Mycorrhiza Establishment Research Articles

Localized elongation of roots of cotton follows establishment of arbuscular mycorrhizas

In an experiment to determine the influence of mycorrhizal colonization on root development, fertilized seedlings of cotton were grown in long tubular pots. Inoculation of soil with an isolate of Glomus mosseae at 10, 25 or 40 cm below the soil surface resulted in spread of arbuscular mycorrhizas up and down the root system, and localized elongation of roots following colonization. Specific root length was not influenced by colonization. Increases in local root density at the point of inoculation were observed, though total root mass in relation to shoot mass declined following initiation of mycorrhizas.

High densities of arbuscular mycorrhizal fungi maintained during long fallows in soils used to grow cotton except when soil is wetted periodically.

Sequential harvests of cotton seedlings grown in soil cores enabled the quantification of the density of arbuscular mycorrhizal fungi to detect the effects of time, cultivation and periodic wetting of the soil. Cotton seedlings grown in soil cores from three locations formed arbuscular mycorrhizas at similar rates when cores were stored dry for up to 18 months. Disturbance of dry cores followed by dry storage for 18 months did not reduce the rate of establishment of mycorrhizas. Periodic wetting and drying of the cores, especially if the cores had first been disturbed, significantly reduced the rate of establishment of mycorrhizas. We suggest that long fallow disorder is possibly caused by falls of rain in clay soils of eastern Australia used to grow cotton. The proportion of the root with mycorrhizas at 3 wk was strongly correlated with the infection at 8 wk. We also suggest that it might be possible to predict maximum levels of infection and early uptake of phosphate of seedlings by determining the proportion of roots that are mycorrhizal 3 wk after emergence of cotton seedlings.

Detection of symbiosis‐related polypeptides during the early stages of the establishment of arbuscular mycorrhiza between Glomus mosseae and Pisum sativum roots

SUMMARYChanges in polypeptide contents following inoculation of roots of Pisum sativum L. (wild type cv. Frisson (myc+, nod+)) with the arbuscular mycorrhizal fungus Glomus mosseae were analysed by two–dimensional gel electrophoresis (2D‐PAGE). A different polypeptide pattern was obtained in a mycorrhiza–resistant pea genotype P2 (myc+, nod−) inoculated with G. mosseae. In order further to characterize the polypeptide modifications detected and to provide evidence for some possible symbiosis–related (SR) proteins, a time course experiment, from appressoria formation to fully developed symbiosis, was carried out on two genotypes allowing fungal colonization: the wild type and its isogenic mutant P56 (myc+, nod−). The same experiment was done with the mycorrhiza‐resistant pea genotype (myc+, nod−). After G. mosseae inoculation, we characterized 12 additional polypeptides in the two mycorrhiza‐compatible pea genotypes which were never observed in root extracts from the mycorrhiza‐resistant mutant. Five polypeptides were first detected in the early stage of the symbiosis (5 d of inoculation) while others were observed later (8 d of inoculation). The induction and accumulation of these polypeptides seem to be more correlated to the establishment of the functional symbiosis than to the recognition stages and appressorium formation. Furthermore, none of the additional polypeptides were detected in the mycorrhiza‐resistant pea genotype. This mutant was more characterized by a great repression of polypeptides. In addition, up‐regulated and down‐regulated polypeptides from the mycorrhiza‐compatible genotypes were different from those of the mycorrhiza‐resistant genotype.

Cellular and molecular approaches in the characterization of symbiotic events in functional arbuscular mycorrhizal associations

