Arbuscular mycorrhizal fungi (AMF) represent an ancient and critical symbiotic partner with the majority of land plants, understood to promote ecosystem productivity and biodiversity and are also important to ecologically sound land management practices. The fungus is thought to be over 400 million years old, and due to a lack of an observable sexual cycle, has been placed into a select group of eukaryotes called 'ancient asexuals', which seemingly defy evolutionary theory by persisting for an extended period of time in the absence of sexual reproduction. Recently however, molecular evidence has accumulated which may suggest AMF harbour a cryptic sexual cycle. In the first chapter of this thesis, entitled "Searching for clues of sexual reproduction in the genomes of arbuscular mycorrhizal fungi", I review evidence supporting this notion of a cryptic sexual cycle in AMF which includes: the presence of recombinational events, meiosis-specific genes as well as mating-specific transcription factors called SexM and SexP of the MATA_HMG protein family which are otherwise found only in the genomes of sexual fungi. In the second chapter, I present the main research of my MSc work where I used bioinformatic, population genetic, molecular and experimental approaches to build on this existing evidence of sexuality in AMF. These findings include the presence of a dramatically expanded family of MAT-HMG genes which are present in several isolates of the Rhizophagus irregularis and also harbour significant allele variation amongst these isolates, some of which resembles variation expected at MAT-genes in other fungi. Q-RT-PCR procedures revealed that at least some of these genes tend to increase in expression during crosses of R. irregularis isolates. We also uncovered the presence of a unique genomic region where at least three of these genes are located in tandem. Finally, several tests of recombination support the presence of intraisolate as well as interisolate recombination events occurring between these MAT-HMG genes.

Many orchids require mycorrhizal symbioses with fungi for their development and survival. Rhizanthella gardneri the Western Australian underground orchid is associated with the companion plant Melaleuca uncinata and its ectomycorrhizal fungus symbiont. Much less is known about the habitat requirements of its sister species, R. slateri, which occurs in Eastern Australia. The absence of chlorophyll from Rhizanthella gardneri and R. slateri results in total dependency on associations with fungal symbionts. Many ecological and biological aspects of these fascinating orchids remained poorly known, including the identity of the fungal associates and the nature of their tripartite associations with Rhizanthella and Melaleuca. Extremely high specificity of these mycorrhizal relationships is likely to be the most important factor explaining the highly specific habitat requirements of underground orchids. The purpose of this study was to conduct further investigations of the role of the mycorrhizal associations of Australian underground orchids by identifying the fungi involved in these associations, optimising their growth in sterile culture and devising efficient means for synthesising their tripartite associations with R. gardneri and M. uncinata. In total, 16 isolates of fungi were successfully obtained from the two underground orchids and used in a series of experiments to understand both the nature of the fungi and their relationship with orchids. The identity of these fungi was established by using conventional morphological and molecular methods. Cultural and morphological studies revealed that all isolates from R. gardneri and R. slateri were binucleate rhizoctonias with affinities to members of the genus Ceratobasidium. However, the teleomorph state that was observed from the R. slateri symbiont during this study more closely resembled a Thanatephorus species. Further identification using ITS sequence comparisons confirmed that mycorrhizal fungi of Rhizanthella belonged to the Rhizoctonia alliance with relatives that include Thanatephorus, Ceratobasidium, or Rhizoctonia from other continents with over 90% similarity. Most of these related fungi are known as plant pathogens, but some were orchid mycorrhizal fungi. However, the isolates from the two underground orchids were most closely related to each other and formed a discrete group relative to other known members of the Rhizoctonia alliance. Sterile culture experiments determined culture media preferences for mycorrhizal fungi from Rhizanthella and other orchids. A fully defined sterile culture medium designed to more closely resemble Australian soil conditions was formulated. This new medium was compared to undefined media containing oats or yeast extract and recommendations for growth of these fungi are provided. The undefined media based on oats provided the best growth of most fungi, but the new Australian soil media was also effective at growing most orchid mycorrhizal fungi and this fully defined media was less prone to contamination and should provide more reproducible results. A comparison of three methods for inoculating M. uncinata with the underground orchid fungi resulted in the production and characterisation of ectomycorrhizal roots and hyphae formed by fungi isolated from R. gardneri and R. slateri. These underground orchid fungi could easily be distinguished from other mycorrhizal fungi (caused by airborne contamination) by the characteristic appearance of these roots and hyphae. A new system for growing and observing tripartite mycorrhizal associations was devised using pots with side viewing windows and the use of transparent seed packets to contain Rhizanthella seeds. This method allowed all the stages of seed germination to be observed in the glasshouse, culminating in the production of underground orchid rhizomes. Seed germination was only successful when seed was placed directly over active M. uncinata ectomycorrhizas confirmed to belong to the correct fungus by microscopic observations through the side of window pots. The importance of these new scientific discoveries concerning the biology and ecology of the underground orchids and their associated fungi for the recovery of these critically endangered orchids are discussed.

