Arbuscular mycorrhizal fungi (AMF) are an ancient group of obligate symbionts, colonizing the roots of over 72% of land plants, increasing the uptake of nutrients from the soil, and providing many fitness benefits to their host plants. The multinucleate and coenocytic nature of AMF have interested researchers for decades, leading to many theories of the evolution, and genetic organization of these organisms. Recent findings propose that AMF carry two types of strains, identified based on putative MAT-loci, as either homokaryotic, carrying multiple and genetically similar nuclei, or dikaryotic with co-existing nuclei deriving from two parental strains. In other fungi, hyphal fusions, or anastomosis, between compatible strains results in nuclear transfer, creating heterokaryotic spore progeny. It has been hypothesized that dikaryotic AMF strains arose from the anastomosis between compatible homokaryons harbouring different nucleotypes. The goal of this research is to determine whether anastomosis events, known to occur in other fungi, like homokaryon-homokaryon, homokaryon-dikaryon (Buller phenomenon), and/or dikaryon-dikaryon nuclear exchanges also occur in AMF. To achieve this, the anastomosis frequencies between 15 crosses of homokaryotic and dikaryotic strains of the model AMF species Rhizophagus irregularis were examined using microscopy and droplet digital PCR (ddPCR) to determine if nuclear transfer between strains is possible. Overall, these experiments build on the existing evidence of compatible interactions between strains of R. irregularis.

The mutualistic associations between Arbuscular mycorrhizal (AM) fungi and plant roots are ancient and ubiquitous across the plant kingdom, where AM fungi provide Phosphorus, Nitrogen, and water to the plant, and receive photosynthetically fixed Carbon in the form of fatty acids and sugars in return. Moreover, AM fungi are associated with increased plant resistance to both abiotic and biotic stressors such as drought and viral pathogens. Frequently used in agriculture, AM fungi are observed to increase crop yields and decrease chemical fertilizer needs for many economically important plant species. The potential to increase AM fungal effectiveness remains a driving force for current research. To determine their role in establishing and/or supporting AM symbiosis, we propose a reverse genetic study of two genes in the model legume Medicago truncatula. Based on RNA sequencing data indicating increased expression during AM symbiosis, we selected one gene that encodes for NAC TF-like protein, which belongs to a large family of plant transcription factors primarily involved in regulating the secretion of defence hormones. The second gene selected, PALM1, was recently discovered to play a role in the regulation of the trifoliate leaf structure of M. truncatula. We hypothesize that the genes under study play mechanistic roles in regulating AM fungal symbiosis and that we will observe a difference between the colonization rates of corresponding gene mutants and control groups. Firstly, we explored the involvement of the PALM1 and NAC TF genes by examining the root developmental phenotype of Medicago truncatula mutants. Secondly, we employed symbiosis assays to investigate the colonization rates of the genes in question. Results indicated that the NAC TF gene had no consistent role in the AM symbiosis, while the PALM1 gene revealed promising results, where significant increases in colonization rates were observed in PALM1 mutants throughout repeated experiments. Future research involves using this study to help pursue more effective ways to use AM fungi symbiosis in sustainable agro ecosystems.

