The symbiotic unit of an arbuscular mycorrhizal fungus and its host is able toachieve and maintain far higher inflow of nutrients than non-mycorrhizal roots. The colonization strategy of the mycobiont within the plant is intrinsic to the symbiosis with respect to both structural adaptations and nutrient exchange. An investigation into the effect of Glomus etunicatum colonization on the structure and phloem transport in Eragrostis curvula (Umgeni) allowed for greater insight into the dynamic of the symbiosis. The combined use of stains (such as Trypan Blue, Chlorazol Black, Safranin and Fast Green), and techniques, (such as freeze-microtome transverse sectioning and permanent slide preparations) contributed to a successful general observation of an intermediate colonization strategy using light microscopy methods. However, clarity into structural detail of mycorrhizal forms required electron microscopy studies. The SEM method used with freeze fracture was a relatively quick and simple method allowing for the observation of surface and internal features. The TEM method allowed for highresolution images providing insight into the variations in the apoplasmic compartmental form, and how this may relate to the function of the symbiosis with regard to fungal coils or arbuscules. The apoplasmic nature of mycorrhizas was substantiated and no symplasmic connections were found between symbionts. Fluorescence studies demonstrated that 5,6-carboxyfluorescein was transported through the phloem into the roots of E. curvula, but remained predominantly in the root phloem. Unloading only occurred in optimal nutrient exchange areas of meristimatic lateral or apical growth regions. It was not possible, using fluorescence techniques and related equipment available, to conclusively establish if there were symplasmic connections between the mycobiont and its host or if bidirectional transfer of nutrients occurred at the same interface.

South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.

Please read the abstract (Summary) in the section 00front of this document / Dissertation (MSc (Botany))--University of Pretoria, 2007. / Plant Science / MSc / Unrestricted

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.

La plupart des espèces végétales terrestres vivent en symbiose avec les champignons mycorhiziens à arbuscules (MA). Il s’agit d’une symbiose très ancienne datant de plus de 400 millions d’années. Les champignons MA sont des champignons du sol qui appartiennent aux Gloméromycètes. Ils sont présents dans la plupart des écosystèmes terrestres. Ainsi, ils peuvent être considérés comme une composante intégrale des racines des plantes. Ils forment dans les cellules racinaires corticales des structures fonctionnelles essentielles appelées arbuscules où ils apportent à la plante des minéraux nutritifs en échange de sucres. L’auxine est une phytohormone impliquée dans de nombreux processus de développement des plantes, y compris la dominance apicale, les tropismes, la structuration vasculaire et la formation de racines latérales. Le principal objectif de notre travail était d’étudier de manière approfondie le rôle de l’auxine dans le processus de développement des mycorhizes. On sait déjà que la symbiose MA stimule la formation de racines latérales dans les plantes hôtes, ce qui pourrait être due à une modification du métabolisme de l’auxine, de son transport ou de sa perception. Les microARNs (miARNs) sont des molécules d’ARN non codantes de ~ 21 nucléotides capables de réprimer l’expression de gènes en ciblant et clivant spécifiquement leur ARNm correspondant. Plusieurs miARNs interagissent avec la signalisation de l’auxine et parmi eux miR393 qui cible les récepteurs à l’auxine. Nous avons étudié le rôle de miR393 dans la colonisation mycorhizienne. Nous mettons en évidence que chez Solanum lycopersicum (Solanacées), Medicago truncatula (Fabaceae) et Oryza sativa (Poaceae), l’expression des précurseurs de miR393 diminue lors de la mycorhization. En outre nous montrons que DR5-GUS, un gène rapporteur de réponse à l’auxine, est préférentiellement exprimé dans les cellules de la racine contenant les arbuscules. En sur-exprimant miR393 dans les racines et donc en régulant négativement l’expression des gènes de récepteurs à l’auxine, nous montrons également que les arbuscules ne se développent pas normalement. En tant que composantes des complexes récepteurs d’auxine, les protéines Aux/IAA jouent un rôle majeur dans la voie de signalisation de l’auxine en réprimant l’activité des facteurs de transcription de type ARF. Nous avons vérifié dans des racines de tomate mycorhizées l’expression de 25 gènes AUX/IAA. Nous nous sommes concentrés sur IAA27 dont l’expression est induite lors des premiers stades de la symbiose MA. Nous observons qu’une répression par ARNi de l’expression de IAA27 dans des plants de tomate conduit à une forte diminution de la colonisation MA et du nombre des arbuscules. Puis nous montrons par des approches différentes que la régulation positive de la mycorhization par IAA27 est liée à la biosynthèse des strigolactones. Globalement, ces résultats appuient fortement l’hypothèse selon laquelle la signalisation de l’auxine joue un rôle important aussi bien dans le stade précoce de la mycorhization que dans la formation des arbuscules. / Most land plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. This is a very ancient symbiosis dating back to 450 million years. AM fungi are soil fungi that belong to the Glomeromycota. They are present in most terrestrial ecosystems. Thus they can be considered as an integral root component of plants. They form essential functional structures called arbuscules in root cortical cells at which mineral nutrients are released to the plant in exchange of sugars. The phytohormone auxin is involved in many developmental processes in plants, including apical dominance, tropisms, vascular patterning and lateral root formation. The main objective of our work was to investigate further the role of auxin in the mycorrhizal developmental process. We already know that AM symbiosis stimulates the lateral root formation in host plants, which could be due to modification of auxin metabolism, transport or perception. The microRNAs (miRNAs) are ~21-nucleotides noncoding RNAs that target corresponding mRNA transcripts for cleavage and transcriptional repression. Several miRNAs interact with auxin signaling and among them miR393 that targets auxin receptors. We investigated the role of miR393 in AM root colonization. In Solanum lycopersicum (Solanaceae), Medicago truncatula (Fabaceae) and Oryza sativa (Poaceae), expression of the precursors of the miR393 was down-regulated during mycorrhization. In addition DR5-GUS, a reporter for auxin response, was found to be preferentially expressed in root cells containing arbuscules. By over-expressing miR393 in roots and therefore down-regulating auxin receptor genes, arbuscules could not develop normally. As components of auxin receptor complexes, Aux/IAA proteins play a major role in auxin signaling pathway by repressing the activity of ARF type transcription factors. We checked the expression of 25 AUX/IAA genes in AM roots. Among them, we focused on IAA27 that was significantly up-regulated during the early stages of AM symbiosis. IAA27 down-regulation in plants led to a strong decrease of AM colonization and arbuscule abundance. We showed by different approaches that the positive regulation of mycorrhization by IAA27 was linked to strigolactone biosynthesis. Overall these results strongly support the hypothesis that auxin signaling plays an important role both in the early stage of mycorrhization and in the arbuscule formation.

