Interactions between Lotus japonicus genotypes and arbuscular mycorrhizal fungi

Abstract

Interactions between three genotypes (Ljsym 71-1, Ljsym 71-2 and Ljsym 72) of Lotus japoicus and one isolate from each of four species of arbuscular mycorrhizal fungi (Glomus sp. R-10, Glomus intraradices, Glomus etunicatum, and Gigaspora margarita) were investigated and compared with the wild-type ‘Gifu’ B-129. All the three genotypes showed no or defective internal colonization after inoculation with these AM fungi. In Ljsym72 mutant, the AM fungi produced deformed appressoria on the root surface, but failed to form any internal structures (internal hyphae, arbuscules and vesicles) except only in Glomus intraradices. The Ljsym71-1 and Ljsym71-2 mutants had more deformed appressoria and occasionally formed internal hyphae, arbuscules and vesicles, depending on AM fungi used. Wild-type ‘Gifu’ (nod⁺myc⁺) plants had typical colonization. The colonization of mutants by several fungi varied and provides a basis for studying recognition and compatibility between plants and mycorrhizal fungal species. These mutants also will be useful in studies of the genetics of the symbiosis between plant species and AM fungi.     

Temporal and spatial variation of arbuscular mycorrhizas in salt marsh plants of the Tagus estuary (Portugal)

The factors which may influence temporal and spatial variation in plant arbuscular mycorrhizal (AM) colonization and propagule occurrence were evaluated in a Portuguese salt marsh poor in plant diversity. Two distinct sites were studied: a more-flooded (low marsh) and a less-flooded zone (high marsh). AM root colonization, AM fungal spore number and inoculum potential, soil edaphic parameters and tidal flooding time periods were analysed. Levels of AM colonization were considerable in Aster tripolium and Inula crithmoides but very low in Puccinellia maritima and non-existent in Spartina maritima, Halimione portulacoides, Arthrocnemum fruticosum and Arthrocnemum perenne. Fungal diversity was very low, with Glomus geosporum dominant at both marsh zones. Colonization showed no spatial variation within marsh zones but temporal variation was observed in the high marsh, dependent on plant phenological phases. In the low marsh, no significantly seasonal variation was observed. Apparently, plant phenological events were diluted by stressful conditions (e.g. flooding, salinity). Spore density was significantly different between marsh zones and showed temporal variation in both zones. This study showed that distribution of mycorrhizas in salt marsh is more dependent on host plant species than on environmental stresses.     

Ethylene Alleviates the Suppressive Effect of Phosphate on Arbuscular Mycorrhiza Formation

Phosphorus is one of the most important macronutrients required for plant growth. Plants have evolved many strategies for inorganic phosphorus (Pi) acquisition, including the symbiotic pathways, involving the formation of mycorrhiza. With regard to arbuscular mycorrhiza (AM), high Pi availability has long been known to negatively affect this association, although the underlying mechanism is unknown. In the present study, the interactive role played by ethylene and Pi in AM regulation was investigated in the tomato-Rhizophagus irregularis symbiosis. AM fungal colonization was analysed in epi, rin and NRO ethylene-responsive mutants under different Pi availability conditions, with a focus on the late stages of the interaction. Although Pi inhibited mycorrhizal parameters in the ethylene-insensitive rin mutant and wild-type cultivars, it did not alter the mycorrhization of the epi tomato mutant, which exhibits a constitutive ethylene-induced response. As with the colonization parameters, root ethylene content and the expression of AM-related and ethylene receptor 6 genes were inhibited by Pi in wild-type cultivars and rin mutants, but were unaffected or slightly activated in epi plants. The application of ethephon offsets the negative impact on the mycorrhizal development caused by the application of Pi. This compensation effect is dose dependent and was ineffective in the NRO mutant, which is more insensitive to the action of ethylene. Our results provide evidence that ethylene signalling negatively affects the suppressive effect of Pi on AM formation and suggests an overlap between this suppressive effect and the regulatory mechanism of Pi-starvation response pathway in plants mediated by ethylene.     