Arbuscular mycorrhizal fungi (AMF) play an important role in nutrient cycling and growth of flax (Linum usitatissimum L.). However, limited information is available regarding the symbiotic association between flax and AMF in field environments. A study was conducted to survey AMF communities colonizing flax grown in Saskatchewan. Additionally, field and growth chamber studies investigated the impact of AMF inoculation on nutrient uptake and growth of flax. Eighteen commercial flax fields were surveyed to assess mycorrhizal colonization of flax and to assess the impact of agricultural practices and soil abiotic factors on AMF activity. The flax root-associated AMF communities were explored using a 454 sequencing method, together with microscopic-based measurements of root AMF colonization and soil spore density. High levels of root colonization were detected in most flax fields. Of the 222 AMF operational taxonomic units (OTUs) identified in flax roots, 181 OTUs clustered as Funneliformis-Rhizophagus, 19 as Claroideoglomus, 14 as Paraglomus, six as Diversisporales and two as Archaeospora. Results suggest that tillage influenced the composition of AMF communities colonizing flax, and reduced relative AMF abundance and species richness. Additionally, AMF community characteristics were related to soil abiotic factors such as pH, EC, available phosphorus and nitrogen. Field experiments were conducted over two years (two sites per year) using a commercial AMF inoculant applied at three rates (0, 1X, and 2X the recommended rate) with or without P fertilizer (16.8 kg ha-1). The response of flax cultivars to AMF inoculation was examined in a growth chamber experiment. In addition, 454 sequencing was employed to examine the impact of AMF inoculation on root-associated AMF communities. Under field conditions, only one site showed increased root colonization with AMF inoculation. Flax responded to AMF inoculation differently under different field conditions. At the two sites with intermediate initial soil P level, evidence of increased above-ground biomass and plant nutrient uptake with AMF inoculation was observed. However, such an effect was not detected when P fertilizer was combined with the inoculation. At a low P site and an irrigated site, P application accounted for all of the increases in plant nutrient uptake and biomass of flax, whereas no responses to AMF inoculation were detected. The 454 sequencing revealed different inoculation-induced changes in the diversity and composition of root-associated AMF communities between sites, which was possibly related to different field environments and native AMF communities. In the growth chamber, AMF inoculation resulted in general increases of plant nutrient uptake among cultivars, but only one cultivar showed enhanced biomass with inoculation. The diversity of AMF communities colonizing different flax cultivars was generally reduced by AMF inoculation. Community composition shifted under AMF inoculation, and the shifts appeared to be cultivar specific. These results suggested that benefits of AMF inoculation in flax production are limited and currently not predictable, and the degree of response is likely dependent on a myriad of soil and environmental conditions.

Podocarpus cunninghamii is an endemic New Zealand conifer that, in pre-human times, formed extensive forest communities across the eastern South Island high country. Anthropogenic disturbances have reduced the distribution of Podocarpus cunninghamii communities such that they now exist mainly as small and isolated remnants within a highly modified, predominantly pastoral landscape. Very little is known of the ecology of high country Podocarpus cunninghamii communities, and without this information it is not possible to develop an ecological basis for their restoration. This thesis explores the ecology of Podocarpus cunninghamii in the eastern South Island high country, investigating factors that potentially affect the restoration of Podocarpus cunninghamii within this environment, with special attention paid to the role of arbuscular mycorrhizal fungi (AMF). Field investigations of Podocarpus cunninghamii communities showed that they contain a high degree of floristic and structural variation determined by soil and climatic variables. Analysis of age and size class distributions suggest that Podocarpus cunninghamii has more than one regeneration strategy, and can regenerate within intact forest following the opening of small canopy gaps or can undergo large-scale recruitment following catastrophic disturbance. Field and glasshouse experiments investigating growth and nutrient responses of Podocarpus cunninghamii to different AMF inoculants found that Podocarpus cunninghamii responses are dependent on both AMF type and grass competition. Finally, investigation of Podocarpus cunninghamii carbon stocks showed that they are less than that of other New Zealand forest types, but are greater than that of grazed pastures. Successful restoration of high country Podocarpus cunninghamii communities will require the incorporation of associated species based on local environmental conditions, and will also need to allow for disturbance processes. AMF may have an important role to play in restoration by reducing seedling production times and by increasing the competitiveness of Podocarpus cunninghamii when in competition with exotic grasses.

Soil phosphorus (P) is often unavailable in SK soils due to immobilization by microbial biomass and complexation with cations. The prohibition of synthetic fertilizer use in organic systems means farmers rely on crop rotation or approved inputs to supply P for crops. Legumes in crop rotation add P to soil through decomposition and deposition, and approved fertilizers such as bone meal (BM), rock phosphate (RP), and composted manure add P to soil through dissolution. Arbuscular mycorrhizal fungi (AMF) improve crop access to soil P. The fungi colonize roots of host crops, allowing roots to reach immobile pockets of soil P. Colonization by AMF is usually decreased or delayed following partial fallow periods, non-host plants, and the addition of soluble P fertilizers. This thesis consists of two studies. For the first, the effects of crop rotation were tested on AMF colonization and soil P dynamics. Colonization by AMF of mycorrhizal crops was examined following a non-mycorrhizal crop, a partial fallow period, and mycorrhizal crops. All crops were colonized evenly (63-70%) at flowering despite non-mycorrhizal and partial fallow periods, and the sequence most depleted in soil N (wheat-barley) had the lowest colonization in August (36%). The second study evaluated soil P and plant N and P after applications of BM, hydroxyapatite (HAP), and sheep manure compost. Compost application increased plant P uptake compared to the control (1.26 vs. 0.71 mg pot-1), while applications of BM and HAP alone did not. Compost application did not affect AMF colonization of wheat (Triticum aestivum L.). Overall this research highlights the importance of legumes and composted manure use in organic systems. Legume use in crop rotation simultaneously increased soil P deposition and may have preserved AMF communities despite fallow periods and non-host crops in rotation. Conditions normally affecting AMF colonization in conventional systems did not apply. The use of composted manure in the greenhouse study resulted in the greatest P uptake and concentrations in wheat. Thus the use of legumes and composted manure may increase P availability to crops directly and indirectly: directly through soil P deposition and perhaps indirectly through the preservation of AMF communities.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

