Test of biocontrol products against fungal pathogens of tomato and lettuce

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Test of biocontrol products against fungal pathogens of

tomato and lettuce

Ruben Lenaerts

To cite this version:

Ruben Lenaerts. Test of biocontrol products against fungal pathogens of tomato and lettuce. Life

Sciences [q-bio]. 2011. �hal-02811377�

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P

REFACE

This master proof is an endpoint as well a starting point. It is the result of a gathering of all interests and knowledge that I accumulated during my education towards the degree of Master of applied Bio-sciences. It is the starting point of a professional career to which I am looking forward as well. It would not have been possible to do that without the help of other people, whom I would like to thank.

It was not always easy doing an internship abroad neither writing this master proof. But my girlfriend, Jana, kept supporting me at all times. Also I would like to thank my parents, without their help it was impossible to study.

I am deeply grateful to my supervisors, Prof. dr. Philippe Nicot and dr. Marc Bardin for taking a role as my scientific mentors. Thank you for believing in me!

For excellent technical assistance I want to thank Claire Troulet and Magali Dufaud, with their help everything in the lab worked out smoothly.

The friendly atmosphere at the pathology department at INRA Avignon has enabled inspiring conversations and unforgettable adventures with several great people: Manzoor, Florian, Valentin, Toufik, Laurence and the whole running crew from INRA Avignon.

In short, thanks to all the people I have met in Avignon, France to give me the best three months of my life!

And last but not least, my internship would have not been possible without the help of my mentor ir. Rudi Aerts.



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A

BSTRACT

Chemical control remains the main measure to reduce the incidence of fungal diseases in various fruits and vegetables. A serious problem against the effective use of these chemicals is the development of resistance by the fungi. The exploitation of biocontrol agents, safer to consumers and the environment, is urgently needed. Furthermore, the demand for reduction in the use of pesticides in agriculture and horticulture increases interest in the possibility of the application of biocontrol agents.

Research is done to look for biocontrol agents against Oidium neolycopersici and

Botrytis cinerea, two pathogenic fungi. The VALORT-project is such a research project

subsidized by the EU, which looks for biocontrol agents against these two pathogenic fungi on tomato and lettuce plants. B. cinerea and O. neolycopersici cause damages and economic losses in southern Europe, the scope of the project. Products tested in the project were chosen based on two reasons. There are some products that proved their efficiency against B. cinerea and O. neolycopersici on other vegetables. Or other products proved their efficiency against other pathogenic fungi. Lab tests were done to determine if the products have an action against the fungi on tomato and lettuce plants. In total 16 products were tested. And after the statistical analyses there were two products that had a potential. These products were Prev-Am and Serenade Max. In the future further research is necessary to test other products and to take the products that proved their efficiency in the lab out on the field.

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C

ONTENTS

PREFACE ... 2 ABSTRACT ... 3 CONTENTS... 4 INTRODUCTION... 6 LIST OF FIGURES ... 7 LIST OF TABLES... 9 1 BIBLIOGRAPHY ... 10

1.1 Biocontrol through the years ... 10

1.1.1 What is biocontrol... 10

1.1.2 The principles of biocontrol ... 10

1.1.3 Biocontrol agents... 11

1.1.4 The future ... 12

1.2 The pathogens ... 12

1.2.1 Botrytis cinerea ... 12

1.2.1.1 Biology... 12

1.2.1.2 The ecology of Botrytis on plant surfaces... 13

1.2.1.3 Inoculum production and dispersal ... 13

1.2.1.4 Penetration... 14 1.2.2 O. neolycopersici ... 14 1.2.2.1 Biology... 14 1.2.2.2 Symptoms ... 14 1.2.2.3 Host-derived signals ... 15 1.2.2.4 Penetration... 15

1.3 Biocontrol: economic aspects... 16

1.3.1 Cost analysis... 16

1.3.1.1 Size of the targeted market ... 16

1.3.1.2 Cost of production ... 16

1.3.1.3 Cost of registration ... 18

1.3.1.4 Business profitability... 19

1.3.1.5 Conclusion and outlook for industry ... 19

1.3.2 Socio-economic aspect: market analysis and outlook ... 19

1.3.2.1 The estimated market of biocontrol in Europe ... 19

2 OBJECTIVES ... 21

3 MATERIAL AND METHODS... 22

3.1 Production of plant material ... 22

3.1.1 Tomato production... 22 3.1.2 Lettuce production... 22 3.2 Fungal strains ... 23 3.2.1 Botrytis cinerea ... 23 3.2.2 O. neolycopersici ... 23 3.3 Products ... 23 3.4 In vitro test... 26

3.4.1 Mycelium growth test ... 26

3.4.1.1 Inoculum production ... 26

3.4.1.2 Effect of biological products on mycelium growth of B. cinerea ... 26

3.4.1.3 Statistical analysis ... 26

3.4.2 Spore germination test... 26

3.4.2.1 Inoculum production ... 26

3.4.2.2 Effect of biological products on spore germination of B. cinerea... 27

3.4.2.3 Statistical analysis ... 27

3.5 Leaf disk tests... 27

3.5.1 Inoculum production ... 27

3.5.1.1 B. cinerea... 27

3.5.1.2 O. neolycopersici ... 27

3.5.2 Application of the products ... 2



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3.5.3.1 Inoculation with B. cinerea ... 30

3.5.3.2 Inoculation with O. neolycopersici... 30

3.5.4 Disk incubation and disease assessment... 31

3.5.5 Statistical analysis ... 32

3.6 Entire plant tests ... 32

3.6.1 Inoculum production ... 32

3.6.2 Application of the products and inoculation of pruning wounds ... 32

3.6.3 Plant incubation and disease assessment... 37

3.6.4 Statistical analysis ... 37

4 RESULTS ... 38

4.1 In vitro test... 38

4.1.1 Mycelium growth test ... 39

4.1.2 Spore germination test... 39

4.2 Leaf disk tests... 40

4.2.1 Tomato and powdery mildew ... 40

4.2.1.1 Test n°1 ... 40 4.2.1.2 Test n° 2 ...42 4.2.1.3 Test n°3 ... 43 4.2.1.4 Test n°4 ... 45 4.2.1.5 Test n°5 ... 46 4.2.1.6 Test n° 6 ...48

