脱落酸对丛枝菌根真菌侵染和产孢的调控效应研究

【目的】揭示脱落酸(ABA)对丛枝菌根(AM)真菌侵染和产孢的影响,建立利用外源ABA促进孢子产量的高效菌剂扩繁方法。【方法】利用番茄毛状根和AM真菌Rhizophagus irregularis DAOM 197198建立双重培养体系,通过外源施用ABA、赤霉素(GA)或者使用ABA、GA的缺陷突变体,染色观察菌根侵染,荧光定量PCR测定丛枝发育和脂质合成运输相关基因的表达,统计丛枝和孢子的数量,从而揭示ABA对AM真菌侵染和产孢的影响。【结果】ABA缺陷突变体not中的F%(侵染频率)、a%(丛枝丰度)、丛枝数量,以及丛枝发育特异性相关基因EXO70A1-like (LOC101253481)、脂质合成运输相关基因RAM2和STR2的表达均显著低于其野生型MT;外源施用ABA显著促进了F%、M%(侵染强度)、丛枝数量、孢子产量,以及脂质合成运输相关基因RAM2和STR2的表达,外源添加ABA处理的孢子产量约为不添加处理的4.5倍;外源GA处理极显著抑制了菌根侵染的所有指标和孢子产量;GA缺陷突变体gib3与其野生型MM的AM真菌侵染之间没有显著差异,但gib3的孢子产量显著高于MM。【结论】ABA通过促进脂质的合成和运输,提高AM真菌的侵染和丛枝形成,进而增加AM真菌的孢子产量。     

A comparison of visualization techniques for recording arbuscular mycorrhizal colonization

The extent of arbuscular mycorrhizal colonization was assessed in 10 field-collected plant species, representing three annual forbs, three perennial forbs, three perennial grasses and one annual grass. Each root system of each plant was split into four portions, and for each portion, mycorrhizal structures were revealed with epifluorescence microscopy (under which only arbuscules are generally visible) and three commonly used stains (Chlorazol Black E, Acid Fuchsin and Trypan Blue). The aim of the study was not to evaluate the efficacy of each method, but to compare results obtained by each under standard laboratory conditions. The recorded colonization levels of arbuscules, total arbuscular mycorrhizal fungal material and total fungal (arbuscular mycorrhizal+non-arbuscular mycorrhizal) material differed significantly between visualization methods in a number of species. However, there were also interactions between stain and plant species, indicating that the performance of a stain is dependent on the plant species being examined. In some cases (e.g. Plantago lanceolata ), each visualization method produced the same colonization level, while in others (e.g. Dactylis glomerata ), each method gave a different result. These data therefore suggest that the level of mycorrhizal colonization recorded in any particular plant species at a particular time is dependent on the technique employed.     

Mycorrhizal and Endophytic Fungal Colonization in Arrhenatherum elatius L. Roots According to the Soil Contamination in Heavy Metals

The aim of this work was to jointly study non-mycorrhizal (dark septate fungi) and mycorrhizal (arbuscular mycorrhizae) colonization along a large range of heavy metal pollution in soil in order to determine the effective contribution of each type of endophytes in relation to heavy metal uptake and tolerance. Hence, eight sites were chosen in the mining area of northern France with respect both to a large range of heavy metal contamination (Cd, Pb, Zn) and monospecific colonization by Arrhenatherum elatius. Root colonization with both arbuscular mycorrhizae (AM) and dark septate fungi (DSF) as well as spore density in rhizospheric soil were estimated in relation to soil characteristics. Mycorrhizal infestation (hyphae, arbuscules and vesicles) was adversely affected by soil pollution almost to exclusion. The intensity of colonization with DSF was very low in presence of AM in non-contaminated soils but higher in polluted soils. The effect of the fungal colonization on the heavy metal tolerance of Arrhenatherum elatius is discussed.     

