The southernmost myco-heterotrophic plant, Arachnitis uniflora: root morphology and anatomy

Root morphology and anatomy of the myco-heterotrophic Arachnitis uniflora (Corsiaceae) were studied in relation to their association with a Glomus species (Glomeromycota). The mycorrhizal features were studied in three distinctive stages of development: (i) shoot and flower restricted to a small, underground bud; (ii) shoot and flower bud up to 1.5 cm; and (iii) shoot and flower already withered. The hyphae penetrate through and between the epidermal and exodermal cells; the exodermis and outer cortical cells become colonized in an inter- and intracellular manner, with some coils being formed in these layers. The fungi colonize the middle cortex, where intracellular vesicles in bundles are abundant. Arbuscules are formed profusely at very early stages of development, while in older stages they almost disappear and abundant vesicles are formed. Except for some details, the pattern of root colonization corresponds to a Paris-type. Presence of storage substances (starch and oil) also was recorded. Starch is produced and stored within root cells, mainly in the outer and inner root cortex. In senescent stages, plant and fungal tissues collapse.     

The myco-heterotrophic Voyria flavescens (Gentianaceae) and its associated fungus

The myco-heterotrophic Gentianaceae Voyria flavescens from the Peruvian Amazon was investigated morphologically and anatomically. Most attention was paid to the subterranean structures and the mycorrhiza. The architecture of the underground organs and the process of root development in particular indicates neoteny. The stellately arranged roots are lacking a pericycle, an endodermis in the conventional meaning, a rhizodermis and a root-cap. All roots emerge exogenously from a so-called ‘rhizoferous axis’. The root-tips show a capacity of exogenous shoot-regeneration. Further it was found that fungal penetration is restricted to dark banded zones of the root’s surface. These zones are the result of a unique mode of apical growth. The associated fungi colonize the entire cortex intracellularly. In the innermost cortical layers the hyphae are longitudinally spreading, whereas they successively degenerate in the outer cortex. In the final phase of disintegration the hyphal coils almost entirely consist of a highly refractive crystalline substance. The mycorrhizal pattern corresponds to a specialized Paris-type vesicular-arbuscular mycorrhiza, lacking both, arbuscles and vesicles. Due to auxiliary cells, being connected to root invading hyphae, an associated fungus could be identified as most likely being a member of Gigasporaceae (Glomeromycota).     

Anatomy and mycotrophy of the achlorophyllous Afrothismia winkleri

Afrothismia winkleri develops fleshy rhizomes, densely covered with small root tubercles, narrowing to filiform roots with age. The exclusively intracellular mycorrhizal fungus has distinct morphologies in different tissues of the plant. In the filiform root the hyphae grow straight and vesicles are borne on short hyphal stalks. The straight hyphae are present in the epidermis of the root tubercles, but change to loosely coiled and swollen hyphae in the rhizome tissue. No penetration from epidermis to root cortex was found. From the rhizome, a separating cell layer permits only one or rarely two hyphal penetrations into the cortex of each root tubercle. The hyphae proceed apically within the root hypodermis in a spiral row of distinctively coiled hyphae, branches of which colonize the inner root cortex. In the inner root cortex the hyphal coils degenerate to amorphous clumps. In older roots the cortex itself also deteriorates, but epidermis, hypodermis, endodermis and central cylinder persist. The mycorrhizal pattern in A. winkleri is interpreted as an elaborate exploitation system whereby the fungus provides carbon and nutrients to the plant and, simultaneously but spatially distinct, its hyphae are used to translocate and store the matter within the plant. Several features indicate that the endophyte is an arbuscular mycorrhizal fungus.     