4.2.2 Tomato and B. Cinerea... 49

4.2.2.1 Test n°1 ... 49

4.2.2.2 Test n°2 ... 51

4.2.2.3 Test n°3 ... 53

4.2.2.4 Test n°4 ... 54

4.2.2.5 Test n°5 ... 55

4.2.3 Lettuce and B. cinerea ... 56

4.2.3.1 Test n°1 ... 57

4.2.3.2 Test n°2 ... 58

4.2.3.3 Test n°3 ... 60

4.2.3.4 Test n°4 ... 62

4.3 Entire plant tests ... 63

4.3.1 Test n°1 ... 63

4.3.2 Test n°2 ... 64

4.3.2.1 Tomato variety Monalbo ... 64

4.3.2.2 Tomato variety Swanson ... 65

4.3.3 Test n°3 ... 65

4.3.4 Test n°4 ... 67

4.3.5 Test n°5 ... 68

4.3.5.1 Tomato variety Monalbo ... 68

4.3.5.2 Tomato variety Swanson ... 69

4.3.6 Test n°6 ... 69 4.3.7 Test n°7 ... 70 4.3.8 Test n°8 ... 71 CONCLUSION ... 74 REFERENCES ... 75 ANNEX 1 ... 78

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I

NTRODUCTION

Chemical control remains the main measure to reduce the incidence of fungal diseases in various fruits and vegetables. A serious problem against the effective use of these chemicals is the development of resistance by the fungi. The application of higher concentrations of chemicals in an attempt to overcome this problem increases the risk of high level toxic residues in the products, which is particularly serious because fruits and vegetables are consumed in a relatively short time after harvest. The exploitation of biocontrol agents, safer to consumers and the environment, is urgently needed. Furthermore, the demand for reduction in the use of pesticides in agriculture and horticulture increases interest in the possibility of the application of biocontrol agents. Tomato and lettuce are two very important vegetables. Botrytis cinerea and Oidium

neolycopersici are 2 pathogenic fungi which cause grey mould and powdery mildew on

tomato plants, respectively and B. cinerea causes grey mould on lettuce plants. There has been done a lot of research against these two fungi.

This master proof takes part of an European project, the VALORT-project. It is a project where in the first year lab tests are done to see if there are biological products that have an efficiency against the fungal pathogens. In the second year field tests will be done with those products that have proved their value.

My part in this project were the lab tests.

After a summarizing literature study about all relevant topics handled in this study, a detailed description of the used materials and methods is given. Results will be discussed ending with the formulation of an overall conclusion.



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IST OF FIGURES

Figure 1: Evolution of the yearly number of publications dedicated to biological

control of plant diseases based on a survey of the CAB Abstracts® database...10

Figure 2.a: Botrytis infection on tomato...13

Figure 2.b. Lettuce leaf with Botrytis infection...13

Figure 3. Powdery mildew of tomato causes by O. neolycopersici...15

Figure 4: Estimated sales of biocontrol products in Europe in 2008 (in Million €)...20

Figure 5: Estimated distribution of biocontrol use among types of crops in 2008 in EU20 Figure 6: Pulverizing a product on a lettuce plant...28

Figure 7: Taking foliar disks with a cork borer...29

Figure 8: Trays of the foliar disk test...30

Figure 9: Inoculation foliar disks with B. cinerea...30

Figure 10: Tube used to inoculate the foliar disks with O. neolycopersici...31

Figure 11: Taking a photo of a tray...31

Figure 12: Application of the products and inoculation pruning wounds...33

Figure 13: In vitro test...39

Figure 14: Spore germination test...39

Figure 15: Tomato and powdery mildew, test n° 1: D-2...40

Figure 16: Tomato and powdery mildew, test n° 1: D0...41

Figure 17: Tomato and powdery mildew, test n° 2: D-2...42

Figure 18: Tomato and powdery mildew, test n° 2: D0...42

Figure 19: Tomato and powdery mildew, test n° 3: D-2...43

Figure 20: Tomato and powdery mildew, test n° 3: D0...44

Figure 21: Tomato and powdery mildew, test n° 4: D-2...45

Figure 22: Tomato and powdery mildew, test n° 4: D0...45

Figure 23: Tomato and powdery mildew, test n° 5: D-2...46

Figure 24: Tomato and powdery mildew, test n° 5: D0...47

Figure 25: Tomato and powdery mildew, test n° 6: D-2...48

Figure 26: Tomato and powdery mildew, test n° 6: D0...48

Figure 27: Tomato and B. cinerea, test n°1: BC1...49

Figure 28: Tomato and B. cinerea, test n°1: BC21...50

Figure 29: Tomato and B. cinerea, test n°2: BC1...51

Figure 30: Tomato and B. cinerea, test n°2: BC21...52

Figure 31: Tomato and B. cinerea, test n°3: BC1...53

Figure 32: Tomato and B. cinerea, test n°3: BC21...53

Figure 33: Tomato and B. cinerea, test n°4: BC1...54

Figure 34: Tomato and B. cinerea, test n°4: BC21...55

Figure 35: Tomato and B. cinerea, test n°5: BC1...55

Figure 36: Tomato and B. cinerea, test n°5: BC21...56

Figure 37: Lettuce and B. cinerea, test n°1: BC1...57

Figure 38: Lettuce and B. cinerea, test n°1: BC21...58

Figure 39: Lettuce and B. cinerea, test n°2: BC1...59

Figure 40: Lettuce and B. cinerea, test n°2: BC21...59

Figure 41: Lettuce and B. cinerea, test n°3: BC1...60

Figure 42: Lettuce and B. cinerea, test n°3: BC21...61

Figure 43: Lettuce and B. cinerea, test n°4: BC1...62

Figure 44: Lettuce and B. cinerea, test n°4: BC21...62

Figure 45: Entire plant test n°1...63

Figure 46: Entire plant test n°2: Monalbo...64

Figure 47: Entire plant test n°2: Swanson...65

Figure 48: Entire plant test n°3: BC1...65

Figure 49: Entire plant test n°3: BC21...66

Figure 50: Entire plant test n°4: BC1...67

Figure 51: Entire plant test n°4: BC21...67

Figure 52: Entire plant test n°5: Monalbo...68

Figure 53: Entire plant test n°5: Swanson...69

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Figure 55: Entire plant test n°7:

BCl...70

Figure 56: Entire plant test n°7: BC21...71

Figure 57: Entire plant test n°8: BC1...72



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IST OF TABLES

Table 1: Scientific papers published between 1973 and 2008 on

biological control against major plant diseases 11

Table 2. Comparison of data on performance of chemical and biological

control 12

Table 3. Compared structure of the production costs for a microbial

biocontrol agent 17

Table 4. Compared potential costs of registration for a microbial

biocontrol agent 18

Table 5. Compared estimated market potential for a microbial biocontrol

agent 18

Table 6. Compared margin structure estimates for the production and

sales of a microbial biocontrol agent (MBCA) and a chemical pesticide 19

Table 7. Products used in the tests 24

Table 8: Mycelium growth test 26

Table 9: Spore germination test 27

Table 10: Biological products applied on tomato plants 28 Table 11: Biological products applied on lettuce plants 29