Medicago truncatula Mtha1-2 mutants loose metabolic responses to mycorrhizal colonization

Bidirectional nutrient transfer is one of the key features of the arbuscular mycorrhizal symbiosis. Recently we were able to identify a Medicago truncatula mutant (mtha1-2) that is defective in the uptake of phosphate from the periarbuscular space due to a lack of the energy providing proton gradient provided by the symbiosis specific proton ATPase MtHA1¹ In order to further characterize the impact of fungal colonization on the plant metabolic status, without the beneficial aspect of improved mineral nutrition, we performed leaf ion analyses in mutant and wildtype plants with and without fungal colonization. Although frequency of fungal colonization was unaltered, the mutant did not show a positive growth response to mycorrhizal colonization. This indicates that nutrient transfer into the plant cell fails in the truncated arbuscules due to lacking expression of a functional MtHA1 protein. The leaves of wildtype plants showed clear metabolic responses to root mycorrhizal colonization, whereas no changes of leaf metabolite levels of mycorrhizal mtha1-2 plants were detected, even though they were colonized. These results show that MtHa1 is indispensable for a functional mycorrhizal symbiosis and, moreover, suggest that fungal root colonization per se does not depend on nutrient transfer to the plant host.     

Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi,Glomus spp. and Gigaspora spp.

The localization of acid and alkaline phosphatases in the intraradical hyphae of the arbuscular mycorrhizal fungi, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (Gm), Gl. etunicatum Becker and Gerd. (Ge) and Gigaspora rosea Nicol. and Schenck (Gir) were compared. Marigold (Tagetes patula L.) and leek (Allium porrum L.) were inoculated with each of the three fungi. The mycorrhizal roots were harvested at 3, 4, 5 and 6 weeks after sowing (WAS), treated with a digestion solution containing cellulase and pectinase, and then stained for phosphatase activities at pH 5.0 and pH 8.5. The development of fungal structures in the host root was also examined. Gm formed fine-branched (mature) arbuscules only at the early phase of infection (3 to 4 WAS). Mature arbuscules of Ge and Gir were observed from the early phase (4 WAS) up to the end of experiment. At pH 5.0, the localization of the phosphatase activities of the three fungi were similar irrespective to host plant species. The activity appeared in mature arbuscules and intercellular hyphae, whereas the collapsed arbuscules were inactive. Ten millimolar NaF, an acid phosphatase inhibitor, inhibited the phosphatase activities of Gm and Ge but did not affect that of Gir. At pH 8.5, a difference among the fungal species was found in the localization of phosphatase activity while that between host species was not. The mature arbuscules were also the active sites in all three species. Only Gir showed the activity in the intercellular hyphae while the two Glomus spp. did not. Five millimolar EDTA inhibited the activity of Gir at pH 8.5 while the activities of Ge and Gm were not affected by either 5 mM EDTA or 10 mM KCN (both are alkaline phosphatase inhibitors).     

Mycotrophy of Annona cherimola and the morphology of its mycorrhizae

The mycotrophic character of Annona cherimola (Magnoliales), a tropical/subtropical plantation crop of interest, is described for the first time. This crop seems to depend on mycorrhizae (arbuscular) for optimal growth, with Glomus deserticola being the most effective endophyte tested. Study of the morphology of the arbuscular mycorrhizae in Annona roots showed exclusively intracellular hyphal development, with cell-to-cell fungal passage and an abundance of arbuscules and coiled hyphae within cells. Intercellular distributive hyphae were not observed. The morphology and the pattern of spread of the mycorrhizal colonization were similar for the different endophytes involved and appeared to be dependent on the host root. Such features of mycorrhizal colonization are characteristic of host species lacking intercellular air channels and have been described for some species of ecological interest, but they are not commonly noted in the mycorrhizal literature, especially that dealing with crop species. Some ecophysiological consequences of this pattern of colonization are discussed.     

