This study focuses on the soil- and water-borne plant pathogen Phytophthora cinnamomi Rands and the phenomenon of P. cinnamomi suppressive soil. In particular, this thesis reports on the outcome of field surveys and glasshouse assays undertaken to locate P. cinnamomi suppressive soils and to confirm the involvement of biological processes in suppression. The potential role of cellulase and laminarinase in suppression was investigated and a molecular technique known as length heterogeneity PCR (LH-PCR) was used to analyse the structure and diversity of bacterial and fungal communities in avocado orchard soils that were suppressive and conducive to P. cinnamomi. Four avocado orchards with P. cinnamomi suppressive soils were identified and soils were ã-irradiated to destroy their suppressive capacity, thus confirming biological suppression. Suppression was also partially transferred to ã-irradiated and conducive soils by mixing with 10% suppressive avocado soils. Cellulase and laminarinase activities measured in avocado orchard soils inoculated with P. cinnamomi were not associated with disease severity in lupin seedlings during glasshouse assays involving the same soil samples. Minor shifts in bacterial and fungal community structure were observed in response to mixing conducive and irradiated soils with suppressive soils. This was associated with decreased disease severity in avocado seedlings in these treatments. The shift in bacterial community structure was partially determined by the appearance and increased abundance of several bacterial 16S rDNA sequences, which were unique to the suppressive soils, in the mixed soil treatments. It is suggested that the bacteria and fungi from which these sequences originated may be involved in suppression and further work should be undertaken to determine their identity and confirm their potential role in the development and maintenance of P. cinnamomi suppressive soils. / Doctor of Philosophy (PhD)

Les bactéries du sol produisant des antifongiques comme le 2,4-diacétylphloroglucinol(DAPG) protègent les racines des plantes vis-à-vis des champignons phytopathogènes. Néanmoins, les conditions de fonctionnement de ces populations bactériennes dans le sol restent très mal connues. Dans certains sols, dits résistants aux maladies, ces bactéries phytoprotectrices sont présentes à des effectifs importants et leur activité est suffisante pour protéger la plante malgré la présence du pathogène. L'objectif de cette thèse a été de comprendre la relation entre la résistance des sols à la maladie de la pourriture noire des racines de tabac, et la fonction de synthèse du DAPG chez les bactéries du genre Pseudomonas. Dans la situation de référence de Morens (Suisse), les sols résistants diffèrent des sols sensibles par la présence de vermiculite, argile capable de relarguer du fer. On sait que la présence de vermiculite améliore la phytoprotection assurée par les Pseudomonas producteurs de DAPG, mais les mécanismes moléculaires sous-jacents restent inconnus. Dans un premier temps, la quantification de ces bactéries par une nouvelle méthode de PCR quantitative développée ici, a confirmé que leurs effectifs sont élevés dans les sols résistants, mais aussi dans les sols sensibles, suggérant que la résistance puise plutôt dépendre d'une plus forte expression de la fonction de synthèse du DAPG. Dans un second temps, l'étude de l'expression des gènes de synthèse du DAPG en système de sol artificiel, à l'aide de la souche rapportrice P. protegens phlA-gfp, a montré que la présence de vermiculite dans le sol se traduit par une plus forte biodisponibilité du fer pour les Pseudomonas, induisant une plus forte expression des gènes de synthèse du DAPG et la protection du tabac. En conclusion, la résistance des sols de Morens à la maladie de la pourriture noire des racines est conditionnée par plusieurs facteurs abiotiques et biotiques, dont la biodisponibilité du fer qui régule l'expression des gènes de synthèse du DAPG chez Pseudomonas / Soil bacteria producing antimicrobial compounds like 2,4-diacetylphloroglucinol (DAPG) protect plants from soil-borne phytopathogens. Nevertheless, the functioning of these bacterial populations in the soil is largely unknown. In certain soils, termed disease- suppressive soils, these bacteria are present at high numbers and their activity is sufficient to assure effective plant protection in the presence of the pathogen. The aim of this thesis was to understand the relation between soil suppressiveness towards black root rot of tobacco, and the 2,4-diacetylphloroglucinol synthesis ability of certain Pseudomonas. In Morens region (Switzerland), suppressive soils differ from conducive soil by the presence of vermiculite, an iron-releasing clay. It is known that DAPG-producing Pseudomonas provide better plant protection in the presence of vermiculite, but the molecular basis of this interaction is still unknown. First, the quantification of these bacteria, through a new real-time PCR method developed here, confirmed that high numbers of DAPG-producing Pseudomonas occur in suppressive soils, as well as in conducive ones, raising the possibility that suppressiveness depends rather on a higher expression of DAPG synthetic genes. Second, expression studies of DAPG synthetic genes using a P. protegens ph/A- gfp reporter strain and artificial soil systems, confirmed that the presence of vermiculite in the soil can translate into higher iron bioavailability for Pseudomonas, triggering higher expression of DAPG synthetic genes and effective plant protection. In conclusion, black root rot suppressiveness of Morens soils is determined by several abiotic and biotic factors, among which iron bioavailability regulating the expression of DAPG synthetic genes in plant-protecting Pseudomonas

