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Morphological and Molecular Characterisation of Plant-Parasitic Nematodes Associated with Pineapple, Roses and Tea in Kenya

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Morphological and Molecular Characterisation of Plant-Parasitic Nematodes Associated with Pineapple, Roses and Tea in Kenya Morphological and molecular characterisation of plant-parasitic nematodes associated with pineapple, roses and tea in Kenya David Kihoro Sirengo Student number: 01800695 Promoter: Prof. Dr. Wim Bert Co-promoter: Dr. Laura Cortada Supervisor: Rolish Singh A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of International Master of Science in Agro- and Environmental Nematology Academic year: 2019 - 2020 Morphological and molecular characterisation of plant-parasitic nematodes associated with pineapple, roses and tea in Kenya David Kihoro Sirengo Nematology Research Unit, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020 Preamble This master thesis is aimed at establishing the diversity and characterization of plant-parasitic nematodes associated with pineapple, cut flowers (roses) and tea from Kiambu county in Kenya. Morphological and molecular characterization of important plant-parasitic nematodes associated with the above-mentioned crops would provide a baseline study for future reference. However, given this thesis was done amid COVID- 19 pandemic, 43 (pineapple 26, roses 35, and tea 9) soil and pumice samples were analysed based on nematode prevalence, density, prominence and identification at genus level. In this study, only two plant- parasitic nematodes from pineapple fields have been characterized through integrative approach. A thorough analysis of both soil and root samples from the sampled geolocated areas in Kiambu, Kenya is needed to provide an overview of all plant-parasitic nematodes in the samples. Having said that and considering the limitations mentioned above, this master thesis has been written using the limited available data and other assignments suggested to fulfil an accepted level of dissertation. The additional off-campus assignments included in this thesis are: 1) resolving taxonomical problem of the cryptic species status of Scutellonema brachyurus from samples obtained from fingermillet, pineapple, soybean and morphological voucher slides from Rwanda and D2D3 and COI sequence from Tanzania 2) providing an overview of terrestrial nematofauna in Kenya using data from current study and literature search in a joint effort with Denis Gitonga. Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020 Summary - A baseline survey of pineapple, roses and tea fields in Kiambu Kenya was carried out between December 2019 - January 2020 to determine the distribution and abundance of plant-parasitic nematodes and to characterize the prevalent genera using combination of morphological features and molecular analyses. A total of 114 soil and root samples; 60 pineapple, 45 roses and 9 tea samples were collected from Kiambu county, Kenya. Nematodes were extracted from both soil and roots following a standardized protocol and identified at genera and some to species level. In pineapple samples, Helicotylenchus dihystera and Meloidogyne javanica were the two most prevalent parasitic species (100%) followed by Pratylenchus species (36%). Helicotylenchus dihystera was characterized based on its morphology and D2-D3 of 28rDNA sequences similar to GenBank records. The two populations agree with the original description and D2D3. All the four M. javanica populations were identical to each other and a Nad5 gene of mtDNA reference informative polymorphic positions similar to sequence (KU372392) by (Janssen et al., 2016). The top three prevalent genera on roses rhizosphere were Meloidogyne spp. (100%), Xiphinema spp. (54%) and Longidorus spp. (46%). The densities were however relatively low: 9 individuals/100 ml, 5 indiv/100 ml and 4 indiv/100 ml of soil for Meloidogyne, Xiphinema and Longidorus respectively. Plant-parasitic nematode were not detected from tea samples. This results sheds lights on the distribution and diversity of parasitic nematodes on pineapples and roses in Kiambu, Kenya. Additionally, based on samples from fingermillet, soybean, pineapple and potato rhizosphere, our findings revealed all newly generated sequences (D2-D3 and COI) of our S. brachyurus type B populations formed a maximally supported clade with other sequences of African S. brachyurus (type B) and were separated from S. brachyurus type A species which are known to be cryptic species based on molecular data reported by Van den Berg et al. (2016). Morphologically, S. brachyurus type B was found to be different from type A species based on the number of lip region annuli 3-4 vs 4-6 annuli. The species delimitation also revealed taxonomic distinctness of S. brachyurus type B supported by a well significant