Limitations on orchid recruitment: not a simple picture

Mycorrhizal fungi have substantial potential to influence plant distribution, especially in specialized orchids and mycoheterotrophic plants. However, little is known about environmental factors that influence the distribution of mycorrhizal fungi. Previous studies using seed packets have been unable to distinguish whether germination patterns resulted from the distribution of appropriate edaphic conditions or the distribution of host fungi, as these cannot be separated using seed packets alone. We used a combination of organic amendments, seed packets and molecular assessment of soil fungi required by three terrestrial orchid species to separate direct and indirect effects of fungi and environmental conditions on both seed germination and subsequent protocorm development. We found that locations with abundant mycorrhizal fungi were most likely to support seed germination and greater growth for all three orchids. Organic amendments affected germination primarily by affecting the abundance of appropriate mycorrhizal fungi. However, fungi associated with the three orchid species were affected differently by the organic amendments and by forest successional stage. The results of this study help contextualize the importance of fungal distribution and abundance to the population dynamics of plants with specific mycorrhizal requirements. Such phenomena may also be important for plants with more general mycorrhizal associations.     

Effect of root exudation on VA mycorrhizal infection at early stages of plant growth

Summary No relationship between the degree of VA mycorrhizal infection and total sugar content in root exudates of several plant species of different degree of mycorrhizal susceptibility were observed during the early stages of plant growth. Even more, the non host plants tested showed higher sugar exudation ability, when expressed as the amount exuded per g of root, at these early periods of their growth, than plants susceptible to mycorrhizal infection.Root exudates from host and non host plants influenced similarly the percentage of spore germination and number of secondary spores under controlled conditions.     

Arbuscular mycorrhizal fungal propagules in a salt marsh

The tolerance of indigenous arbuscular mycorrhizal fungi (AMF) to stressful soil conditions and the relative contribution of spores of these fungi to plant colonization were examined in a Portuguese salt marsh. Glomus geosporum is dominant in this salt marsh. Using tetrazolium as a vital stain, a high proportion of field-collected spores were found to be metabolically active at all sampling dates. Spore germination tests showed that salt marsh spores were not affected by increasing levels of salinity, in contrast to two non-marsh spore isolates, and had a significantly higher ability to germinate under increased levels of salinity (20) than in the absence of or at low salinity (10). Germination of salt marsh spores was not affected by soil water levels above field capacity, in contrast to one of the two non-marsh spore isolates. For the evaluation of infectivity, a bioassay was established with undisturbed soil cores (containing all types of AM fungal propagules) and soil cores containing only spores as AM fungal propagules. Different types of propagules were able to initiate and to expand the root colonization of a native plant species, but spores were slower than mycelium and/or root fragments in colonizing host roots. The AM fungal adaptation shown by this study may explain the maintenance of AMF in salt marshes.     

Mycorrhizal specificity does not limit the distribution of an endangered orchid species

What factors determine the distribution of a species is a central question in ecology and conservation biology. In general, the distribution of plant species is assumed to be controlled by dispersal or environmentally controlled recruitment. For plant species which are critically dependent on mycorrhizal symbionts for germination and seedling establishment, specificity in mycorrhizal associations and availability of suitable mycorrhizal fungi can be expected to have a major impact on successful colonization and establishment and thus ultimately on a species distribution. We combined seed germination experiments with soil analyses and fungal assessments using 454 amplicon pyrosequencing to test the relative importance of dispersal limitation, mycorrhizal availability and local growth conditions on the distribution of the orchid species Liparis loeselii, which, despite being widely distributed, is rare and endangered in Europe. We compared local soil conditions, seed germination and mycorrhizal availability in the soil between locations in northern Belgium and France where L. loeselii occurs naturally and locations where conditions appear suitable, but where adults of the species are absent. Our results indicated that mycorrhizal communities associating with L. loeselii varied among sites and plant life cycle stages, but the observed variations did not affect seed germination, which occurred regardless of current L. loeselii presence and was significantly affected by soil moisture content. These results indicate that L. loeselii is a mycorrhizal generalist capable of opportunistically associating with a variety of fungal partners to induce seed germination. They also indicate that availability of fungal associates is not necessarily the determining factor driving the distribution of mycorrhizal plant species.     