Table 12: Products used by entire plant tests 34

Table 13: Tomato and powdery mildew, test n° 1: efficacy indices 41 Table 14: Tomato and powdery mildew, test n° 2: efficacy indices 43 Table 15: Tomato and powdery mildew, test n° 3: efficacy indices 44 Table 16: Tomato and powdery mildew, test n° 4: efficacy indices 46 Table 17: Tomato and powdery mildew, test n° 5: efficacy indices 47 Table 18: Tomato and powdery mildew, test n° 5: efficacy indices 49

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Nu m b e r of pub li c a ti ons pe r y e a r 1

Bibliography





1.1

Biocontrol throughtheyears



1.1.1

Whatisbiocontrol



Biocontrol is a fascinating and sometimes misunderstood aspect of agriculture. It is a mix of applied and basic science including traditional exploration. The most common definition of biocontrol is: ‘The use of natural enemies to reduce the population size of pest species.’ Although biocontrol is not new to agricultural practice, modern efforts of it have taken place for about 100 years. (McKimmie, 2000) In 1888, the first large scale use of biocontrol started with the release of Rodolia ladybird beetles to control a scale insect in citrus in California. (van Lenteren, 2007) Because of the environmental and economic concerns, biocontrol of pests has received great interest recently as an alternative to conventional pesticides. (McKimmie, 2000) Biocontrol agents saved in some cases the staple food supply of several countries ‘”a crop vital to the economic survival of growers in a region. (Lazarovits et al., 2007)

In the last 35 years, there is a lot of scientific literature published concerning studies on biocontrol against diseases and pests of agricultural crops. A recent survey of the CAB Abstracts®_{database (Nicot & Bardin, 2010, unpublished) showed that the yearly} number of publications increased from 20 in 1973 to over 700 per year since 2004. (figure 1) 900 800 700 600 500 400 300 200 100 0 1970 1975 1980 1985 1990 1995 2000 2005 2010 Publication year

Figure 1: Evolution of the yearly number of publications dedicated to biological control of plant diseases based on a survey of the CAB Abstracts® database. (Nicot & Bardin, 2010, unpublished)

1.1.2

Theprinciplesofbiocontrol



There are three different approaches to biocontrol. Namely augmentative, conservation and classical biocontrol. Augmentative biocontrol involves adding natural enemies. (McKimmie, 2000) It has seen many successes. In greenhouses, for example, pest management trough biocontrol has become the foundation of integrated pest

management programmes. One of the reasons was that there was a desperate need to control pests that rapidly developed resistance to chemical pesticides within closed ecosystems. (Lazarovits et al., 2007) “Conservation biocontrol involves manipulation of the environment to enhance the survival, fecundity, longevity, and behaviour of natural



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Fusarium 34 818 1 925 5.5 Rhizoctonia 10 744 1 278 11.9 Verticillium 7 585 592 7.8 Pythium 5 772 821 14.2 Sclerotinia 5 545 456 8.2 Air-borne: rusts 29 505 360 1.2 powdery mildews 18 026 251 1.4 Alternaria 12 766 415 3.3 anthracnose 12 390 351 2.8 Botrytis 9 295 705 7.5 downy mildews 8 456 80 1.0 Phytophthora infestans 5 303 61 1.1 Monilia rot 1 861 81 4.3

enemies to increase their effectiveness” (Landis et al., 2000). Conservation

biocontrol is probably the oldest approach to biocontrol. But it is also under-explored. (Lazarovits et al., 2007) And finally, with classical biocontrol one seeks for natural enemies in the geographic area(s) where the pest originated. When a natural enemy is found, it can be introduced to the place(s) where the pest has become a problem. (McKimmie, 2000)

1.1.3

Biocontrolagents



Reliable field performance, cost-effective mass production and formulation, adequate market size and a simple registration procedure are the most important requirements for successfully developing biocontrol agents. The potential market size for biologicals depends on the following, most unpredictable elements:

1. The absence or failure of the available, conventional means of control. The control against soilborne pathogens is more difficult using chemicals than the control against above-ground pathogens. This is reflected in the research input and the number of biocontrol products against soilborne diseases. This can be demonstrated with the results of a survey of the CAB Abstracts® database conducted by Nicot & Bardin (2010, unpublished) shown in table 1. An example of failure is the poor efficacy of chemical active ingredients due to developing fungicide-resistance in the pathogen population.

Table 1: Scientific papers published between 1973 and 2008 on biological control against major plant diseases (from CAB Abstracts® database; Nicot & Bardin, 2010, unpublished).

Disease or plant pathogen Total number

of references References on biological control

% Soil-borne:

Venturia 3 870 104 2.7

2. The public opinion towards pesticides. People nowadays demand pesticide- free fruits and vegetables. And there is also the implementation of

environmentally friendly crop production methods. But it is difficult to predict how much this demand will grow in the near future.

3. Some competing chemical products are withdrawed by the registration authorities. Because there’s a need for reregistrating soil desinfectants, biological will have their opportunities. For example, the market prospects for microbial seed coating largely depend on which chemicals are allowed in this particular sector.

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1.1.4

Thefuture



Agricultural research needs to be redirected to a systems approach due to several facts. The first fact is that the earth will have to feed about 11 billion human beings in the near future. The second fact is that fossil energy is running out, and thus also the conventional synthetic pesticides. And finally, the third and last fact is that man cannot continue to pollute the environment and reduce biodiversity at the same dramatic rate as during the past 100 years. In this way, pest management will be a guiding theme instead of being the marginal issue it was during the past 60 years. Modern pest management will strongly depend on biocontrol, because it is the most sustainable, cheapest and environmentally safest pest management method. (table 2) It is expected that in the year 2050 biocontrol will make up 35-40% of all crop protection methods. (van Lenteren, 2007)

Table 2. Comparison of data on performance of chemical and biological control

_ Chemical control* Biological control

_ Number of ingredients tested > 3,5 million 2,000

Success ratio 1 : 200,000 1 : 10

Developmental costs 150 million US$ 2 million US$

Developmental time 10 years 10 years

Benefit / cost ratio 2 : 1 20 : 1

Risks of resistance large small

Specificity very small very large

Harmful side-effects many nil/few

_ *Data for chemical control originate from material provided by the pesticide industry; data as per 2005. In 1980 10,000 compounds were tested per year, in 2004 this had increased to 500,000 per year (Stenzel, 2004)

