The Medicago truncatula sucrose synthase gene MtSucS1 is activated both in the infected region of root nodules and in the cortex of roots colonized by arbuscular mycorrhizal fungi

The MtSucS1 gene encodes a sucrose synthase (EC 2.4.1.13) in the model legume Medicago truncatula. To determine the expression pattern of this gene in different organs and in particular during root endosymbioses, we transformed M. truncatula with specific regions of MtSucS1 fused to the gusAint reporter gene. These fusions directed an induction to the vasculature of leaves, stems, and roots as well as to flowers, developing seeds, young pods, and germinating seedlings. In root nodules, strong promoter activity occurred in the infected cells of the nitrogen-fixing zone but was additionally observed in the meristematic region, the prefixing zone, and the inner cortex, including the vasculature. Concerning endomycorrhizal roots, the MtSucS1 promoter mediated strongest expression in cortical cells harboring arbuscules. Specifically in highly colonized root sections, GUS-staining was furthermore detected in the surrounding cortical cells, irrespective of a direct contact with fungal structures. In accordance with the presence of an orthologous PsSus1 gene, we observed a comparable regulation of MtSucS1 expression in the grain legume Pisum sativum in response to microbial symbionts. Unlike other members of the MtSucS gene family, the presence of rhizobial or Glomus microsymbionts significantly altered and enhanced MtSucS1 gene expression, leading us to propose that MtSucS1 is involved in generating sink-strength, not only in root nodules but also in mycorrhizal roots.     

Occurrence and localization of apocarotenoids in arbuscular mycorrhizal plant roots

The core structure of the yellow pigment from arbuscular mycorrhizal (AM) maize roots contains the apocarotenoids mycorradicin (an acyclic C14 polyene) and blumenol C cellobioside (a C13 cyclohexenone diglucoside). The pigment seems to be a mixture of different esterification products of these apocarotenoids. It is insoluble in water and accumulates as hydrophobic droplets in the vacuoles of root cortical cells. Screening 58 species from 36 different plant families, we detected mycorradicin in mycorrhizal roots of all Liliopsida analyzed and of a considerable number of Rosopsida, but also species were found in which mycorradicin was undetectable in mycorrhizal roots. Kinetic experiments and microscopic analyses indicate that accumulation of the yellow pigment is correlated with the concomitant degradation of arbuscules and the extensive plastid network covering these haustorium-like fungal structures. The role of the apocarotenoids in mycorrhizal roots is still unknown. The potential C40 carotenoid precursors, however, are more likely to be of functional importance in the development and functioning of arbuscules.     

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.     

Colonization of roots by arbuscular mycorrhizal fungi using different sources of inoculum

Arbuscular mycorrhizal fungi (AMF) form a number of different infective propagules that are used to form new mycorrhizal associations. These are spores, extraradical hyphae and infected roots. However, not all fungi are equally capable of colonizing roots with all of the above-mentioned propagules and there is conflicting evidence of major differences in colonization strategy between members of the Glomineae and Gigasporineae. In this study, we tested the abilities of eight fungal species from four different genera to colonize roots using three different types of inoculum. Glomus and Acaulospora isolates colonized from all inoculum types, whereas Gigaspora and Scutellospora isolates colonized mainly from spores and to a limited degree from root fragments. Extraradical hyphae were not suitable propagules for the species of Gigaspora and Scutellospora tested. This indicates that AMF have different colonization strategies and that this is largely differentiated at the suborder level. It is unclear why there is such a difference among the fungi in inoculum types. Future research should examine differences in the anatomy and physiology to discern a mechanism for such differences in life-history strategies.     

Hydrolytic enzymes and ability of arbuscular mycorrhizal fungi to colonize roots

The production of hydrolytic enzymes from external mycelia associated with roots and colonized soybean roots (Glycine max L.) inoculated with different arbuscular-mycorrhizal (AM) fungi of the genus GLOMUS:, and the possible relationship between these activities and the capacity of the AM fungi to colonize plant roots was studied. There were differences in root colonization and plant growth between the GLOMUS: strains, and also between two isolates of G. mosseae. Hydrolytic activities in the root and external mycelia associated with roots differed in the AM fungi tested. Correlations were only found between the endoxyloglucanase activity of the external mycelia associated with roots of the AM fungi tested and the percentage root colonization or plant growth. However, hydrolytic activities of roots colonized by the different endophytes correlated with those of external mycelia. The hydrolytic activities were not qualitatively different because the endoxyloglucanase from AM colonized roots and the external mycelia did not show a high degree of polymorphism in the different species of fungus tested. The possible role of the hydrolytic activity of external hyphae of AM fungi was discussed as a factor affecting fungal ability to colonize the root and influence plant growth.     