The specificity of fungal isolates from endemic Western Australian orchid species and hybrids in the tribe Diurideae was investigated using symbiotic seed germination and analysis of the fungal DNA by amplified fragment length polymorphism (AFLP). The distribution of the fungal isolates in the field was also assessed using two different seed baiting techniques. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. Two groups of orchids in the tribe Diurideae were studied. Firstly, a number of Caladenia species, their natural hybrids and close relatives from the southwest of Western Australia were selected because orchid species from the genus Caladenia are considered to have among the most specific mycorrhizal relationships known in the orchid family – an ideal situation for the investigation of mycorrhizal specificity. Secondly, species of Drakaea and close relatives, from the southwest of Western Australia and elsewhere in Australia, which are never common in nature and occur in highly specialised habitats, were selected to investigate the influence of habitat on specificity. Seed from the common species Caladenia arenicola germinated on fungal isolates from adult plants of both C. arenicola and its rare and endangered relative C. huegelii, while seed from C. huegelii only germinated on its own fungal isolates. The AFLP analysis grouped the fungal isolates into three categories: nonefficaceous fungi, C. huegelii type fungi, and C. arenicola type fungi. The group of C. huegelii type fungi included some fungal isolates from C. arenicola. An analysis of the AFLP fingerprints of C. arenicola fungal isolates from different collection locations showed that some, but not all, populations were genetically distinct, and that one population in particular was very variable. Despite being thought to have very specific mycorrhizal relationships, Caladenia species hybridise frequently and prolifically in nature, often forming self-perpetuating hybrid lineages. Five natural hybrids within Caladenia and its closest relatives were investigated. Symbiotic cross-germination studies of parental and hybrid seed on fungi from the species and the naturally occurring hybrids were compared with AFLP analyses of the fungal isolates to answer the question of which fungi the hybrids use. The germination study found that, while hybrid seeds can utilise the fungi from either parental species under laboratory conditions, it is likely that the natural hybrids in situ utilise the fungus of only one parental species. Supporting these observations, the AFLP analyses indicated that while the parental species always possessed genetically distinct fungal strains, the hybrids may share the mycorrhizal fungus of one parental species or possess a genetically distinct fungal strain which is more closely related to the fungus of one parental species than the other. The work on Caladenia hybrids revealed that C. falcata has a broadly compatible fungus that germinated seeds of C. falcata, the hybrid C. falcata x longicauda, and species with different degrees of taxonomic affinity to C. falcata. In general, germination was greater from species that were more closely related to C. falcata: seeds from Caladenia species generally germinated well on most C. falcata isolates; species from same subtribe (Caladeniinae) germinated well to the stage of trichome development on only some of the fungal isolates and rarely developed further; and seeds from species from different subtribes (Diuridinae, Prasophyllinae, Thelymitrinae) or tribes (Orchideae, Cranichideae) either germinated well to the stage of trichome development but did not develop further, or did not germinate at all. The AFLP analysis of the fungal isolates revealed that the fungi from each location were genetically distinct. In situ seed baiting was used to study the introduction, growth and persistence of orchid mycorrhizal fungi. A mycorrhizal fungus from Caladenia arenicola was introduced to sites within an area from which the orchid and fungus were absent, adjacent to a natural population of C. arenicola. In the first growing season, the fungus grew up to 50 cm from its introduction point, usually persisted over the summer drought into the second season and even into the third season, stimulating germination and growth to tuber formation of the seeds in the baits. Watering the inoculated areas significantly increased seed germination. Mycorrhizal relationships in Drakaeinae were less specific than in Caladeniinae. A study of the species Spiculaea ciliata revealed that this species, when germinated symbiotically, develops very rapidly and has photosynthetic protocorms, unlike all other members of the Drakaeinae. An AFLP analysis of the fungal isolates of this species grouped the isolates according to whether they had been isolated from adult plants or reisolated from protocorms produced in vitro. Isolates were genetically distinct when compared before germination and after reisolation. A cross-species symbiotic germination study of seeds of three Drakaea species and one Paracaleana species against fungal isolates from the same species and several other Drakaeinae species revealed lower specificity in this group than previously thought. A number of fungal isolates from Drakaea and Paracaleana species germinated two or more seed types, while all seed types germinated on fungal isolates from other species and the seed of Drakaea thynniphila germinated to some extent on every fungal isolate tested. An AFLP analysis of the Drakaeinae fungal isolates supported this information, revealing little genetic differentiation between the fungi of different orchid species. An ex situ seed baiting technique was used to examine the role of mycorrhizal fungi in microniche specialisation in the narrow endemic Drakaea. Soil samples from within and outside two Drakaea populations were tested for germination of the relevant seed types. In both cases, germination was significantly higher on soil samples from within than outside the populations, suggesting that the relevant mycorrhizal fungi may be restricted to the same microniches as the Drakaea species. The presence of similar fungi at distant, disjunct locations may be related to the extreme age and geological stabilityof the Western Australian landscape. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. It appears that the mycorrhizal relationships in these groups of orchids are not as specific as was previously thought. For reintroduction work, a broad sampling strategy is necessary, as it cannot be assumed that the same orchid species has the same fungus at different locations. A broadly compatible fungus may be of considerable utility in conservation work, such as in situations where a specific fungus appears to have poor saprophytic competence or where soil conditions have been altered. Seed baiting studies provide additional data on fungal distribution in situ. In general, molecular data do not provide information about efficacy or fungal distribution, so research programs that combine symbiotic germination studies with seed baiting investigations and genetic analyses of the fungi will provide the maximum benefit for designing more effective conservation programs.

