Seedborne bacteria of onion : a study on pathogenicity and diversity

Abstract

Onion (Allium cepa L) is widely used in cooking; they add flavour to dishes such as salad, soup and stew. Onions are the third most popular vegetable in South Africa, after potatoes being first and tomatoes being second. South Africa is one of the biggest producer of onion seeds worldwide. Seeds are key input for improving agricultural yield and protecting food security. Providing farmers with quality seeds remains critical to unsure national food security in the country. Seeds quality can be defined in terms of some of the following characteristics: seed vigour, free from seedborne diseases and noxious weeds and should be of proper age. South Africa is one of the biggest producer of onion seeds worldwide. Total volumes of 724.80 tons of onions were produced in the 2014/2015 growing season, of which 657.07 tons were for the export market. Research showed that sales of the top seeds continued to increase, with short day onion seed leading the sales board in South Africa in 2019. The first chapter of this thesis is a review of the literature, discussing seedborne pathogens of onion focusing on Pseudomonas and Pantoea species, the mechanisms that these pathogens uses to infect seed. Detection and identification of seed borne pathogens were discussed. Identifying the causal agent of the disease benefits in determining correct approaches to manage or eliminate the pathogen before planting or in the field. Seed treatment aim to promote good seedling establishment, to minimise yield loss, to maintain and improve their quality and to avoid the spread of harmful pathogens. The use of culture-independent methods to examine microbial communities in seeds provides information on diversity of the bacterial communities on and within seed. Plant pathogenic bacteria have developed a number of different mechanisms, which result in disease in the host. Six different secretion systems have been characterised, i.e. T1SS to T6SS. These secretion systems shown to play different roles related to virulence, fitness, colonisation and survival. Full genome sequencing provide information on genetic variations that could lead to disease or can increase the risk of disease development, even in asymptomatic seeds. For example, in chapter 2, we characterised strains of Pseudomonas syringae, which were isolated from onion plants and seeds. We also included some strains isolated from leek because the strains were isolated from leek plants showing similar symptoms with that observed on onion plants. Biolog GNIII and MLST analysis of four housekeeping genes (cts, gapA, gyrB and rpoD) were used to identify the strains. Both Biolog GNIII dendrogram and MLST analysis showed a cluster supporting the existence of a new pathovar of P. syringae and the other corresponding to P. syringae pv. porri. Pathogenicity of the strains were determined in the glasshouse on onion, leek, chive and spring onion. Pathogenicity results revealed that P. syringae pv. porri strains induces symptoms on onion, leek, and spring onion. The strains of P. syringae of unknown pathovar induced symptoms only on onion. Thus, a new pathovar of P. syringae sensu lato, which causes leaf blight of onion, was named pathovar allii. The strains of P. syringae pv. allii differ from the type strain of P. syringae CFBP 1392T and P. syringae pv. porristrains by their ability to produce acid from erythritol and not utilise 3-methyl glucose, D-sorbitol and α-keto butyric acid. This study showed that cts (citrate synthase) primers should be used as a quick and accurate means of identifying new strains of Pseudomonas syringae to pathovar level. In this chapter 3, culture-dependent and culture-independent methods were used to evaluate bacterial community of onion seeds in a single cultivar obtained from Northern Cape and Western Cape Province. Culture-independent 16S rRNA-based approach was used because of its ability to detect unculturable bacterial colonisers, as well as those bacteria that are in such low abundance or grow slowly that they could be missed by culture dependent based protocol. The culture-dependent and -independent analyses used in this study indicated that the majority of bacteria associated with fungicide-treated and non-treated onion seed lots were members of the phylum Proteobacteria. The culture-independent approach identified widely recognised plant pathogens or endophytes (e.g. Pantoea, Pseudomonas, Streptomyces, Stenotrophomonas; Sphingomonas), also some genera that contain species that are potential human pathogens (e.g. Providencia, Enterococcus, Sphingobacterium). Culturable isolates included representatives of Acinetobacteria, Enterobacter, Erwinia, Microbacterium, Pantoea and Pseudomonas. The impact of planting uncertified or untreated seeds will results in loss of yield that will affect the economy. In chapter 4, whole genome comparative analysis of pathogenic and non-pathogenic Pantoea agglomerans was performed to identify genomic differences between pathogenic and nonpathogenic strains in order to reveal possible genetic factors important for emergence of pathogenicity on onion. Genomic analysis revealed that strain BD 1274 of P. agglomerans, which is pathogenic on onion, has a larger genome (4, 968 508 bp) than the non-pathogenic strain BD 1212 (4, 875 404 bp), confirming prior observations that non-pathogens lack the fourth plasmid carrying the T4SS genes responsible for pathogenicity or the four toxin production proteins. Unique genes were identified that differentiated the non-pathogenic and pathogenic P. agglomerans strains. Unique genes present in the pathogenic strain only might play a role in bacterial colonisation, fitness, survival and pathogenicity of that strain. The protein encoding the T3SS in the non-pathogenic strain might be important for colonisation and bacterial fitness in onion plants. In contrast, In contrast, conjugal transfer might play a major role in the pathogenicity to onion of strain BD 1274 of P. agglomerans. In addition, the toxins identified only in the pathogenic strain could play a significant role in the onion virulence of this strain and the sequences of these toxins can be used for rapid detection of Pantoea agglomerans.