Short-term response of arbuscular mycorrhizal association to spider mite herbivory

We examined effects of aboveground herbivory by spider mites (Tetranychus urticae) on colonization and activity of arbuscular mycorrhizal fungi (AMF; Gigaspora margarita) using potted plants (Lotus japonicus). We evaluated changes in arbuscular mycorrhizal (AM) association two ways: (1) conventional trypan blue staining of mycorrhizal hyphae to examine AMF biomass in roots (mycorrhizal colonization) and (2) vital staining for a mycorrhizal enzyme (succinate dehydrogenase, SDH) to examine mycorrhizal activity (SDH activity). Mycorrhizal colonization and SDH activity started to increase 4 days after aboveground herbivory, and returned to the initial levels in the absence of mite herbivory in 7 and 12 days, respectively. These results suggest that the change in AM association in response to mite herbivory is a short-term response.     

Occurrence of arbuscular mycorrhizal fungi in bromeliad species from the tropical Atlantic forest biome in Brazil

The mycorrhizal status of epiphytic, rupicolous, and terrestrial bromeliad species from the Brazilian Atlantic Rain Forest has been examined. Roots of 13 species of bromeliads were analyzed for the presence of mycorrhizal structures such as arbuscules, hyphae, and vesicles as well as other fungal structures. Rhizosphere soil was sampled to identify arbuscular mycorrhizal fungal (AMF) species associated only with terrestrial bromeliad species. Most specimens collected were epiphytic bromeliads in the genera Aechmea, Bilbergia, Nidularium, Tillandsia, and Vriesea. Differentiating structures of AMF were found in only three species of bromeliads. The pattern of mycorrhizal colonization was mainly internal, and external mycelium and arbuscules were observed only in the terrestrial Nidularium procerum. Root endophytes with dark brown septate mycelium, thin external hyphae, and Rhizoctonia-like sclerotia were also detected in some root segments. A total of ten spore morphotypes were recovered from the rhizosphere of N. procerum, with Acaulospora mellea, A. foveata, and Glomus sp. being the most common species recovered. Our study demonstrated that most of the epiphytic species are not associated with AMF. We attribute this mainly to the exposed bare root conditions found in epiphytic bromeliads.     

The mycorrhizal status and colonization of 26 tree species growing in urban and rural environments

Urban environments are highly disturbed and fragmented ecosystems that commonly have lower mycorrhizal fungal species richness and diversity compared to rural or natural ecosystems. In this study, we assessed whether the mycorrhizal status and colonization of trees are influenced by the overall environment (rural vs. urban) they are growing in. Soil cores were collected from the rhizosphere of trees growing in urban and rural environments around southern Ontario. Roots were extracted from the soil cores to determine whether the trees were colonized by arbuscular mycorrhizal fungi, ectomycorrhizal fungi, or both, and to quantify the percent colonization of each type of mycorrhizal fungi. All 26 tree species were colonized by arbuscular mycorrhizal fungi, and seven tree species were dually colonized by arbuscular mycorrhizal and ectomycorrhizal fungi. Overall, arbuscular mycorrhizal and ectomycorrhizal fungal colonization was significantly (p < 0.001) lower in trees growing in urban compared to rural environments. It is not clear what ‘urban’ factors are responsible for the reduction in mycorrhizal fungal colonization; more research is needed to determine whether inoculating urban trees with mycorrhizal fungi would increase colonization levels and growth of the trees.     