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.     

四种蕨类植物根际土壤中VA菌根真菌孢子种群组成和季相变化

对４ 种移栽到温室中的蕨类植物根际土壤中的ＶＡ 菌根真菌孢子种群组成和季相变化进行了研究, 结果发现, ＶＡ菌根真菌孢子的产生具有明显的宿主依赖性和季相变化。在相同气候条件下, 不同植物根际土壤中的ＶＡ菌根真菌种群组成不同; 同种ＶＡ 菌根真菌在不同宿主植物根际土壤中, 孢子的丰富度有很大的差异。本文对影响ＶＡ菌根真菌孢子种群组成和季相变化的因素进行了讨论。     

Effect of liming on spore germination,germ tube growth and root colonization by vesicular-arbuscular mycorrhizal fungi

Summary The effect of soil acidity on spore germination, germ tube growth and root colonization of vesicular-arbuscular mycorrhizal (VAM) fungi was examined using a Florida Ultisol. Soil samples were treated with 0, 4, 8 and 12 meq Ca/MgCO₃/100 g soil and each lime level received 0, 240, and 720 ppm P as superphosphate. Corn (Zea mays L.) was planted in the soil treatments, inoculated with eitherGlomus mosseae orGigaspora margarita spores and grown for 31 days. Acid soil inhibits mycorrhizal formation byG. mosseae through its strong fungistatic effect against the spores. The dolomitic lime increased mycorrhizal formation by both fungal species.G. margarita is much less sensitive to acidic conditions thanG. mosseae. Al ions are a very important component of the fungistatic property against the VAM symbiosis. VAM fungus adaptation may be important for plants growing on infertile acid soils if soil inoculation with these fungi is to contribute significantly to low-input technology for tropical agricultural systems.     

Mycorrhizal diversity,seed germination and long‐term changes in population size across nine populations of the terrestrial orchid Neottia ovata

In plant species that rely on mycorrhizal symbioses for germination and seedling establishment, seedling recruitment and temporal changes in abundance can be expected to depend on fungal community composition and local environmental conditions. However, disentangling the precise factors that determine recruitment success in species that critically rely on mycorrhizal fungi represents a major challenge. In this study, we used seed germination experiments, 454 amplicon pyrosequencing and assessment of soil conditions to investigate the factors driving changes in local abundance in 28 populations of the orchid Neottia ovata. Comparison of population sizes measured in 2003 and 2013 showed that nearly 60% of the studied populations had declined in size (average growth rate across all populations: ?0.01). Investigation of the mycorrhizal fungi in both the roots and soil revealed a total of 68 species of putatively mycorrhizal fungi, 21 of which occurred exclusively in roots, 25 that occurred solely in soil and 22 that were observed in both the soil and roots. Seed germination was limited and significantly and positively related to soil moisture content and soil pH, but not to fungal community composition. Large populations or populations with high population growth rates showed significantly higher germination than small populations or populations declining in size, but no significant relationships were found between population size or growth and mycorrhizal diversity. Overall, these results indicate that temporal changes in abundance were related to the ability of seeds to germinate, but at the same time they provided limited evidence that variation in fungal communities played an important role in determining population dynamics.     

