Heavy-Metal Stress and Developmental Patterns of Arbuscular Mycorrhizal Fungi

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices, had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices. A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.     

磷和镉对根内球囊霉Glomus intraradices孢子萌发、菌丝生长和外生菌丝内聚磷酸累积的影响

丛枝菌根真菌(AMF)能够显著提高植物对重金属的抗性,菌丝内聚磷酸(Polyphosphate,PolyP)可能参与了这种抗性的形成。试验以Glomus intraradices孢子为试材,对灭菌条件进行了优化,并进一步研究了不同P和Cd2+水平对孢子萌发、菌丝生长、分支和外生菌丝中聚磷酸含量的影响。结果表明,孢子在1%氯胺T+0.02%链霉素+0.01%庆大霉素+1/100(V/V)吐温-20中灭菌5min的效果最好。孢子萌发率、菌丝分支和菌丝长度随着Cd2+浓度的增加不断降低;当Cd2+浓度达到0.1mmol/L时,孢子萌发率降低为0%,表明Glomus intraradices的孢子萌发对Cd2+的耐受极限为0.1mmol/L;1mmol/L的P促进菌丝分支增加,却降低了萌发率,但对菌丝生长没有影响;在培养23d以后,三者基本不再变化。外生菌丝内的聚磷酸含量随着P的升高而增加;在Cd2+胁迫作用下,聚磷酸的含量降低而菌丝密度随着聚磷酸的升高而升高,表明聚磷酸在减弱重金属毒性方面起了重要作用。     

Ultrastructural localization of heavy metals in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi, obligate symbionts of most plant species, are able to accumulate heavy metals, thereby, protecting plants from metal toxicity. In this study, the ultrastructural localization of Zn, Cu, and Cd in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic cultures was investigated. Zinc, Cu, or Cd was applied to the extraradical mycelium to final concentrations of 7.5, 5.0, or 0.45 mmol/L, respectively. Samples were collected at time 0, 8 h, and 7 days after metal application and were prepared for rapid freezing and freeze substitution. Metal content in different subcellular locations (wall, cytoplasm, and vacuoles), both in hyphae and spores, was determined by energy-dispersive X-ray spectroscopy. In all treatments and fungal structures analysed, heavy metals accumulated mainly in the fungal cell wall and in the vacuoles, while minor changes in metal concentrations were detected in the cytoplasm. Incorporation of Zn into the fungus occurred during the first 8 h after metal addition with no subsequent accumulation. On the other hand, Cu steadily accumulated in the spore vacuoles over time, whereas Cd steadily accumulated in the hyphal vacuoles. These results suggest that binding of metals to the cell walls and compartmentalization in vacuoles may be essential mechanisms for metal detoxification.     

Fungal lipid accumulation and development of mycelial structures by two arbuscular mycorrhizal fungi

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1omega5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1omega5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1omega5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.     

Characterization of a Glomus intraradices gene encoding a putative Zn transporter of the cation diffusion facilitator family

A full-length cDNA (GintZnT1) encoding a putative Zn transporter was isolated from the extraradical mycelium of Glomus intraradices. Based on its sequence analysis, GintZnT1 was classified as a member of the cation diffusion facilitator (CDF) family of heavy metal transporters. Functional analysis of GintZnT1 was performed by heterologous expression in yeast mutants defective in different CDFs. Although Zn sensitivity of the mutants was not reverted, an effect of GintZnT1 on the labile regulatory Zn pool was detected by using a Zn-regulated beta-galactosidase reporter gene. GintZnT1 expression was studied in the extraradical mycelium obtained from a symbiotic root organ culture. Gin +/- ZnT1 was up-regulated in the extraradical mycelium of G. intraradices upon short-time exposure to Zn and when the mycelia were developed in 75 microM Zn supplemented plates. These data suggest a role of GintZnT1 in Zn compartmentalization and in the protection of G. intraradices against Zn stress.     