Arbuscular mycorrhizas represent the most widespread, and probably most ancient, type of plant–fungus association in which the large majority of terrestrial plants must have evolved with compatibility systems towards the fungal symbionts. Cellular interactions leading to reciprocal morphofunctional integration between symbionts during mycorrhiza establishment are complex. Some plant genes and cellular events may be shared with nodulation processes, but there is evidence of molecular modifications specific to arbuscular mycorrhiza formation. Plant defence responses, which are normally weakly activated during the symbiotic state, are strongly elicited by arbuscular mycorrhizal fungi in genetically altered, resistant hosts suggesting control over defence gene expression during establishment of a successful symbiosis. Modifications are also induced in the fungal symbionts during colonization of host tissues, with changes in wall metabolism and protein expression. Nothing is known of the genetic make-up of arbuscular mycorrhizal fungi which are recalcitrant to pure culture. Recent cloning of DNA from these fungi opens the possibility of identifying functional genes in order to study their regulation and role in symbiosis establishment. Key words: arbuscular mycorrhiza, reciprocal symbiosis, molecular mechanisms, plant determinants, fungal molecules.

Ericoid mycorrhizal fungi: cellular and molecular bases of their interactions with the host plant

A number of soil-borne fungi are able to form typical ericoid mycorrhizae with plants belonging to Ericales. Together with Hymenoscyphus ericae, the first isolate from roots of ericaceous plants, other fungal species belonging to the genus Oidiodendron and many sterile mycelia have been recognized as mycorrhizal by several authors. A high genetic diversity was even found when a population of ericoid mycorrhizal fungi isolated from a single plant of Calluna vulgaris was analysed with morphological and molecular techniques. Ericoid fungi have a relevant saprotrophic potential, as they can degrade several organic polymers present in the soil matrices. Different cell wall degrading enzymes, which are part of this arsenal and are produced in vitro by several ericoid fungi, have been investigated biochemically. Immunocytochemical studies on the production of pectin degrading enzymes during the infection process of host and non-host plants suggest that regulation mechanisms for the production of cell wall degrading enzymes in vivo may be a crucial step for the establishment of successful mycorrhiza with host plants. Key words: ericoid mycorrhizae, cell wall degrading enzymes, polygalacturonase, DNA-RAPD techniques.

Root soluble carbohydrates of Afzelia africana Sm. seedlings and modifications of mycorrhiza establishment in response to the excision of cotyledons

Stem length, number of secondary lateral roots, shoot dry weight and reducing sugar concentrations of root were significantly reduced when translocation of reserves from cotyledons to the roots of Afzelia africana seedlings was interrupted by complete or partial cotyledon excision. The sucrose but not the glucose concentration of lateral roots also decreased significantly after complete cotyledon excision. Hartig net development rather than fungal sheath formation was affected after inoculation with the early fungal isolate E1 and by both late-stage fungal isolates L1 and L2 after partial or complete cotyledon excision. However, mycorrhizal colonization by the early fungal isolate E2 was not affected by cotyledonary reserves, suggesting that this fungal isolate has a lower carbohydrate requirement than fungal isolates E1, L1 and L2. The late-stage fungal isolates L1 and L2 induced a hypersensitivity reaction by epidermal cell walls of the host plant after complete cotyledon excision, suggesting they are more dependent than the early fungal isolate E1 on available root carbohydrate substrates for ectomycorrhizal colonization. These results are discussed in the light of the hypothesis that early and late-stage fungi were different carbohydrate requirements, and that the time sequence of colonization was related to the root carbohydrate status, which increased with time.

Root soluble carbohydrates of Afzelia africana Sm. seedlings and modifications of mycorrhiza establishment in response to the excision of cotyledons

Root soluble carbohydrates of Afzelia africana Sm. seedlings and modifications of mycorrhiza establishment in response to the excision of cotyledons

Effect of arbuscular mycorrhiza on the growth and development of micropropagated Annona cherimola plants

Annona cherimola Mill., cherimoya, is a tropical plantation crop of interest in fruit culture. Micropropagation techniques have been developed due to the need to increase productivity through clonal selection. Because of the mycorrhizal dependence exhibited by this crop for optimal growth and the recognized role of mycorrhiza establishment for the survival and development of most of the plants produced in vitro, the effect of mycorrhiza inoculation on the development of micropropagated plants of Annona cherimola was investigated. Mycorrhizal inoculation was assayed at two different stages of the micropropagation process: (i) immediately after the in vitro phase, before starting the acclimatization period, and (ii) after the acclimatization phase, before starting the post-acclimatization period under greenhouse conditions. Plantlet survival was about 50 % after the acclimatization period. Plant growth and development profited remarkably from mycorrhiza establishment. Most of the arbuscular mycorrhizal fungi (AMF) assayed greatly increased shoot and root biomass and leaf area. Micropropagated Annona plants seem to be more dependent on mycorrhiza formation for optimal growth than plants derived from seeds. The greatest effects of AMF on plant growth were observed when they were introduced after the acclimatization period.

