South Africa is the world's second largest exporter of fresh citrus and is ranked 14th in citrus production. Fungal pathogens such as Phytophthora and Pythium cause economic losses as a result of root rot and brown rot. Mycorrhizal fungi are specialized members of the fungal community forming a mutualistic relationship with plant roots. Mycorrhizal fungal structures are known to associate with other soil microorganisms and these may contribute to improved plant growth. A diverse group of bacteria that interact with the mycorrhizal fungi are known as Mycorrhizal Helper Bacteria (MHB). The aim of this study was to investigate the role of arbuscular mycorrhiza and associated bacteria isolated from spores and determine whether they had any plant growth promoting potential. A total of 19 bacteria were isolated from arbuscular mycorrhizal spores and were molecularly identified as belonging to several Bacillus, Micrococcus, Onchrobactrum and Staphylococcus sp. All bacterial isolates were tested for plant growth promotion abilities. One Bacillus isolate was able to solubilise phosphate. Four isolates Micrococcus sp, Micrococcus leteus, Ochrobacterum sp and Ochrobacterum antropi were able to produce Indole Acetic Acid and three isolates showed potential to reduce growth of Phytophthora nicotianae, P. citrocola and P. citrophthora in in vitro plate cultures. Further tests using culture supernatants of the Bacillus sp, Micrococcus sp and Bacillus cereus confirmed their ability to inhibit or reduce growth of the three Phytophthora species in a 96 well bioassay. Bacillus sp and Bacillus cereus were able to inhibit Phytophthora spp by 95 to 100 % and Micrococcus spp was able to decrease pathogen growth by 60 to 94 %. These bacterial isolates were further evaluated for plant growth promoting abilities on citrus rough lemon seedlings alone or in combination with arbuscular mycorrhizal inoculum. Bacterial and mycorrhizal inoculants influence the increase in shoot and root biomass. Bacillus cereus in combination with mycorrhizal inoculum significantly increased seedling shoot to root ratio while root biomass was significantly increased with mycorrhizal inoculation. Due to the short duration of the trial mycorrhizal colonisation could not be assessed. It is evident that selected combinations of bacteria and mycorrhizal fungi could promote citrus seedling growth and potentially improve seedling health. Further studies under nursery conditions are recommended.

Orientador: Sandra de Moraes Gimenes Bosco / Resumo: Pythium insidiosum é o agente etiológico da pitiose, uma infecção granulomatosa crônica, com prevalência em regiões de clima tropical e subtropical que acomete mamíferos, principalmente equinos, cães e humanos. Este micro-organismo é um falso fungo que apresenta uma ampla distribuição geográfica, sendo muito prevalente na América do Sul (pitiose equina e canina) e na Tailândia (pitiose humana). Estudos moleculares têm permitido diagnóstico precoce e melhor compreensão das relações filogenéticas, dividindo o patógeno em três clados (I, II e III ou A, B e C). Entretanto essas informações são ainda bastante limitadas e novas regiões gênicas poderiam ajudar a esclarecer a história evolutiva dessa espécie. Assim sendo, este trabalho visou estabelecer as relações filogenéticas entre 52 isolados de P. insidiosum, americanos e asiáticos, por meio do sequenciamento de três novas regiões gênicas: a região do fator de alongamento da tradução (Tef-1α), α e β tubulina, além da padronização da técnica de Pulse Filed Gel Electrophoresis (PFGE) para esta espécie. A região do Tef-1α mostrou-se menos polimórfica em relação a α e β tubulina, entretanto separou as cepas aqui trabalhadas em dois clados distintos, sendo um composto apenas de cepas classificadas previamente como sendo do clado III (asiáticas), e agrupou todas as cepas americanas junto às cepas do clado II. A filogenia baseada no gene da β tubulina separou os isolados nos três clados esperados. Em relação à α tubulina houve diferenc... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pythium insidiosum is the etiological agent of pythiosis, a chronic granulomatous infection, prevalent in tropical and subtropical regions that affects mammals, especially horses, dogs and humans. This is a fungus-like microorganism that has a wide geographical distribution and is very prevalent in South America (equine and canine pythiosis) and Thailand (human pythiosis). Molecular studies have allowed early diagnosis and better understand of phylogenetic relationships, and the pathogen is currently divided into three clades (I, II and III or A, B and C). However, this information is still quite limited for this pathogen and new gene regions could help understanding the evolutionary history of this species. Therefore, this study aimed to establish the phylogenetic relationships among 52 american and asian P. insidiosum isolates by sequencing three new gene regions: the translation elongation factor (Tef-1α), α and β tubulin, in addition to the standardization of the Pulse Filed Gel Electrophoresis (PFGE) technique for this species. The Tef-1α region showed little polymorphic in relation to α and β tubulin, however it separated the strains here studied into two distinct clades, being composed only of strains previously classified as clade III (Asian), and grouped all the American strains next to the clade II strains. The β tubulin gene-based phylogeny separated the isolates into the three expected clades. Regarding α tubulin there was differentiation of isolates in clades I a... (Complete abstract click electronic access below) / Mestre