Nitrogen transfer and assimilation between the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith and Ri T-DNA roots of Daucus carota L. in an in vitro compartmented system

Nitrogen metabolism was examined in monoxenic cultures of carrot roots (Daucus carota L.) colonized with the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith. Glutamine synthetase and glutamate dehydrogenase activities were significantly increased in mycorrhizal roots for which only the extraradical mycelium had exclusive access to NH4NO3 in a distinct hyphal compartment inaccessible to the roots. This was in comparison with the water controls but was similar to the enzyme activities of non-arbuscular-mycorrhizal (non-AM) roots that had direct access to NH4NO3. In addition, glutamate dehydrogenase activity was significantly enhanced in AM roots compared with non-AM roots. Carrot roots took up 15NH4+ more efficiently than 15NO3-, and the extraradical hyphae transfered 15NH4+ to host roots from the hyphal compartment but did not transfer 15NO3-. The extraradical mycelium was shown, for the first time, to have a different glutamine synthetase monomer than roots. Our overall results highlight the active role of AM fungi in nitrogen uptake, transfer, and assimilation in their symbiotic root association.     

Different nitrogen sources modulate activity but not expression of glutamine synthetase in arbuscular mycorrhizal fungi

Glutamine synthetase (GS) is a central enzyme of nitrogen metabolism that allows assimilation of nitrogen and biosynthesis of glutamine. We isolated the cDNA encoding GS from two arbuscular mycorrhizal fungi, Glomus mosseae (GmGln1) and Glomus intraradices (GiGln1). The deduced protein orthologues have a high degree of similarity (92%) with each other as well as with GSs from other fungi. GmGln1 was constitutively expressed during all stages of the fungal life cycle, i.e., spore germination, intraradical and extraradical mycelium. Feeding experiments with different nitrogen sources did not induce any change in the mRNA level of both genes independent of the symbiotic status of the fungus. However, GS activity of extraradical hypahe in G. intraradices was considerably modulated in response to different nitrogen sources. Thus, in a N re-supplementation time-course experiment, GS activity responded quickly to addition of nitrate, ammonium or glutamine. Re-feeding with ammonium produced a general increase in GS activity when compared with hyphae grown in nitrate as a sole N source.     

Symbiotic interaction of endophytic bacteria with arbuscular mycorrhizal fungi and its antagonistic effect on Ganoderma boninense

Endophytic bacteria (Pseudomonas aeruginosa UPMP3 and Burkholderia cepacia UMPB3), isolated from within roots of oil palm (Elaeis guineensis Jacq.) were tested for their presymbiotic effects on two arbuscular mcorrhizal fungi, Glomus intraradices UT126 and Glomus clarum BR152B). These endophytic bacteria were also tested for antagonistic effects on Ganoderma boninense PER 71, a white wood rot fungal pathogen that causes a serious disease in oil palm. Spore germination and hyphal length of each arbuscular mycorrhizal fungal (AMF) pairing with endophytic bacteria was found to be significantly higher than spores plated in the absence of bacteria. Scanning electron microscopy (SEM) showed that the endophytic bacteria were scattered, resting or embedded on the surface hyaline layer or on the degraded walls of AMF spores, possibly feeding on the outer hyaline spore wall. The antagonistic effect of the endophytic bacteria was expressed as severe morphological abnormalities in the hyphal structures of G. boninense PER 71. The effects of the endophytic bacteria on G. boninense PER 71 hyphal structures were observed clearly under SEM. Severe inter-twisting, distortion, lysis and shriveling of the hyphal structures were observed. This study found that the effect of endophytic bacteria on G. intraradices UT126 and G. clarum BR152B resembled that of a mycorrhiza helper bacteria (MHB) association because the association significantly promoted AMF spore germination and hyphal length. However, the endophytic bacteria were extremely damaging to G. boninense PER 71.     

Branched absorbing structures (BAS): a feature of the extraradical mycelium of symbiotic arbuscular mycorrhizal fungi

The present work describes the morphogenesis and cytological characteristics of 'branched absorbing structures' (BAS, formely named arbuscule-like structures, ALS), small groups of dichotomous hyphae formed by the extraradical mycelium of arbuscular mycorrhizal (AM) fungi. Monoxenic cultures of the AM fungus Glomus intraradices Smith & Schenck and tomato ( Lycopersicum esculentum Mill.) roots allowed the continuous, non-destructive study of BAS development. These structures were not observed in axenic cultures of the fungus under different nutritional conditions or in unsuccessful (asymbiotic) monoxenic cultures. However, extraradical mycelium of G. intraradices formed BAS immediately after fungal penetration of the host root and establishment of the symbiosis. The average BAS development time was 7 d under our culture conditions, after which they degenerated, becoming empty septate structures. Certain BAS were closely associated with spore formation, appearing at the spore's substending hypha. Branches of these spore-associated BAS (spore-BAS) usually formed spores. Electron microscopy studies revealed that BAS and arbuscules show several ultrastructural similarities. The possible role of BAS in nutrient uptake by the mycorrhizal plant is discussed.     

