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Effect of nitrogen fertilization on the efficacy of biocontrol agents.
In order to enhance biocontrol activity of antagonists against fungal pathogens, certain strategies, such as adding calcium salts, carbohydrates, amino acids and other nitrogen compounds to biocontrl treatments are proposed Janisiewicz et al., (1992). How does N act on the pathogen- antagonism interactions. Foliar fertilizers cause changes in the development and antagonism of Trichoderma spp. The kind of changes depends on the fungal isolate and fertilizer composition. T. harzianum isolate, proved the most sensitive to the chosen fertilizers. Among the selected fertilizers Mikrovit Cu acted most unfavorably, since it most strongly inhibited mycelial growth and T. harzianum spore germination, and diminished the antagonism of Trichoderma isolates towards B. cinerea and R. solani Duzniewska, (2008). Soil nitrogen fertilization enhances aroma expression, but it also increases vine vigour and susceptibility to grey rot Lacroux et al., (2008) .
Efficacy of biocontrol agents against Botrytis cinerea
Replacement of fungicides with biocontrol of foliar diseases is an alternative mean of managing plant pathogens. One of the most important biocontrol agent is Trichoderma spp. against B. cinerea in tomato. The Trichoderma harzianum isolate Jn 14 reduced the disease by 77 % when applied as spore suspension on tomato and bean plants (Barakat and Al-Masri, 2005). T. harzianum were tested for the control of vegetable diseases relatively good control of P. cubensis, B. cinerea and R. solani was achieved (Bedlan, 1997). Application of antagonistic Trichoderma spp. to leaves of pepper or tomato resulted in a decrease in disease severity caused by B. cinerea. Moreover, an antagonistic strain of Fusarium sp. significantly reduced the incidence of stem lesions (caused by B. cinerea) on tomatoes. The yeast strain PBGY1 and Trichoderma harzianum T39 (Trichodex) were effective under a range of conditions in tomato and cucumber, while integration of the biological control agent with a suitable climatic regime can increase the overall control of B. cinerea (Dik and Wubben, 2001). The application of Trichoderma and Gliocladium as biological control agents reduced tomato leaf receptivity (infection and sporulation) to grey mould. The application of these antagonists reduced the conidial germination of B. cinerea on tomato leaves (Hmouni et al., 2005). Strain L13 of Microdochium dimerum was shown in earlier work to be efficient as a protectant of tomato pruning wounds against B. cinerea at crop level in a wide range of environmental conditions, the biocontrol agent provided efficient protection of foliage against B. cinerea (Nicot et al., 2003).
This fungus was used in tests on lettuce leaves. The inoculum consisted of 5 mm diameter mycelial disks excised from the growing margin of three day old colonies produced on PDA medium in the same growth chamber as for B. cinerea.
As this powdery mildew fungus is an obligate parasite, the isolate used in this work was maintained on tomato plants. For the duration of the work, six potted plants (cultivar Monalbo) were produced every two weeks, and the fungus was periodically transferred to fresh substrate. The inoculum for the trial consisted of spores produced two weeks after inoculation of the young potted plants. Mildewed leaflets were collected and shaken in 500 mL of sterile water. Containing 0.1 % Tween 80. The concentration of the spore suspension was then adjusted to 104sp/ml with the help of a haemocytometer. To avoid losing spore viability in water, the inoculum was prepared rapidly and used immediately to spray tomato leaves.
One strain of Trichoderma harzianum (T1) and one strain of Microdochium dimerum (L 13) were used in this study for tests on the effect of nitrogen fertilization on the interaction between the host plant (tomato), the parasite (B. cinerea) and the biocontrol agent. They were selected for their known protective effect of leaf pruning wounds on tomatoes against B. cinerea (studied in previous work in the laboratory) and differences in their presumed mode of action (nutrient competition for M. dimerum and antibiosis for T. harzianum).