Polyamines stimulate hyphal branching and infection in the early stage of <Emphasis Type="Italic">Glomus etunicatum</Emphasis> colonization

Polyamines are known to strongly stimulate hyphal growth in arbuscular mycorrhizal fungi. The effect of the polyamines putrescine, spermidine and spermine on spore germination, hyphal elongation and branching by the AM fungus Glomus etunicatum was investigated in this study. The effect of spermine on infection and the development of the host and of daughter spores was further investigated using the dual monoaxenic culture system comprised of Gl. etunicatum fungal cultures in Ri T-DNA transformed carrot hairy roots. Spermidine and spermine showed positive effects on germination and all three polyamines significantly promoted hyphal growth. Hyphal branching was also strongly stimulated by treatment with polyamines, such as an increase in the number of branches. Infection during the early stages of the in vitro co-culture life cycle was enhanced in the presence of spermine, and daughter spores appeared at earlier timepoints compared to the control. Our results demonstrate that polyamines stimulate germination and hyphal branching in the early stage of AM fungal colonization. Moreover, results from the investigations conducted in the fungus-root co-culture suggest that polyamines may be involved in establishing the symbiotic relationship between root and fungus.     

兰科植物种子共生萌发真菌多样性及共生萌发机制研究进展

自然环境下,兰科植物种子细小无胚乳,需要和适宜的真菌共生才能萌发,因而与真菌有天然的共生关系。自身繁殖率低加之近年来栖息地环境破坏导致兰科植物资源更加濒危,而通过筛选适合的真菌进行种子的共生萌发可以有效地实现兰科植物的种质保育及濒危种类野生居群的生态恢复。本文对地生型、附生型以及腐生型等兰科植物已发现的萌发真菌的多样性进行了系统地梳理,发现担子菌门的胶膜菌科、角担菌科以及蜡壳耳目真菌为已报道共生萌发真菌的主要类群;同时对兰科植物种子的共生萌发机制,包括形态学机制、营养机制和分子机制等方面的相关研究进行了归纳论述,但是当前关于兰科植物和真菌互作机制方面的研究还相对较少,许多问题需要进一步明确。本文对共生萌发真菌在兰科植物保育和繁育中的应用以及共生萌发机制的研究等方面具有一定的参考价值。     

Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae)

Tropical orchids constitute the greater part of orchid diversity, but little is known about their obligate mycorrhizal relationships. The specificity of these interactions and associated fungal distributions could influence orchid distributions and diversity. We investigated the mycorrhizal specificity of the tropical epiphytic orchid Ionopsis utricularioides across an extensive geographical range. DNA ITS sequence variation was surveyed in both plants and mycorrhizal fungi. Phylogeographic relationships were estimated for the mycorrhizal fungi. Orchid functional outcomes were determined through in vitro seed germination and seedling growth with a broad phylogenetic representation of fungi. Most fungal isolates derived from one clade of Ceratobasidium (anamorphs assignable to Ceratorhiza), with 78% within a narrower phylogenetic group, clade B. No correlation was found between the distributions of orchid and fungal genotypes. All fungal isolates significantly enhanced seed germination, while fungi in clade B significantly enhanced seedling growth. These results show that I. utricularioides associates with a phylogenetically narrow, effective fungal clade over a broad distribution. This preference for a widespread mycorrhizae may partly explain the ample distribution and abundance of I. utricularioides and contrasts with local mycorrhizal diversification seen in some nonphotosynthetic orchids. Enhanced orchid function with a particular fungal subclade suggests mycorrhizal specificity can increase orchid fitness.     

Soil salinity delays germination and limits growth of hyphae from propagules of arbuscular mycorrhizal fungi

Colonisation of plant roots by some arbuscular mycorrhizal (AM) fungi is reduced in the presence of sodium chloride (NaCl), probably due to a direct effect of NaCl on the fungi. However, there appear to be differences between the fungi in their ability to colonise plants in the presence of NaCl. This experiment tested the hypothesis that propagules of different isolates and species of AM fungi from saline and nonsaline soils would differ in their ability to germinate and grow in the presence of NaCl in the soil solution. Spores or pieces of root colonised by a range of AM fungi were incubated between filters buried in soil to which NaCl had been added at concentrations of 0, 150 or 300 mM in the soil solution. At regular intervals, filters were removed from the soil and both the percentage of propagules which had germinated and the length of proliferating hyphae were determined. Germination of spores of AM fungi studied was delayed in the presence of NaCl, but the fungi differed in the extent to which germination was inhibited. Two isolates of Scutellospora calospora reached maximum germination in 300 mM NaCl, but neither of two isolates of Acaulospora laevis germinated in the presence of NaCl. Germination of spores of the other fungi, including some isolated from saline soil, fell between these extremes. For some fungi, the specific rate of hyphal extension was reduced by NaCl. For others, the specific rate of growth was similar in the presence of NaCl to that in the control treatment, but overall production of hyphae was reduced in the NaCl treatments because germination was reduced.     