Competition and substrate colonization strategies of three polyxenically grown arbuscular mycorrhizal fungi

Intra- and extraradical colonization competition and hyphal interactions among arbuscular mycorrhizal fungi (AMF) Glomus intraradices, Glomus proliferum and Gigaspora margarita were investigated in two in vitro experimental systems. AMF were polyxenically cultured with a Ri T-DNA transformed carrot root organ culture (ROC) in either big Petri plates containing three culture compartments and a common hyphal compartment (i.e. an independent host root for each AMF) or two by two in the culture compartment of regular bicompartmented Petri dishes (i.e. a common host root and a common hyphal compartment). Maps of the extraradical mycelial development of the three AMF were obtained. Two distinct substrate colonization strategies (Glomus-type and Gigaspora-type) were identified, reflecting intrinsic differences among AMF genera/families. Our data reveal a general lack of antagonism between the isolates when extraradical hyphae explore and exploit the substrate outside the root influence zone; however certain growth restrictions were imposed by Gi. margarita extraradical mycelium when developing near the host root and by G. proliferum intraradical hyphae. This work highlights once more the appropriateness of AM in vitro culture systems to perform in vivo studies on the biology of this symbiosis and opens new avenues to the formulation of in vitro AMF inoculants.     

The Glyoxylate Cycle in an Arbuscular Mycorrhizal Fungus. Carbon Flux and Gene Expression

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of (13)C labeling of germinating spores and extraradical mycelium with (13)C(2)-acetate and (13)C(2)-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle.     

Fungal Lipid Accumulation and Development of Mycelial Structures by Two Arbuscular Mycorrhizal Fungi

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1ω5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1ω5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1ω5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.     

Differential morphogenesis of the extraradical mycelium of an arbuscular mycorrhizal fungus grown monoxenically on spatially heterogeneous culture media

A new in vitro experimental system was developed to study the morphogenesis of discrete regions of a single extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, growing simultaneously in six different agar-based media. The media were (i) unamended water agar (WA), (ii) WA+PO(4)(3-) (PO(4)(3-)), (iii) WA+NO(3)(-) (NO(3)(-)), (iv) WA+NH(4)(+) (NH(4)(+)), (v) WA+NH(4)(+)+MES (NH(4)(+)+MES) and (vi) minimal medium (M, complete nutrients). Each medium was amended with the pH indicator bromocresol purple. The extraradical mycelium of the fungus showed between-treatment differences in morphogenesis, architecture, formation of branched absorbing structures (BAS) and sporulation. Extraradical hyphae that developed in WA or PO(4)(3-) compartments exhibited an economic development pattern, in which runner hyphae radially extended the external colony. Extraradical hyphal growth in the NO(3)(-) compartments was characterized by increased formation of runner hyphae, BAS and spores and an alkalinization of the medium. In the two NH(4)(+)-amended media (NH(4)(+), NH(4)(+)+MES), sporulation was suppressed and considerable morphological changes were noted. These results show the plasticity of G. intraradices that lets it efficiently exploit an heterogeneous substrate.     

