.3 Nitrogen increases the susceptibility of crops to B. cinerea
4.4 Reduction of plant susceptibility to Botrytis cinerea with nitrogen fertilization
4.5 High N nutrition increase the susceptibility of crops during storage
5. Effect of nitrogen nutrition on fungal sporulation
5.1 Effect of type of substrates on the sporulation of different fungi
5.2 Effect of nutrient substrate on aggressiveness of fungi
5.3 Effect of nitrogen supply on sugars, acids carotinoids and phenolic compounds.
5.4 Effect of nitrogen nutrition on growth, transpiration and nutrient uptake of the
6 Effect of nutrition on the enhancement of biological control
7 Possible mechanisms involved in disease suppression by nitrogen
7.1 Physiology of the plant
7.2 Pathogen growth and virulence
7.3 Modification of the biotic or a biotic environment
7.4 Effect of nitrogen on gene expression of fungal pathogens
Chapter II: Contrasted response of Botrytis cinerea isolates developing on tomato plants grown under different nitrogen nutrition regimes
Chapeter III: Can plant sugars mediate the effect of nitrogen fertilization on lettuce susceptibility to two necrotrophic pathogens : Botrytis cinerea and Sclerotinia sclerotiorum ?
Chapter 1V: Nitrogen fertilization of the host plant influences production and pathogenicity of Botrytis cinerea secondary inoculum défini.
Chapter V: Enhancement of biocontrol efficacy against Botrytis cinerea through the manipulation of
nitrogen fertilization of tomato plants Signet non défini.
Chapter VI: Cyto-histological evidences for the influence of nitrogen fertilization on the efficacy of biological control against Botrytis cinerea in tomato.
General discussion
Conclusions and Perspectives
References

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