Nested multiplex PCR—A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Nested multiplex PCR——A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Evidence for leaf endophyte regulation of root symbionts: effect of <Emphasis Type="Italic">Neotyphodium</Emphasis> endophytes on the pre-infective state of mycorrhizal fungi

Neotyphodium endophytes and arbuscular mycorrhizal (AM) fungi are common constituents of natural grasslands. The plant–endophyte symbiosis can introduce changes in soil conditions that affect the density and activity of different functional groups of soil organisms. In the present work we performed in vitro assays to evaluate the effect of root and endophyte exudates on the pre-infective state of mycorrhizal fungi (Gigaspora margarita and G. rosea). Plant roots of Bromus setifolius from populations of Patagonia, and four strains of Neotyphodium were used to obtain the exudates. Root exudates of infected plants, at a high concentration, significantly increased AMF hyphal branches and length relative to exudates from naturally endophyte free plants. The effect of Neotyphodium endophyte exudates on AMF mycelial length varied depending on strain and the concentration used, suggesting a differential interaction between endophyte and AMF species. AMF hyphal branches were increased by Neotyphodium fungal exudates in both mycorrhizal species. A few previous studies have suggested that Neotyphodium endophytes can reduce mycorrhizal sporulation and colonization of host roots in commonly-cultivated agronomic hosts. In this study we report the opposite effect in B. setifolius. This study reports the direct and positive effect of root exudates from plants in symbiosis with Neotyphodium, on AMF pre-infective state. Further, identical effects were detected using exudates from Neotyphodium endophytes.     

Symbiosis-stimulated chitinase isoenzymes of soybean (Glycine mas (L.) Merr.)

Isoforms of endochitinase in soybean were studied in relation to root symbiosis. Five selected cultivars differing in their nodulation potential were inoculated with two strains of Bradyrhizobium japonicum, the broad host-range Rhizobium sp. NGR234, and with the mycorrhizal fungus Glomus mosseae. Total chitinase activity in nodules was up to 7-fold higher than in uninoculated roots and in mycorrhizal roots. The chitinase activity in nodules varied depending on the strain-cultivar combination. On semi-native polyacrylamide gels, four acidic isoforms were identified. Two isoforms (CH 2 and CH 4) were constitutively present in al analysed tissues. The other two isoforms (CH 1 and CH 3) were strongly induced in nodules and were simulated in mycorrhizal roots as compared to uninoculated roots. The induction of CH 1 varied in nodules depending on the soybean cultivar. This isoform was also stimulated in uninfected roots when they were treated with tri-iodobenzoic acid, rhizobial lipochitooloigosaccharides (Nod factors) and chitotetraose. CH 3 was not affected by these stimuli indicating that this isoform could represent a marker for enzymes induced in later stages of the symbiotic interactions.Key words: (Brady)rhizobium, chitinase isoenzymes, mycorrhiza, (restricted) nodulation, Nod factors      

菌根化育苗对大棚黄瓜生长发育和果实品质的影响

以Glomus versiforme （GV）、Glomus mosseae（GM）和Glomus intraradices（GI）3种丛枝菌根菌及其混合菌种GM+GV、GM+GI、GV+GI和GM+GV+GI接种黄瓜幼苗,进行菌根化育苗,研究菌根化育苗对黄瓜的生长发育、产量和品质的影响.结果表明,接种的丛枝菌根菌均能与黄瓜幼苗根系形成菌根,侵染率高达41.74%～55.69%.其中,GV、GM、GM+GV、GM+GV+GI 和GV+GI是黄瓜的优势菌种,与对照相比,壮苗指数提高了58.14%～123.6%,前期产量增加了21.71%～37.87%,总产量增加了19.72%～34.41%.接种GM+GV和GV+GI处理可显著地提高黄瓜果实中Vc的含量,分别比对照提高了22.84%和21.95%;接种GV、GV+GI和GI处理可显著提高黄瓜果实中可溶性糖含量,分别比对照提高了13.79%、10.20%和8.25%;接种GV和GM+GI处理黄瓜果实中的氨基酸含量,分别比对照提高了47.66%和23.19%,蛋白质含量提高了17.67%～34.79%.     