菌根真菌对白及种子萌发和幼苗生根的作用

自然条件下,兰科植物需要依赖菌根真菌获得营养才能萌发。本研究对白及根和原球茎中分离的4株菌根真菌（WQ17-33、WQ17-43、JST-3和SL15-7）进行分子鉴定,并评价光照和黑暗条件下不同菌株促白及种子萌发和幼苗生根的效果。结果表明,4个菌株分别隶属于鬼伞属Coprinus、胶膜菌属Tulasnella、腊壳菌属Sebacina和Serendipita。在种子萌发前期（未形成叶子）进行暗培养较光照对菌株JST-13和SL15-7处理组原球茎阶段萌发具有显著的促进作用。不同菌株共生萌发效果不同,菌株SL15-7较其他处理原球茎和幼苗发育阶段的萌发率高。菌株JST-13和SL15-7处理组形成的幼苗较其他处理组强壮,定殖的菌丝团也较多,其幼苗生根效果也较对照组好。该研究表明白及可与多种不同类群的菌根真菌菌株形成共生关系,这些真菌在促进白及种子萌发和生根能力方面存在差异。     

Interaction between Earthworms and Arbuscular Mycorrhizal Fungi in Plants: A Review

Different kinds of soil animals and microorganisms inhabit the plant rhizosphere, which function closely to plant roots. Of them, arbuscular mycorrhizal fungi (AMF) and earthworms play a critical role in sustaining the soil-plant health. Earthworms and AMF belong to the soil community and are soil beneficial organisms at different trophic levels. Both of them improve soil fertility and structural development, collectively promoting plant growth and nutrient acquisition capacity. Earthworm activities redistribute mycorrhizal fungi spores and give diversified effects on root mycorrhizal fungal colonization. Dual inoculation with both earthworms and AMF strongly magnifies the response on plant growth through increased soil enzyme activities and changes in soil nutrient availability, collectively mitigating the negative effects of heavy metal pollution in plants and soils. This thus enhances phytoremediation and plant disease resistance. This review simply outlines the effects of earthworms and AMF on the soil-plant relationship. The effects of earthworms on root AMF colonization and activities are also analyzed. This paper also summarizes the interaction between earthworms and AMF on plants along with suggested future research.     

Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra-radical hyphae

Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc(-) phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc(-) characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.     

Growth response of Atriplex nummularia to inoculation with arbuscular mycorrhizal fungi at different salinity levels

Chenopods are generally regarded as non-host plants for mycorrhizal fungi and are believed not to benefit from colonization by mycorrhizal fungi. Perennial Atriplex nummularia Lindl., growing under field conditions, showed a relatively high level of colonization by mycorrhizal fungi (10–30% of root length colonized) in spring and summer. Accordingly, two glasshouse experiments were designed to assess the effects of inoculation with mycorrhizal fungi (with a single species or a mixture of different species) on growth, nutrient uptake, and rhizosphere bacterial community composition of A. nummularia at high and low salinity levels (2.2 and 12 dSm^–1). Only low and patchy colonization by mycorrhizal fungi (1–2 of root length colonized) was detected in inoculated plants under glasshouse conditions which was unaffected by salinity. Despite the low colonization, inoculation increased plant growth and affected nutrient uptake at both salinity levels. The effects were higher at an early stage of plant development (6weeks) than at a later stage (9–10 weeks). Salinity affected the bacterial community composition in the rhizosphere as examined by ribosomal intergenic spacer amplification (RISA) of 16S rDNA, digitization of the band patterns and multivariate analysis. The effects of inoculation with mycorrhizal fungi on growth of A. nummularia may be attributed to (i) direct effects of mycorrhizal fungi on plant nutrient uptake and/or (ii) indirect effects via mycorrhizal-induced changes in the bacterial community composition.     