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

Arbuscular mycorrhizas (AM) are the plant root-fungus interactions that are most widespread mycorrhiza in nature. As classically defined, there are two major AM morphologies named after the plant genera in which they were first described: Arum- (intercellular hyphae with arbuscules mainly in inner root cortex), Paris- (extensive hyphal coils in outer root cortex), as well as intermediate morphotypes. In this study, dandelions and chives harvested in Saskatoon (SK, Canada) were examined for AM colonization and morphological types. A Multiple Quantitation Method (MQM) was used for assessing fungal colonization intensity using magnified epifluorescence images of lactofuchsin stained roots, plus details analyzed by high-resolution confocal fluorescence imaging. The results showed that host plants harbored diverse endorhizal fungi, including arbuscular mycorrhizal fungi (AMF), septate endophytes (SE) and fine endophytes (FE), with varying abundances. The soil properties were assessed with respect to P status, organic matter and pH, but there was no correlation with the fungal abundance in this study. Both dandelion and chive roots had Arum- and Paris-type AM. In order to assess the applicability of a current model, I studied quantitative relationship between the cell packing pattern and AM morphotype. Cross sections of host roots were analyzed with Image J software to calculate the proportion of air spaces. The abundance of arbuscules (Arum-type) and hyphal coils (Paris-type) were significantly different in chive and dandelion roots. However, there was no difference in the proportion of air spaces in the inner or outer cortex. Therefore, host root cell packing does not appear to influence AM morphotype at least in the samples in this study. AM fungal diversity was preliminarily investigated by nested PCR with group specific primers, showing multiple PCR bands within root samples, and indicating the potential complexity of AMF groups. Further work to sequence the PCR products is needed to elucidate the AMF groups present.

There are no seeding rates established for organic production of field pea and lentil in Saskatchewan and organic producers must rely upon rates recommended for conventional production of these crops. These seeding rates may not be suitable for organic production as the two systems differ in the use of inputs and in pest management. The objectives of this study were to determine an optimal seeding rate for organic production of field pea and lentil in Saskatchewan considering a number of factors, including yield, weed suppression, soil nitrogen (N) and phosphorus (P) concentrations, soil water storage, colonization of crop roots by arbuscular mycorrhizal fungi (AMF), plant P uptake, and profitability. A field experiment was conducted to determine the optimal seeding rates of field pea and lentil. Field pea seeding rates were 10, 25, 62, 156 and 250 plants m-2 and lentil seeding rates were 15, 38, 94, 235 and 375 plants m-2. Sites were established at Vonda, Vanscoy and Delisle, SK using a randomized complete block designs with summerfallow and green manure treatments included for each crop. Seed yield increased with increasing seeding rate for both crops, up to 1725 kg ha-1 for field pea and 1290 kg ha-1 for lentil. Weed biomass at physiological maturity decreased with increasing seeding rate for both crops. In field pea, weeds were reduced in weight by 68%, while lentil reduced weed biomass by 59% between the lowest and highest seeding rates. <p>Post-harvest soil phosphate-P levels did not change consistently between treatments, indicating that there was no trend in soil P concentration with seeding rate. Post-harvest soil inorganic N, however, was higher for the summerfallow and green manure treatments than for the seeding rate treatments in both crops. Inorganic N was higher at some sites for the highest two seeding rates in field pea. Soil water storage following harvest was not affected by treatment.<p>Colonization of crop roots by AMF increased for lentil with increasing seeding rate, but the same trend was not observed in field pea. A growth chamber experiment to study the rate of colonization of field pea between 10 and 50 d after emergence did not show any differences in AMF colonization between seeding rates. Colonization levels were high (70 to 85%) for both crops in both the field and growth chamber. Arbuscular mycorrhizal fungi colonization and seeding rate had no effect on plant P concentration for either field pea or lentil. Both crops became increasingly profitable as seeding rate increased. Field pea reached a maximum return at 200 plants m-2 and lentil return increased to the highest seeding rate of 375 plants m-2. Organic farmers should increase seeding rates of these crops to increase returns and provide better weed suppression.