The differential behavior of arbuscular mycorrhizal fungi in interaction with Astragalus sinicus L. under salt stress

Three arbuscular mycorrhizal (AM) fungi (Glomus mosseae, Glomus claroideum, and Glomus intraradices) were compared for their root colonizing ability and activity in the root of Astragalus sinicus L. under salt-stressed soil conditions. Mycorrhizal formation, activity of fungal succinate dehydrogenase, and alkaline phosphatase, as well as plant biomass, were evaluated after 7 weeks of plant growth. Increasing the concentration of NaCl in soil generally decreased the dry weight of shoots and roots. Inoculation with AM fungi significantly alleviated inhibitory effect of salt stress. G. intraradices was the most efficient AM fungus compared with the other two fungi in terms of root colonization and enzyme activity. Nested PCR revealed that in root system of plants inoculated with a mix of the three AM fungi and grown under salt stress, the majority of mycorrhizal root fragments were colonized by one or two AM fungi, and some roots were colonized by all the three. Compared to inoculation alone, the frequency of G. mosseae in roots increased in the presence of the other two fungal species and highest level of NaCl, suggesting a synergistic interaction between these fungi under salt stress.     

Arbuscular mycorrhizal fungi alter thymol derivative contents of Inula ensifolia L.

Individuals of Inula ensifolia L. (Asteraceae), a valuable xerothermic plant species with potential therapeutic value, were inoculated under laboratory conditions with different strains of arbuscular mycorrhizal fungi (AMF): (1) Glomus intraradices UNIJAG PL-Bot, (2) G. intraradices UNIJAG PL-Kap, (3) Glomus clarum UNIJAG PL13-2, and (4) AMF crude inoculum from natural stands of I. ensifolia. We found AMF species specificity in the stimulation of thymol derivative production in the roots of I. ensifolia. There was an increase in thymol derivative contents in roots after G. clarum inoculation and at the same time the decreased production of these metabolites in the G. intraradices treatments. Moreover, no correlation between the extent of AMF colonization and the effects of the fungal symbionts on the plant was observed. A multilevel analysis of chlorophyll a fluorescence transients (JIP test) permitted an evaluation of plant vitality, expressed in photosynthetic performance index, influenced by the applied AMF strains, which was found to be in good agreement with the results concerning thymol derivative production. The mechanisms by which AMF trigger changes in phytochemical concentration in plant tissues and their consequences for practice are discussed.     

Fungal root symbionts and their relationship with fine root proportion in native plants from the Bolivian Andean highlands above 3,700?m elevation

Here, we examined the colonization by fungal root symbionts in the cultivated Andean grain Chenopodium quinoa and in 12 species that dominate plant communities in the Bolivian Altiplano above 3,700 m elevation and explore for the possible relationships between fungal colonization and fine root proportion. The 12 most abundant species in the study area were consistently colonized by AMF and DSE. In contrast, the annual Andean grain C. quinoa showed negligible or absence of mycorrhizal fungi colonizing roots. On the other hand, C. quinoa, Junelia seriphioides and Chersodoma jodopappa were infected to a varying degree by the root pathogen Olpidium sp. We observed no relationship between AMF and DSE colonization and proportion of fine roots in the root system, but instead, the ratio between DSE and AMF colonization (ratio DSE/AMF) negatively related with proportion of fine roots. Our findings support the hypothesis regarding the importance of DSE at high altitudes and suggest a functional relationship between the rate of DSE/AMF and proportion of fine roots. The colonization by the root pathogen Olpidium sp. in C. quinoa deserves further study since this Andean grain is increasingly important for the local economy in these marginal areas.     

Long-term tracing of Rhizophagus irregularis isolate BEG140 inoculated on Phalaris arundinacea in a coal mine spoil bank, using mitochondrial large subunit rDNA markers

During the last decade, the application of arbuscular mycorrhizal fungi (AMF) as bioenhancers has increased significantly. However, until now, it has been difficult to verify the inoculation success in terms of fungal symbiont establishment in roots of inoculated plants because specific fungal strains could not be detected within colonized roots. Using mitochondrial large subunit ribosomal DNA, we show that Rhizophagus irregularis (formerly known as Glomus intraradices) isolate BEG140 consists of two different haplotypes. We developed nested PCR assays to specifically trace each of the two haplotypes in the roots of Phalaris arundinacea from a field experiment in a spoil bank of a former coal mine, where BEG140 was used as inoculant. We revealed that despite the relatively high diversity of native R. irregularis strains, R. irregularis BEG140 survived and proliferated successfully in the field experiment and was found significantly more often in the inoculated than control plots. This work is the first one to show tracing of an inoculated AMF isolate in the roots of target plants and to verify its survival and propagation in the field. These results will have implications for basic research on the ecology of AMF at the intraspecific level as well as for commercial users of mycorrhizal inoculation.     