1.2

Thepathogens



1.2.1

Botrytiscinerea



1.2.1.1Biology



B. cinerea (and other Botrytis species) are important pathogens of nursery plants,

vegetables, ornamental, field and orchard crops and stored and transported agricultural products. (Elad et al., 2007) Botrytis cinerea causes serious losses in more than 200 crop species worldwide. (Williamson et al., 2007) Therefore there’s invested a considerable effort in protecting the agricultural produce against Botrytis before and after harvest. In recent years the market size for anti-Botrytis products has been € 11- 19 million per year. Over the last 125 years, an increasing number of specialists in diverse fields have investigated Botrytis spp. The fields included chemistry, biochemistry, molecular and cell biology, genetics, morphology and histology, taxonomy, host-parasite interaction, ecology and epidemiology. (Jarvis, 1977; Coley- Smith et al., 1980; Verhoeff et al., 1992) Botrytis spp. have been the subject of an immense number of published studies. (Elad et al., 2007) B. cinerea has become the most extensively studied necrotrophic fungal pathogen. (Williamson et al., 2007)

Botrytis spp. are known as high humidity pathogens and their conidia germinate at high

humidity. A film of water on the susceptible plant tissue is mostly present when there’s an infection, but the pathogen is also able to infect plants when no film of water exists on the plant surfaces. (Williamson et al., 1995; Elad, 2000) Botrytis spp. are commonly isolated from upper plant parts (leaves, flowers, fruits, buds and stems), and in some cases from upper root parts and stem bases, although they can be isolated from soils and from seeds, bulbs and corms. (Elad et al., 2007)



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In figure 2.a you can see a stem infection of a tomato plant with Botrytis cinerea and in figure 2.b you can see an infected lettuce leaf with Botrytis cinerea.

Figure 2.a Botrytis infection on tomato (INRA, Avignon)

2.b Lettuce leaf with Botrytis infection (BBC Gardener’s World, 2010)

1.2.1.2TheecologyofBotrytisonplantsurfaces



The most Botrytis spp. have a necrotrophic life style occurring as pathogens infecting a single specific host or closely related host. But B. cinerea infects numerous hosts plants. When these fungi have infected and killed the host tissues, they can survive and sporulate as saprophytes on the necrotic tissue, or produce long-term survival

structures, such as sclerotia. These sclerotia can be associated with living plants or with plant debris lying on or buried in soil. For B. cinerea the primary inoculum is most likely generated within the crop (Johnson and Powelson, 1983), but the potential for

incoming primary inoculum from a different crop or weed host is greater than for the host-specific pathogens, because the host range is extremely wide. Therefore plants will be affected by the phasing of crop growth and harvest within a district or region.

Botrytis spp. exist in the different habitats as mycelia, micro- and macro-conidia,

chlamydospores, sclerotia, apothecia and ascospores and these are dispersed by diverse means (Coley-Smith et al., 1980). It is surprising that the majority of published work about B. cinerea describes infection arising from suspensions of conidia in water droplets, although the fungus releases its macroconidia mainly in dry air currents. (Elad et al., 2007)

1.2.1.3Inoculumproductionanddispersal



For B. cinerea, inoculum is always present in the field and production, liberation and dispersal of inoculum is an ongoing process (Coley-Smith et al., 1980). There are various factors essential for high propagule numbers in the air: a viable, productive inoculum source, conditions favourable for propagule production, and for their dispersal at the source site. (Elad et al., 2007)

To infect a host, the pathogen must conquer space (Zadoks and Schein, 1979), that is to move from the primary source and land on susceptible tissue. Each part of the fungus thallus can serve as a dispersal unit. These propagules are dispersed by wind, rain and insects. (Elad et al., 2007)

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1.2.1.4Penetration



Botrytis pathogens are well known for their ability to form either spreading lesions in host tissues, or latent infections in young fruit and seeds. (Elad et al., 2007) There are different routes how the pathogen can enter the host.

- Penetration through specialized host structures

- Penetration through undamaged host tissue and natural openings - Penetration through wounds

(Elad et al., 2007)

1.2.2

O.neolycopersici



1.2.2.1Biology



O. neolycopersici is a relatively new powdery mildew disease. It was first reported in

the UK in 1986 (Fletcher et al., 1988), but it has now spread world-wide. (other name in Australia, see further) O. neolycopersici is a specific tomato powdery mildew. Its true identity was uncertain due to the lack of a sexual stage and varying reports of its structure, particularly whether conidia were formed singly or in chains. Confusion remained over classification based on morphological characteristics. There was a difference in fungi outside Australia and in Australia. This was confirmed by Kiss et al. (2001) from a study of tomato powdery mildew fungi from Europe, North and South America and Asia. In this study, Kiss et al. (2001) recognized that all recent outbreaks of tomato powdery mildew reported outside Australia were caused by a species that formed conidia singly, or, in high relative humidity, in pseudo-chains of 2-6 conidia, and so created a new species, O neolycopersici, for this pathogen. The Australian isolates were called O lycopersici, which always form conidia chains. (Jones et al., 2001)

O neolycopersici has become recently a problem and it has spread rapidly around the

world. The increased movement of plants through the international horticulture trade could be responsible, but so too could the aerial dispersal of conidia and their subsequent survival on numerous alternative hosts. (Jones et al., 2001)

1.2.2.2Symptoms



As noted earlier, O neolycopersici is a fungus which infects tomatoes and it is a highly polyphageous powdery mildew fungus. It causes powdery white lesions on the adaxial tomato leaf surface (figure 3). It can also infect abaxial surfaces, petioles and the calyx but the fruit remains uninfected. Whipps et al. (1998) noticed that severe infections lead to leaf chlorosis, premature senescence and a marked reduction in fruit size and quality. Glasshouse-grown tomatoes are currently threatened by O neolycopersici and it is of increasing importance on field-grown tomato crops. (Jones et al., 2001)



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Figure 3. Powdery mildew of tomato caused by O neolycopersici (Extension, 2011)

1.2.2.3HostͲderived signals



O neolycopersici is an obligate biotroph. So the development of mature, sporulating

colonies depends upon successful initial penetration of the host and then continued haustorial formation and functioning. Jones (2001) revealed with preliminary analysis of differentiation on various artificial substrata that hydrophobic surfaces and cellulose, as well as host wax, induce germling morphogenesis in O neolycopersici. Furthermore, at the peak time for germination and for host penetration, at 4 and 11 hours after

inoculation respectively, small peaks in O neolycopersici cutinase activity were monitored as well in vivo (on tomato leaf discs) as in vitro (in multiwall plate assays). This data suggest a role for cutinase activity in either the perception of host-derived cutin

breakdown products, found in a range of plant pathogenis fungi including other powdery mildews (Fan and Koller, 1998; Francis et al., 1996; Fric and Wolf, 1994; Rumbolz et al., 2000) or else in the use of enzymic means to breach the host (Maiti and Kolattukudy, 1979; Podila et al., 1995; Rogers et al., 1994). (Jones et al., 2001)