The fungi were cultured on potato dextrose agar in the same growth chamber as described above. The spores of T. harzianum were collected in sterile distilled water from the surface of 14 day old cultures. For M. dimerum a one week old culture was used. Each suspension was filtered through a 30 µm mesh sterile filter to remove mycelium fragments and adjusted to the desired concentration (107 spores per ml for both biocontrol agents) with the help of a hemacytometer.
Botrytis cinerea on leaf pruning wounds
In the first experiment (first batch of plant production in May), the objective was to compare the effect of plant nutrition on their susceptibility to six different isolates of B. cinerea. Six sets of five plants were randomly chosen in the greenhouse for each nitrogen treatment (150 plants in total) and each set was inoculated with one of the six strains of B. cinerea. For each plant, the third, fourth, fifth and sixth leaves were cut with pruning scissors, leaving 5-10 mm petiole stubs on the stem. The four wounds were each inoculated with a 10 µl aliquot of spore suspension. The third and fifth leaves were inoculated with a spore suspension containing 107 sp/ml and the fourth and sixth leaves with inoculum containing 106 sp/ml.
In the second experiment (second batch of plant production in July), the objective was to compare the effect of plant nutrition on the efficacy of two biocontrol agents against each of two strains of B. cinerea. In this experiment, two sets of 15 plants were randomly chosen in the greenhouse for each nitrogen treatment (150 plants in total) and each set was inoculated with either strain BC 1 or BC 21 of B. cinerea. For each plant, four leaves were excised as described above and the third and fifth leaves were inoculated with a spore suspension containing 107 sp/ml while the fourth and sixth leaves were inoculated with 106 sp/ml. For each set of 15 inoculated plants per fertilization regime, five plants were used as an inoculated control and 10 plants were further treated with a biocontrol agent.
Biocontrol agents and Botrytis cinerea on leaf pruning wounds
The biocontrol agents were applied five minutes after the inoculation with B. cinerea. On each pruning wound, 10µL of spore suspension (containing 107 sp/mL) of either M. dimerum or T. harzianum were deposited. For each set of 15 Botrytis-inoculated plants per fertilization regime, five plants were treated with M. dimerum and five plants were treated with T. harzianum.
Oidium neolycopersici on leaves
For each batch of plant production in the greenhouse, one set of five plants per fertilization regime (25 plants per experiment) was used for tests with O. neolycopersici. For these two replicate experiments (one in May and one in July), the inoculum (spore suspension containing 104 sp/mL) was applied with the help of a compressed-air sprayer. All leaves of each plant were sprayed until run off.
Botrytis cinerea on leaves
Five sets of five plants were randomly chosen in the greenhouse for each nitrogen treatment (125 plants in total) and each set was inoculated with one of the five strains of B. cinerea. For each plant, three leaves were selected for inoculation and a mycelial disk (5 mm diameter) was placed in the centre of each leaf.
Sclerotinia sclerotiorum on leaves
One set of five plants was randomly chosen in the greenhouse for each nitrogen treatment (25 plants in total) and each set was inoculated with S. sclerotiorum. For each plant, three leaves were selected for inoculation and a mycelial disk (5 mm diameter) was placed in the centre of each leaf.
Incubation and quantification of disease development
Following inoculation, the plants were transferred to a growth chamber in conditions conducive to disease development (21 °C, RH above 85 %) with a 14h photoperiod. During this period, the plants were irrigated manually, using the same fertilization solutions as those used before inoculation.
Assessment of Botrytis stem lesions on tomato
Each inoculated wound was examined daily, between day 3 and day 7 after inoculation, for infection of the petiole stubs by B. cinerea and consequent development of stem lesions. The incidence of stem lesions and the length of developing lesions (mm) were recorded daily. To summarize the kinetics of stem canker development, the area under the disease progress curve (AUDPC) was calculated with values measured between day 3 and day 7 after inoculation.