Growth and element assimilation was investigated in post oak seedlings exposed to four different treatment combinations of fertilization and ectomycorrhizal inoculation. Element concentration in excised leaves was analyzed via particle induced X-ray emission spectrometry with a 1.8 MeV proton macrobeam. Mean growth was significantly different across the treatment groups as well as mean concentration of Mg, Al, S, K, Ca, Fe, Cu, and Zn. The data suggest that fertilization rather than mycorrhizal inoculation had a stronger influence on plant growth and nutrient uptake. A follow up study was conducted with a 3 MeV microbeam. A 850 μm2 scanned area of a post oak leaf produced topographical maps of 11 elements.

The aim with this study was to investigate factors that affect the occurrence of mycorrhizal fungi species of nature conservation concern in sand pine forests in Norrbotten County in Sweden. In 2019, the occurrence of fruit-bodies of the species in focus was inventoried in 21 forest stands in three different areas located in three municipalities on land that SCA Skog owns and manages in Norrbotten County. The fungi included in study had been noted in the studied areas in previous inventories between the years 2013–2016, which makes it possible to compare variation in fruit body production between years. Specifically, the study tested whether mean age and diameter of trees, and base area and size of forest stands affect the occurrence of fruit bodies of the studied mycorrhiza species in the forest stands. A multiple regression analysis found a positive significant relationship between the number of species that occurred and the size and base area in the forest stands, while there was no effect of mean age and mean diameter of the trees. This indicates that it is important to consider the size and base area of forest stands to maintain mycorrhizal fungi when planning management in dry sandy pine forests. However, because fruit body production is highly variable among years further research is required to get a better understanding of how tree structure affect mycorrhizal fungi.

6 Abstract This study aimed to compare the mycorrhizal fungal communities inhabiting the roots of invasive Pinus strobus L. and native Pinus sylvestris L. We also compared carbon allocation into ectomycorrhizal fungal (EcMf) and other structures of the two pine species. The aim was to assess the influence of mycorrhiza on the invasive potential of P. strobus in the protected areas of National Park Bohemian Switzerland. The two field experiments were conducted on three locations of each species. We estimated the EcM extramatrical mycelium (EMM) production by measuring the ergosterol content in sterile sand filled mesh-bags. Next measured variables were: biomass of ectomycorrhizal and saprotrophic sporocarps, fine roots biomass and leaf litter biomass to compare the one season production of each measured variables. The results revealed a major difference in EcM sporocarps production, whereas on the P. sylvestris sites was the production 100% higher. Same results came from the fine roots measurements: P. sylvestris had a higher fine roots production, which may be also related with the production of EMM, which was about 60% higher as well. The EcMf species richness on the P. strobus root-tips was as high as the native pine, but the species composition was different. The P. strobus prefers EcMf species with...

No description available.

