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Identification and Characterisation of Some Phytopathogens Infecting South African Indigenous Ornamental Plants

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Identification and Characterisation of Some Phytopathogens Infecting South African Indigenous Ornamental Plants IDENTIFICATION AND CHARACTERISATION OF SOME PHYTOPATHOGENS INFECTING SOUTH AFRICAN INDIGENOUS ORNAMENTAL PLANTS C.A. Becker BSc (UKZN); BSc (Hons) (UKZN) Submitted in partial fulfilment of the requirements for the degree of Master of Science In the Discipline of Plant Pathology School of Agricultural, Earth and Environmental Sciences College of Agriculture, Engineering and Science University of KwaZulu-Natal, Pietermaritzburg South Africa November 2014 DISSERTATION SUMMARY South Africa is endowed with a large number of native plant species and many of the country’s plants have become important floricultural crops both locally and internationally. Three genera of South African monocotyledonous plants are particularly prominent floricultural crops of high value to the industry, namely Clivia, Strelitzia and Zantedeschia. A single species from each of these genera have stood out in the industry and become the most well-known representatives of their respective genera. These plants are Clivia miniata, Strelitzia reginae and Zantedeschia aethiopica. The reasons that each are of value to the industry are as follows: C. miniata has large orange flowers and glossy evergreen leaves and is able to grow well in shaded areas and in pots. Strelitzia reginae has bold flowers and foliage, which are highly desirable to consumers, and can be grown as a pot plant or in the garden. Zantedeschia aethiopica has attractive white flowers and is grown for as a cut flower, pot plant or in the garden. A number of highly apparent disease symptoms were observed on plants of C. miniata, S. reginae and Z. aethiopica growing mainly in KwaZulu-Natal, South Africa. Further samples of diseased plant material were also obtained from the Gauteng and Mpumalanga provinces to supplement the samples obtained from plant producers in KwaZulu-Natal. Six apparent disease complexes formed the focus of the studies presented in this dissertation. Three distinct diseases afflicting C. miniata and related Clivia species were observed and studied, two distinct diseases of S. reginae were observed and studied and one disease of Z. aethiopica was documented and studied. The three diseases observed on C. miniata were: (i) an anthracnose disease causing symptoms of leaf spotting and necrosis; (ii) a soft rot of the leaves and stems of afflicted plants and (iii) foliar streaking and mottling speculated to be caused by viral infection. The anthracnose disease was attributed to infection of C. miniata by a species of Colletotrichum. A fungal isolate was obtained from senescing, symptomatic leaf material and identified as a species of Colletotrichum, based on its cultural and morphological characteristics. Evaluation of Koch’s postulates with the isolate demonstrated its pathogenicity towards C. miniata and morphological characterisation of the conidia of the isolate identified it as C. karstii. The morphological identification was corroborated by a Maximum Likelihood (ML) phylogenetic analysis of the ITS region of the isolate, which indicated well-supported clustering with other C. karstii isolates. This is the first report of C. karstii causing an anthracnose disease of C. miniata in South Africa. i Two outbreaks of the soft rot disease of C. miniata were observed in commercial nurseries where C. miniata was being grown, one in Howick, KwaZulu-Natal and the other in White River, Mpumalanga, South Africa. A bacterial isolate from each outbreak was demonstrated to cause identical soft rot symptoms on inoculated plants, in fulfilment of Koch’s postulates. Both bacterial isolates exhibited cultural and morphological characteristics similar to members of the bacterial genus Pseudomonas. A BLAST search of the 16S rRNA region of the White River isolate in the Ribosomal Database Project database was performed and indicated similarities to a number of Pseudomonas species, corroborating the morphological assessments of the isolate. This is the first report of soft rot of C. miniata caused by a Pseudomonas species in South Africa. The virus-like disease symptoms studied were observed on C. caulescens, C. miniata and numerous interspecific hybrid plants. Transmission electron microscopy (TEM) of sap samples obtained from symptomatic showed the presence of filamentous viral particles in the leaf sap preparations and attempts to purify viral samples from symptomatic leaf material were successfully carried out. Reverse transcription polymerase chain reaction (RT-PCR) was implemented