1.2.2.4Penetration



Does O neolycopersici breach the host cuticle and epidermal cell wall by enzymic activity, by mechanical force, or by a combination of both? Enzymes may play a role in penetration. This is suggested by the small peak in cutinase activity at the time of penetration and the appearance of a smooth-edged penetration hole. (Jones et al., 2000; Jones, 2001) Nevertheless, development of increased turgor pressure in appressoria has been found to be the key feature of penetration in a range of plant pathogenic fungi including powdery mildews (de Jong et al., 1997; Howard et al., 1991; Money and Howard, 1996; Money et al., 1998; Money, 1995; Pryce-Jones et al., 1999). There are strong data supporting the use of mechanical force in penetration for O

neolycopersici. Cell turgor measurements revealed that mature O neolycopersici

appressoria produce a maximum turgor pressure of approximately 3 MPa, coincident with host cell penetration from the appressoria, at 11 hours after inoculation. This was determined by cytorrhysis and plasmolysis experiments. Blumeria graminis appressoria also have a maximum turgor pressure around 3 MPa, so the turgor pressure of O

neolycopersici is in near concordance (Pryce-Jones et al., 1999). But it is significantly

lower than the 8 MPa recorded in the melanized appressoria of the rice blast fungus

Magnaporthe grisea (Howard et al., 1991). It remains a mystery how the powdery

mildew fungi withstand such osmotic pressure because there is no evidence to support the presence of melanin in the hyaline appressoria of either O neolycopersici or

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1.3

Biocontrol:economicaspects



(Nicot & Bardin, 2010, unpublished)

1.3.1

Costanalysis



The industrial and commercial development of biological is facing different constraints which are particularly difficult to overcome due to the size of the involved companies and the early development stage of the market. Although these biologicals are needed as an alternative to chemical pesticides in both organic farming and integrated pest management systems.

These constraints can be classified within 4 categories: - Size of the targeted market

- Cost of production - Costs of registration - Business profitability

In this chapter, the situation will be considered regarding microbial biocontrol agents (MBCAs), using the real case of a well defined product that cannot be mentioned here due to proprietary rights.

1.3.1.1Sizeofthetargetedmarket



The markets, for which MBCAs are developed for, are rather small, if not niche. The total value of MBCAs sold worldwide amounted in 2008 to € 620 million (€ 122 million in Europe) including products with insecticidal or fungicidal effects. There’s a big difference when you compare this value with the sales of chemical insecticides and fungicides amounting to a total of € 21 000 million.

MBCAs are presently still used in speciality crops, greenhouses and covered crops.

Bacillus thuringiensis products are an exception on this, because they can be used in

larger crops such as grapes, forestry or even cereals.

The size of the speciality crops is not growing anymore or at a very reduced rate. The development of organic faster farming is the only optimistic perspective where MBCAs can find a good market.

Additionally, the potential market is widely fragmented within a long list of crops such as carrots, petersillium, onions, etc, usually referred to as “minor crops”. Even large chemical companies refrain from the investments that would cover the needs because the markets are so smalle. Due to the specificity of their products, manufacturers of MCBAs are obliged to invest and cover costs where scale economy can never be reached.

1.3.1.2Costofproduction



Unlike the synthesis of chemicals, the production of MBCAs requires a complicated and extremely expensive process of production. This process can be divided into 4 phases:

-fermentation -extraction -purification

-formulating and packaging



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1) Fermentation

This first production step has to be undertaken either with solid or with liquid phase technology. The liquid phase fermentation is usually simple and cost effective, but the process is more risky because the produced spores are more fragile. The solid fermentation on the other hand will produce stronger spores, but it becomes more difficult to increase the production volume. 2) Extraction

Like in the first step, there is a very strong difference between the MBCAs produced in liquid or in solid fermentation.

The advantage to the liquid fermentation is that the extraction will be rather easy by filtration, but the product will need to be dried, which is a very long, energy-demanding and expensive process.

Starting from a solid fermentation process, a mechanical extraction is used. Such a process is rather harmful for the spores. It is again energy-

demanding and it is extremely difficult to extract more than 60% of the spores from a substrate. In this case the productivity becomes rather poor. 3) Purification

This step is very important to ensure the stability of the MBCAs produced. The industrially produced MBCAs always contain impurities. Although this impurities are biologically inactive they may become critical over time and create risks of degradation, inactivation, etc.

In all situations the purification step requires a high level of sophistication and expensive processes.

4) Formulation and packaging

Due to their living state, the formulation and packaging of MBCAs is an extremely difficult step and in any case more expensive than the equivalent process for chemicals. The choice of co-formulants, adjuvants and packaging material, which must secure the quality of the MBCAs and their vitality, is again a source of problems and heavy costs.

It also has to be secured that no contamination will occur during the four steps mentioned. All the safety measures are very expensive to carry out, but they are necessary in order to ensure the quality of the product brought to the market. Table 3 shows us, by the consequence of all these extra expenses and technical difficulties, that the MBCAs used for this analysis were more than 4 times more expensive to produce than an equivalent chemical pesticide.

Table 3. Compared structure of the production costs for a microbial biocontrol agent (MBCA) and a chemical insecticide (IBMA; Nicot & Bardin, 2010, unpublished).

Typical MBCA Comments

Sales value 100 100

Type of production cost

Raw materials %* 8 29 40% lost material for MBCA by

solid fermentation process

Packaging 1 2

Energy and miscellaneous 1 2

Manpower 5 9

Consumables 2 3

Amortisation 4 11

TOTAL 13 56

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1.3.1.3Costofregistration



The regulations for registration have initially been set up to reduce the risks attached to molecules and the regulator is trying to extrapolate these requirements for the

registration of living organisms.

Nicot & Bardin (2010, unpublished) show in table 4 that the estimated cost for the registration of a microbial biocontrol agent is currently lower than that for a chemical pesticide. However, Nicot & Bardin (2010, unpublished) show in table 5 that the size of this investment is still very high for a company in comparison with the market potential. This evaluation indicates that the introduction on the market of a MBCA is about 4 times less effective than its chemical equivalent.