Effects of Alliaria petiolata (garlic mustard; Brassicaceae) on mycorrhizal colonization and community structure in three herbaceous plants in a mixed deciduous forest

Herbaceous plant species are important components of forest ecosystems, and their persistence in forests may be affected by invasive plant species that reduce mycorrhizal colonization of plant roots. I examined the effect of the invasive plant Alliaria petiolata on arbuscular mycorrhizal fungi (AMF) colonizing the roots of three forest plant species. AMF root colonization and community structure was examined from plants that were growing either in the absence or presence of Alliaria under natural forest conditions. AMF root colonization varied among the plant species but was not significantly affected by Alliaria. With molecular methods, ～12 different taxa of AMF could be distinguished among the root samples, and these taxa belonged to the genera Acaulospora and Glomus, with Glomus dominating AMF communities. There were significant differences between the community of AMF colonizing roots of Maianthemum racemosum and Trillium grandiflorum, but only AMF communities of Maianthemum roots were significantly affected by Alliaria. Indicator species analysis found that an Acaulospora species type was a significant indicator of Maianthemum plants grown in the absence of Alliaria. These results suggest invasive plants like Alliaria may selectively suppress AMF fungi, and this suppression can affect AMF communities colonizing the roots of some native plant species.     

Distribution of arbuscular mycorrhizal fungi in marshy and shoreline vegetation of Deepar Beel Ramsar Site of Assam,India

Arbuscular mycorrhizal fungal (AMF) symbiosis was thought to be rare in wetland plant roots, although several recent studies suggested that this association might be important in wetland ecosystems. In this research work we have studied the distribution of AMF in the marshy and shoreline vegetation of Deepar Beel Ramsar site of Assam, India. The study reveals the percentage of mycorrhizal colonization in the roots of different plant species which were observed from 20.89 to 86.47% and particularly found larger among the members of the family Poaceae. The Vetiveria zizanioides L. from the family Cyperaceae showed the highest (86.47%) percentage of root colonization, however, only one plant species viz. Scirpus lateriflorus Gmel. from the same family was found to be nonmycorrhizal. The rhizospheric soil samples of most of the plant species were found to be dominated by Glomus morphotypes. All total 18 AMF morphotypes were recorded which comprises four genera viz. Glomus (66.67%), Acaulospora (16.66%), Gigaspora (11.11%) and Scutellospora (5.56%). The observation of diversity of AMF in 25 different plant species among the wetland plants gives a glimpse of AMF diversity and their host selectivity in the said ecosystem.     

Reduced aboveground tree growth associated with higher arbuscular mycorrhizal fungal diversity in tropical forest restoration

Establishing diverse mycorrhizal fungal communities is considered important for forest recovery, yet mycorrhizae may have complex effects on tree growth depending on the composition of fungal species present. In an effort to understand the role of mycorrhizal fungi community in forest restoration in southern Costa Rica, we sampled the arbuscular mycorrhizal fungal (AMF) community across eight sites that were planted with the same species (Inga edulis, Erythrina poeppigiana, Terminalia amazonia, and Vochysia guatemalensis) but varied twofold to fourfold in overall tree growth rates. The AMF community was measured in multiple ways: as percent colonization of host tree roots, by DNA isolation of the fungal species associated with the roots, and through spore density, volume, and identity in both the wet and dry seasons. Consistent with prior tropical restoration research, the majority of fungal species belonged to the genus Glomus and genus Acaulospora, accounting for more than half of the species and relative abundance found on trees roots and over 95% of spore density across all sites. Greater AMF diversity correlated with lower soil organic matter, carbon, and nitrogen concentrations and longer durations of prior pasture use across sites. Contrary to previous literature findings, AMF species diversity and spore densities were inversely related to tree growth, which may have arisen from trees facultatively increasing their associations with AMF in lower soil fertility sites. Changes to AMF community composition also may have led to variation in disturbance susceptibility, host tree nutrient acquisition, and tree growth. These results highlight the potential importance of fungal–tree–soil interactions in forest recovery and suggest that fungal community dynamics could have important implications for tree growth in disturbed soils.     