The Allium tricoccum Aiton (ramps, aka wild leeks), a native spring ephemeral, is a cultural keystone species in Appalachia, a mountainous physiographic region encompassing 205,000 square miles of the eastern United States. People in Appalachia have long harvested ramps in the wild. However, growing demand for the plant in and outside the region has increased harvesting, resulting in threats to native populations. Agroforestry cultivation techniques and technical support for sustained-yield forest farming practices are needed to conserve ramps and meet increasing demand. Various techniques for assessing suitable production practices for ramps were explored in this dissertation, particularly examining best-suited ramp ecotypes, mycorrhizal treatment, and habitat suitability determination. In the first study, bulbs and seeds from diverse Appalachian locations were gathered and transplanted to a common experimental site to investigate the effects of different ramp ecotypes on growth, survival, and stress responses. Plant characteristics and stress measurements were recorded before transplantation and post-transplantation assessments. Specifically, the study investigated the germination rate of three ramp seed ecotypes and the growth, survival, and stress responses of eight native ramp bulb ecotypes and three commercially obtained seedling samples. Results indicated that above-ground growth, survival, and stress response on the ramp ecotypes differed significantly. The second study explored the impact of mycorrhizae on ramps and was evaluated by assessing the impact of Arbuscular Mycorrhizal Fungi (AMF) inoculation on Allium tricoccum. Four measurements, collectively referred to as parameter categories, were assessed. These included measures of 1) above-ground plant growth: leaf length, and leaf width; 2) stress measurement: transplant stress after a few days of transplant, and photosynthetic performance stress after a year of transplant; 3) survival analysis; and 4) mycorrhizal colonization rate. For each parameter category, three treatment comparison categories were conducted: 1) Positive control treatments: bulbs were planted from their native environment without treatment; 2) Negative controls: bulbs were treated with fungicide before planting to eradicate existing AMF in roots; and 3) The test group: bulbs were inoculated with commercial AMF (Atriva 500). Results indicated that mycorrhizal inoculation could increase ramp leaf length (P≤0.03). However, the impact varied by ecotype, highlighting the importance of considering local environmental conditions and ramp ecotype. Mycorrhizal inoculation did not impact ramp growth at the seedling stage. Mycorrhizal treatment increased the transplanted ramp's survival and stress tolerance (P≤0.001). The third study used multi-criteria decision-making (MCDM), the Analytic Hierarchical Process (AHP), and weighted linear combinations to model suitable habitats for ramps production. Ten habitat criteria were chosen (including five soil properties, three topographic parameters, and two land use properties) to assess the potential for growing ramps in seven counties in Virginia, West Virginia, Pennsylvania, and North Carolina. The percentage of highly suitable areas for ramps production in the studied counties ranges from 21.5% in Haywood County to 49.6% in Macon County. Similarly, moderately suitable areas range from 36.7% in Macon County to 54.5% in Lawrence County. Ground truthing was performed to validate the model. Ramp patch locations within each county were geocoded in the final suitability maps. Existing ramp patches were within the model's estimate of moderate to high site suitability ranges, suggesting the model is valid. Results of the study suggest that site suitability modeling could be useful for producers interested in growing ramps in forest farm settings across Appalachia. / Doctor of Philosophy / The ramp, also known as the wild leek, is an important food, medicinal and cultural resource for the people of Appalachia. However, increasing demand for ramps beyond this region has led to over-harvesting and threatens the plant's native populations. Appropriate cultivation techniques and technical support for sustained-yield forest farming practices are needed to conserve ramps and meet growing demand. This dissertation explores various techniques for assessing suitable production practices for ramps, including examining ramp ecotypes, mycorrhizal treatments, and habitat suitability determination. The first study evaluated the ecotypic variation among ramps collected from different geographic regions and their impact on plant performance. Ramp ecotypes displayed differences in above-ground growth, survival, and stress response. The second study examined the impact of mycorrhizae on ramps and found that mycorrhizal inoculation can increase ramp leaf length and survival and stress tolerance of transplanted ramps. However, this impact varied by ecotype, highlighting the importance of considering local environmental conditions and ramp ecotype. The third study involved developing a model to identify suitable habitats for growing ramps. The model was tested for seven counties across Virginia, West Virginia, Pennsylvania, and North Carolina. The percentage of land predicted as highly suitable for ramps production ranged from 21.5% in Haywood County, NC, to 49.6% in Macon County, NC. Moderately suitable lands ranged from 36.7% in Macon County, NC to 54.5% in Lawrence County, PA. Ground truthing confirmed the model's accuracy as geocoded existing ramp patch locations fell within the estimated suitable ranges. Site suitability modeling could be useful for people interested in growing ramps in forest farm settings across Appalachia. Overall, this research provides insights into best practices for ramp cultivation that help conserve a cultural keystone species and meet the growing demand for ramps.