Arbuscular mycorrhizal colonization in field-collected terrestrial cordate gametophytes of pre-polypod leptosporangiate ferns (Osmundaceae,Gleicheniaceae, Plagiogyriaceae,Cyatheaceae)

To determine the mycorrhizal status of pteridophyte gametophytes in diverse taxa, the mycorrhizal colonization of wild gametophytes was investigated in terrestrial cordate gametophytes of pre-polypod leptosporangiate ferns, i.e., one species of Osmundaceae (Osmunda banksiifolia), two species of Gleicheniaceae (Diplopterygium glaucum, Dicranopteris linearis), and four species of Cyatheales including tree ferns (Plagiogyriaceae: Plagiogyria japonica, Plagiogyria euphlebia; Cyatheaceae: Cyathea podophylla, Cyathea lepifera). Microscopic observations revealed that 58 to 97 % of gametophytes in all species were colonized with arbuscular mycorrhizal (AM) fungi. Fungal colonization was limited to the multilayered midrib (cushion) tissue in all gametophytes examined. Molecular identification using fungal SSU rDNA sequences indicated that the AM fungi in gametophytes primarily belonged to the Glomeraceae, but also included the Claroideoglomeraceae, Gigasporaceae, Acaulosporaceae, and Archaeosporales. This study provides the first evidence for AM fungal colonization of wild gametophytes in the Plagiogyriaceae and Cyatheaceae. Taxonomically divergent photosynthetic gametophytes are similarly colonized by AM fungi, suggesting that mycorrhizal associations with AM fungi could widely occur in terrestrial pteridophyte gametophytes.     

Do novel weapons that degrade mycorrhizal mutualisms promote species invasion?

Non-native plants often dominate novel habitats where they did not co-evolve with the local species. The novel weapons hypothesis suggests that non-native plants bring competitive traits against which native species have not adapted defenses. Novel weapons may directly affect plant competitors by inhibiting germination or growth, or indirectly by attacking competitor plant mutualists (degraded mutualisms hypothesis). Japanese knotweed (Fallopia japonica) and European buckthorn (Rhamnus cathartica) are widespread plant invaders that produce potent secondary compounds that negatively impact plant competitors. We tested whether their impacts were consistent with a direct effect on the tree seedlings (novel weapons) or an indirect attack via degradation of seedling mutualists (degraded mutualism). We compared recruitment and performance using three Ulmus congeners and three Betula congeners treated with allelopathic root macerations from allopatric and sympatric ranges. Moreover, given that the allelopathic species would be less likely to degrade their own fungal symbiont types, we used arbuscular mycorrhizal (AMF) and ectomycorrhizal (ECM) tree species to investigate the effects of F. japonica (no mycorrhizal association) and Rhamnus cathartica (ECM association) on the different fungal types. We also investigated the effects of F. japonica and R. cathartica exudates on AMF root colonization. Our results suggest that the allelopathic plant exudates impact seedlings directly by inhibiting germination and indirectly by degrading fungal mutualists. Novel weapons inhibited allopatric seedling germination but sympatric species were unaffected. However, seedling survivorship and growth appeared more dependent on mycorrhizal fungi, and mycorrhizal fungi were inhibited by allopatric species. These results suggest that novel weapons promote plant invasion by directly inhibiting allopatric competitor germination and indirectly by inhibiting mutualist fungi necessary for growth and survival.     