Characterization of Mycorrhizas Formed by Glomus sp. on Roots of Hypernodulating Mutants of Lotus japonicus

Lotus japonicus hypernodulating mutants, Ljsym78-1 and Ljsym78-2, by the arbuscular mycorrhizal fungus Glomus sp. was characterized. The mutants are defective in systemic autoregulation of nodulation and nitrate inhibition, and form an excess of nodules and lateral roots. The percent root length colonized by the arbuscular mycorrhizal fungi was significantly higher for the mutant than wild-type roots. Detailed assessment of the colonization indicated that the percentage of colonization by arbuscules was increased, but that by external hyphae, internal hyphae and vesicles was decreased, in the mutant roots compared with the wild-type. The succinate dehydrogenase activity of arbuscules, external hyphae and internal hyphae showed similar trends. In addition, the majority of individual arbuscules that formed on the mutant roots had a well-developed and seemingly tough morphology. The results suggest that mutation at the Ljsym78 locus positively stimulates the growth and activity of arbuscules, but leads to reduced growth and activity of hyphae. We report the first identification of Lotus japonicus mutants that show significantly increased arbuscule formation and termed these mutants Arb⁺⁺. Received 8 August 2000/ Accepted in revised form 19 October 2000     

A purple acid phosphatase,GmPAP33, participates in arbuscule degeneration during arbuscular mycorrhizal symbiosis in soybean

Arbuscules are the central structures of the symbiotic association between terrestrial plants and arbuscular mycorrhizal (AM) fungi. However, arbuscules are also ephemeral structures, and following development, these structures are soon digested and ultimately disappear. Currently, little is known regarding the mechanism underlying the digestion of senescent arbuscules. Here, biochemical and functional analyses were integrated to test the hypothesis that a purple acid phosphatase, GmPAP33, controls the hydrolysis of phospholipids during arbuscule degeneration. The expression of GmPAP33 was enhanced by AM fungal inoculation independent of the P conditions in soybean roots. Promoter‐β‐glucuronidase (GUS) reporter assays revealed that the expression of GmPAP33 was mainly localized to arbuscule‐containing cells during symbiosis. The recombinant GmPAP33 exhibited high hydrolytic activity towards phospholipids, phosphatidylcholine, and phosphatidic acid. Furthermore, soybean plants overexpressing GmPAP33 exhibited increased percentages of large arbuscules and improved yield and P content compared with wild‐type plants when inoculated with AM fungi. Mycorrhizal RNAi plants had high phospholipid levels and a large percentage of small arbuscules. These results in combination with the subcellular localization of GmPAP33 at the plasma membrane indicate that GmPAP33 participates in arbuscule degeneration during AM symbiosis via involvement in phospholipid hydrolysis.     

Fertilization and forb:graminoid ratio affect arbuscular mycorrhiza in seedlings but not adult plants of Plantago lanceolata

Aims

Nutrients play a key role in arbuscular mycorrhizal (AM) symbiosis. We quantified the response of AM symbiosis of seedlings and adult plants of Plantago lanceolata to fertilization under field conditions in managed grasslands differing in nutrient availability and soil moisture.

Methods

The AM symbiosis was measured as the total extent of AM fungal colonization and frequency of arbuscules or vesicles, and as the relative proportions of morphotypes. We further examined the effects of the surrounding vegetation upon AM symbiosis.

Results

Fertilization decreased total AM colonization and relative arbuscular frequency of the whole mycorrhizal community and of Acaulospora and “fine endophyte” morphotypes in seedling roots, but it had no effect upon the mycorrhiza in adult plants. The decline in arbuscular frequency in seedling roots due to fertilization was greater at the sites with higher nutrient availability and lower N:P ratio. Seedlings surrounded by more forbs had a greater total AM colonization and higher vesicular frequency.

Conclusions

Increased nutrient availability in the initial stages of seedling development has a prominent effect upon AM symbiosis development, but these effects seem to diminish over the long term, as evidenced by the results obtained for adult plants and from the limited effects of parameters characterizing long-term nutrient availability.     

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.     