in attempts to amplify viral nucleic acids from leaf samples and purified virus preparations. These attempts were, however, unsuccessful in generating amplicons for phylogenetic analyses. Attempts were made to study possible viral transmission methods, through the mechanical inoculation of Clivia plants and Nicotiana tabacum. These transmission studies were inconclusive as none of the inoculated plants developed any symptoms. The possibility that these viruses are novel entities was raised by the results of the research carried on this subject. The two diseases affecting S. reginae were: (i) foliar spotting and necrosis and (ii) a flower blight. The foliar disease was attributed to infection by a species of Pestalotiopsis, a genus of known phytopathogen fungi. Morphological characterisation of the versicolorous conidia of the Pestalotiopsis isolate shown to be pathogenic on S. reginae indicated affinities to P. clavispora. A neighbour-joining (NJ) phylogenetic analysis of the ITS sequence data for the isolate placed it within a well-supported clade comprised of Pestalotiopsis species with versicolorous conidia. The relationships between the Pestalotiopsis species within the versicolorous clade was, however, not well resolved by the ITS-based phylogenetic inference and concatenated phylogenetic analysis of the ITS, β-tubulin and tef1 sequence data for this isolate will be required for accurate taxonomic placement within the genus. This is the first report of a leaf blight of S. reginae caused by a Pestalotiopsis species in South Africa. ii The flower blight observed to afflict S. reginae was demonstrated to be caused by a phytopathogenic bacterium, belonging to the bacterial genus Pantoea. The morphological and biochemical characteristics of the isolate were consistent with various Pantoea species. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) and a BLAST analysis of the 16S rRNA region of the isolate’s genome indicated affinities to P. agglomerans, a documented phytopathogen. This is the first report of a Pantoea species as a phytopathogen of S. reginae and also the first report of a floral disease of S. reginae in South Africa. The disease symptoms commonly observed on Z. aethiopica in KwaZulu-Natal were a conspicuous foliar blight of infected leaves. The etiology and symptoms of the foliar disease observed in Pietermaritzburg were similar to those previously reported to be caused by the bacterium Xanthomonas campestris pv. zantedeschiae. Koch’s postulates were successfully carried out with the bacterial strain consistently isolated from symptomatic leaf material. PCR amplification of the 16S rRNA region isolate indicated similarities to various species of Pseudomonas, chiefly P. putida, but not to any X. campestris isolates. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) of the peptidic profile of the isolate corroborated the 16S rRNA based assessment of its identity as a Pseudomonas sp but did not provide a strong indication of species identity. This is the first report of a foliar blight of Z. aethiopica caused by a Pseudomonas sp. in South Africa. Cultures of all the successfully isolated phytopathogens were deposited at the South African Agricultural Research Council Plant Protection Research Institute (ARC-PPRI) with the following accessions numbers: C. karstii PPRI 16882, Pseudomonas sp. BD 1294 and Pseudomonas sp. BD 1295 [Clivia pathogens]; Pestalotiopsis sp. PPRI 16883 and Pantoea sp. BD 1293 [Strelitzia pathogens] and Pseudomonas sp. BD 1296 [Zantedeschia pathogen]. The research carried out and presented in this dissertation highlights a number of new records of plant diseases and their associated phytopathogens for South Africa. It is indicative of the need for further studies of phytopathogens infecting floricultural crops in South Africa in order to update the field and provide management strategies for floricultural practitioners in the country. iii DECLARATION I, CALVIN ALEXANDER BECKER, declare that (i) The research reported in this dissertation, except where otherwise indicated, is my original work. (ii) This dissertation has not been submitted for any degree or examination at any other university. (iii) This dissertation does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. (iv) This dissertation does not contain other person’s writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a) their words have been re-written but the general information attributed to them has been referenced; b) where their exact words have been used, their writing has been placed inside quotation marks, and referenced. (v) This dissertation does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the dissertation and in the References sections. Signed……………………………………………
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