Thesis (Ph.D.)-- University of Western Sydney, Macarthur, Dept. of Biological Sciences, 1997.

Thesis (Ph.D.)--University of Adelaide, Dept. of Soil and Water, 2002? / Bibliography: leaves 172-197.

Thesis (M.Sc.) (Hons.) -- University of Western Sydney, Nepean, 1998. / Bibliography : p. 89-102.

Thesis (Ph. D.)--University of Montana, 2007. / Title from title screen. Description based on contents viewed Aug. 12, 2008. Includes bibliographical references.

Thèse (D.R.Min.) -- Université du Québec à Chicoutimi, programme en association avec l'Université du Québec à Montréal, 2006. / Bibliogr.: f. 150. Document électronique également accessible en format PDF. CaQCU

A seca é um dos principais problemas que afetam a produção do feijoeiro. A despeito da importância de caracteres fenotípicos radiculares, muitos dos esforços de melhoramento genético da cultura tem focado na seleção de cultivares com maior produção de grãos. A simbiose estabelecida entre plantas e FMA aumentam o potencial de captação de água no solo através das extensas redes formadas pelas hifas e alteram vias metabólicas vitais para a manutenção das relações hídricas da planta. O modelo de interação feijoeiro (BAT 477) colonizado por uma mistura de FMA (Glomus clarum, Acaulospora scrobiculata e Gigaspora rosea) foi submetido a um déficit hídrico de 96 h durante o pré-florescimento. O transcritoma global de raízes inoculadas e não-inoculadas, sujeitas ou não à seca, foi comparado por RNA-Seq. Um conjunto de 71 transcritos foram induzidos por FMA durante a seca. Comparando-se os tratamentos estresse e controle, 12.086 unigenes foram regulados em plantas inoculadas e 11.938 em não-inoculadas, refletindo o alto potencial de tolerância da linhagem BAT 477 e indicando que a presença de FMA produz uma regulação fina no perfil de expressão de genes regularmente envolvidos na resposta da planta ao estresse. Foram selecionados 15 fatores de transcrição e seus perfis de expressão foram caracterizados por RT-qPCR tomando-se três períodos, 48, 72 e 96 h de déficit hídrico. Plantas inoculadas ativaram a expressão destes genes mais tardiamente (após 72 h), refletindo melhorias nas condições hídricas da planta que adiam a percepção do estresse. Adicionalmente, a expressão de 23 transcritos foi avaliada em três amostras teciduais diferentes obtidas por microscopia de microdissecção a laser. Glucan 1,3 ?-Glucosidase e PIP2,3, foram detectados somente em células do córtex radicular contendo arbúsculos indicando uma possível indução tecido específica dependente da presença dos fungos. Análises complementares apontaram a regulação de 171 unigenes envolvidos na resposta das FMA ao estresse. Estes resultados validam a hipótese inicial de que a inoculação com FMA altera os perfis de expressão de genes vitais para a resposta da planta ao déficit hídrico / Drought is one of the main problems that affect common bean\'s production. Despite the importance of root fenological characters, breeding efforts for the culture have focused on the selection of cultivars for grain yield. The symbiosis stablished between AMF and plants enhances the potential of water absorption from the soil through an extensive net formed by hyphae and alters vital metabolic pathways involved in the maintenance of the water relations in plants. The interaction model common bean (BAT 477) colonized by a mixture of AMF (Glomus clarum, Acaulospora scrobiculata and Gigaspora rosea) was exposed to a water deficit regime of 96 h during pre-flowering. Global transcriptome from inoculated and non-inoculated roots, exposed or not to drought, were compared through RNA-Seq. A set of 71 transcripts was induced by AMF during drought. Comparing both stress and control treatments, 12,086 unigenes were regulated in inoculated plants, and 11,938 in non-inoculated, reflecting the great tolerance potencial of the lineage BAT 477 and indicating that the presence of AMF produces a fine tune regulation on the expression of genes regularly involved on the drought response of the plant. It was selected 15 transcription factors and their expression profiles were characterized through RT-qPCR taking 3 periods, 48, 72 and 96 h of water deficit. AM plants activated earlier (after 72 h) the expression of these genes, reflecting improvements on the water conditions of the plant that delay the stress perception. Additionally, the expression of 23 transcripts was evaluated on three different tissue samples obtained through laser microdissection microscopy. Glucan 1,3 ?-Glucosidase and PIP2,3, were detected only in cortical cells containing arbuscules, pointing to a possible tissue specific induction dependent of the presence of the fungus. Additional analysis point to the regulation of 171 unigenes involved on the response of the AMF to drought. These results corroborate the initial hypothesis that the inoculation with AMF alters the gene expression profiles of genes that are vital for water deficit response in plants