Ethylene Biosynthesis and Activities of Chitinase and ß-1,3-Glucanase in the Roots of Host and Non-Host Plants of Vesicular-Arbuscular Mycorrhizal Fungi after Inoculation with Glomus mosseae

Summary Ethylene production and activities of chitinase and s-1,3-glucanase, two parameters often induced in the plant's response to pathogenic fungi, were measured in the roots of various plants after inoculation with the vesicular-arbuscular (VA) mycorrhizal fungus Glomus mosseae and after mock-inoculation in the absence of VA mycorrhizal fungi. Tomato, a host plant for VA mycorrhizal fungi, was compared in this respect with three species of non-host plants, rape, spinach and lupin. In the case of rape, two cultivars were examined, cv. Jet Neuf (rape 0) and Arabella (rape 00) with normal and low levels of glucosinolates, respectively. Only roots of rape 00, spinach and lupin showed a slightly enhanced ethylene production after inoculation. In roots of tomato, spinach and lupin, chitinase activity was not affected during the first days after inoculation but was depressed afterwards in comparison to mock-inoculated controls. In roots of rape 0 and rape 00, chitinase was weakly induced upon inoculation. Levels of s-1,3-glucanase were not altered in tomato roots during VA mycorrhiza establishment, but they were slightly enhanced in all non-host plants in an initial stage after inoculation. In a later stage, most of the inoculated non-host plants showed less activity than the mock-inoculated controls. We conclude that both host and non-host plants perceive the VA mycorrhizal fungus and respond to its presence. Since the different non-host plants showed different response patterns, none of the reactions studied can be taken as a general indicator for the inability of plants to entertain the VA mycorrhizal symbiosis.

Comparative structure of vesicular-arbuscular mycorrhizas and ectomycorrhizas

During the establishment of vesicular-arbuscular mycorrhizas, fungal hyphae contact the root surface, form appressoria and initiate the internal colonization phase. Structural changes occur in the cell wall, the cytoplasm and the nucleus as the fungus progresses from a presymbiotic to a symbiotic phase. Nuclei in spores are in G1 whereas in intraradical hyphae they are in G1 and G2. Changes in nuclear organization are evident in various stages in the colonization process. Dramatic changes in both symbionts occur as the nutrient exchange interface is established between arbuscules and root cortical cells. An interfacial matrix, consisting of molecules common to the primary wall of the cortical cell, separates the cortical cell plasma membrane from the fungal cell wall. Ectomycorrhizas are characterized structurally by the presence of a mantle of fungal hyphae enclosing the root and usually an Hartig net of intercellular hyphae characterized by labyrinthine branching. As hyphae contact the root surface, they may respond by increasing their diameter and switching from apical growth to precocious branching. The site of initial contact of hyphae may be either the root cap or the ‘mycorrhiza infection zone’. The mantle varies considerably in structure depending on both the plant and fungus genome. In some ectomycorrhizas, the mantle may be a barrier to apoplastic transport, and in most it may store polyphosphate, glycogen, lipids and perhaps protein.