濒危药用植物桃儿七根的显微结构及其菌根真菌分布研究

本文研究了桃儿七Sinopodophyllum hexandrum根的显微结构及其真菌分布。结果表明,桃儿七的根为根状茎,节状,不定根形成的须根系发达。根的结构主要由表皮、皮层、维管柱三部分构成,其中,皮层所占比例最大,超过80%。根的木质部有四原型和五原型两种类型,五原型较为常见;四原型的根和五原型的根在皮层细胞形态上存在一定差异。在桃儿七的不定根和其上的侧根观察到真菌菌丝分布,其数量和种类与根的直径有关,在不定根较细(先端)的部位真菌以暗色有隔内生真菌(DSE真菌)为主,侵染率为77.9%;而较粗根中真菌菌丝为无隔菌丝为主,分布很少且仅存在于皮层细胞的一至二层,不侵染皮层深部和维管柱。不定根侧根中真菌以丛枝菌根真菌为主,丛枝菌根常常占据大部分的皮层细胞,侵染率高达90%以上。桃儿七根中没有发现根毛存在,因此,侧根中共生的丛枝菌根真菌可能是桃儿七养分和水分吸收的主要途径。     

Wide geographical and ecological distribution of nitrogen and carbon gains from fungi in pyroloids and monotropoids (Ericaceae) and in orchids

* Stable isotope abundance analyses recently revealed that some European green orchids and pyroloids (Ericaceae) are partially myco-heterotrophic, exploiting mycorrhizal fungi for organic carbon and nitrogen. Here we investigate related species to assess their nutritional mode across various forest and climate types in Germany and California. * C- and N-isotope signatures of five green pyroloids, three green orchids and several obligate myco-heterotrophic species (including the putatively fully myco-heterotrophic Pyrola aphylla) were analysed to quantify the green plants' nutrient gain from their fungal partners and to investigate the constancy of enrichment in (13)C and (15)N of fully myco-heterotrophic plants from diverse taxa and locations relative to neighbouring autotrophic plants. * All green pyroloid and one orchid species showed significant (15)N enrichment, confirming incorporation of fungi-derived N compounds while heterotrophic C gain was detected only under low irradiance in Orthilia secunda. Pyrola aphylla had an isotope signature equivalent to those of fully myco-heterotrophic plants. * It is demonstrated that primarily N gain from mycorrhizal fungi occurred in all taxonomic groups investigated across a wide range of geographical and ecological contexts. The (13)C and (15)N enrichment of obligate myco-heterotrophic plants relative to accompanying autotrophic plants turned out as a fairly constant parameter.     

Rapid in vitro ectomycorrhizal infection onPinus densiflora roots byTricholoma matsutake

The root systems of 11-wk-oldPinus densiflora seedlings were inoculated with a hyphal suspension ofTricholoma matsutake and aseptically incubated for 4 wk in a forest soil without supplying exogenous carbohydrates. One week following inoculation, fungal hyphae had colonized the root surface and bound soil particles together establishing a root-substrate continuum. Fungal hyphae were visible within the main root cortex following clearing bleaching and staining. In the ensuing days, fungal colonization was observed within elongating lateral roots in which Hartig net formation was confirmed 4 wk after inoculation. This is the first report of rapid ectomycorrhizal infection ofP. densiflora seedings byT. matsutake.     

Root Anatomy and Mycotrophy of the Achlorophyllous Voyria tenella Hook. (Gentianaceae)

The central cylinder of the root of Voynet tenella consists of up to ten central, non-lignified, tracheidal xylem elements surrounded by some parenchymatic tissue and 5–7 groups of phloem. A pericycle could not be discerned. Even though the endodermis carries a faint suberin lamella it cannot be discerned anatomically without special staining. The cells of the 1–3 cortex layers next to the endodermis are elongated longitudinally, the subsequent cortex parenchyma is multi-layered and consists of isodiametric cells. The cells of the 2–3 layered outer dermal tissue are smaller than those of the adjacent cortex, their walls carry a suberin lamella and the outermost of them constantly scale off. The dermal tissue is interpreted as a multilayered exodermis. The fungal colonization in roots of Voyria tenella remarkably differs from any known mycorrhizal pattern. After having penetrated the dermal tissue, the always intracellularly-growing hyphae head straight towards the inner cortex layers, where they spread along the central cylinder. Ramifications from these inner-spreading hyphae then colonize the cortex parenchyma from the inside and they develop dense hyphal coils. Eventually, the coiled hyphae swell and collapse, resulting in amorphous clumps of fungal material. This mycorrhizal pattern is referred to as an intraradical fungus garden. Arguments are given to call the mycorrhiza in Voyria tenella a specialized arbuscular mycorrhiza. Phylogenetic and ecological implications of the observations and the results are discussed.     

