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Streptomycetes As Biological Control Agents and Plant Growth-Promoting Bacteria

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Streptomycetes As Biological Control Agents and Plant Growth-Promoting Bacteria UNIVERSITÀ DEGLI STUDI DI MILANO Department of food, environmental and nutritional sciences Corso di dottorato in Chimica, biochimica ed ecologia degli antiparassitari Philosophy Doctorate course in Chemistry, biochemistry and ecology of pesticides MARIA BONALDI PhD thesis Streptomycetes as Biological Control Agents and Plant Growth-Promoting Bacteria Settore scientifico disciplinare AGR/12 SUPERVISOR: Prof. Paolo Cortesi CO-SUPERVISOR: Prof. Marco Saracchi COORDINATOR: Prof. Daniele Daffonchio A.A. 2013/2014 ii Non basta guardare, occorre guardare con occhi che vogliono vedere, che credono in quello che vedono Galileo Galilei iii Abstract Developing no-chemical strategies for the control of soil borne pathogens is one of the major issues for the cultivation of leafy vegetables. The application of Biological Control Agents (BCAs) represents a valuable approach and nowadays some biocontrol products are available on the market for greenhouse and field applications. However, these products often show lack of consistency and variable results mainly due to the poor knowledge about their biology and modes of applications and how the agroecosystem components modulate their efficacy. Streptomycetes are soil inhabitants and have an important ecological role in the turn-over of organic matter; they can also establish beneficial relationships with plant roots enhancing host growth and protection against pathogens through the production of bioactive compounds, lytic enzymes, phytohormones and siderophores. This PhD project aimed to study streptomycetes as BCA and Plant Growth Promoting Bacteria for their use to manage soil borne fungal epidemics in horticulture. A collection of 200 endophytic streptomycete strains isolated from roots was used in this work. To be able to compare the activity of every strain against the pathogens, the dual culture assay was optimized for some representative fungal pathogens based on mycelium radial growth rate in vitro. Subsequently, the optimized method was applied to screen the collection. Some strains showed strong inhibitory activity, but it was specific for one target pathogen and in few cases comprised more than one pathogen. Based on the promising results obtained from the in vitro assays for Sclerotinia sclerotiorum, further studies were focused on the activity of ten strains used for biological control of lettuce drop in different conditions. In particular, it was studied the effect of the application timing of the antagonist and the pathogen, and the amount of the streptomycete used to improve lettuce drop protection. The survival analysis applied to the data of the growth chamber experiments showed that when lettuce was sown one week after the growth substrate inoculation with the pathogen and antagonists, disease control improved, and Streptomyces spp. FT05W, SW06W and SW29W reduced the risk of disease incidence by 42%. On the contrary, no beneficial effect was observed when lettuce was sown the same day of the growth substrate inoculation. Streptomycetes spore concentration significantly influenced lettuce drop protection, but this effect was strain-dependent. Based on these results we planned appropriate field experiment to confirm the results obtained, however, in the field we did not observed significant differences in lettuce protection. Therefore we speculated that moving from controlled to a more complex agroecosystem environment the streptomycete antagonistic activity could fade away probably due to unfavorable interaction in a more complex microflora. iv Indole-3-acetic acid and the siderophore production were observed for Streptomyces spp. CVM02R and SW29W in in vitro assays, but in field experiments no significant PGP effect on lettuce was obtained at harvest assessing the head weights of plants. The colonization of lettuce rhizosphere and root tissues was investigated using the EGFP labelled strain Streptomyces sp. ZEA17I. This strain showed both rhizospheric and endophytic competences, characters necessary for its successful use for biological control. In addition we showed that applying the strains as spore suspension in the growth substrate resulted in significantly higher roots and rhizosphere colonization than when delivered as seed coating. In conclusion, the results obtained in this study showed that bacteria of the genus Streptomycetes appear valuable candidates for the biological control of soil borne fungal pathogens. However, the complex interactions among the host plant, the antagonist and the pathogen occurring in the agroecosystem are mostly unknown and could generate contradictory results for different environments. Therefore, we think that further studies on simplified models are necessary in order to understand the mechanism on which biological control is based, in order to improve streptomycete activity as BCA for the management of fungal soil borne epidemics. Keywords: soil borne pathogens, Sclerotinia sclerotiorum, lettuce, streptomycetes, biological control, colonization. v Table of contents Abstract ............................................................................................................................................. iv Table of contents ............................................................................................................................... vi List of figures .................................................................................................................................... xi List of tables ..................................................................................................................................... xii Introduction ........................................................................................................................................ 1 1 Horticulture in Italy ................................................................................................................... 1 1.1 Overview ............................................................................................................................... 1 1.2 Salad crops ............................................................................................................................. 1 1.3 Ready-to-eat salads ................................................................................................................ 3 1.4 Features of horticultural farming ........................................................................................... 4 2 Soil borne pathogens .................................................................................................................. 7 2.1 Soil borne pathogens: biology and epidemiology ................................................................. 7 2.1.1 Inoculum ......................................................................................................................... 7 2.1.2 Invasion and colonization ............................................................................................... 7 2.1.3 Host range ...................................................................................................................... 8 2.1.4 Symptoms ....................................................................................................................... 8 2.2 Soil borne diseases of lettuce and related salad crops ........................................................... 9 2.3 Basal drop ............................................................................................................................ 10 2.3.1 Causal agents ................................................................................................................ 10 2.3.2 Disease cycle and pathogenesis .................................................................................... 10 2.3.3 Symptoms ..................................................................................................................... 11 3 Soil borne diseases management ............................................................................................. 14 3.1 Integrated Pest/Disease Management .................................................................................. 14 3.2 Chemical strategies: fumigants ............................................................................................ 14 3.3 Non-chemical strategies ...................................................................................................... 15 3.3.1 Crop rotation ................................................................................................................ 15 vi 3.3.2 Soil solarization ............................................................................................................ 15 3.3.3 Biofumigation ............................................................................................................... 16 3.3.4 Organic amendments and compost .............................................................................. 17 4 Biological control of soil borne pathogens ............................................................................. 18 4.1 Definition ............................................................................................................................. 18 4.2 Suppressive soils.................................................................................................................. 18 4.3 Modes of action of BCAs .................................................................................................... 19 4.4 Limits of biological control ................................................................................................
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