Rhizoctonia solani and Pythium spp. are aggressive soil-borne fungal pathogens responsible for seed rot and seedling damping-off of many crops. With increased environmental and public concern over the use of chemicals, biological control of these diseases has been attracting more attention. However, success with this strategy depends on the development of effective antagonists, which requires repeated in vitro and in vivo tests. Bacillus spp. were isolated from a soil sample obtained from a field where sorghum and tef had been grown for at least two years. Potential Bacillus isolates were screened for their ability to inhibit in vitro growth of R. solani and Pythium sp. Among 80 isolates tested, endospore forming Bacillus spp. H44 and H51 gave highest antifungal activity against the two test-pathogens in three consecutive tests. Results demonstrated that both H44 and H51 have potential as biocontrol agents against diseases caused by these two pathogenic fungi. The interaction between three isolates of Trichoderma (T. harzianum Eco-T, Trichoderma spp. SY3 and SY4) and Pythium sp. were investigated using in vitro bioassays together with environmental scanning electron microscopy (ESEM). Visual observation on the dual culture tests revealed that hyphal growth of Pythium was inhibited by these antagonists soon after contact between the two organisms within 3-4 days of incubation. The ESEM investigations showed that all three isolates of Trichoderma grew toward the pathogen, attached firmly, coiled around and penetrated the hyphae of the pathogen, leading to the collapse and disintegration of the host's cell wall. Degradation of the host cell wall was postulated as being due to the production of lytic enzymes. Based on these observations, antibiosis (only by Eco-T) and mycoparasitism (by all three isolates) were the mechanisms of action by which in vitro growth of Pythium sp. was suppressed by these Trichoderma isolates. The reduction of seedling diseases caused by R. solani and a pythium sp. were evaluated by applying the antagonists as seed coating and drenching antagonistic Bacillus spp. (B81, H44 and H51) and Trichoderma (T. harzianum Eco-T and Trichoderma spp. SY3 and SY4). On both crops, R. solani and Pythium sp. affected stand and growth of seedlings severely. With the exceptions of H51, applications all of isoltes to seeds reduced damping-off caused by R. solani in both crops. Application of Eco-T, H44 and SY3 to sorghum controlled R. solani and Pythium sp. effectively by yielding similar results to that of Previcur®. On tef, biological treatments with Eco-T and SY4 reduced seedling damping-off caused by R. solani and Pythium sp., respectively, by providing seedling results similar to the standard fungicides, Benlate® and Previcur®. Most other treatments gave substantial control of the two pathogens on tef. Overall, Bacillus sp. H44 and T harzianum Eco-T were the best biocontrol agents from their respective groups in reducing damping-off by the two pathogens. In all instances, effects of application method on performance of biocontrol agents and adhesive on emergence and growth of seedlings were not significant. A field trial was conducted at Ukulinga Research Farm at the University of Natal, Pietermaritzburg, South Africa, to determine efficacy of biological and chemical treatments on growth promotion and reduction of damping-off incited by R. solani and Pythium sp., and to evaluate the effects of a seed coating material, carboxymethyl cellulose (CMC), on seedling emergence and disease incidence. Seeds of sorghum and tef were treated with suspensions of antagonistic Bacillus H44 or T harzianum Eco-T, or sprayed with fungicides, Benlate® or Previcur®. Application of Benlate® and Previcur® during planting significantly increased the final stand and growth of sorghum seedlings. Seed treatments with both H44 and Eco-T substantially controlled damping-off caused by Pythium, resulting in greater dry weights of seedlings than the standard fungicide. However, they had negative effects when they were tested for their growth stimulation and control of R. solani. The CMC had no significant effect on germination and disease levels. These results showed that these antagonists can be used as biocontrol agents against Pythium sp. However, repeated trials and better understanding of the interactions among the antagonists, the pathogens, the crop and their environment are needed to enhance control efficiency and growth promotion of these antagonists. Some of these biocontrol agents used in this study have the potential to diseases caused by R. solani and Pythium sp. However, a thorough understanding of the host, pathogen, the antagonist and the environment and the interactions among each other is needed for successful disease control using these antagonists. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

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