Prepenetration apparatus assembly precedes and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota

Arbuscular mycorrhizas (AM) are widespread, ancient endosymbiotic associations that contribute significantly to soil nutrient uptake in plants. We have previously shown that initial fungal penetration of the host root is mediated via a specialized cytoplasmic assembly called the prepenetration apparatus (PPA), which directs AM hyphae through the epidermis (Genre et al., 2005). In vivo confocal microscopy studies performed on Medicago truncatula and Daucus carota, host plants with different patterns of AM colonization, now reveal that subsequent intracellular growth across the root outer cortex is also PPA dependent. On the other hand, inner root cortical colonization leading to arbuscule development involves more varied and complex PPA-related mechanisms. In particular, a striking alignment of polarized PPAs can be observed in adjacent inner cortical cells of D. carota, correlating with the intracellular root colonization strategy of this plant. Ultrastructural analysis of these PPA-containing cells reveals intense membrane trafficking coupled with nuclear enlargement and remodeling, typical features of arbusculated cells. Taken together, these findings imply that prepenetration responses are both conserved and modulated throughout the AM symbiosis as a function of the different stages of fungal accommodation and the host-specific pattern of root colonization. We propose a model for intracellular AM fungal accommodation integrating peri-arbuscular interface formation and the regulation of functional arbuscule development.     

Benzothiadiazole-Mediated Induced Resistance to Fusarium oxysporum f. sp. radicis-lycopersici in Tomato

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.     

'Prepackaged symbioses': propagules on roots of the myco-heterotrophic plant Arachnitis uniflora

Arachnitis uniflora, a myco-heterotrophic plant species, has fleshy tuberous roots colonized by the arbuscular mycorrhizal fungal genus Glomus (Phylum Glomeromycota). These roots produce apical and lateral propagules, both reported here for the first time. The objective of the study was to characterize the ontogeny and structure of the propagules, and to determine their function. Scanning electron microscopy, laser scanning confocal microscopy and light microscopy were used to study the ontogeny and structure of the propagules. Propagules developed either from cortical parenchyma cells or from cells immediately beneath the root cap; they developed a shoot meristem and cells in the basal region which were colonized by various fungal structures including hyphae and vesicles. These propagules may detach from the roots, establishing new plants.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

赤松不定根和愈伤组织与松口蘑外生菌根菌的相互作用

本实验报道了以简单的离体培养方式来诱导赤松不定根和愈伤组织与松口蘑的菌根反应。不定根和愈伤组织均起源于无菌苗的下胚轴,接种2周后,菌丝体开始包围不定根。接种3周后,菌丝体出现在不定根皮层细胞间,哈蒂氏网型的形成也同时被确认。在愈伤组织培养物中,细胞间也能观察到菌丝体及拟-哈蒂氏网结构。这是第一个离体条件下成功地诱导赤松培养组织与松口蘑形成外生菌根的报道。     

Anatomical Structures of the VA Mycorrhiza in the Apocynaceae (Gentianales)