Dark Septate Root Endophytic Fungus <Emphasis Type="Italic">Nectria haematococca</Emphasis> Improves Tomato Growth Under Water Limiting Conditions

The ascomycetous dark septate endophytic (DSE) fungi characterized by their melanized hyphae can confer abiotic stress tolerance in their associated plants in addition to improving plant growth and health. In this study inoculation of the DSE fungus Nectria haematococca Berk. & Broome significantly improved all the plant growth parameters like the plant height, stem girth, leaf characteristics and plant biomass of drought-stressed tomato. Root characters like the total root length, primary root diameter, 2nd order root number and diameter, root hair number and length were also significantly influenced by the fungal inoculation. Nevertheless, N. haematococca inoculation did not affect root colonization by native arbuscular mycorrhizal (AM) fungi and no significant correlation existed between the AM and DSE fungal variables examined. The proline accumulation in shoots of N. haematococca inoculated plants was significantly higher than uninoculated plants. The present study clearly indicates for the first time the ability of the DSE fungus, N. haematococca in inducing the drought stress tolerance and promoting the growth of the host plant under water stress.     

Contribution of different arbuscular mycorrhizal fungal inoculum to <Emphasis Type="Italic">Elymus nutans</Emphasis> under nitrogen addition

Arbuscular mycorrhizal (AM) fungi are known to promote plant growth and nutrient uptake, but their role in nitrogen (N) uptake still remains unclear. Therefore, a pot experiment was set up to evaluate the impacts of N addition and AM inoculation (Diversispora eburnea, Claroideoglomus etunicatum, Paraglomus occultum, and their mixture) on AM root colonization, plant biomass, N and P nutrition in Elymus nutans. Our results showed that AM root colonization was unaffected by N addition but was significantly affected by different AM fungal species. D. eburnea and C. etunicatum showed significant higher root colonization than P. occultum. The E. nutans exhibited the highest biomass when inoculated with D. eburnea and significantly higher than non-mycorrhizal (the control) regardless of N addition. Under N addition treatment, D. eburnea significantly enhanced P content of roots, N content of shoots and roots, while AM mixture significantly enhanced shoot P content compared with non-mycorrhizal. However, N and P content in shoots and roots did not significantly vary among treatments when no N was added. In addition, inoculation with C. etunicatum and P. occultum showed no significant effect on plant biomass, N and P content regardless of N addition. In conclusion, this study revealed that the plant response to N addition depends on AM fungal species and also confirmed that significant functional diversity exists among AM fungal species.     

Metal concentrations and mycorrhizal status of plants colonizing copper mine tailings: potential for revegetation

A field survey of metal concentrations and mycorrhizal status of plants growing on copper mine tailings was conducted in Anhui Province, China. Available phosphorus and organic matter in the tailings were very low. High concentrations of Pb, Zn, As and Cd as well as Cu were observed on some sites. The dominant plants growing on mine tailings belonged to the families Gramineae and Compositae, and the most widely distributed plant species wereImperata cylindrica, Cynodon dactylon andPaspalum distichum. Coreopsis drummondii also grew well on the arid sites but not on wet sites. Very low or zero arbuscular mycorrhizal (AM) fungal colonization was observed in most of the plants, but extensive mycorrhizal colonization was recorded in the roots ofC. drummondii andC. dactylon. Metal concentrations in plant tissues indicated that /.cylindrica andP. distichum utilized avoidance mechanisms to survive at high metal concentrations. The investigation suggests that remediation and revegetation of heavy metal contaminated sites might be facilitated by selection of tolerant plant species. Isolation of tolerant AM fungi may also be warranted.     

Land-use change impact on mycorrhizal symbiosis in female and male plants of wild <Emphasis Type="Italic">Carica papaya</Emphasis> (Caricaceae)