This study examined the rehabilitation potential of AM fungi with organic and inorganic fertilisers under pot and field trial conditions as well as their interaction with rhizospheric organisms and specific functional groups. In addition, the study highlighted the effects of land-use management on AM fungal populations in soil and the mycorrhizal status of some selected plants from one of the study sites. The study focussed on two sites that differ in operational activities and these included a mined area that was to be rehabilitated and a commercial farming site. A pot trial was conducted using an overburdened soil resulting from kaolin clay mining. Pots were seeded with Cynodon dactylon and treated with either Organic Tea or NPK (3:1:5) fertiliser, with or without AM fungal inoculum. The compatibility of these fertilisers with AM fungi was assessed by plant growth and percentage root colonisation. Maximum shoot height and plant biomass were observed at the 28th week with NPK (3:1:5) fertiliser supporting mycorrhizal colonisation by 80%. The result indicated the potential of AM fungi to be used in rehabilitation with minimal phosphate fertiliser. Similarly, a field trial was set-up using 17 x 17 m[superscript 2] plots in the mining site that were treated with the same organic and inorganic fertilisers as well as with AM fungal inoculum in different combinations. The interaction between AM fungi and soil microbial population was determined using culture dependent and culture independent techniques. The culture dependent technique involved the use of soil dilution and plating on general purpose and selective media. The result showed that there was no change in the total culturable bacterial number in the untreated and AM fungal treated plots, while a change in species composition was observed in the functional groups. Different functional groups identified included nitrogen fixing bacteria, pseudomonads, actinomycetes, phosphate solubilisers and the fungal counterparts. Gram-positive bacteria were observed as the predominant phenotypic type, while nitrogen fixers and actinomycetes were the predominant functional groups. Species identified from each functional group were Pseudomonas fulva, Bacillus megaterium, Streptomyces and actinomycetales bacteria. Meanwhile, fungi such as Ampelomyces, Fusarium, Penicillium, Aspergillus, Cephalosporium and Exserohilium were identified morphologically and molecularly. Furthermore, the mining site had a significantly higher bacterial number than the farming site thereby indicating the effects of land-use management on culturable bacterial numbers. The culture independent technique was carried out by cloning of the bacterial 16S rDNA and sequencing. Identified clones were Bradyrhizobium, Propionibacterium and Sporichthya. A cladogram constructed with the nucleotides sequences of identified functional species, clones and closely related nucleotide sequences from the Genbank indicated that nucleotide sequences differed in terms of the method used. The activity and establishment of the introduced AM fungal population was determined by spore enumeration, infectivity assay, percentage root colonisation and assessment of glomalin concentrations. The results indicated that the two land use types affected AM fungal populations. However, the establishment of AM fungi in the farming site was more successful than in the mining site as indicated by the higher infectivity pontential. Selected host plants, which were collected around the mine area, were observed to be mainly colonised by AM fungi and these were identified as Pentzia incana, Elytropappus rhinocerotis, Euphorbia meloformis, Selago corymbosa, Albuca canadensis and Helichrysum rosum. These plant species were able to thrive under harsh environmental conditions, thereby indicating their potential use as rehabilitation host plants. Generally, the findings of this study has provided an insight into the interaction between arbuscular mycorrhizal fungi and other soil microorganisms in two fields with differing land use management practices.