Roots of 19 Apocynaceae species were studied anatomically with respect to their symbiosis with VAM-fungi. In plants collected from the field, VAM-fungi were established in the root cortex. Also, inoculations with different Glomus species on the cultured plants are very successful in the infection and colonization of the root cortex. After penetration of the rhizodermis, the special exodermal short cells become colonized by winding hyphae. Then, in the root cortex of many Apocynaceae species, the VAM-fungi produce intercellular running hyphae which leads to extensive colonization of the root. Arbuscules develop on intracellular running hyphae, whereas vesicles develop mainly on intercellular hyphae. Except for some special details, this is the most common type of colonization of VAM fungi in flowering plants. But in Amsonia tabernaemontana, Nerium oleander, and Thevetia peruviana, another type of colonization could be observed. In these species, the colonization of the hyphae within the root cortex is only possible by intracellular growth. Intercellular running hyphae in the root are lacking. Therefore, after penetration the colonization in the cortex is cluster-like and strictly limited. Only by additional penetrations from hyphae in the soil, will roots show heavy infestations. This type of growth of the VAM fungi in the root is well known from the Gentianaceae and was explained as a structural incompatibility. In Catharanthus roseus, Pachypodium lamerei, and Trachelospermum jasminoides, intermediate stages of both types of colonization could be described. The results are discussed in the search for possible stimulants for structural incompatibility.     

A New Fungal Endophyte,Scolecobasidium humicola,Promotes Tomato Growth under Organic Nitrogen Conditions

A new fungal endophyte, Scolecobasidium humicola, was identified as a common dark septate endophytic fungal (DSE) species under both natural and agricultural conditions. This fungus was found to grow endophylically in the roots of tomato seedlings. Light microscopy of cross-sections of colonized tomato roots showed that the intercellular, pigmented hyphae of the fungus were mostly limited to the epidermal layer and formed outer mantle-like structures. Two isolates of S. humicola, H2-2 and F1-3, have shown the ability to increase plant biomass with an organic nitrogen source. This finding is the first report of S. humicola as an endophyte and could help to improve plant growth with organic nitrogen sources.     

A dorsiventral mycorrhizal root in the achlorophyllous<Emphasis Type="Italic"> Sciaphila polygyna</Emphasis> (Triuridaceae)

The star-like root system of the achlorophyllous Sciaphila polygyna (Triuridaceae) consists of roots up to 1.4 mm thick and 1 cm long seemingly radiating from a single origin. Internally, the roots show a bilateral symmetry when viewed in cross-section: the third root cell layer contains rather loose coils of the aseptate mycorrhizal fungus from the dorsal to the lateral sides, in contrast to the extremely dense coils of thin hyphae in its ventral part. Additionally, the hyphae develop vesicle-like swellings mainly in the central part of the dorsal side as well as the lateral parts of the third layer. The fourth root layer is anatomically heteromorphic, having exceptionally large cells, reaching up to 320×130 m in size (giant cells), in the lower lateral parts. The root-colonizing hyphae only degenerate in the fourth layer, most readily in the giant cells, where they may swell to 24 m in diameter, collapse and end as amorphous clumps. Hyphae in the third layer keep their definite structure. The structures are interpreted to be the result of a dynamic reaction of the root to the actual fungal penetration points in order to maximize the benefit from the subsequent colonization by compartmentation of the root tissue. The function of the third layer is to host the fungus and keep it alive within its cells, while mainly the giant cells serve for its digestion. Many indications suggest an arbuscular mycorrhiza for this association. Similarities and differences to other myco-heterotrophic species are discussed.     

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular fungal pegs within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.     

Role of chemotaxis toward fusaric acid in colonization of hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.     

天麻种子／原球茎和营养繁殖茎被菌根真菌定殖后的细胞分化

天麻（Gastrodia elata Bl.)种子可与紫萁小菇（Mycena osmundicola Lange),兰小菇（M.orchidicola Fan et Guo)等一类小菇属真菌共生萌发形成原球茎。侵入种皮的菌丝集结在柄状细胞外周的胚柄残迹中,首先侵入胚的柄状细胞,然后自柄状细胞侵入其他原胚细胞。原胚细胞发生功能分化,形成菌丝结细胞和消化细胞,初被菌丝定殖的原胚细胞具有消化菌丝的功能,随后,部分原胚细胞逐渐被菌丝充满,充育成菌丝结细胞。菌丝由菌丝结细胞进一步侵入消化细胞后最终被消化。由原球茎分化形成的营养繁殖茎（以下简称营繁茎）进一步被蜜环菌（Armilariella mellea(Vahl.Fr.)Karst.)定植,蜜环菌与紫箕小菇的菌丝同时存在于营繁茎中,但两菌相遇时都停止蔓延,互不交错侵染。     

