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Characterization of Wheat Nematodes from Cultivars in South Africa

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Characterization of Wheat Nematodes from Cultivars in South Africa Characterization of wheat nematodes from cultivars in South Africa SQN Lamula orcid.org/0000-0001-7140-8327 Thesis accepted in fulfilment of the requirement for the degree Doctor of Philosophy in Environmental Sciences at the North- West University Supervisor: Prof T Tsilo Co-supervisor: Prof MMO Thekisoe Assistant promoter: Dr A Mbatyoti Graduation: May 2020 28728556 DEDICATION To my grandmother Dlalisile Dlada, mother Nokuthula Bongi Dladla, aunt, Dumisile Dladla, and the rest of my family. i ACKNWOLEDGEMENTS To the almighty GOD for the strength, ability, knowledge, opportunity and perseverance you have given me from the beginning of this project till this day. My sincere gratitude goes to my promotors and mentors: Firstly, Prof. Oriel Thekisoe for the unrelenting support that he has given me for a long time. Under your guidance, leadership and patience, has lead me to acquire more knowledge about academics and also life in general. Secondly, Prof. Toi Tsilo for the undying patience, believing in me and providing the opportunity and encouraging me to be a better hard working person. Without either of you, the completion of this project would have not been possible. Dr. Antoinette Swart and Dr. Mariette Marais for their assistance and guidance in identifying nematode species detected in this project. Mr. Timmy Baloyi with his technical and logistics support for the project. Henzel Saul and his team (HA Hatting, C Miles and M da Graca) for their immense contribution in accessing and obtaining samples from commercial farmers in Western Cape. Mofalali Makuoane, Richard Taylor and Teboho Oliphant for assisting in sample collection from other provinces. My family; mother for her support, aunt (Dumisile Dladla) and grandmother (Dlalisile Dladal) for always being there and supported me financially and emotionally. Furthermore, I wish to appreciate my colleagues, Lehlohonolo “Sanchez” Mofokeng, Malitaba Mlangeni, and Clara-Lee van Wyk for their assistance during the project and for all the fun we had in the last two years. I wish to appreciation to Mr Denis Komape for the assistance with development of the maps. May I also express deep gratitude to all the people whom I cannot mention their names but they contributed to this work in one way or the other. I am also very grateful to the financial support I have received from the National Research Foundation of South Africa, Winter Cereal Trust and North-West University Postgraduate Bursaries. ii ABSTRACT Plant-parasitic nematodes (PPNs) naturally live in soil and attack small roots, but some species inhabit and feed in bulbs, buds, stems, leaves, or flowers. This leads to plant weakness and they often appear to suffer from drought, excessive soil moisture, sunburn or frost, as well as mineral deficiency or imbalance. According to the South African Plant-Parasitic Nematode Survey (SPPNS) and National Collection Nematodes (NCN) databases, 453 plant-feeding nematodes have been recorded in South Africa and species identified from wheat have not been included on the database as a result of not being georeferenced and the need to update information on NCN database. The aim of the current study is to characterize and document wheat nematodes that occur in South African wheat producing areas with an emphasis on morphological and molecular identification. A total of 776 composite rhizosphere soil and root samples was collected from 56 localities over two seasons in 7 provinces; the Free State (FS), KwaZulu-Natal (KZN), Northern Cape (NC), Mpumalanga (MP), Limpopo (L), North West (NW) and Western Cape (WC) provinces. The majority of samples collected were in WC (452), FS (120) and NC (160), as these are the major producers of wheat in South Africa. Nematodes were extracted from soil and root samples by modified decanting and sieving baermann-funnel technique, followed by the adapted sugar centrifugal-floatation. Nematodes were extracted from kernels by means of soaking the samples in tap water for 24 hours and decanting the extract through a 20 μm sieve. Nematode species were identified basis on morphological features, while prominence values (PV), frequency of occurrences and abundances were calculated for each genus. Individuals from the following genera were identified from the following provinces: Free State: Pratylenchus, Spiral (Rotylenchus, Scutellonema and Helicotylenchus), Criconema and Dolichodorus; KwaZulu-Natal: Melodogyne, Pratylenchus, Criconema, Helycotylenchus (Spiral) and Longidorus; Northern Cape: Pratylenchus, Criconema and Spiral; Western Cape: Pratylenchus, Rotylenchus, Scutellonema, Helicotylenchus, Coslenchus, Tylenchus and Xiphinema. The predominant genera identified across the localities