施肥对垂穗披碱草根系中丛枝菌根真菌的影响

采用传统染色与克隆测序的方法,研究了8年不同施肥(氮磷)梯度对垂穗披碱草根系中丛枝菌根(AM)侵染率和AM真菌群落的影响.结果表明: 随施肥浓度升高, 垂穗披碱草根系单位根长AM总侵染率从67.5%下降至7.3%,丛枝侵染率从5.2%降至0.1%.根系共检测出24个AM真菌分子种,但随着施肥浓度上升,AM真菌的平均物种丰富度从6种下降至2.6种.不同施肥处理对AM真菌群落结构有显著影响,土壤速效磷和根系氮含量与AM真菌群落呈极显著相关.氮磷有效性随施肥梯度逐渐上升,且与AM侵染率和AM真菌物种丰富度呈显著负相关.施高浓度氮磷肥对AM共生体有明显的抑制作用,导致AM真菌物种多样性丧失.     

Molecular identification of arbuscular mycorrhizal fungi in roots: Perspectives and problems

Molecular identification methods are about to revolutionize studies on ecology of arbuscular mycorrhiza. These techniques offer the unique opportunity to investigate communities of arbuscular mycorrhizal fungi (AMF) within roots. Recent technical advances are reviewed, discussing their drawbacks and advantages. An experimental approach to analyze AMF communities within roots using a molecular identification method is presented. Sample results from the analysis of trap cultures from a current project are shown.     

一种改进的丛枝菌根染色方法

研究改进了Vierheilig等描述的AM菌根染色法:将根样于20%KOH溶液中60℃水浴透明40-120 min,5%醋酸酸化5min后,用5%醋酸墨水染色液(派克纯黑书写墨水Quink),于60℃水浴染色30 min,清水浸泡脱色(14h)后即可镜检。根皮层细胞内AM真菌的丛枝结构清晰可见,并且能够明确地分辨AM真菌与其它未知真菌。此外,Quink初染后,再经过SudanⅣ复染(60℃、60 min),70%乙醇脱色5min,暗隔真菌的透明菌丝内所积聚的脂类颗粒被SudanⅣ染上鲜红色,在复式显微镜下能够观察到此类透明菌丝在根皮层组织内的存在状况。采用甘油明胶为封固剂制片,根的染色效果可以保存长久。此项技术可以对同一种植物的多个根样进行同步的透明和染色处理,而且操作简便、低毒性、成本低廉、染色效果极佳,适用于野生和栽培草本植物AM菌根的染色和制片观察。     

Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt marshes

The occurrence of arbuscular mycorrhizal fungi (AMF) was assessed by both morphological and molecular criteria in two salt marshes: (i) a NaCl site of the island Terschelling, Atlantic Coast, the Netherlands and (ii) a K₂CO₃ marsh at Schreyahn, Northern Germany. The overall biodiversity of AMF, based on sequence analysis, was comparably low in roots at both sites. However, the morphological spore analyses from soil samples of both sites exhibited a higher AMF biodiversity. Glomus geosporum was the only fungus of the Glomerales that was detected both as spores in soil samples and in roots of the AMF-colonized salt plants Aster tripolium and Puccinellia sp. at both saline sites and on all sampling dates (one exception). In roots, sequences of Glomus intraradices prevailed, but this fungus could not be identified unambiguously from DNA of soil spores. Likewise, Glomus sp. uncultured, only deposited as sequence in the database, was widely detected by DNA sequencing in root samples. All attempts to obtain the corresponding sequences from spores isolated from soil samples failed consistently. A small sized Archaeospora sp. was detected, either/or by morphological and molecular analyses, in roots or soil spores, in dead AMF spores or orobatid mites. The study noted inconsistencies between morphological characterization and identification by DNA sequencing of the 5.8S rDNA-ITS2 region or part of the 18S rDNA gene. The distribution of AMF unlikely followed the salt gradient at both sites, in contrast to the zone formation of plant species. Zygotes of the alga Vaucheria erythrospora (Xanthophyceae) were retrieved and should not be misidentified with AMF spores.     