Impact of Bacillus subtilis JA, a biocontrol strain of fungal plant pathogens, on arbuscular mycorrhiza formation in Zea mays

Summary  In the present study, the influence of Bacillus subtilis JA on arbuscular mycorrhizal fungi (AMF) was evaluated by either pot culture or in vitro conditions, respectively. Under the pot culture conditions, the inoculation of B. subtilis JA decreased the frequency (% F) of the root colonization by indigenous arbuscular mycorrhizal fungi and the shoot dry weight of maize (Zea mays L.), but had no apparent effect on the intensity (% I) of AM fungal root colonization. The unknown volatile emitted from the B. subtilis JA in vitro significantly inhibited spore germination and the hyphal growth in the dual-compartment experiments. Moreover, the data from the direct interaction between B. subtilis JA and Glomus etunicatum showed that soluble antifungal lipopeptides influenced the development of AMF. Therefore, the application of antifungal Bacillus strains should take the compatibility with the indigenous beneficial fungi into consideration.     

Differential influence of native and introduced arbuscular mycorrhizal fungi on growth of dominant and subordinate plants

The impact of arbuscular mycorrhizal fungi (AMF) on plant ecosystems has been intensively reported. In this research, we explored the difference between native and introduced AMF in promoting the growth of dominant and subordinate plant species. In glasshouse experiments, dominants and subordinates from subtropical grasslands were colonized by native AMF or introduced AMF, Glomus versiforme. The biomass revealed that mycorrhizal dependencies (MD) on the native AMF of the dominants were much higher than those of the subordinates, while MD on the introduced AMF changed following the replacement of native AMF with introduced AMF. A close relationship between biomass promotion and increase in phosphorus uptake was observed, indicating the important role of AMF-enhanced nutrient acquisition by roots. Our results show that plant community structures are partly determined by MD on native AMF, and could be modified by introducing exogenous AMF species.     

Functional diversity of arbuscular mycorrhizal fungal isolates in relation to extraradical mycelial networks

We investigated the functional significance of extraradical mycorrhizal networks produced by geographically different isolates of the arbuscular mycorrhizal fungal (AMF) species Glomus mosseae and Glomus intraradices. A two-dimensional experimental system was used to visualize and quantify intact extraradical mycelium (ERM) spreading from Medicago sativa roots. Growth, phosphorus (P) and nitrogen (N) nutrition were assessed in M. sativa plants grown in microcosms. The AMF isolates were characterized by differences in extent and interconnectedness of ERM. Phenotypic fungal variables, such as total hyphal length, hyphal density, hyphal length per mm of total or colonized root length, were positively correlated with M. sativa growth response variables, such as total shoot biomass and plant P content. The utilization of an experimental system in which size, growth rate, viability and interconnectedness of ERM extending from mycorrhizal roots are easily quantified under realistic conditions allows the simultaneous evaluation of different isolates and provides data with a predictive value for selection of efficient AMF.     

Effect of arbuscular mycorrhizal fungi on the acclimatization of micropropagated banana plantlets

 The effect of arbuscular mycorrhizal fungi (AMF) on micropropagated banana plantlets was evaluated during the acclimatization period. Plants were inoculated with Acaulospora scrobiculata, Glomus clarum or Glomus etunicatum. After cultivation in a greenhouse for 3 months, height, leaf area, fresh weight and dry matter of root and shoots, level of AMF colonization, nutrient level, photosynthesis and transpiration rate, water potential and stomatal conductance were measured. The number of AMF spores produced in each treatment was also determined. Plantlets inoculated with AMF had greater height, leaf area and fresh weight of shoots and roots, as well as higher rates of photosynthesis and transpiration than controls. Plants inoculated with Glomus were superior in most of the evaluated parameters. Accepted: 24 May 1999     

Nonlegumes, Legumes, and Root Nodules Harbor Different Arbuscular Mycorrhizal Fungal Communities