Table 4. Compared potential costs of registration for a microbial biocontrol agent (MBCA) and a chemical pesticide (IBMA; Nicot & Bardin, 2010, unpublished)

Area Study Type _{Chemical (€)}Cost for _{MBCA (€)}Cost for

Toxicity of the active

substance

Acute studies (6 tests) 140 000 140 000

Sub-acute (rat study) 140 000 120 000

Mutagenicity 40 000 may be waived

Toxicity on cultured cells 10 000 not required

Toxicity of the

formulation Acute studiesToxicity on cultured cells 140 00010 000 not required140 000

Environmental

fate Soil, water, air 200 000 70 000

Biology Mode of action etc 150 000 *50 000

Ecotoxicology of active substance

Birds, fish, bees, algae, daphnia,

earthworm 60 000 40 000

Beneficials 20 000 may be waived

Ecotoxicology of formulation

Birds, fish, bees, algae,daphnia,

earthworm 60 000 40 000

Beneficials 20 000

Residues 8 trials / crop 80 000 may be waived

Development of analytical methods 100 000 **variable

Formulation Physical properties, shelf life, etc. 200 000 220 000

Efficacy 8 field trials 40 000 40 000

TOTAL 1 410 000 860 000

* cost of strain identification

** e.g. development of strain-specific markers

Table 5. Compared estimated market potential for a microbial biocontrol agent (MBCA) and for a chemical pesticide (IBMA; Nicot & Bardin, 2010, unpublished)

Year Estimated sales value ( Mio €)

Chemical pesticide MBCA

1 0.1 0.05

2 1.2 0.15

3 6.0 0.90

4 15.0 1.50

5 35.0 3.50

Total early sales 57.3 6.10

Plateau sales 120.0 15.00 Registration costs 1.410 0.860 Ratio registration/ early sales 2.4 % 14.0 % Ratio registration/ Plateau sales 1.1 % 5.7 %



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1.3.1.4Businessprofitability



Nicot & Bardin (2010, unpublished) demonstrate in table 6 that there are large differences between chemical pesticides and microbial biocontrol agents when you compare the estimated production and other costs, relative to the sales value at plateau level. The estimated profit of the chemical industry is nearly 10 times bigger. Table 6. Compared margin structure estimates for the production and sales of a microbial

biocontrol agent (MBCA) and a chemical pesticide (IBMA; Nicot & Bardin, 2010, unpublished)

%* Chemical pesticide MBCA

Sales value at plateau level 100 100

Costs of production 13 56

Gross margin 87 44

Cost of sales 21 15

Cost of research 8 12

Cost of administration 4 3

Earnings before investments taxes and

amortisation (EBITA) 54 14

Profit after taxes, provisions and

amortisation 18 2

* costs and margins are expressed as percent of the sales value of the commercial product

1.3.1.5Conclusionandoutlookforindustry



These data show clearly that the profitability of a biocontrol business is much less attractive than that of chemical pesticides. This may explain why the large chemical companies decided in the 90’s to retreat from this business. Although these companies show presently some new signs of interest, they seem to remain basically reluctant to re-enter despite the new attractiveness of a fast growing biocontrol market.

The smaller companies which have invested in this business and try to overcome their financial problems have only two alternatives:

- Either develop, often at lost, into larger markets (grapevine, field crops etc), if they can. In order to sustain these efforts, they will need a strong support from venture capital companies;

- or enter into venture agreements with other manufacturers/suppliers, in order to build up a product portfolio which will make them successful in the future.

1.3.2

SocioǦeconomicaspect:marketanalysisandoutlook



The market for biocontrol agents appears to be extremely small. The sales in Europe in 2008 were estimated on € 200 million. This is not much, compared with the € 7 000 million turnover achieved with chemical pesticides. However, very important efforts have been undertaken for the development of biocontrol agents. The Organization for

Economic Co-operation and Development estimated that $ 5 000 million have been spent worldwide in public research for biocontrol during the last 40 years. This amounts to a yearly average of $ 500 million. Compared with the $ 600 million spent yearly in research by the agrochemical industry, there isn’t much difference, but the result is not so good.

1.3.2.1TheestimatedmarketofbiocontrolinEurope



Nicot and Bardin (2010, unpublished) show with the results of a survey in figure 4 an estimation of the total biological market in ha and in value in Europe. And in figure 5 its partition among different crops.

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These data confirm that in 2008, the main use of biologicals was in protected crops, followed by grapevine and fruit production. Nearly 40% of the estimated biocontrol market consisted in sales of beneficial insects, compared to 25% for microorganisms and 21% for semiochemicals.

Figure 4: Estimated sales of biocontrol products in Europe in 2008 (in Million €). The estimates were obtained by extrapolating use patterns in a representative sample of EU farmers. (Nicot & Bardin, 2010, unpublished)

Figure 5: Estimated distribution of biocontrol use among types of crops in 2008 in Europe. (Nicot & Bardin, 2010, unpublished)



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2

Objectives

Tests were performed on tomato plants and lettuce plants, because these two crops are used in a rotation cycle in Southern-Europe, the geographical area of the VALORT- project, the scope of my internship.

The products used in the tests proved their efficiency on other vegetables or against other pathogens. Some products have a direct effect and other products an indirect effect. And there are also products who believe to have a direct and an indirect effect. When they have an indirect effect, this means that they have an induction of resistance on the plant. As it were they prepare the plant for an attack of a pathogen. In the tests this means that you add the products 2 days before you do the inoculation of the plants.

Trianum-P, Trianum-G, Microdochium dimerum L13, Prev-Am, Semafort, Enzicur, Chitoplant, Serenade Max and Sonata believe to have a direct effect.

And Bion, Hexanoic acid, Stifenia, Serenade Max, Trianum-P, Trianum-G, Siliforce and Milsana believe to have an indirect effect.

All products are tested indirect and direct to see if they have an action against B.

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3

Material and methods

3.1

Productionofplantmaterial



Tests were performed on tomato plants and lettuce plants, because these two crops are used in a rotation cycle in Southern-Europe, the geographical area of the VALORT- project, the scope of my internship.

3.1.1

Tomatoproduction



Solanum lycopersicum cv Monalbo

Batches of tomato plants were grown in pest-free greenhouse facilities of INRA, Avignon, France. Seeds were sown in a germination mix (Klasmann-Deilmann GmbH, Geeste, Germany) composed of white and frozen through black sphagnum peat + buffered coco. Based on the manufacturer’s indication, its pH was 6.0 and it contained 0.7 kg/m3_{fresh weight of a NPK 14:16:18 fertilizer, 30% (w/w) dry matter and 85%} (w/w of dry matter) organic matter. Its conductivity was 25 mS/m and its retention capacity for water was 80%.

