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Controlling the Meloidogyne Disease Complex in Ugandan Tomatoes

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Controlling the Meloidogyne Disease Complex in Ugandan Tomatoes Adrian Wolfgang BSc Controlling the Meloidogyne disease complex in Ugandan tomatoes MASTER’S THESIS To achieve the university degree of Master of Science Master degree program: Ecology and Evolutionary Biology Submitted to Karl-Franzens University Graz Supervisor Priv.-Doz. Mag. Dr.rer.nat. Günther Raspotnig Institute of Biology Graz, April 2018 In cooperation with: ❆✩✩✪✫❆✬✪✭ 2 AFFIDAVIT Ich erkläre ehrenwörtlich, dass ich die vorliegende Arbeit selbständig und ohne fremde Hilfe verfasst, andere als die angegebenen Quellen nicht benutzt und die den Quellen wörtlich oder inhaltlich entnommenen Stellen als solche kenntlich gemacht habe. Die Arbeit wurde bisher in gleicher oder ähnlicher Form keiner anderen inländischen oder ausländischen Prüfungsbehörde vorgelegt und auch noch nicht veröffentlicht. Die vorliegende Fassung entspricht der eingereichten elektronischen Version. I declare that I have authored this thesis independently, that I have not used other than the declared sources/resources, and that I have explicitly indicated all material which has been quoted either literally or by content from the sources used. The text document uploaded to UNIGRAZonline is identical to the present master’s thesis. ________________________ _____________________________________ Date Signature 3 Table of contents Table of contents ........................................................................................................................ 4 0.1 Abstract ........................................................................................................................... 6 0.2 Zusammenfassung ........................................................................................................... 7 1.0 Introduction ..................................................................................................................... 8 1.1 Present situation of Agriculture in Uganda ................................................................. 8 1.2 State and challenges of tomato farming in Uganda .................................................... 8 1.3 Root-knot nematodes - Meloidogyne spp. .................................................................. 9 1.4 The impact of root-knot nematodes on host plants ................................................. 11 1.5 Plant health and microbiome .................................................................................... 12 1.6 Volatile organic compounds (VOCs) of bacteria as RKN control ............................... 12 1.7 Aims of this study ...................................................................................................... 13 2.0 Materials and methods ................................................................................................ 14 2.1 Sampling .................................................................................................................... 15 2.2 Bacterial isolates and DNA extraction ....................................................................... 16 2.3 DNA preparation for Amplicon analysis .................................................................... 16 2.4 Amplicon analysis ...................................................................................................... 17 2.5 Bacterial antagonistic activity against fungal pathogens .......................................... 18 2.6 Screening for nVOCs-producing strains ..................................................................... 20 2.7 GC-MS of nVOC-producing bacteria .......................................................................... 21 2.8 Testing nematicdal effect of single VOCs .................................................................. 22 2.9 Breeding of root-knot nematodes ............................................................................. 22 2.10 J2 larvae extraction .................................................................................................... 23 2.11 Molecular and morphological determination of RKNs .............................................. 24 3.0 Results ........................................................................................................................... 25 3.1 Physical soil composition ........................................................................................... 25 3.2 Cultivable bacteria from tomato root endosphere ................................................... 25 4 3.3 Abundant bacterial community in soil and rhizosphere ........................................... 26 3.4 Bacterial diversity of the tropical RKN disease complex ........................................... 27 3.5 Differences between infected and non-infected tomato plant roots ....................... 30 3.6 Antifungal isolates ..................................................................................................... 31 3.7 Nematicidal volatile producing strains ...................................................................... 33 3.8 Chemistry of bacterial volatiles ................................................................................. 34 3.9 Abundance of nematicidal bacterial genera in tomato plants .................................. 36 3.10 Testing single compounds for nematicidal activity ................................................... 37 3.11 Abundant nematode species ..................................................................................... 38 4.0 Discussion ...................................................................................................................... 40 4.1 The microbiome of tomato roots in Uganda ............................................................. 40 4.2 Genera with RKN-controlling properties within Ugandan tomatoes ........................ 40 4.3 Bacterial community shift in tomato roots due to RKN infection ............................. 41 4.4 Potential of nVOCs-producing bacterial strains ........................................................ 43 4.5 Nematicidal volatiles ................................................................................................. 45 4.6 Managing the root-knot nematode disease complex with BCAs .............................. 46 4.7 Determination problems of abundant RKN species .................................................. 47 4.8 Future prospects ........................................................................................................ 47 5.0 References ..................................................................................................................... 49 6.0 Acknowledgements: ...................................................................................................... 58 7.0 Appendix ........................................................................................................................ 59 8.0 Supplementary data ...................................................................................................... 62 5 0.1 Abstract Root-knot nematodes (RKNs, Meloidogyne spp.) are one of the major polyphagous pests in tropical climates. Their effect on crops includes a change in the physiological state of the root system that increases the severity of fungal infections. For effective crop protection, management methods that control both fungi and RKNs are crucial. This study focuses on the influence of RKN infections on the root endosphere microbiome and bacteria that may be suitable for biocontrol by the production of nematicidal volatile organic compounds (nVOCs). RKNs were extracted from roots of tomato plants originating from two sites in Uganda: an open field with virgin soil in Luwero and a RKN breeding bed at the IITA research center in Namulonge. RKNs were identified using morphological and molecular techniques as M. javanica and M. incognita. Bacterial strains were isolated from the rhizosphere, healthy and diseased endosphere of 20 different tomato plants. In addition, a 16S rDNA amplicon analysis of the microbiome in soil, rhizosphere, healthy and diseased roots was carried out. A set of 260 randomly selected bacterial strains were tested whether they produce nVOCs. Furthermore, all bacterial strains were tested in dual cultures for antagonistic activity towards Botrytis cinerea, Fusarium verticilloides, F. oxysporum, Sclerotium rolfsii and Verticillium dahliae. Six nVOC-producing strains from the genera Pseudomonas, Comamonas and Variovorax could be detected. Their relative abundance was highest in rhizosphere (Pseudomonas spp.: 22.01%; Comamonas spp.: 0.34%) or diseased root endosphere (Variovorax spp.: 0.97%) VOCs of nematicidal strains were analyzed using GC-MS and present alkenes and different pyrazines were tested as single compounds for their nematicidal activity. Pyrazines with nematicidal properties could be found. Five fungal antagonists were identified as Bacillus amyloliquefaciens, B. methylotrophicus and B. velezensis. Bacillus spp. reach their highest relative abundance (4.1%) in non-infected root parts. Antagonists of V. dahliae were only found in rhizosphere isolates; they were less efficient against the other fungal pathogens and vice versa. Microbiome composition of diseased roots does not differ qualitatively but quantitatively from healthy roots. Pasteuriaceae and Rhizobiaceae are more abundant while Enterobacteriaceae and Burkholderiaceae are less abundant in diseased roots. Since bacterial antagonists of V. dahliae, the other tested fungal pathogens and RKN differ, biocontrol of RKNs should focus on bacterial control consortia. 6 0.2 Zusammenfassung
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