It has been acknowledged that land-use change has negative effects on genetic diversity and sex ratio in dioecious species, but less attention has been paid on the influence that land-use change has on the biotic interactions, especially between dioecious species and arbuscular mycorrhizal (AM) fungi. AM mutualism involves reciprocal transfer of carbohydrates and mineral nutrients between the host plant’s roots and these fungi. Here, we report spatial and temporal variation in AM colonization in dioecious wild Carica papaya plants growing in sites with different land use intensity. We tagged, recorded the basal stem circumference and collected roots of reproductive female and male Carica papaya plants in three wild sites during dry and rainy seasons of western Mexico. We also collected soil samples in each site to conduct soil chemical analyses. The sexes of C. papaya did not show significant differences in the frequency (percentage of root colonized by intraradical fungal structures) and abundance (length of intraradical hyphae) of AM fungi but the higher AM colonization was observed during the dry season, and in the site with the lowest disturbance. There was no relationship between soil chemistry and AM colonization. Overall, our findings suggest that land-use intensity has a negative effect on AM colonization and we discuss the consequences of habitat loss for the reproductive female and male plants, the implications of decreasing AM colonization for wild Carica papaya plants an important species that provides a source of genetic variation for the C. papaya varieties.     

AM fungal communities inhabiting the roots of submerged aquatic plant <Emphasis Type="Italic">Lobelia dortmanna</Emphasis> are diverse and include a high proportion of novel taxa

While the arbuscular mycorrhizal (AM) symbiosis is known to be widespread in terrestrial ecosystems, there is growing evidence that aquatic plants also form the symbiosis. It has been suggested that symbiosis with AM fungi may represent an important adaptation for isoëtid plants growing on nutrient-poor sediments in oligotrophic lakes. In this study, we address AM fungal root colonization intensity, richness and community composition (based on small subunit (SSU) ribosomal RNA (rRNA) gene sequencing) in five populations of the isoëtid plant species Lobelia dortmanna inhabiting oligotrophic lakes in Southern Sweden. We found that the roots of L. dortmanna hosted rich AM fungal communities and about 15 % of the detected molecular taxa were previously unrecorded. AM fungal root colonization intensity and taxon richness varied along an environmental gradient, being higher in oligotrophic and lower in mesotrophic lakes. The overall phylogenetic structure of this aquatic fungal community differed from that described in terrestrial systems: The roots of L. dortmanna hosted more Archaeosporaceae and fewer Glomeraceae taxa than would be expected based on global data from terrestrial AM fungal communities.     

Nitrogen limitation impairs plant control over the arbuscular mycorrhizal symbiosis in response to phosphorus and shading in two European sand dune species

The symbiosis of plants with arbuscular mycorrhizal fungi (AMF) may become parasitic if the cost:benefit ratio (carbon:phosphorus ratio) increases. In case of mycorrhizal parasitism, a plant may prevent growth depression through the reduction of root colonization as a form of control over the symbiosis. In this greenhouse study, we attempted to manipulate the cost:benefit ratio of the arbuscular mycorrhizal symbiosis by shading and/or phosphorus (P) fertilization in the differentially mycotrophic plant species Hieracium pilosella and Corynephorus canescens. By repeated sampling of soil cores, we assessed the temporal progress of plant investment towards mycorrhizal structures as a measure of plant control over the AMF. Unexpectedly, we found no obvious treatment effects on mycorrhizal growth dependency (MGD), most likely caused by constant N-limitation in AM plants being enhanced by P-fertilization and shade probably not exacerbating plant C-budget for AMF. This highlights the importance of N:P:C stoichiometry for the outcome of the symbiosis. Nevertheless, we found possible control mechanisms in shaded H. pilosella, with considerably higher resource investments into root than into hyphal growth, while root colonization was only marginally suppressed. This control only manifested after 4 weeks of growth under potentially detrimental conditions, emphasizing the importance of time in plant control over the arbuscular mycorrhizal symbiosis. In contrast, the less mycotrophic C. canescens did not exhibit obvious changes in mycorrhizal investments in reaction to shading and P-fertilization, possibly because the low mycotrophy and AMF colonization already imposes a functioning control mechanism in this species. Our study suggests that highly mycotrophic plants may have a stronger need to keep AMF in check than less mycotrophic plants, which may have implications for the role of mycotrophy in the outcome of symbiotic interactions in natural situations.     