Rosenberg’s PAB of 1.2E-14 and 1.9E- 8 for D2-D3 and COI respectively, revealing it is a different species from S. brachyurus type A species. Finally, a total of 10 taxa from current study and 116 taxa from literature on Kenyan nematofauna has been revealed. The ten taxa identified were; Criconema spp., Helicotylenchus dihystera, Meloidogyne javanica, Paratylenchus spp., Rotylenchus robustus, Rotylenchulus macrosoma, Trichodorus spp., Tylenchulus spp, and Scutellonema brachyurus. R. robustus, R. macrosoma and S. brachyurus are new records in Kenya. This checklist is a baseline for future records of new nematofauna identified and described in Kenya. Keywords – COI, D2-D3, cryptic species, diversity, Kenya, plant-parasitic nematode, pineapple Agriculture, a principal source of employment and a significant contributor to GDP, remains vital to the Kenyan economy (World Bank, 2019). Nearly 75% of Kenyans live in rural areas and are actively engaged in the production, processing, and marketing of agricultural products and produce. With an increasing population growth in sub-Saharan Africa (SSA), which is expected to grow even more by 2050 (United Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020 Nations, 2017), food security issues need to be addressed and warrants immense increase in crop production through the intensification of cropping systems. However, this will result in the aggravation of pest and disease pressure (Lopes-Caitar et al., 2019). The horticultural industry and cash crops occupy the most prominent position in terms of foreign exchange-earners and source of livelihoods to many Kenyans (Ongeri, 2014). Over the past decade, Kenya’s horticultural industry has received a great deal of attention due to the rapid and sustained growth of its export to Europe. The sector produces fruits, flowers, vegetables, and herbs, which generates approximately $US 1 billion annually. Currently, the horticultural industry is ranked third after tourism and tea (World Bank, 2019). Pineapple, cut flowers, and tea sub-sectors are among the top export produce. However, these crops are exposed to abiotic and biotic constraints resulting in substantial yield losses. Intensive monoculture of these crops is becoming vulnerable to damage from biotic stressors including plant-parasitic nematodes (PPN), especially root-knot nematodes (RKN, Meloidogyne sp.) (Jones et al., 2013). Plant-parasitic nematodes are soil-dwelling micro-organisms with over 4100 species described (Decraemer & Hunt, 2006). Being present in all agricultural land, PPN account for 10% yield losses globally (De Waele & Elsen, 2007), ranging from $US 80 billion (Abad et al., 2008) to $US 157 billion annually (Nicol et al., 2011). Due to their nonspecific symptoms, horticultural damage caused by PPN remains unnoticed and therefore, underestimated (Lopes-Caitar et al., 2019). Research on diagnostic of PPN in SSA is gradually improving as expertise develops and reliable modern techniques becomes available (Coyne et al., 2018). Damage due to PPN, their behaviour, and management options associated with many crops, including pineapple, cut flowers and tea, have received little attention in Kenya and in other SSA countries. Tea production in Kenya is undertaken by both large-scale and smallholder farmers. According to the tea directorate, there are over half a million registered smallholder tea farmers. Following its favorable climatic conditions and acid soils that foster healthy tea bushes, Kenya ranks first in the export of black tea to United Kingdom, Egypt, Pakistan, Afghanistan, and the United Arab Emirates (Kibet et al., 2013). According to Gnanapragasam and Mohotti (2018), several PPN have been recovered from the roots of tea and/or from the rhizosphere of tea plantation globally. However, only a handful have been found to cause economic damage. They include Hemicriconemoides kanayaensis, Meloidogyne sp., Pratylenchus sp., and Radopholus similis. In Kenya, little research has been conducted concerning PPN of tea. Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020 Following tea in export earning is the flower industry which contributes about US$ 1billion annually into the economy (Alessandro et al., 2015). Roses, orchids, carnations, hypericum, lilies, and gladioli are the main flowers grown in different parts of Kenya, including Kiambu where cut flowers produced are mainly for export. Pests and diseases are a considerable challenge in flower production (Rikken, 2011), including PPN (Handayati & Sihombing, 2017). Globally, Meloidogyne sp. contributes significantly to crop damage and yield reduction (Koenning et al., 2004). In addition, foliar nematodes such as Ditylenchus sp., Aphelenchus sp., and Aphelenchoides sp. cause qualitative damage to cut flowers (Langat et al., 2008). Losses due to PPN in cut flowers is estimated
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