Assessment of powdery mildew symptoms on tomato
Disease symptoms were assessed 14 days after inoculation. On each plant, every leaflet of the 3rd 4th 5th and 6th leaves was examined. On the first repetition of the experiment (May), the severity of powdery mildew was rated for each leaflet on a scale of 0 to 9, adapted from a method developed by the laboratory for melon (see annex 1). The scores for all leaflets of a leaf were then averaged to provide four replicate disease ratings per plant.
In addition, a photo of each leaf was taken with a digital camera, and the percentage of mildewed leaf area was assessed with the help of image analysis software developed by the American Phytopathological Society for disease quantification (Assess 2, APS Press, St Paul, Minnesota). For the second repetition of the experiment (July), following a comparison of results obtained with both disease assessment methods, only the image analysis method was used.
Assessment of leaf symptoms on lettuce (Botrytis and Sclerotinia)
Both B. cinerea and S. sclerotiorum induce soft rot lesions on the lettuce leaves. The incidence and severity of disease was assessed on day 4 and day 6 after inoculation for B. cinerea and on day 6 for S. sclerotiorum.
For each leaf inoculated with B. cinerea, the percentage of diseased leaf area was assessed visually. In addition on day 6 after inoculation, the leaves were detached from the plants and a photo was taken for each leaf with a digital camera. The image analysis software (Assess 2.0) was then used to quantify the total leaf area (in mm²) and the percentage of diseased leaf area. For plants inoculated with S. clerotiorum, disease assessment was only carried out with the help of photos and image software analysis.
ANOVA/MANOVA module of statistica software was used to analyse all the data. For tomato & lettuce the length of lesion, latency period, lesion growth rate, leaf area with necrosis and the Area under the Disease Progress Curve AUDPC (computed between day 3 and 7) on day 7 after inoculation of B. cinerea & for O. neolycopersici disease severity percent was analysed.
Table of contents :
REVIEW OF LITERATURE
2. Botrytis cinerea :
4. Host range:
7. Effect of nitrogen fertilization on susceptibility of plants to Botrytis cinerea
8. Effect of nitrogen fertilization on the efficacy of biocontrol agents
9. Efficacy of biocontrol agents against Botrytis cinerea
MATERIAL AND METHODS
1. Production of plant material
2. Assessment of plant development and tissue content
3. Plant pathogens and inoculum production
3.1. Botrytis cinerea
3.2. Spore suspensions
3.4. Sclerotinia sclerotiorum
3.5. Oidium neolycopersici
3.6. Biocontrol agents
4.1.1. Botrytis cinerea on leaf pruning wounds
4.1.2. Biocontrol agents and Botrytis cinerea on leaf pruning wounds
4.1.3. Oidium neolycopersici on leaves
4.2.1. Botrytis cinerea on leaves
4.2.2. Sclerotinia sclerotiorum on leaves
5. Incubation and quantification of disease development
5.1. Incubation of plants after inoculation
5.2. Assessment of Botrytis stem lesions on tomato
5.3. Assessment of powdery mildew symptoms on tomato
5.4. Assessment of leaf symptoms on lettuce (Botrytis and Sclerotinia)
6. Data analysis
1. Effect of N fertilization on the mineral contents in the tissues of the plants
2. Effect of N fertilization on the susceptibility of tomato plants
2.1 Powdery mildew
2.2 Botrytis cinerea
2.2.1. Latency period (days)
2.2.2. Lesion growth rate (mm/day)
2.2.3. Kinetics of disease development
3. Effect of N fertilization on the efficacy of biocontrol agents against B. cinerea.
3.1 Tricoderma harzianum
3.1.1 Latency period (days)
3.1.1 Lesion expansion rate
3.2 Microdochium dimerium (strain L 13)
3.2.1 Latency period
3.2.2 Lesion expansion rate
4 Effect of N fertilization on the susceptibility of Lettuce to B. cinerea
4.1 Leaf area necrosis percent
4.2 Lesion expansion rate (mm2)
CONCLUSION AND PERSPECTIVES