The one-week old seedlings were then transplanted to disposable pots (9x9x8 cm) containing 450 ml (120g) of a type SP533 potting mix (Klasmann-Deilmann GmbH, Geeste, Germany) composed of white and frozen through black sphagnum peat. Based on the manufacturer’s indication, its pH was 6.2 and it contained 1,5 kg/m3_fresh weight of a NPK 14:16:18 fertilizer, 30% (w/w) dry matter and 85% (w/w of dry matter) organic matter. Its conductivity was 40 mS/m and its retention capacity for water was 70%.

Following transplant, the plants were fertigated several times daily (with a frequency adapted to evaporative needs) with a solution obtained by mixing 98.2% water and 0.8% (v/v) of a fertilizer stock solution prepared by dissolving, per liter of water, 48 g of a NPK 16:8:28 fertilizer (EDDHA Optiplan, Duclos, Lunel-Viel, France). To adjust the pH, the final fertigation solution also contained 1% (v/v) of a stock acid solution prepared by diluting, per litreliter of water, 0.06 L of a nitric acid solution containing 60% nitric acid (JO.ProChim, Vedène, France).

Solanum lycopersicum cv Swanson

Batches of tomato plants were grown in pest-free greenhouse facilities of INRA, Avignon, France. Seeds were sown in 1 cm3_{Rockwool plugs (Grodan B.V., Roermond,} the Netherlands). One week after sowing, the plugs, each containing one plantlet, were transferred on to Rockwool blocks of 7.5 x 7.5 x 6 cm (Grodan B.V., Roermond, the Netherlands).

Following transplant, the plants were fertigated the same way as cv Monalbo.

3.1.2

Lettuceproduction



Lactuca sativa cv Trocadero, cv Montilia, cv Mariska, cv Vanguard, cv Vanguard 75

Batches of lettuce plants were grown in pest-free greenhouse facilities of INRA, Avignon, France. Seeds were sown in a germination mix (Klasmann-Deilmann GmbH, Geeste, Germany) composed of white and frozen through black sphagnum peat + buffered coco. Based on the manufacturer’s indication, its pH was 6.0 and it contained 0.7 kg/m3_{fresh weight of a NPK 14:16:18 fertilizer, 30% (w/w) dry matter and 85%}



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(w/w of dry matter) organic matter. Its conductivity was 25 mS/m and its retention capacity for water was 80%.

The one-week old seedlings were then transplanted to disposable pots (9x9x8 cm) containing 450 ml (120g) of the same potting mix used by Solanum lycopersicum cv

Monalbo.

Following transplant, the plants were fertigated the same way as the tomato plants.

3.2

Fungalstrains



3.2.1

Botrytiscinerea



Two single-spore isolates of B. cinerea were selected from a collection maintained in the laboratory at INRA, Avignon, France. The choise was made on the basis of difference in aggressiveness to tomato plants and lettuce plants. BC1, isolated on tomato plants in 1989 at Plougastel, France is more aggressive than the second isolate, BC21, isolated on strawberry plants in 1991 at Carpentras, France. For the duration of this work, the isolates were maintained in stock cultures stored at -20°C in a 0.06 M phosphate buffer containing 20% (V/V) of glycerol.

3.2.2

O.neolycopersici



O. neolycopersici, isolated on tomato plants in 2010 at Montfavet, Vaucluse, France was

selected because this fungus attacks tomato plants.

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 every two weeks to this new batch of four-week-old tomato plants. Inoculation was done by taking a heavily mildewed leaf from the old batch of plants and shaking it above the batch of fresh plants. The plants were maintained in a growth chamber in conditions conducive to disease development (21°C, relative humidity above 85%) with a 14h photoperiod.

3.3

Products



The products used in the tests contained biological compounds or mico-organisms. Some products are known for their direct action and other products for their indirect action. Apart from this fact, the products were used for all types of tests. This way we could see if products known for their direct action have an indirect effect against the fungi on tomato and lettuce and vice versa.

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T abl e 7. P ro d u cts u se d in th e te st s Tr ia num -G T ri cho d er m a ha rz an ium T 2 2 K op p er t B .V . Le ttu ce SD Bc T ri anum -P T ric h od er ma h a rz an iu m T 22 Ko pp er t B .V. T om at o PW , FS Bc , O n Na me Mi c ro-org a ni sm Co mp a n y LPeltat un cte App lic at io n * T ar g e t* * Co m p ou n d Fr uc tos e Si gma-A ldr ic h C h emi e G m bH T omat o PW , FS Bc , O n He xa n oi c a ci d Th er m o F is h er S ci en tific I n c. To ma to Le ttu ce PW , F S , S D B c, O n M ic rod oc h iu m d im er u m L13 To m at o PWB c Bi o n /5 0 W G Ac ib en zo la r- S-m et h yl S yn g en ta AG T om at o Le ttu ce PW , FS Bc , O n C h it op la n t C hit os an Ch iP ro G m b H T om at o Le ttu ce PW , FS Bc , O n E nzi cu r La ct op er ox id as e (m ilk enz ym e) , ka lium jo di d e, ka lium th ioc yn at e Ko p p er t B .V . To m at o Le ttu ce PW , FS Bc , O n M ilsana Fa llo p ia s ak h alin e n sis Bi of a G m bH T omat o Le ttu ce PW , FS Bc , O n Pr ev -A m B ora x, c ol d -p re ss ed or an g e oi l a n d va ri ou s bi od egr ada bl e sur fa ct an ts Sa m abi ol To m at o Le ttu ce PW , FS Bc , O n Semaf or t Al g ae e xt ra ct Trib o Te ch n ol og ie s To m at o PW , FS Bc , O n Ser ena de Ma x B ac ill us s u b ti lli s Q S T 713 Agr aques t I n c. T omat o PW , FS Bc S ili fo rc e B io a va ila b le si lic ic ac id A g ro -S o lu tio n s B .V . T oma to Le ttu ce PW , FS Bc , O n So l-A ct if Ch it os an + ch it in Fr an ce Ch it in e Le tt u ce SD Bc So na ta B ac ill u s p u m ilu s Q S T 280 8 Agr aques t I n c. T omat o Le ttu ce PW , FS Bc S ti fen ia Ex tr ac t f ro m f enu gr ee k Sa m abi ol To m at o PW , FS Bc , O n

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*: PW = p run ing wound, FS = fo!iar spray ,50 = soif dren ch **:Be= B o tryti s ci ne rea , On= O idi um neo lycope rsi ci

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3.4

Invitrotest



3.4.1

Myceliumgrowthtest



3.4.1.1Inoculumproduction



Strains BC1 and BC21 of B. cinerea were used throughout the study. Inoculum of both strains was produced in Petri dishes on Potato Dextrose Agar medium (PDA: 39 g l-1_; Difco laboratory, Detroit, Michigan). A 2-μl drop of the stock cultures was placed in the middle of a Petri dish and 3 Petri dishes were prepared for each strain. The inoculum was incubated under cool white fluorescent light in a growth chamber (21°C, 14h photoperiod).