大豆毛状根-VA菌根真菌双重培养体系的建立

以大豆毛状根为宿主,接种VA菌根真菌珠状巨孢囊霉（Gigaspora margarita）,经过3.5个月的双重培养,观察到VA菌根真菌珠状巨孢囊霉对大豆毛状根的侵染,辅助细胞形成,并获得VA菌根真菌成熟孢子,在无菌条件下建立了大豆毛状根-VA菌根真菌双重培养体系,为研究菌根真菌侵染大豆根部形成共生体系及相关分子机制提供了一种有效的研究方法。     

Acacias respond to additions of phosphorus and to inoculation with VA mycorrhizal fungi in soils stockpiled during mineral sand mining

Three pot experiments were conducted to test the hypothesis that the growth ofAcacia spp. in stockpiled soil from two mineral sand mines, could be increased by the addition of phosphorus (P) or inoculation with VA mycorrhizal fungi. In soils from North Stradbroke Island, the dry weight of shoots ofAcacia concurrens was increased by P and by VA mycorrhizal fungi in tailings sand, while in less adsorptive topsoil dry weight was only increased at low levels of applied P. WhenA. concurrens was grown in a layer of topsoil placed over tailings sand, shoot dry weight increased, in response to inoculation with VA mycorrhizal fungi banded between the soil layers.In topsoil from Eneabba, the dry weight of shoots at low rates of applied P was increased by up to 4 times by inoculation with VA mycorrhizal fungi. The response to inoculation in both experiments was due to increases in the uptake of P by the plants.Species of VA mycorrhizal fungi differed in their ability to increase plant growth. However, in soils from both sites, the same fungal species were effective.     

The development of vesicular-arbuscular mycorrhizal infection in plant root systems

Summary Many crop plants carry VA mycorrhizal infection. The fungi benefit their host principally by increasing the rate of phosphorus uptake from soils low in available phosphorus.Infection of seedling root systems occurs from soil-borne propagules and/or from neighbouring mycorrhizal roots. When, under the right conditions, the propagules germinate, a mycelium is produced which cannot growth extensively until mycorrhizal infection is established.The level of initial (primary) infection is related to propagule density. The relation can be described by a mathematical model which takes account of propagule germination rates, rates of hyphal growth through the soil and rates of root growth.Subsequent spread of secondary infection is by growth, along and between roots, of hyphae originating from established infection units and by the formation of new infection units. This spread can be modelled.If certain simplifying assumptions are made, the effects on the host of developing mycorrhizal infection can also be modelled and satisfactory predictions of infection spread and the mycorrhizal growth response obtained.In conclusion, some implications for agriculture and forestry are briefly discussed.     

Plant hormones as signals in arbuscular mycorrhizal symbiosis

Abstract

Arbuscular mycorrhizal (AM) fungi are non-specific symbionts developing mutual and beneficial symbiosis with most terrestrial plants. Because of the obligatory nature of the symbiosis, the presence of the host plant during the onset and proceeding of symbiosis is necessary. However, AM fungal spores are able to germinate in the absence of the host plant. The fungi detect the presence of the host plant through some signal communications. Among the signal molecules, which can affect mycorrhizal symbiosis are plant hormones, which may positively or adversely affect the symbiosis. In this review article, some of the most recent findings regarding the signaling effects of plant hormones, on mycorrhizal fungal symbiosis are reviewed. This may be useful for the production of plants, which are more responsive to mycorrhizal symbiosis under stress.