Screening and selecting arbuscular mycorrhizal fungi for inoculating micropropagated apple rootstocks in acid soils

Santa Catarina state is the largest producer of apples in Brazil. Soils in this region have low pH and high levels of aluminum and manganese, requiring high inputs of fertilizers and amendments increasing costs of apple production. Inoculation of arbuscular mycorrhizal fungi can improve the establishment of micropropagated apple plants in such adverse soil conditions. Soil samples were collected from apple orchards in the Caçador, Fraiburgo and São Joaquim regions to develop a corn bioassay to identify mycorrhizal communities with high infectivity. Eleven fungal species were identified from one Caçador soil with the highest infectivity. Glomus etunicatum SCT110, Scutellospora pellucida SCT111, Acaulospora scrobiculata SCT112 and Scutellospora heterogama SCT113 were brought into single-species culture and used in a plant growth and nutrient uptake experiment using micropropagated apple (Malus prunifolia), cultivated at three soil pH. Colonization by fungal isolates significantly affected plant height, shoot and root dry weights, and root:shoot ratio. Soil pH also significantly affected all growth parameters except shoot dry weight. Mycorrhizal inoculation also significantly altered tissue concentrations of P, Zn, Cu, Ca, S, Na, N, K, Fe and Al. Association with mycorrhizal fungi increased P concentration and also decreased Al concentrations in the shoots. Overall, G. etunicatum and S. pellucida were the most effective isolates to promote plant growth and nutrient uptake. Inoculation of apple rootstock with selected fungal isolates during the acclimatization stage represents a useful strategy for producing micropropagated apples that can withstand acidic soil conditions.     

Distribution of <Emphasis Type="Italic">Petrosavia sakuraii</Emphasis> (Petrosaviaceae), a rare mycoheterotrophic plant,may be determined by the abundance of its mycobionts

Petrosavia sakuraii (Petrosaviaceae) is a rare, mycoheterotrophic plant species that has a specific symbiotic interaction with a narrow clade of arbuscular mycorrhizal (AM) fungi. In the present study, we tested the hypothesis that the distribution and abundance of mycobionts in two P. sakuraii habitats, Nagiso and Sengenyama (central Honshu, Japan), determine the distribution pattern of this rare plant. Nagiso is a thriving habitat with hundreds of P. sakuraii individuals per 100 m², whereas Sengenyama is a sparsely populated habitat with fewer than 10 individuals per 100 m². AM fungal communities associated with tree roots were compared at 20-cm distances from P. sakuraii shoots between the two habitats by molecular identification of AM fungal partial sequences of the small subunit ribosomal RNA gene. The percentage of AM fungal sequences showing over 99 % identity with those of the dominant P. sakuraii mycobionts was high (54.9 %) in Nagiso, but low (13.2 %) in Sengenyama. Accordingly, the abundance of P. sakuraii seems to reflect the proportion of potential mycobionts. It is likely that P. sakuraii mycobionts are not rare in Japanese warm temperate forests since 11.2 % of AM fungal sequences previously obtained from a deciduous broad-leaved forest devoid of P. sakuraii in Mizuho, central Honshu, Japan, were >99 % identical to those of the dominant P. sakuraii mycobionts. Thus, results suggest that the abundant mycobionts may be required for sufficient propagation of P. sakuraii, and this quantitative trait of AM fungal communities required for P. sakuraii may explain the rarity of this plant.     

Effect of AMF inoculum from field isolates on the yield of green pepper,parsley, carrot,and tomato