Influence of Mycorrhizal Inoculation, Inundation Period, Salinity, and Phosphorus Availability on the Growth of Two Salt Marsh Grasses, Spartina alterniflora Lois. and Spartina cynosuroides (L.) Roth., in Nursery Systems

Restoration of salt marsh ecosystems is an important concern in the eastern United States to mitigate damage caused by industrial development. Little attention has been directed to the mycorrhizal influence on plantings of salt marsh species to stabilize estuarine sediments and establish cover. In our study, seedlings of two salt marsh grasses, Spartina alterniflora and Spartina cynosuroides, were grown in soil with a commercial, mixed species inoculum of arbuscular mycorrhizal fungi. Plants were grown in experimental “ebb and flow” boxes, simulating three levels of tidal inundation, to which two levels of applied phosphorus (P) and two levels of salinity were imposed. After 2.5 months, S. alterniflora was poorly colonized by arbuscular mycorrhizae, developing only fungal hyphae and no arbuscules, but S. cynosuroides became moderately colonized. Mycorrhizal inoculation marginally improved growth and P and nitrogen (N) content of both plant species at low levels of P supply but significantly increased tillering in both plant species. This factor could be beneficial in enhancing ground cover during restoration procedures. Greater P availability increased the mycorrhizal status of S. cynosuroides and improved P nutrition of both plant species, despite a reduction in the root‐to‐shoot ratio. Increasing salinity reduced mycorrhizal colonization of S. alterniflora but not of S. cynosuroides. Growth and nutrient content of S. alterniflora was improved at higher levels of salinity, but only increased nutrient content in S. cynosuroides. Increased duration of tidal inundation decreased plant growth in both species, but tissue P and N concentrations were highest with the longest time of inundation in both species.     

柑橘丛枝菌根形态结构观察

以田间栽培的‘国庆1号’温州蜜柑(Citrus unshiu ‘Guoqing No.1’)/枳(Poncirus trifoliata)和‘国庆4号’温州蜜柑(C.unshiu ‘Guoqing No.4’)/枳为试材观察丛枝菌根的形态结构。结果表明,泡囊、丛枝、侵入点、根内菌丝、根外菌丝、根外厚垣孢子等结构出现在柑橘根系中,说明柑橘属于典型的丛枝菌根植物。丛枝菌根结构中泡囊出现概率较小,丛枝随处可见,侵入点多样化。丛枝菌根的结构主要存在柑橘根段的伸长区和成熟区,首次观察到根冠和分生区也有侵染。     

In vitro mycorrhization of the rubber tree Hevea brasiliensis Müll Arg

In vitro cultivation systems of arbuscular mycorrhizal fungi are useful tools to study the interaction between plants and their fungal symbiont, and also to develop new biotechnologies. Plantlets of the latex-producing species Hevea brasiliensis clone PB 260 were grown in a dense extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 developed from a mycelium donor plant (Medicago truncatula A17). The factors indole-3-butyric acid (IBA), 2-morpholineoethanesulfonic acid monohydrate (MES) buffer, and carbon dioxide (CO₂) were tested on root development and colonization by the fungus. No colonization was observed in the presence of plantlets pre-treated with IBA. The highest levels of root colonization were obtained when plantlets were mycorrhized under a high CO₂ concentration (1,000 μmol?mol^?1) with MES (10 mM) added to the growth medium. Widespread root colonization (with presence of arbuscules, intraradical mycelium, and spores/vesicles) was predominantly observed in newly produced roots. Therefore, it appears essential to improve root initiation and growth for improving in vitro mycorrhization of H. brasiliensis. We demonstrated the potential of the “mycelium donor plant” in vitro culture system to produce colonized H. brasiliensis plantlets before their transfer to ex vitro conditions.     