植物磷营养状况对丛枝菌根真菌生长及代谢活性的调控*

采用四室隔网培养装置,以玉米为宿主植物,通过在植物生长室设0、50、250和500 mgPkg-1 4个施磷水平,研究了植株体内的磷营养状况对AM真菌Glomus sinuosum和Glomus intraradices生长及活性的影响。研究发现在不施磷条件下,接种AM真菌G. intraradices显著促进了植物生长和磷的吸收;低磷条件(50 mgPkg-1)下,接种菌根真菌显著促进了植物对磷的吸收,但对植物生长没有明显的影响;而在高磷条件(250 mgPkg-1 和500 mgPkg-1)下,接种菌根真菌不但没有促进植物的生长和磷的吸收,反而对其有抑制作用。随着施磷水平的提高, AM真菌根内菌丝的碱性磷酸酶活性显著下降;与不施磷相比,低量(50 mgPkg-1)供磷增加了AM真菌土壤中根外菌丝的密度,高磷(250 mgPkg-1 和500 mgPkg-1)降低了土壤中根外菌丝的密度。上述结果说明:⑴ 给宿主植物施用磷肥引起的植物磷营养状况的改变,对AM真菌生长和代谢活性具有一定的调控作用;⑵ G. sinuosum和G. intraradices两种AM真菌的生长和代谢活性对施磷水平的响应程度无显著性差异;⑶ 高磷抑制AM真菌生长和代谢活性,使真菌吸磷量减少,可能是造成菌根效应降低的原因之一     

Expressed genes in the extraradical hyphae of an arbuscular mycorrhizal fungus, Glomus intraradices, in the symbiotic phase

To collect extraradical hyphae of arbuscular mycorrhizal (AM) fungi for RNA isolation, a PVDF membrane was laid on the hyphal compartment of a two-compartment culture system of transformed carrot hairy roots and Glomus intraradices. Extraradical hyphae free from host tissue were easily collected, and their RNA was rapidly extracted with a modified acid guanidinium thiocyanate-phenol-chloroform method. A 3'-RACE (rapid amplification of cDNA ends) of a known gene indicated that this protocol enabled the isolation of mRNA molecules as small as 2.3 kb. The cDNA libraries of an AM fungus from the aseptic extraradical hyphae in a symbiotic state were constructed for the first time. Three-fourth of 150 ESTs (expressed sequence tags) indicated low or no similarities to known sequences from other organisms.     

Cd-tolerant arbuscular mycorrhizal (AM) fungi from heavy-metal polluted soils

Spores of arbuscular mycorrhizal (AM) fungi were isolated from two heavy-metal polluted soils in France via trap culture with leek (Allium porrum L.). Preliminary identification showed that the predominant spore type of both cultures (P2 and Cd40) belongs to the Glomus mosseae group. Their sensitivity to cadmium was compared to a laboratory reference strain (G. mosseae) by in vitro germination tests with cadmium nitrate solutions at a range of concentrations (0 to 100 mg L^–1) as well as extracts from a metal-polluted and unpolluted soils. Both cultures of AM fungi from heavy-metal polluted soils were more tolerant to cadmium than the G. mosseae reference strain. The graphically estimated EC₅₀ was 0.8 mg L^–1 Cd (concentration added to the test device) for G. mosseae and 7 mg L^–1 for P2 culture, corresponding to effective Cd concentrations of approximately 50–70 g L^–1 and 200–500 g L^–1, respectively. The extract of the metal-polluted soil P2 decreased germination of spores from the reference G. mosseae but not from P2 culture. However, the extracts of two unpolluted soils with different physico-chemical characteristics did not affect G. mosseae, whereas germination of P2 spores was markedly decreased in the presence of one of the extracts. These results indicate a potential adaptation of AM fungi to elevated metal concentrations in soil. The tested spores may be considered as metal-tolerant ecotypes. Spore germination results in presence of soil extracts show the difficulty of assessing the ecotoxic effect of metals on AM fungi without considering other soil factors that may interfere in spore germination and hyphal extension.     

AM真菌在煤矿废弃物中生态适应性的初步研究

以粉煤灰、草炭、蛭石、河沙为培养基质,分别对4种不同AM真菌:Glomus mosseae,G.diaphanum,G.intraradices和G.versiforme的生态适应性进行研究。结果表明,菌根的侵染率、孢子密度和菌丝长度分别与菌根真菌种类、培养基质状况及寄主植物种类有关。4种基质的扩繁效果顺序为:河沙>粉煤灰>草炭>蛭石。G.mosseae和G.diaphanum在基质中的产孢量和菌丝长度优于G.intraradices和G.versiforme,可作为优势菌株。4种菌根真菌在粉煤灰中对寄主的侵染率均达到60%以上,粉煤灰作为菌根真菌培养基质具有更大潜力和实际应用价值。