Fungal root colonization responses in natural grasslands after long-term exposure to elevated atmospheric CO2

Arbuscular mycorrhizae, ubiquitous mutualistic symbioses between plant roots and fungi in the order Glomales, are believed to be important controllers of plant responses to global change, in particular to elevated atmospheric CO₂. In order to test if any effects on the symbiosis can persist after long-term treatment, we examined root colonization by arbuscular mycorrhizal (AM) and other fungi of several plant species from two grassland communities after continuous exposure to elevated atmospheric CO₂ for six growing seasons in the field. For plant species from both a sandstone and a serpentine annual grassland there was evidence for changes in fungal root colonization, with changes occurring as a function of plant host species. We documented decreases in percentage nonmycorrhizal fungal root colonization in elevated CO₂ for several plant species. Total AM root colonization (%) only increased significantly for one out of the five plant species in each grassland. However, when dividing AM fungal hyphae into two groups of hyphae (fine endophyte and coarse endophyte), we could document significant responses of AM fungi that were hidden when only total percentage colonization was measured. We also documented changes in elevated CO₂ in the percentage of root colonized by both AM hyphal types simultaneously. Our results demonstrate that changes in fungal root colonization can occur after long-term CO₂ enrichment, and that the level of resolution of the study of AM fungal responses may have to be increased to uncover significant changes to the CO₂ treatment. This study is also one of the first to document compositional changes in the AM fungi colonizing roots of plants grown in elevated CO₂. Although it is difficult to relate the structural data directly to functional changes, possible implications of the observed changes for plant communities are discussed.     

Gibberellin regulates infection and colonization of host roots by arbuscular mycorrhizal fungi

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from this symbiotic interaction, and both regulate AM development. Our recent work revealed that gibberellic acids (GAs) are required for AM development in the legume Lotus japonicus. GA signaling interact with symbiosis signaling pathways, directing AM fungal colonization in host roots. Expression analysis showed that genes for GA biosynthesis and metabolism were induced in host roots around AM fungal hyphae, suggesting that the GA signaling changes with both location and time during AM development. The fluctuating GA concentrations sometimes positively and sometimes negatively affect the expression of AM-induced genes that regulate AM fungal infection and colonization.     

Influence of Arbuscular Mycorrhizal (AM) Symbiosis on Phosphorus Leaching through Soil Cores

Two experiments with soil cores were carried out to investigate the effects of arbuscular mycorrhizal (AM) fungal colonization on mobility of phosphorus (P) during leaching of repacked columns of a soil with a loamy sand texture. Trifolium subterraneum plants inoculated with an AM fungus or not inoculated were grown in cores with low or high P concentrations for 8 or 10 weeks in the glasshouse. Cores were then irrigated with 2500 mL water and the leachate collected. Plant growth and the amounts of P removed by plants, remaining in soil as available P and removed dissolved in leachate were measured. Mycorrhizal fungal colonization and development of external hyphae were also determined. Inoculation and/or P application significantly increased plant growth and plant P removal and decreased P leaching. In low P soils AM fungal colonization significantly increased plant P uptake and decreased soil available P and total dissolved P in leachates. Lower P leaching from cores with AM plants under low P conditions was related to enhancement of plant growth and to scavenging and removal of P from the soil by roots and/or external hyphae. When P was applied AM effects were not observed and available P remaining in the soil after leaching was much higher, regardless of AM fungal colonization.     

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.     

Cellular Events Involved in Survival of Individual Arbuscular Mycorrhizal Symbionts Growing in the Absence of the Host

A survival strategy operating in the absence of the host was shown in obligately biotrophic arbuscular mycorrhizal (AM) symbionts. When no host-derived signals from the surrounding environment were perceived by germinating spores, fungal hyphae underwent a programmed growth arrest and resource reallocation, allowing long-term maintenance of viability and host infection capability. The early stages of mycelial growth of AM fungi were studied by a combination of time-lapse and video-enhanced light microscopy, image analysis, and immunodetection, with the aim of acquiring knowledge of cell events leading to the arrest of mycelial growth. The time-course of growth arrest was resolved by precisely timing the growth rate and magnitude of the mycelium originating from individual spores of Glomus caledonium. Extensive mycelial growth was observed during the first 15 days; thereafter, fungal hyphae showed retraction of protoplasm from the tips, with formation of retraction septa separating viable from empty hyphal segments. This active process involved migration of nuclei and cellular organelles and appeared to be functional in the ability of the fungus to survive in the absence of a host. Immunodetection of cytoskeletal proteins, metabolic activity, and the retention of infectivity of germinated spores confirmed the developmental data. The highest amounts of tubulins were detected when hyphal growth had ceased but when retraction of protoplasm was most active. This was consistent with the role of the cytoskeleton during protoplasm retraction. Succinate dehydrogenase activity in hyphae proximal to the mother spore was still detectable in 6-month-old mycelium, which remained viable and able to form appressoria and produce symbiotic structures.     

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.     