Legumes are an important plant functional group since they can form a tripartite symbiosis with nitrogen-fixing Rhizobium bacteria and phosphorus-acquiring arbuscular mycorrhizal fungi (AMF). However, not much is known about AMF community composition in legumes and their root nodules. In this study, we analyzed the AMF community composition in the roots of three nonlegumes and in the roots and root nodules of three legumes growing in a natural dune grassland. We amplified a portion of the small-subunit ribosomal DNA and analyzed it by using restriction fragment length polymorphism and direct sequencing. We found differences in AMF communities between legumes and nonlegumes and between legume roots and root nodules. Different plant species also contained different AMF communities, with different AMF diversity. One AMF sequence type was much more abundant in legumes than in nonlegumes (39 and 13%, respectively). Root nodules contained characteristic AMF communities that were different from those in legume roots, even though the communities were similar in nodules from different legume species. One AMF sequence type was found almost exclusively in root nodules. Legumes and root nodules have relatively high nitrogen concentrations and high phosphorus demands. Accordingly, the presence of legume- and nodule-related AMF can be explained by the specific nutritional requirements of legumes or by host-specific interactions among legumes, root nodules, and AMF. In summary, we found that AMF communities vary between plant functional groups (legumes and nonlegumes), between plant species, and between parts of a root system (roots and root nodules).     

A method for assessing arbuscular mycorrhizal fungi group distribution in tree roots by intergenic transcribed sequence variation

We identified five taxonomic groups of arbuscular mycorrhizal fungi (AMF) inside roots of young trees of six species of legumes and six species of non-legumes from a field site in southern Costa Rica using an AMF group-specific PCR assay of the intergenic transcribed sequence and 18S rRNA gene fragment. Assay specificity was verified by cloning and sequencing representatives from four of the five AMF groups. We found no difference in overall AMF diversity levels between legumes and non-legumes or between plant species. Some groups of AMF may associate more frequently with legumes than others, as Glomus Group A (Glomus mosseae/intradices group) representatives were detected more frequently in legumes than non-legumes relative to Glomus Group B (Glomus etunicatum/claroideum) representatives.     

Synergistic interactions between Glomus mosseae and Bradyrhizobium japonicum in enhancing proton release from nodules and hyphae

Soybean (Glycine max L. Merr.) seedlings were inoculated with Glomus mosseae (GM) and Bradyrhizobium japonicum (BJ) together or separately to study the effect of interactions on net H⁺ effluxes of nodules or extraradical hyphae by in vivo vibrating electrode techniques. GM promoted three-fold the H⁺ effluxes of nodules on mycorrhizal lateral roots and BJ increased eight-fold the net H⁺ effluxes of hyphae developing in the vicinity of nodules on lateral roots. Increments in plant P content were positively and linearly correlated with the net H⁺ efflux of nodules and hyphae. It is concluded that increased H⁺ effluxes of nodules resulted from enhanced nitrogenase activities induced by the presence of the AM fungus in lateral roots. The results point to additive effects of interactions between mycorrhizal fungi and rhizobia in increasing the extent of acidification of the “nodulesphere” and the hyposphere.     

Contrasting impacts of defoliation on root colonization by arbuscular mycorrhizal and dark septate endophytic fungi of Medicago sativa

Individual plants typically interact with multiple mutualists and enemies simultaneously. Plant roots encounter both arbuscular mycorrhizal (AM) and dark septate endophytic (DSE) fungi, while the leaves are exposed to herbivores. AMF are usually beneficial symbionts, while the functional role of DSE is largely unknown. Leaf herbivory may have a negative effect on root symbiotic fungi due to decreased carbon availability. However, evidence for this is ambiguous and no inoculation-based experiment on joint effects of herbivory on AM and DSE has been done to date. We investigated how artificial defoliation impacts root colonization by AM (Glomus intraradices) and DSE (Phialocephala fortinii) fungi and growth of Medicago sativa host in a factorial laboratory experiment. Defoliation affected fungi differentially, causing a decrease in arbuscular colonization and a slight increase in DSE-type colonization. However, the presence of one fungal species had no effect on colonization by the other or on plant growth. Defoliation reduced plant biomass, with this effect independent of the fungal treatments. Inoculation by either fungal species reduced root/shoot ratios, with this effect independent of the defoliation treatments. These results suggest AM colonization is limited by host carbon availability, while DSE may benefit from root dieback or exudation associated with defoliation. Reductions in root allocation associated with fungal inoculation combined with a lack of effect of fungi on plant biomass suggest DSE and AMF may be functional equivalent to the plant within this study. Combined, our results indicate different controls of colonization, but no apparent functional consequences between AM and DSE association in plant roots in this experimental setup.