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 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.

Projected climate change may alter soil carbon dioxide (CO2) efflux from terrestrial ecosystems; yet disentangling effect of plant species from climate drivers remains a key challenge. We explored the effects of the dominant plant species, warming, and precipitation distribution on soil CO2 efflux, its underlying components, and mycorrhizal abundance in southern post oak savannah. Post oak savannah in the south-central US are dominated by three contrasting plant functional types: Schizachyrium scoparium (Michx.) Nash. (little bluestem) a C4 grass, Quercus stellata Wangenh.(post oak)a C3 deciduous tree, and Juniperus virginiana L. (eastern redcedar) a C3 evergreen tree. Monocultures and tree-grass plots were warmed using infrared heaters and precipitation events were manipulated to intensify summer drought and augment cool season precipitation. Soil CO2 efflux, the root, bacterial and hyphal components of CO2 efflux, and mycorrhizal abundance were measured. Soil CO2 efflux varied with seasonal changes in soil VWC and temperature, with higher soil CO2 efflux rates in the spring and lower rates in both the cooler winter season and at the end of the dry summer period. There was no relationship between root length density or root mass density and soil CO2 efflux during the short term precipitation distribution campaigns. Partitioning of root, fungal, and bacterial component contribution to soil CO2 efflux indicated a substantial contribution of bacterial respiration to soil CO2 efflux within this system. There was no relationship between microbial biomass [microbial dissolved organic carbon (DOC)] and soil CO2 efflux, or root length (or mass) density and microbial biomass. This suggests that species and climatic effects on root and microbial activity drive soil CO2 efflux. As plant species within this system differed in their association with mycorrhizal fungi and had a strong effect on the individual components of soil CO2 efflux, we conclude that shifts in vegetation cover and growth and the response of vegetation to long term warming and potential future extreme precipitation events (e.g., large preciptation events, prolonged drought) will be major drivers of changes in soil carbon (C) dynamics and associated soil CO2 efflux.

Thesis (Ph. D.)--University of California, Davis and San Diego State University, 2003. / Includes bibliographical references (leaves 142-144). Also available via the World Wide Web. (Restricted to UC campuses).

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

Cereal crops such as maize and sorghum are economically important in South Africa (SA) as a staple food diet. In order to meet the needs of South Africa’s growing population, higher yields in crop production need to be attained. However, the two major stress factors that affect yield production and require primary attention are nutrient deficiencies and pest infestations. Research is now being focused on certain endophytes that have become a valuable tool for agriculture as they protect crops against the above-mentioned stresses. The endophyte focused on in this study was Arbuscular Mycorrhizal fungi (AMF). This research was aimed at developing an in vitro culture system for SA cereal crops to enable interaction studies of endophytes. This dissertation is divided into two parts; the first part focused on the development of an in vitro culture system, the assessment of sorghum plant growth and exudate production in the presence of the Glomus intraradices strain. The results indicated that sorghum produces the required root exudates in the second stage of growth. Using high pressure liquid chromatography with mass spectrometry (HPLC/MS), it was noted that sorghum produced phytochemicals as chemoattractants for the respective endophytes. However, it was documented that when the plant underwent certain stresses they produced exudates, which acted as phytotoxic compounds that destroyed symbiotic organisms around sorghum rhizophere. The second part focused on optimization of the surface sterilization of maize seeds. The results indicated that maize contained unidentified endophytes, which negatively affected plant development. Surface sterilization of maize seeds was accomplished. The successful in vitro development can be used for future use to study plant development. Understanding plant development and interaction with symbiotic endophytes would not only be of great benefit but would also make it easier to create a biocontrol agent in vitro, which would bring about high crop yields at cost-effective prices and would be less labour intensive. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

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.