Specific PCR primers to identify arbuscular mycorrhizal fungi within colonized roots

 A set of PCR primers targeted at five major phylogenetic subgroups of arbuscular mycorrhizal fungi (Glomales) was designed to facilitate specific amplification of internal transcribed spacers and 18 S rRNA gene fragments from colonized roots in the absence of spores. The subgroups include the recently discovered deeply divergent lineages of Glomales, which could not be detected by previously reported PCR primers, and the former genus Sclerocystis. Restriction fragment length polymorphism patterns presented allow identification of presently known members of these groups. The resulting PCR products can be used to identify the fungal symbionts at the genus or species level by restriction digests or DNA sequencing. A novel DNA extraction method allows visual control of the analyzed roots by staining procedures after analysis by PCR. Accepted: 2 April 2000     

Microwave-assisted technology for the clearing and staining of arbuscular mycorrhizal fungi in roots

The use of microwave irradiation as a source of energy to clear and stain intra-radical arbuscular mycorrhizal fungi propagules has been tested on a variety of indigenous and cultivated herbaceous plants. The aim of the study was to evaluate the efficiency of microwave irradiation on root softening, fungi tissue staining, and preservation of DNA integrity for subsequent molecular analyses. The proposed methodology has been adapted from the standard procedures used to detect and quantify mycorrhizal root colonization levels. Using a domestic microwave oven, tissue clearing and staining required together between 30 s and 1.5 min of microwave treatment to be completed, depending the diameter size of the roots. The well-performing chemical stains tested were acid fuchsin, trypan blue, and aniline blue. The acid fuchsin clearing and staining processes, as performed, were also demonstrated to preserve DNA integrity for further molecular analyses. Irradiation by microwaves has been used with success in our laboratory within the frame of several studies. It offers considerable time saving over traditional method, reducing processing times from several hours to a few minutes while decreasing considerably the amount of chemicals and energy required to perform analyses.     

Morphological types of arbuscular mycorrhizal fungi in roots of weeds on vacant land

Morphological types of arbuscular mycorrhizal (AM) fungi in weeds of vacant land were examined in spring and autumn. In total, 33 plant species belonging to 28 genera in 13 families were examined. The number of plant species with Arum-type AM was higher than those with Paris- or intermediate types in both seasons. Thus, Arum-type colonization may be beneficial for fast-growing plant species on vacant land. There was a strong relationship between plant identity and AM morphological type, as the colonization types were mostly distinguished at the plant family level.     

Organic fertilizers alter the composition of pathogens and arbuscular mycorrhizal fungi in maize roots

Roots of agricultural crops, including maize, are hosts of different microorganisms, many beneficial, like plant growth and health‐promoting arbuscular mycorrhizal fungi (AMF), as well as pathogens including Pythium, Polymyxa and Microdochium. To improve crop nutrition and health, profound knowledge is required regarding how agricultural practices affect field populations of root‐associated microorganisms. Hence, the objective of this work was to evaluate the effect of crop genotype and organic fertilizers on the plant growth performance of maize and their root‐associated microorganisms. The experiment was conducted as a fully factorial greenhouse pot experiment with maize cultivars (two land races and two hybrids) and organic fertilizers (green manure, cow manure and compost) as the two main factors. Plants were harvested 8 weeks after sowing. In general, the different maize cultivars responded similarly to the applications of the organic fertilizers. Cow manure and compost increased plant growth, whereas green manure had limited effect on plant growth. Root colonization with AMF was reduced by green manure with rape. Infection with the root pathogens Pythium and Polymyxa was reduced by all organic fertilizers, whereas in contrast, infection with Microdochium increased with the majority of the organic fertilizers applied. In conclusion, both maize genotype and organic fertilizers affect the abundance of AMF and root pathogens in maize, which should be considered when developing management strategies of these root‐inhabiting microorganisms.     

Apical meristems of tomato roots and their modifications induced by arbuscular mycorrhizal and soilborne pathogenic fungi

Functional morphological patterns in root apices of tomato ( Lycopersicon esculentum ) dependent on growth, ageing and infection by the arbuscular mycorrhizal (AM) fungus Glomus mosseae and/or by the soilborne pathogenic fungus Phytophthora nicotianae var parasitica ( P. parasitica ) were studied. Uninfected root apices were characterized by closed, tri-layered meristems with nonreticulate nuclei; however, some apices of each treatment lost their meristematic nature, stopped growing and differentiated, becoming 'parenchymatized'. The pathogenic fungus reduced the apex diameter and the number of mitotically active and viable apices inducing plasmolysis, cell and nucleus degeneration, and necrosis. The AM fungus, on the other hand, produced an increase in apex size and reduced the percentage of necrosis both in uninfected roots and in roots infected by P. parasitica . Thus, the AM fungus protected the apices from the pathogenic infection, allowing normal root growth. Furthermore, larger apices, which produce thicker roots, might indirectly contribute to plant protection. Increased volumes of colonizable tissues favour the spreading of the symbiont, and P. parasitica hyphae are always excluded from arbuscule-containing cells.