A comparative study of the development and reproduction of Meloidogyne enterolobii and other thermophilic South African Meloidogyne species RL Collett orcid.org 0000-0003-0287-6404 Dissertation accepted in fulfilment of the requirements for the degree Master of Science in Environmental Sciences with Integrated Pest Management at the North-West University Supervisor: Prof H Fourie Co-supervisor: Dr M Marais Co-supervisor: Dr MS Daneel Graduation October 2020 22847650 ACKNOWLEDGEMENTS First, I would like to thank and give praise to our Heavenly Father for all His guidance, strength, and granting me this life. I would like to thank my family, each one of them. My mother and father, without whom I could not be here and no words can explain how grateful I am for everything you two do. For all the support (emotionally, financially, and spiritually) and encouraging me to pursue a bright and positive future. Thank you to my departed grandmother, Annetjie van Niekerk, who instilled in me the deep desire for knowledge, education and to achieve my full potential. I would also like to thank my grandfather, Nico van Niekerk, who from an early age enkindled my fascination with nature, biology, and agriculture. To my supervisor and co-supervisors Prof. Driekie, Dr Mieke, and Dr Mariette (I can not set in words all that I want to say), thank you for the guidance, support, hope, positivity and wisdom that you have provided during this project. I will always be grateful for the life lessons and knowledge you have given me throughout these years. You are all truly an inspiration. Thank you to each staff member and student of Eco Rehab and the NWU Potchefstroom campus for your support, kindness, aid, and friendship for all these past years. You all remain an inspiration to me and will remain edged in my memories. I would aspecially give thanks to Mrs Helena Strydom for her support and hard work at Eco Rehab. Also Meagan Martin for her assitance in the lab and her enduring friendship. I would like to thank those close nematologists who have also adviced me and motivated me during the period of this project namely Dr Suria Bekker and Dr Nancy Ntidi. To the funders of the NRF and SACTA, I am ever greatful for the finacial support. i DECLARATION DECLARATION BY THE CANDIDATE I, Raymond L Collett, declare that the work presented in this MSc thesis is my own work, that it is not been submitted for any degree or examination at any other University and that all the sources I have used or cited have been acknowledged by the complete reference. Signature…………… Date…27/05/2020……………… DECLARATION AND APPROVAL BY SUPERVISORS We declare that the work presented in this thesis was carried out by the candidate under our supervision and we approve this submission. Prof Hendrika (Driekie) Fourie Unit for Environmental Sciences and Management, North West University, Private Bag, X6001, Potchefstroom, 2520, South Africa. Signature Date 30 May 2020 Dr Mariette Marais Agricultural Research Council –Plant Health and Protection, Private Bag X134, Queenswood, 0121, South Africa. Signature …………………………….. Date 2020.05.27. Dr Mieke S Daneel Agricultural Research Council – Tropical and Subtropical Crops, Private Bag X12208, Mombela, 1200, South Africa. Signature …………………………….. Date …27/05/2020……………. ii ABSTRACT Meloidogyne enterolobii, a highly pathogenic root-knot nematode species, infects fruit, grain, oilseed, ornamental and vegetable crops causing severe damage to agri-and horticultural crops worldwide. The species is infamous for its ability to render host plant resistance ineffective since it damages crops exhibiting resistance against other thermophilic species; M. incognita and M. javanica. This study commenced with an extensive desk-top study; a collective review (consulting 274 articles) about the global distribution, biology, pathogenicity and management of M. enterolobii, with special reference to sub-Saharan Africa. The research aim of the glasshouse study was to determine the life-stage development, life-cycle duration and reproduction potential of a South African M. enterolobii population compared to its counterpart species, M. incognita and M. javanica. Seedlings of three crops, maize (‘P- 2432-R’), soybean (‘DM-5953-RSF’) and tomato (‘Moneymaker’), were inoculated with motile second-stage juveniles (J2) of each species. Ambient temperature regimes maintained in the glasshouse were 19-32 ºC, 15-32 ºC and 18-32 ºC for the maize, soybean and tomato experiments, respectively, over 25 days. Random isolation of 20 life stages of each species from root systems of crop seedlings followed at time intervals of 3, 5, 10, 15, 20, and 25 days after inoculation (DAI), including five replicates of each crop and species. Infected crop roots were removed, for each time interval, and the nematode life stages stained using the sodium-hypochlorite-acid-fuchsin method. Egg-masses were present on the crop’s root surfaces 20 DAI and were first stained with eosin-Y before the life stages inside the roots were stained with the sodium-hypochlorite-acid-fuchsin method. Ten egg- masses were randomly removed from each crop seedling’s root system, for each of the species, and the number of eggs per egg-mass counted. Data were subjected to Factorial Analyses of Variance and the degree days (DD) were calculated for each species. Morphological and molecular identification verified the identity of the three species used. Significant (P≤0.05) differences existed for the number of each of the life stages, of each species, among some of the time intervals. Meloioigyne enterolobii developed more rapidly from one life-stage to the other compared to the other two species. Although females were observed for all three species 15 DAI, single eggs were observed for M. enterolobii only. Egg masses were, however, produced by females of all three species 20 and 25 DAI. The presence of second J2 generations of M. enterolobii and M. javanica from 20 DAI compared to those of M. incognita (recorded from 25 DAI only) confirmed the quicker development of M. enterolobii as well as M. javanica. Ultimately, the shorter DD needed by M. enterolobii to complete its life cycle in roots of all three crop genotypes compared to those of M. javanica iii and M. incognita represents novel information, both fundamentally and for its applicability. An improved advisory approach to farmers can now, for example, focus on rather using crop genotypes with shorter growing periods. Other management strategies can also be streamlined to focus on combatting M. enterolobii by, for example, interfering with its rapid life-cycle duration. Keywords: Life cycle, maize, Meloidogyne enterolobii, M. incongita, M. javanica, reproduction, South Africa, soybean, sub-Saharan Africa, tomato iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ..................................................................................................... i DECLARATION ................................................................................................................... ii DECLARATION BY THE CANDIDATE ............................................................................ ii DECLARATION AND APPROVAL BY SUPERVISORS ................................................... ii ABSTRACT ......................................................................................................................... iii CHAPTER 1: GENERAL INTRODUCTION ......................................................................... 1 1.1 Problem statement ..................................................................................................... 2 1.2 Research aims and objectives .................................................................................... 3 1.3. References ................................................................................................................ 5 CHAPTER 2: A REVIEW ON MELOIDOGYNE ENTEROLOBII: A VIRULENT ROOT-KNOT NEMATODE SPECIES THREATENING CROP PRODUCTION WITH PARTICULAR REFERENCE TO SUB-SAHARAN AND SOUTH AFRICA .................................................. 9 2.1 From origin to a global threat: insight into research done on M. enterolobii................ 9 2.1.1 Symptoms .......................................................................................................... 16 2.1.2 Damage potential/pathogenicity and crop losses ............................................... 17 2.1.3 Identification ....................................................................................................... 19 2.1.4 Biology, reproduction and life cycle .................................................................... 29 2.1.5 Host plants ......................................................................................................... 31 2.2 Management strategies ............................................................................................ 37 2.2.1. Genetic host plant resistance ............................................................................ 38 2.2.2 Biological control ............................................................................................ 45 2.2.3 Cultural control ................................................................................................... 45 2.2.4 Chemical control ............................................................................................ 46 2.2.5 An overall perspective of management .............................................................. 48 2.3. Focus on sub-Saharan Africa .................................................................................