3.4.1.2EffectofbiologicalproductsonmyceliumgrowthofB.cinerea



The efficacy of 5 biological products was tested on mycelium growth for 2 isolates of B.

cinerea. The biological products were added at concentrations recommended by the

manufacturers or by scientific literature, to PDA medium (table 8). The mycelium growth was assessed by placing in the center of a Petri dish a 4-mm mycelium plug taken from the periphery of a 4-day-old colony. The inoculated dishes were incubated under cool white fluorescent light in a growth chamber (21°C; 14h photoperiod) and colony diameter was measured 48, 60 and 72 hours after inoculation. 3 Petri dishes were inoculated for each isolate and for each product.

Table 8: Mycelium growth test

Product Dose Source

Prev-Am 0,8% (V/V) Samabiol

Stifenia 1% (m/V) Samabiol

Milsana 0,3% (V/V) Biofa GmbH

Siliforce 400ml ha-1 _{* Agro-Solutions B.V} Hexanoic acid 20 mM Leyva et al., 2008 * = application of 1000 l ha-1

3.4.1.3Statisticalanalysis



Statistical analyses were carried out, using the ANOVA/MANOVA module of Statistica (Statsoft Inc., Tulsa, OK, USA).

ANOVA tests were carried out on the daily mycelium growth, comparing all treatments together with the untreated control, for each isolate. In the presence of a significant treatment effect (p<0,05), Newman and Keuls tests were carried out for a multiple comparison of the means for all treatments

3.4.2

Sporegerminationtest



3.4.2.1Inoculumproduction



Strains BC1 and BC21 of B. cinerea were used throughout the study. Inoculum of both strains was produced in Petri dishes on PDA medium. 2 μl of the stock cultures was placed in the middle of a Petri dish and 1 Petri dish was prepared for each strain. The



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Product Dose Source

Prev-Am 0,8% (V/V) Samabiol

Stifenia 1% (m/V) Samabiol

Milsana 0,3% (V/V) Biofa GmbH

Siliforce 400ml ha-1 _{* Agro-Solutions B.V} Hexanoic acid 20 mM Leyva et al., 2008

inoculum was incubated under cool white fluorescent light in a growth chamber (21°C, 14h photoperiod). Spores were collected in 5 ml of water on 14-day-old cultures. The spore suspensions were vortexed with glass beads (2.5 mm diameter) to separate the spores and then filtered through sterile 30 μm mesh filters to remove mycelial fragments. Spore concentration was determined with the help of a Malassez haemocytometer and adjusted to 105_{spores ml}-1 _{with sterile water.}

3.4.2.2Effectofbiologicalproductsonsporegermination ofB.cinerea



The efficacy of 5 biological products was tested on spore germination for 2 isolates of B.

cinerea. The biological products were added at concentrations recommended by the

manufacturers or by scientific literature, to PDA medium (table 9) The spore germination was assessed by placing 5 times 5 μl of the spore suspension on a Petri dish. The inoculated dishes were incubated under cool white fluorescent light in a growth chamber (21°C; 14h photoperiod) and spore germination was determined 24 hours after inoculation. 1 Petri dish was inoculated for each isolate and for each product.

Table 9: Spore germination test

* = application of 1000 l ha-1

3.4.2.3Statisticalanalysis



Statistical analyses were carried out, using the ANOVA/MANOVA module of Statistica (Statsoft Inc., Tulsa, OK, USA).

ANOVA tests were carried out on the percentage of germination for both isolates, comparing all treatments together with the untreated control, for each isolate. In the presence of a significant treatment effect (p<0,05), Newman and Keuls tests were carried out for a multiple comparison of the means for all treatments

3.5

Leafdisktests



3.5.1

Inoculumproduction



3.5.1.1B.cinerea



See 2.4.1.1

3.5.1.2O.neolycopersici



Inoculum of O. neolycopersici was already available on the batch of tomato plants, maintained in the growth chamber (21°C, RH above 85%) with a 14h photoperiod.

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TestofbiocontrolproductsagainstfungalpathogensoftomatoandlettucedeLenaerts

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3.5.2

Applicationoftheproducts



Tomato plants cv Monalbo were used 4 or 5 weeks after sowing, at a stage were they carried 5-7 leaves. Lettuce plants cv Trocadero, cv Montilia, cv Mariska, cv Vanguard,

cv Vanguard 75 were used 4 or 5 weeks after sowing, at a stage were they carried 8-10

leaves.

The efficacy of biological products against B. cinerea and O. neolycopersici was tested on leaf disks of tomato plants and lettuce plants. When the biological products were pulverized, they were either applied on the entire plant 2 days before inoculation or just before inoculation on other plants with the help of a compressed-air sprayer untill run- off (figure 6).

Depending on the test and the availability of plants, the products were applied on 1 to 4 plants by each pulverization, with an average applied quantity of 25 ml for tomato plants and 8.4 ml for lettuce plants. When the biological products were mixed with the potting soil (type SP533 potting mix, Klasmann-Deilmann GmbH, Geeste, Germany), they were applied 14 days before inoculation on 14 plants per product. At inoculation day 7 of these 14 plants were pulverized with the same products or with products with the same compounds as the products mixed with the potting soil. The biological products were added or pulverized at concentrations recommended by the

manufacturers or by scientific literature. More details are shown in table 10 and 11.

Figure 6: Pulverizing a product on a lettuce plant Table 10: Biological products applied on tomato plants

Product Source Dose Target**

Trianum-P 30 g / 100 l On (2), Bc Milsana 0.3 % (V/V) On (3), Bc Enzicur Part 1: 1 ml l -1 Part 2: 1 ml l-1 Addit: 2.5 ml l-1 On (3), Bc Siliforce 400 ml ha-1_{* On (2), Bc} Bion/50WG 0.05 kg ha-1_{* On (2), Bc} Semafort 1 % (V/V) On (2), Bc Fructose 100 ppm On (2), Bc

Hexanoic acid 16 mM On, Bc

20 mM On

Chitoplant 1 % (m/V) On (3), Bc

Prev-Am 0.8 % (V/V) On (4), Bc