H2O2参与AM真菌与烟草共生过程

以烟草((Nicotiana tabacum,品种CF90NF)为寄主,苗期接种丛枝菌根(AM)真菌摩西球囊霉(Glomus mosseae,G.m),测定G.m与烟草共生过程中烟草根部H2O2含量以及多胺氧化酶(PAO)和过氧化物酶(POD)活性;研究外源H2O2对G.m侵染烟草的影响以及H2O2清除剂和合成抑制剂对烟草侧根H2O2含量及烟草侧根和菌丝中H2O2荧光强度的影响,以探究H2O2在AM真菌侵染烟草过程中的作用。结果表明,接种G.m 20d后烟草侧根中出现H2O2含量的猝发,一定浓度的外源H2O2促进G.m对烟草的侵染,而H2O2清除剂抗坏血酸(AsA)显著削弱烟草侧根和菌丝中的H2O2荧光强度,降低G.m对烟草的侵染率,表明H2O2参与G.m与烟草共生过程;在G.m与烟草共生过程中,PAO和POD活性显著升高,PAO抑制剂二氨基十二烷(DADD)和POD抑制剂水杨羟肟酸(SHAM)显著降低烟草侧根中H2O2荧光强度,对菌丝中H2O2荧光强度无显著影响,表明烟草根部和G.m均可产生H2O2,PAO和POD参与烟草侧根中H2O2的合成,菌丝中可能存在其他来源的H2O2。     

A Microscopic Characterization of the Infection of Green and Red Pepper Fruits by an Isolate of Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is a common pathogenic fungus in many plants. To investigate the specificity of the isolate C. gloeosporioides to green fruits, fungal behaviours and anthracnose development on green and red pepper fruits were compared using light and stereo microscopic techniques. When the isolate of C. gloeosporioides was inoculated on both green and red fruits, conidial germination, appressoria, and infection hyphae were observed on both fruits within 24 h after inoculation. The fungal invasion and colonization continued to the epidermal cells of green fruits, but not to those of red ones. Initial anthracnose symptoms were detected only on green fruits at 2 days after inoculation resulting in typical sunken necrosis within 5 days after inoculation. Thus the specificity of the isolate to green fruits may be due to successful invasion and colonization of the infection hyphae from appressoria into the epidermal cells through epicuticular layers of green pepper fnats, but not on red ones.     

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.     

Medicago truncatula Vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism formed between vascular plants and fungi of the Glomeromycota. In this endosymbiosis, fungal hyphae enter the roots, growing through epidermal cells to the cortex where they establish differentiated hyphae called arbuscules in the cortical cells. Reprogramming of the plant epidermal and cortical cells occurs to enable intracellular growth of the fungal symbiont; however, the plant genes underlying this process are largely unknown. Here, through the use of RNAi, we demonstrate that the expression of a Medicago truncatula gene named Vapyrin is essential for arbuscule formation, and also for efficient epidermal penetration by AM fungi. Vapyrin is induced transiently in the epidermis coincident with hyphal penetration, and then in the cortex during arbuscule formation. The Vapyrin protein is cytoplasmic, and in cells containing AM fungal hyphae, the protein accumulates in small puncta that move through the cytoplasm. Vapyrin is a novel protein composed of two domains that mediate protein–protein interactions: an N‐terminal VAMP‐associated protein (VAP)/major sperm protein (MSP) domain and a C‐terminal ankyrin‐repeat domain. Putative Vapyrin orthologs exist widely in the plant kingdom, but not in Arabidopsis, or in non‐plant species. The data suggest a role for Vapyrin in cellular remodeling to support the intracellular development of fungal hyphae during AM symbiosis.     

The Jasmonic Acid Signalling Pathway Restricts the Development of the Arbuscular Mycorrhizal Association in Tomato

The role of the jasmonate signalling pathway in modulating the establishment of the arbuscular mycorrhiza (AM) symbiosis between tomato plants and Glomus intraradices fungus was studied. The consequences of AM formation due to the blockage of the jasmonate signalling pathway were studied in experiments with plant mutants impaired in JA perception. The tomato jai-1 mutant (jasmonic acid insensitive 1) failed to regulate colonization and was more susceptible to fungal infection, showing accelerated colonization. The frequency and the intensity of fungal colonization were greatly increased in the jai-1 insensitive mutant plants. In parallel, the systemic effects on mycorrhization due to the activation of the jasmonate signalling pathway by foliar application of MeJA were evaluated and histochemical and molecular parameters of mycorrhizal intensity and efficiency were measured. Histochemical determination of fungal infectivity and fungal alkaline phosphatase activity reveal that the systemic application of MeJA was effective in reducing mycorrhization and mainly affected fungal phosphate metabolism and arbuscule formation, analyzed by the expression of GiALP and the AM-specific gene LePT4, respectively. The results of the present study clearly show that JA participates in the susceptibility of tomato to infection by arbuscular mycorrhizal fungi, and it seems that arbuscular colonization in tomato is tightly controlled by the jasmonate signalling pathway.     