Take-all, caused by the soilborne fungus, Gaeumannomyces graminis var. tritici (Ggt), is an important root disease of wheat that can be reduced by take-all decline (TAD) in successive wheat crops, due to general and/or specific suppression. A study of 112 New Zealand wheat soils in 2003 had shown that Ggt DNA concentrations (analysed using real-time PCR) increased with successive years of wheat crops (1-3 y) and generally reflected take-all severity in subsequent crops. However, some wheat soils with high Ggt DNA concentrations had low take-all, suggesting presence of TAD. This study investigated 26 such soils for presence of TAD and possible suppressive mechanisms, and characterised the microorganisms from wheat roots and rhizosphere using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A preliminary pot trial of 29 soils (including three from ryegrass fields) amended with 12.5% w/w Ggt inoculum, screened their suppressiveness against take-all in a growth chamber. Results indicated that the inoculum level was too high to detect the differences between soils and that the environmental conditions used were unsuitable. Comparison between the Ggt DNA concentrations of the same soils collected in 2003 and in 2004 (collected for the pot trial), showed that most soils cropped with 2, 3 and 4 y of successive wheat had reduced Ggt DNA concentrations (by 195-2911 pg g-1 soil), and their disease incidences revealed 11 of the 29 test soils with potential take-all suppressiveness. Further pot trials improved the protocols, such that they were able to differentiate the magnitudes of suppressiveness among the soils. The first of the subsequent trials, using 4% w/w Ggt inoculum level, controlled conditions at 16°C, 80% RH with alternate 12 h light/dark conditions, and watering the plants twice weekly to field capacity (FC), screened 13 soils for their suppressiveness against take-all. The 13 soils consisted of 11 from the preliminary trial, one wheat soil that had been cropped with 9 y of wheat (considered likely to be suppressive), and a conducive ryegrass soil. The results revealed that 10 of these soils were suppressive to take-all. However, in only four of them were the effects related to high levels of microbial/biological involvement in the suppression, which were assessed in an experiment that first sterilised the soils. In a repeat trial using five of the soils H1, H3, M2, P7 (previously cropped with 3, 3, 4 and 9 y successive wheat, respectively) and H15 (previously cropped with 5 y of ryegrass), three of them (H1, H3 and M2) had reduced Ggt DNA concentrations (>1000 pg g-1 soil reductions), and were confirmed to be suppressive to take-all. A pot trial, in which 1% of each soil was transferred into a γ-irradiated base soil amended with 0.1% Ggt inoculum, indicated that soils H1 and H3 (3 y wheat) were specific in their suppressiveness, and M2 (4 y wheat) was general in its suppressiveness. The microbial communities within the rhizosphere and roots of plants grown in the soils, which demonstrated conduciveness, specific or general suppressiveness to take-all, were characterised using PCR-DGGE, and identities of the distinguishing microorganisms (which differentiated the soils) identified by sequence analysis. Results showed similar clusters of microorganisms associated with conducive and suppressive soils, both for specific and general suppression. Further excision, re-amplification, cloning and sequencing of the distinguishing bands showed that some actinomycetes (Streptomyces bingchengensis, Terrabacter sp. and Nocardioides sp.), ascomycetes (Fusarium lateritium and Microdochium bolleyi) and an unidentified fungus, were associated with the suppressive soils (specific and general). Others, such as the proteobacteria (Pseudomonas putida and P. fluorescens), an actinomycete (Nocardioides oleivorans), ascomycete (Gibberella zeae), and basidiomycete (Penicillium allii), were unique in the specific suppressiveness. This indicated commonality of some microorganisms in the take-all suppressive soils, with a selected distinguishing group responsible for specific suppressiveness. General suppressiveness was considered to be due to no specific microorganisms, as seen in soil M2. An attempt to induce TAD by growing successive wheat crops in pots of Ggt-infested soils was unsuccessful with no TAD effects shown, possibly due to variable Ggt DNA concentrations in the soils and addition of nutrients during the experiment. Increasing numbers of Pseudomonas fluorescens CFU in the rhizosphere of plants, during successive wheat crops was independent of the Ggt DNA concentrations and disease incidence, suggesting that increases in P. fluorescens numbers were associated with wheat monoculture. This study has demonstrated that TAD in New Zealand was due to both specific and general suppressiveness, and has identified the distinguishing microorganisms associated with the suppression. Since most of these distinguishing microorganisms are known to show antagonistic activities against Ggt or other soilborne pathogens, they are likely to act as antagonists of Ggt in the field. Future work should focus on validating their effects either individually, or interactively, on Ggt in plate and pot assays and under field conditions.