Structure and histochemistry of mycorrhizae synthesized between Arbutus menziesii (Ericaceae) and two basidiomycetes, Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae)

Arbutoid mycorrhizae were synthesized in growth pouches between Arbutus menziesii Pursch. (Pacific madrone) and two broad host range basidiomycete fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Piloderma bicolor (Peck) Julich. P. tinctorius induced the formation of dense, pinnate mycorrhizal root clusters enveloped by a thick fungal mantle. P. bicolor mycorrhizae were usually unbranched, and had a thin or non-existent mantle. Both associations had the well-developed para-epidermal Hartig nets and intracellular penetration of host epidermal cells by hyphae typical of arbutoid interactions. A. menziesii roots developed a suberized exodermis which acted as a barrier to cortical cell penetration by the fungi. Ultrastructurally, the suberin appeared non-lamellar, but this may have been due to the imbedding resin. Histochemical analyses indicated that phenolic substances present in epidermal cells may be an important factor in mycorrhiza establishment. Analyses with X-ray energy dispersive spectroscopy showed that some of the granular inclusions present in fungal hyphae of the mantle and Hartig net were polyphosphate. Other inclusions were either protein or polysaccharides.

Collembolan effects on establishment of vesicular-arbuscular mycorrhizae in soybean ( Glycine max)

Collembolan effects on establishment of vesicular-arbuscular mycorrhizae in soybean ( Glycine max)

Cell differentiation and mycorrhizal infection in Dactylorhiza majalis (Rchb. f.) Hunt & Summerh. (Orchidaceae) during germination in vitro

SUMMARYSeeds of Dactylorhiza majalis (Rchb. f.) Hunt & Summerhayes were sown in vitro with a compatible fungus. A mycorrhiza was established after infection through the rhizoids of the germinating seedling. Infection through the suspensor did occur, but these hyphae did not form pelotons, and embryo cells in contact with the hyphae were filled with a tannin‐like substance.Germination, and establishment of mycorrhiza, took about 14 days in vitro. From day 9 the protein reserves were hydrolysed, the protein vacuoles coalesced and starch accumulated in plastids. Certain epidermal cells developed nuclei about eight times original volume and produced rhizoids which emerged from day 11. After infection the hyphae formed pelotons in central cells with enlarged nuclei (16–64 times original volume). The intracellular hyphae developed close contacts with the hypertrophied host nuclei. Collapsed pelotons could be observed in cells from day 12, one day after infection. Meristematic activity in the uninfected chalazal end of the seedling began on day 12. On day 28 the first vascular tissue started to develop and on day 35 the beginning of a leafy shoot could be detected.

Cell surface inCalluna vulgaris L. hair roots

Calluna vulgaris possesses small roots called “hair roots”, which in natural conditions are colonized by symbiotic mycorrhizal fungi. A specialized cell surface-consisting of the cell wall and the overlaying mucilage-has been hypothesized to be important for the establishment of ericoid mycorrhizae. In this work the cell surface of hair roots of plants growing in sterile conditions has been characterized by using in situ techniques, integrated when possible, by biochemical analysis. The mucilage is abundant around the apex, while it becomes thinner and thinner on the differentiated parts. Sugar residues such as mannose, glucose and galactose are regularly distributed along the whole root length, while N-acetylglucosamine residues are limited to the differentiated part of the hair root. Cellobiohydrolase-gold complex, used to reveal β-1, 4-glucans, regularly labels mucilage and cell walls of apical and differentiated regions. Polygalacturonic acids revealed by monoclonal antibodies are found at the surface of the cap cells and on the cell walls of the inner tissues in the differentiated zones, but never at the surface of the epidermal cells. The labeling continuity between mucilage and cell walls demonstrates that some molecules such as β-1, 4-glucans are common to the two compartments, but probably have a different status of aggregation. On the contrary, other molecules, such as N-acetylglucosamine or polygalacturonic acid display a precise pattern of localization following root differentiation.

Effect of soil humus cover on the establishment and development of mycorrhiza on containerisedpinus sylvestrisL. andpinus contortassp.latifoliaEngelm. after outplanting

The presence of humus was evaluated as a factor affecting the establishment and development of mycorrhiza in Scots pine (Pinus sylvestris L.) and lodgepole pine (Pinus contorta ssp. latifolia Engelm.) during the first growing season after outplanting on two clear‐cut sites. Humus proved to be of minor importance for the formation of mycorrhizae. Instead, the colonisation showed a patchy pattern, indicating spatial variation in the amounts of active inoculum. This variation may reflect differences in soil microhabitat and perhaps the mycorrhizal distribution in the logged stands. Total mycorrhizal short‐root frequency increased from 10% at outplanting in June to 40–65% in October. The nursery mycorrhiza accounted for 80–95% of the total mycorrhiza at the end of the investigation. However, by this time 60–90% of the seedlings had, to some extent, indigenous mycorrhiza with fungi from the clear‐cut sites. Piloderma croceum tended to be formed at a higher rate in the presence of humus than in its absence while another mycorrhizal type increased faster in the mineral soil.