according to PV- value were Pratylenchus (PV=575), Spiral (PV=309), Tylenchus (PV=348), Criconema (PV=377), Aphelenchus (PV=131) and Xiphinema (PV=32). Cedara in KwaZulu-Natal had a high number of Meloidogyne (420 second-stage juveniles /200 g soil) compared iii to the other sampled localities. Western Cape (Malmesbury) showed a high number of Pratylenchus sp. (8750/200 g soil). In root samples, the PV of Meloidogyne in Cedara was 183 followed by De Vlei (PV=943), Tygerhoek (PV=490), Kopporfontein (PV=134) and Wellington (PV=57) for Pratylenchus sp. No plant-parasitic nematodes were found in kernel samples. However, a bacterivore species of Panagrolaimus was identified from wheat kernels from Clarens in the Free State, is first record both in South Africa and worldwide. DNA was extracted from 320 nematodes using chilex method. PCR was used for amplification of 18S rRNA, ITS1, D2-D3 and CO1 genes and positive PCR products were sequenced. This study analysed 20 sequences of the D2–D3 expansion segments of the 28S rRNA gene, 80 sequences of the 18S rRNA and 91 sequences of ITS1 gene. Gene sequences of nematodes obtained from this study matched with their related species when subject to BLASTn on NCBI data. Phylogenetic trees constructed with 18S rRNA and ITS1 rDNA genes have shown that nematodes detected in this study including Helicotylenchus dihystera, Amplimerlinius paraglobigerus, Bitylenchus maximus, Merlinius joctus, Paralongidorus bikanerensis, Hoplolaimus galeatus and Rotylenchus unisexus each formed clades with strains of the same species obtained from the GenBank using the 18S rRNA gene. The H, dihystera, H. pseudorobustus, R. brevicaudatus, B. ventrosignatus and Mesocriconema sphaerocephalum also clustered with the strains of the same species obtained in the GenBank when using the ITS1 gene clusters with their related species from other countries. Keywords: Nematodes, Wheat, morphology, molecular, PCR, South Africa iv RESEARCH OUTPUTS Lamula S.Q.N. Thekisoe O.M.M., Fourie H., Tsilo T.J. 2017. Nematodes Associated with nematodes associated with wheat crops in South Africa: A revision and the way forward. The 21st symposium of the nematological society of Southern Africa: Fairmont Zimbali resort, Balito Kwazulu Natal 14th – 18th May 2017. (Poster) Lamula S.Q.N. Thekisoe O.M.M., Fourie H., Tsilo T.J. 2018. Plant-parasitic nematodes associated with wheat crops in South Africa, UESM prestigious PhD conference, 30 August, 2018, Potchefstroom, South Africa. (Oral) Lamula S.Q.N., Thekisoe O.M.M., Tsilo T.J. 2019. Characterization of nematodes associated with rhizosphere soil and roots of South African wheat cultivars. 3rd new voices symposium. The grain building, Pretoria, 11 September 2019. (Oral) v TABLE OF CONTENTS DEDICATION ............................................................................................................................. I ACKNWOLEDGEMENTS ......................................................................................................... II ABSTRACT ............................................................................................................................. III RESEARCH OUTPUTS ............................................................................................................ V TABLE OF CONTENTS ........................................................................................................... VI LIST OF PLATES ................................................................................................................. XVII CHAPTER ONE ...................................................................................................................... 20 INTRODUCTION ..................................................................................................................... 20 1.1 Background of the study................................................................................ 20 1.2 Problem statement ......................................................................................... 22 1.3 Research aim .................................................................................................. 23 1.4 Objectives ....................................................................................................... 23 1.5 Hypothesis ...................................................................................................... 23 1.6 Thesis outline ................................................................................................. 23 CHAPTER TWO ...................................................................................................................... 25 LITERATURE REVIEW ........................................................................................................... 25 2.1 Wheat around the globe ................................................................................
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