贵州栽培笃斯越橘DSE真菌、AM真菌与ERM真菌空间分布研究

为探明贵州省栽培笃斯越橘根系深色有隔内生真菌（dark septate endophytes,DSE）、丛枝菌根（arbuscular mycorrhiza,AM）真菌与欧石南菌根（ericoid mycorrhiza,ERM）真菌的定殖及地理分布情况,揭示共生真菌在栽培笃斯越橘生长中的地位,本研究在贵州省笃斯越橘主栽区麻江县、凤岗县和高坡乡分别选取主栽品种圆蓝、粉蓝、奥尼尔和莱格西的根样及根围土样,观测不同地区不同品种根样DSE真菌、AM真菌和ERM真菌的定殖结构和定殖率,并测定土样土壤理化性质,分析不同真菌与土壤因子相关性。结果表明,3个地区的4个笃斯越橘品种均有DSE真菌、AM真菌和ERM真菌定殖,栽培笃斯越橘能与3类真菌形成共生关系,平均定殖率分别为61.11%、25.55%和22.50%。DSE真菌定殖率：高坡（62.50%）>麻江（61.66%）>凤岗（59.16%）;AM真菌定殖率：凤岗（34.14%）>麻江（25.83%）>高坡（16.66%）;ERM真菌定殖率：高坡（35.00%）>凤岗（20.00%）>麻江（12.5%）。相关性分析表明,DSE真菌中的菌丝与微菌核的定殖率呈负相关,AM真菌的总定殖率及定殖强度与微菌核的定殖率呈极显著正相关,与DSE真菌菌丝的定殖率呈负相关。ERM真菌总定殖率与DSE真菌菌丝的定殖率及AM真菌定殖强度呈极显著正相关,与DSE真菌总定殖率呈显著正相关。土壤有效磷与DSE真菌和ERM真菌总定殖率呈显著正相关,与AM真菌定殖率呈显著负相关。土壤铵态氮与DSE真菌中微菌核结构定殖率及AM真菌定殖率呈极显著正相关,与ERM真菌定殖率呈极显著负相关。土壤pH值与DSE定殖强度呈显著负相关,与ERM定殖强度呈极显著正相关。本研究分析比较贵州省3个笃斯越橘种植基地不同品种栽培笃斯越橘DSE真菌、AM真菌和ERM真菌的定殖及其与土壤理化性质之间的相关性,为贵州省栽培笃斯越橘的管理和发展提供技术基础和理论依据。     

Fungal endophytes in Astromyelon-type (Sphenophyta,Equisetales, Calamitaceae) roots from the Upper Pennsylvanian of France

A distinctive fungal endophyte, Cashhickia acuminata nov. gen. et sp., is described from permineralized calamite roots from the Upper Pennsylvanian Grand-Croix cherts of France. Heavily infected roots contain numerous intracellular hyphae in the outer cortex that arise from a meshwork-like mycelium extending between cortical cells. All intracellular hyphae are oriented toward the root center; none occur on the inner periclinal host cell walls. Other roots of the same type show localized infection by this fungus in which isolated cortical cells contain or give rise to intracellular fungal growth. Within the cortical cells are host responses in the form of callosities that indicate the roots were alive at the time of infection. Other endophytes are present in the same host tissue but are less frequent. The discovery of this association provides the first detailed account on the morphology of a Carboniferous fungal root endophyte, as well as the spatial distribution within the host, and infection pathways within the cortical tissues.