Vesicular-arbuscular mycorrhizae in taconite tailings. I. Incidence and spread of endogonaceous fungi following reclamation

The incidence and spread of vesicular-arbuscular (VA) mycorrhizae in reclaimed and unreclaimed taconite tailings were assessed. A corn bioassay was used to determined the relative levels of infective propagules of VA mycorrhizal fungi in tailing samples collected before and periodically after reclamation of a 144-ha taconite tailing settling basin in Wisconsin, U.S.A. Samples were collected at three distances from the western berm of the settling basin to attempt to monitor the spatial establishment and spread of mycorrhizae in the basin. There was a significant temporal effect, but no distinguishable spatial effect on the population densities of VA mycorrhizal propagules in the tailing samples. The relative levels of mycorrhizal propagules in samples collected before reclamation were not significantly different from the levels in sterilized controls. Tailings collected 1, 8 and 20 weeks after reclamation had progressively higher densities of infective propagules. These results contrast with the very slow rates of spread reported for studies conducted in semi-arid sites in North America. It was suggested that VA mycorrhizal fungi are able to become established more rapidly in mesic sites than semi-arid sites because mesic sites are generally more conducive to host plant growth.

Groupings of ectomycorrhizal fungi of birch and pine, based on establishment of mycorrhizas on seedlings from spores in unsterile soils

Birch (Betulapendula) seedlings were grown in pots of unsterile brown earth, vermiculite-peat mixture or coal spoil supplemented with freshly deposited basidiospores of ectomycorrhizal fungi. Species of Hebeloma, Laccaria and Inocybe established mycorrhizas from spores and prevented mycorrhiza formation by other fungi occurring naturally in (or possibly contaminating) the rooting media. Species of Lactarius, Cortinarius, Leccinum and Russula, and Amanita muscaria, Elaphomyces muricatus, Scleroderma citrinum and Suillus luteus did not establish mycorrhizas from spores. Paxillus involutus formed mycorrhizas from spores in coal spoil but only poorly in brown earth, demonstrating some substrate-dependency in mycorrhizal establishment. However, spores of Lactarius turpis and Leccinum scabrum from fruit bodies on coal spoil did not establish mycorrhizas on birch seedlings in coal spoil, whereas those of P. involutus did so. Also, seedlings planted in intact coal spoil samples from beneath fruit bodies of Amanita muscaria, Leccinum roseofractum and Scleroderma citrinum did not develop mycorrhizas of these fungi, whereas samples from beneath fruit bodies of Inocybe lacera and P. involutus did so. Thus, soil-effects did not obscure a major difference between two groups of mycorrhizal fungi, i.e. those that could or could not establish mycorrhizas on seedlings from spores added to unsterile soil. At least for the fungi tested, these seem to be generic attributes, and they correlate with groups termed early- and late-stage in successions of mycorrhizas. In limited tests, Hebeloma crustuliniforme established mycorrhizas from spores on pine seedlings whereas Suillus luteus did not do so, which agreed with the results for birch.

PRODUCTION OF INDOLE‐3‐E THANOL AND INDOLE‐3‐A CETIC ACID BY THE MYCORRHIZAL FUNGUS OF OPHRYS LUTEA (ORCHID ACEAE)

SummaryThe GC/MS analysis of trimethylsilyl derivatives of extracts from the culture medium and from the mycelium of Ophrys lutea Cav., a mycorrhizal fungus grown in a liquid culture medium, revealed the ability of this fungus to synthesize indole‐3‐ethanol and indole‐3‐acetic acid.The identification of these compounds in the liquid medium and in the mycelial extracts suggested their transference from the fungus to the host during the establishment of mycorrhizas.