There is accumulating evidence that arbuscular mycorrhizal fungi (AMF) produce a glycoprotein called glomalin, which has the potential to increase soil carbon (C) and nitrogen (N) storage, thereby reducing soil emissions of carbon dioxide (CO2) and nitrous oxide (N2O) into the atmosphere. However, other soil microorganisms such as plant growth-promoting rhizobacteria (PGPR) that interact with AMF could indirectly influence glomalin production. The objectives of this study were to determine the effects of AMF and PGPR interactions on glomalin production and identify possible combinations of these organisms that could enhance C and N storage in the rhizosphere. The effects of AMF and PGPR interactions on pea (Pisum sativum L.) growth and correlations between glomalin production and plant growth also were assessed.<p> A series of growth chamber and laboratory experiments were conducted to examine the effect of fungal and host plant species on glomalin production by comparing the amounts of glomalin produced by Glomus clarum, G. intraradices, and G. mosseae in association with corn (Zea mays L.), in addition to examining differences in the ability of corn, pea, and wheat (Triticum aestivum L.) to support glomalin production by G. intraradices. There were no significant differences in glomalin production [measured in the rhizosphere as Bradford-reactive soil protein (BRSP)] by the three AMF species, whereas host plant significantly affected glomalin production. Specifically, higher BRSP concentrations were found in the rhizosphere of corn as compared to pea and wheat.<p> Additionally, the effect of long-term storage on the growth promoting traits of the PGPR strains selected; namely, Pseudomonas cepacia R55 and R85, P. aeruginosa R75, P. putida R105, and P. fluorescence R111 were investigated. These bacterial strains previously had been identified as PGPR, but had since undergone approximately twenty years of storage at -80¢ªC; thus, it was necessary to confirm that these strains had retained their plant growth promoting characteristics. Apparently, long-term storage had no significant adverse effect on the PGPR strains as all strains increased the total biomass of wheat significantly and demonstrated antagonism against fungal pathogens.<p> The possibility that spore-associated bacteria (SAB) could influence AMF associations, thereby affecting glomalin production, and subsequent crop yield potential was assessed. This was achieved by first isolating bacteria from disinfested spores of the AMF species and determining their potential as PGPR for wheat. According to fatty acid methyl ester (FAME) profiles, four genera of bacteria were isolated from AMF spores namely; Arthrobacter, Bacillus, Micrococcus, and Paenibacillus, of which Bacillus species were the most common SAB. None of these isolates, however, showed growth promoting abilities on wheat.<p> Based on the preliminary findings, the combined effects of the three AMF species and the five PGPR strains were examined on plant growth and glomalin production under gnotobiotic conditions using pea as the host plant. Interactions between G. intraradices and R75, R85, or R105 resulted in increased BRSP concentration in the mycorrhizosphere of pea. Additionally, significant interactions were observed between the AMF species and PGPR strains on BRSP concentration in pea rhizosphere under non-sterile conditions. As observed under sterile conditions, the co-inoculation of pea with G. intraradices and R75 or R85 increased BRSP concentrations in the rhizosphere of pea grown in non-sterile soil, although interaction effects were not significantly different from the control or when G. intraradices was applied alone. Significant AMF and PGPR interactions were observed to affect AMF colonization; however, the combination of these organisms did not significantly affect pea growth, nutrient uptake, and C and N storage in the plant rhizosphere. No correlations were detected between glomalin-related soil protein (GRSP), pea growth, nutrient concentrations in the plant tissue, and soil organic C and N content. This study demonstrated that although the potential exists to manipulate certain AMF and PGPR to enhance glomalin production, co-inoculation of AMF and PGPR did not enhance plant growth or C and N storage beyond that achieved by inoculation of either organism.