Measurement of the viability of arbuscular-mycorrhizal fungi using three different stains; relation to growth and metabolic activities of soybean plants

Histochemical staining of alkaline phosphatase (ALP) and succinate dehydrogenase (SDH) activities in four arbuscular mycorrhizal fungi (Glomus intraradices, G. fasciculatum, G. monosporum and G. mosseae) and their relation to growth and metabolic activities of soybean plants were investigated in a greenhouse experiment. In general, mycorrhizal inoculation significantly increased the growth responses, phosphorus and nitrogen contents, acid and alkaline phosphatases as well as total soluble protein of soybean compared to non-mycorrhizal plants. Stimulation was related to the viability of each mycorrhizal fungus. The localization of succinate dehydrogenase (as a vital stain of metabolically active fungus) and alkaline phosphatase activity (as a potential marker of efficiency of the symbiosis) in the arbuscular mycorrhizal fungi were variable. The activity appeared in young arbuscles and intercellular hyphae, whereas the collapsed arbuscules were inactive. The histochemical staining results demonstrated that the activity of alkaline phosphatase fungi was lower than succinate dehydrogenase. The use of nitroblue tetrazolium chloride as a vital stain for SDH activity showed that all mycorrhizal infection revealed by trypan blue staining was not physiologically active. Thus, the possible utilization of these enzymes to assess the activity of mycorrhizal fungi and its relation with effectively for plant growth and mineral contents is discussed.     

Arbuscules of vesicular-arbuscular mycorrhizal fungi inhabit an acidic compartment within plant roots

 The most widespread type of mycorrhiza is the so-called vesicular-arbuscular mycorrhiza. In this endomycorrhiza, fungal hyphae penetrate plant cell walls in the root cortex. There they form densely branched arbuscules. Fungus and plant plasma membrane are separated by a common interfacial apoplast. The pH of the compartment between the symbionts is of pivotal importance for nutrient transfer. Histochemical experiments were conducted to check for an acidic nature of the interface in the model system Glomus versiforme (Karst.) Berch-Allium porrum L. Two chemically different acidotropic dyes (neutral red and LysoSensor Green DND-189) stained the arbuscules intensely. The staining of arbuscules could be eliminated by addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) or treatments leading to membrane rupture. Therefore, the staining of the arbuscules was based on the ion-trap mechanism, which indicates acidic, membrane-bound compartments. Microscopic examination of stained arbuscules at high optical resolution revealed a peripheral accumulation of the dye. Since plasmolysis rapidly destained the arbuscules, it is concluded that the dyes accumulate in the arbuscular interface, indicating the highly acidic nature of this compartment. The findings are discussed with respect to their relevance for the nutrient transfer in mycorrhizas. In addition, evidence for a discontinuity in the arbuscular interface between the stem and the branches of the arbuscule is given. Received: 15 September 1999 / Accepted: 20 February 2000     

Comparative study of mycorrhizal susceptibility and anatomy of four palm species

A morphological and anatomical study of the root systems of the palm species Brahea armata S. Watson, Chamaerops humilis L., Phoenix canariensis Chabaud and Phoenix dactylifera L. has been carried out to determine possible mycorrhizal colonization sites. Furthermore, the arbuscular mycorrhizal (AM) anatomical types formed by the four palm species in association with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe have been examined. The presence of a continuous sclerenchymatic ring in the outer cortex and aerenchyma in the inner cortex that are anatomical indicators of mycorrhizal nonsusceptibility in all four palm species is observed. The root systems of B. armata and C. humilis present only one group of third-order roots, while the third-order roots of P. canariensis and P. dactylifera may be divided into five different groups: short thick roots, mycorrhizal thickened roots, fine short roots, fine long roots, and pneumatorhizas. Third-order and some second-order roots of B. armata and C. humilis are susceptible to colonization by AM fungi, while only the mycorrhizal thickened roots form mycorrhizas with arbuscules in the Phoenix species. The root system of the Phoenix species also presents AM colonization in fine roots with only intraradical hyphae and spores, but without arbuscules, and pseudomantles of spores anchored in the pneumatorings of the second-order roots, which are described for the first time. The mycorrhizas formed by the four palm species are of an intermediate type, between the Arum and the Paris types, and are characterized by intercalary arbusculate coils and not only by intracellular but also by intercellular fungal growth. Our study suggests that a different degree of adaptation may exist among palm mycorrhizas toward the slow growth of palms and low spore numbers in the soil where they grow.