EFFECTS OF SOIL TRENCHING AND CORING ON THE FORMATION OF ECTOMYCORRHIZAS ON BIRCH SEEDLINGS GROWN AROUND MATURE TREES.

Initially non-mycorrhizal seedlings of birch (Betula pendula Roth.) were grown for a season around two 11-year-old birch trees on an experimental site south of Edinburgh. Some seedlings (non-isolated) were planted in undisturbed positions, others (trenched) in volumes of soil isolated from the mature tree by trenches, and yet others (cored) in cores of soil (8 × 10 cm diameter) that had been removed and replaced immediately. Mycorrhizas of Lactarius pubescens (Fr. ex Krombh.) Fr., Lactarius glyciosmus (Fr. ex Fr.) Fr., and Leccinum scabrum (Bull. ex Fr.) S. F. Gray and Leccinum roseofracta Watl. developed abundantly on the non-isolated seedlings but usually very poorly on the trenched and cored seedlings. That some development of Leccinum mycorrhizas occurred in trenched areas is attributed to incomplete severance of roots. In contrast, mycorrhizas of other fungi, for example Hebeloma spp. and Inocybe spp., developed most abundantly on trenched and cored seedlings rather than on non-isolated seedlings. The results demonstrate an important difference between types of ectomycorrhizal fungus. Lactarius and Leccinum spp. seem to depend on a continuing supply of photosynthate from a mature tree in order to colonize seedling roots, whereas several other mycorrhizal fungi show no such dependence. The results also indicate an important role for mycelial strands in establishment of mycorrhizas by some fungi (Lactarius pubescens and Leccinum spp.) and thus parallel previous reports on the importance of mycelial strands in colonization of substrata by saprophytic and pathogenic fungi.

INFLUENCE OF PHOSPHATE SOURCE ON VESICULAR‐ARBUSCULAR MYCORRHIZAE OF BOUTELOUA GRACILIS

SUMMARYNon‐mycorrhizal and vesicular‐arbuscular mycorrhizal Bouteloua gracilis infected with Glomus fasciculatus were grown in defined media containing different phosphate sources and compared with respect to phosphate content and form, biomass, chlorophyll concentration, and root phosphatase activity. The phosphate sources were sodium monobasic phosphate, a mixture of sodium monobasic phosphate and calcium phytate, and calcium phytate. Inositol and inositol plus calcium were added to the sodium phosphate medium as additional treatments. Mycorrhizal infection was highest in roots of plants grown in the presence of phytate (75%). Lower root infection levels were noted in plants from the sodium phosphate (19%) and mixed phosphate (22%) media. No penetration by fungi occurred in plants from the sodium phosphate plus inositol or inositol and calcium media. Dry wts of non‐mycorrhizal plants were highest when grown in media containing phytate and sodium phosphate plus inositol and calcium followed in decreasing order by sodium phosphate plus inositol, mixed phosphates, and sodium phosphate. Mycorrhizal infection increased leaf dry wt in plants from the sodium phosphate medium and root dry wt from the phytate medium. Phosphate concentrations in the plants were highest when grown in mixed phosphate medium followed by sodium phosphate and phytate. Mycorrhizal infection always increased significantly leaf phosphate concentrations but increased root phosphate concentrations only in the phytate medium. Phosphates were found predominantly as organically‐bound compounds in leaves of mycorrhizal plants whereas in leaves of non‐mycorrhizal plants, most of the phosphate was inorganic. Chlorophyll concentrations increased significantly with mycorrhizal infection with no change in a/b ratios. Mycorrhizal plants grown in the phytate medium had substantially higher alkaline phosphatase activity than did non‐mycorrhizal plants; acid phosphatase activity was not affected by mycorrhizal condition.These results suggest that form of the phosphate in the root environment influences mycorrhizal establishment and effect of mycorrhizae on plant growth.