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。     

Full Establishment of Arbuscular Mycorrhizal Symbiosis in Rice Occurs Independently of Enzymatic Jasmonate Biosynthesis

Development of the mutualistic arbuscular mycorrhiza (AM) symbiosis between most land plants and fungi of the Glomeromycota is regulated by phytohormones. The role of jasmonate (JA) in AM colonization has been investigated in the dicotyledons Medicago truncatula, tomato and Nicotiana attenuata and contradicting results have been obtained with respect to a neutral, promotive or inhibitory effect of JA on AM colonization. Furthermore, it is currently unknown whether JA plays a role in AM colonization of monocotyledonous roots. Therefore we examined whether JA biosynthesis is required for AM colonization of the monocot rice. To this end we employed the rice mutant constitutive photomorphogenesis 2 (cpm2), which is deficient in JA biosynthesis. Through a time course experiment the amount and morphology of fungal colonization did not differ between wild-type and cpm2 roots. Furthermore, no significant difference in the expression of AM marker genes was detected between wild type and cpm2. However, treatment of wild-type roots with 50 μM JA lead to a decrease of AM colonization and this was correlated with induction of the defense gene PR4. These results indicate that JA is not required for AM colonization of rice but high levels of JA in the roots suppress AM development likely through the induction of defense.     

The proliferation of fungal hyphae in soils supporting mycorrhizal and non-mycorrhizal plants

This study investigated the impact of mycorrhizal plants, non-mycorrhizal plants and soil organic matter on the relative abundance of soil hyphae perceived to belong to indigenous arbuscular mycorrhizal (AM) plants. The mycorrhizal plants corn (Zea mays L.) and barley (Hordeum vulgare L.) and a non-mycorrhizal plant, canola (Brassica napus L.), were grown in unsterilized soil in pots inoculated with mycorrhizal corn root fragments. The abundance of hyphae was measured after 5 weeks and the response of fungal growth to the addition of corn residues in the absence of plants was assessed. The abundance of hyphae was higher in the presence of the mycorrhizal plants than in the other treatments. AM hyphae present under mycorrhizal plants accounted for more than 83% of the measured hyphae. The levels of root colonization of 32% in corn and 27% in barley confirmed the mycorrhizal status of the experimental plants. Only a few points of entry were observed in canola, the non-host plant. The percentage of mycorrhizal colonization was positively related (R ²?=?0.85) to the abundance of soil hyphae, indicating that AM hyphae were the major component of the soil hyphae in the presence of mycorrhizal plants in this study.     

Effect of the arbuscular mycorrhizal symbiosis upon uptake of cesium and other cations by plants

Pot experiments were set up to determine the species-specific uptake of cesium (Cs) by mycorrhizal (AM) and non-mycorrhizal (non-AM) plants. Using stable Cs and K application, side-effects of mineral fertilization (K) on AM development and uptake of Cs and the other cations Na, Ca and Mg were investigated. AM colonization by the fungus Glomus mosseae led to a significant decrease in shoot Cs content of Agrostis tenuis from the first (4 weeks) to the third harvest (8 weeks). With regard to the root system, statistically significant differences were observed from the first (4 weeks) to the second harvest (6 weeks). Supply of additional K produced a significant decrease in Cs uptake by both AM and non-AM plants over a 10-week period. In the case of AM plant shoots, K fertilization did not very effectively reduce Cs uptake by A. tenuis. Cs contents of fertilized AM roots were similar to non-AM controls. Potassium application resulted in an increase in K content and a slight reduction in Na and Mg contents of shoots and roots. Without K fertilization, the Na content of non-AM controls was significantly enhanced over AM shoots. Shoot and root Ca contents were generally higher without than with K addition. Negative side-effects of K fertilization as a countermeasure to Cs uptake were not observed in relation to AM development. The intensity of colonization by G. mosseae was not significantly depressed by K treatment. AM development in plants appeared to decrease Cs uptake, at least at moderate nutrient levels. It is possible that Cs is sequestered by AM extraradical fungal hyphae and consequently not transferred to the plant to the extent found in non-AM roots. Accepted: 6 November 2000