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Endophytes in Maize (Zea Mays) in New Zealand

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Endophytes in Maize (Zea Mays) in New Zealand Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Endophytes in Maize (Zea mays) in New Zealand A thesis submitted in partial fulfilment of the requirements for the Degree of Master of Science at Lincoln University by Jennifer Joy Brookes Lincoln University 2017 Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Master of Science. Abstract Endophytes in Maize (Zea mays) in New Zealand by Jennifer Joy Brookes The aim of this study was to isolate fungal endophytes from maize in New Zealand (NZ) and to select endophytes with potential to reduce insect pests and/or plant diseases. Culture methods were used to isolate 322 isolates of fungi belonging to four phyla from maize (Zea mays L.) plants. Plants were sampled over two growing seasons (2014 and 2015) in two regions of NZ. Morphological and molecular (ITS rDNA sequencing) techniques were used to identify the fungi. The most common genera recovered were Fusarium, followed by Alternaria, Trichoderma, Epicoccum, Mucor, Penicillium and Cladosoprium spp. Of the Acomycota isolates, 33 genera from 6 classes were recovered. Basidiomyctes were represented by two classes and Zycomycota by one class and a superphylum, Heterokonta, was represented with one class. To determine fungi with potential as biocontrol agents, several assay approaches were taken. Initially, most fungi were used to challenge a plant pathogen on media plates in dual culture experiments. This allowed selection of 21 promising isolates which were inoculated into maize plants by seed coating, then used in plant disease assay and a caterpillar feeding challenge assay. A final eight isolates were selected as the most promising for conferring beneficial traits on plants: Sordaria fimicola, Mucor racemosus, Mucor fragilis, Trichoderma atroviride, Penicillium brasilianum, Fusarium equiseti, Fusarium acuminatum and Fusarium proliferatum. These isolates have shown potential as BCAs against disease and/or insects in laboratory assays. Keywords: Endophytes, BCA, maize, fungi, screening, bioassay, dual culture, in planta, Setosphaeria turcica, Helicoverpa armigera, Beauveria, Fusarium, Trichoderma, Alternaria, Mucor, Epicoccum ii Acknowledgements I want to extend a very big thank you to my supervisors, Prof. T Glare and Dr. M. Rostas, and to Dave Saville, who provided all the statisticial assistance. A big thank you to my children, Charles, Scott and Robert, and my parents, Ron and Philippa Rivers, for their patience through my course work and thesis. Many thanks to my friends and co-workers for their support and encouragement through my Masters at the Bio-Protection Research Centre (BPRC), Lincoln University. A big thank you to my office mate, Josefina Narciso for her support and shoulder and in the latter part, Jin-Hua Li for her assistance in the lab. To everyone else at the BPRC a big thank you so much for your help during the last four years I have being doing my Masters part time. Its done!!! This has been an incredible steep learning curve, one which I have thoroughly enjoyed. I hope to continue and be able to add to this knowledge. iii Table of Contents Abstract .................................................................................................................................. ii Acknowledgements ................................................................................................................ iii Table of Contents ................................................................................................................... iv List of Tables .......................................................................................................................... vi List of Figures .........................................................................................................................vii Chapter 1 Introduction ............................................................................................................ 1 1.1 Maize in agriculture ................................................................................................................. 1 1.2 Important pathogens affecting maize in NZ ............................................................................ 2 1.3 Insect pests of NZ maize .......................................................................................................... 5 1.4 Endophytes .............................................................................................................................. 8 1.4.1 Maize endophytes .................................................................................................... 10 1.4.2 Endophytes and disease ........................................................................................... 12 1.4.3 Endophytes and insects ............................................................................................ 13 1.5 Research objectives ............................................................................................................... 13 Chapter 2 General methods ................................................................................................... 15 2.1 Introduction ........................................................................................................................... 15 2.2 Sampling field collected maize plants for endophytes .......................................................... 15 2.2.1 Recovery of putative endophytes from maize ......................................................... 15 2.2.2 Surface sterilisation of maize tissues ........................................................................ 17 2.2.3 Media ........................................................................................................................ 19 2.2.4 Identification of recovered fungi from PDA ............................................................. 20 2.2.5 DNA extraction ......................................................................................................... 21 2.2.6 Polymerase chain reaction ....................................................................................... 21 2.2.7 DNA analysis ............................................................................................................. 22 2.3 In vitro bioassay of recovered putative endophytes against a plant pathogenic fungus ..... 22 2.4 In planta maize challenge methods ....................................................................................... 24 2.4.1 Seed coating ............................................................................................................. 24 2.4.2 Plant disease assay ................................................................................................... 24 2.4.3 Application of pathogen S. turcica. .......................................................................... 26 2.5 Development of an insect bioassay ....................................................................................... 28 2.6 Presence of putative endophytes in seed ............................................................................. 29 2.6.1 Surface sterilising of seeds ....................................................................................... 29 Chapter 3 Results .................................................................................................................. 30 3.1 Isolation of fungi from surface sterilised maize .................................................................... 30 3.1.1 Microscopy results .................................................................................................... 31 3.1 1 Difficult isolate identifications ...................................................................................... 37 3.2 Dual culture results ............................................................................................................... 39 3.3 In planta ................................................................................................................................. 42 3.4 Insect bioassays ..................................................................................................................... 48 3.5 Endophyte recovery from plants ........................................................................................... 54 iv Chapter 4 Discussion ............................................................................................................. 56 4.1 Main findings ......................................................................................................................... 56 4.2 Maize screening ..................................................................................................................... 56 4.3 Culture methods .................................................................................................................... 58 4.4 Detection of endophyte in plant tissue ................................................................................. 59 4.5 Colonisation
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