Inoculum of an indigenous mixture of arbuscular mycorrhizal fungi (AMF) containingGlomus mosseae, Glomus fasciculatum, Glomus etunicatum, Glomus intraradices andScutellospora sp. was applied to four of the most frequently used crop species in Slovenia: green pepper (Capsicum annuum), parsley (Petroselinum crispum), carrot (Daucus carrota) and tomato (Lycopersicon esculentum). A simple, feasible, and effective protocol for application of AMF biotechnology in horticulture was adopted.Mycorrhizal inoculation significantly increased the plant biomass parameters of pepper, and parsley and the root biomass of carrots. Statistically significant correlations between biomass parameters of pepper, parsley, and the root biomass of carrots with mycorrhizal colonization parameters (mycorrhizal frequency (F%), global mycorrhizal intensity (M%) and arbuscular richness (A%) were calculated. A significant increase in chlorophyll content was observed in mycorrhizal parsley and a significant increase in carotenoids was observed in mycorrhizal parsley, carrots, and tomato fruits. A significant increase in titratable acidity of fruits from inoculated tomato plants indicates prolonged fruiting period of mycorrhizal tomatoes. In addition, inoculation with an indigenous AMF mixture significantly increased the mycorrhizal potential of soil and thus the growth of non-inoculated plants in the second season. Thus, the results confirmed the potential of applying mycorrhizal biotechnology in sustainable horticulture.     

Biodiversity of arbuscular mycorrhizal fungi in the drawdown zone of the Three Gorges Reservoir under different fertilization histories

Impoundment of the Three Gorges Reservoir (TGR) has dramatically influenced the riparian environment and shaped a new drawdown zone, which has experienced long-term winter conditions and short periods of summer flooding. The community structure and diversity of arbuscular mycorrhizal (AM) fungi (AMF) were investigated in three areas with different fertilization histories [Area A (5 years of fertilization), Area B (3 years of fertilization) and Area C (no fertilization)] in the drawdown zone of the TGR. Altogether, 50 AMF species were identified; the genera Acaulospora, Funneliformis and Glomus were predominant. The AM fungal community differed among areas A, B and C. A higher isolation frequency and relative abundance of Acaulospora, Ambispora, Entrophospora and Paraglomus were observed in areas A and B; however, Claroideoglomus, Diversispora, Sclerocystis and Septoglomus were more abundant in Area C. The highest spore density occurred in Area C, and was slightly lower in Area A and lowest in Area B. Conversely, species richness and diversity indices (Shannon–Wiener and evenness indices) were the highest in Area A, followed by areas C and B. Based on nonmetric multidimensional scaling analyses, the distribution of AMF was influenced by plant host, fertilization practice and environmental factors. Among them, the soil physicochemical properties were the main drivers affecting AMF, in which three edaphic attributes (carbon/nitrogen ratio, available phosphorus and potassium content) were significantly correlated (P < 0.001) with the AM fungal community composition in the three areas of the drawdown zone of the TGR.     

The ectomycorrhizal status of a tropical black bolete, <Emphasis Type="Italic">Phlebopus portentosus</Emphasis>, assessed using mycorrhizal synthesis and isotopic analysis

Phlebopus portentosus is one of the most popular wild edible mushrooms in Thailand and can produce sporocarps in the culture without a host plant. However, it is still unclear whether Phlebopus portentosus is a saprotrophic, parasitic, or ectomycorrhizal (ECM) fungus. In this study, Phlebopus portentosus sporocarps were collected from northern Thailand and identified based on morphological and molecular characteristics. We combined mycorrhizal synthesis and stable isotopic analysis to investigate the trophic status of this fungus. In a greenhouse experiment, ECM-like structures were observed in Pinus kesiya at 1 year after inoculation with fungal mycelium, and the association of Phlebopus portentosus and other plant species showed superficial growth over the root surface. Fungus-colonized root tips were described morphologically and colonization confirmed by molecular methods. In stable isotope measurements, the δ¹³C and δ¹⁵N of natural samples of Phlebopus portentosus differed from saprotrophic fungi. Based on the isotopic patterns of Phlebopus portentosus and its ability to form ECM-like structures in greenhouse experiments, we conclude that Phlebopus portentosus could be an ECM fungus.     

Ultrastructural studies and molecular characterization of root-associated fungi of $$\textit{}$$ (D. Don) Szlach.: a threatened and medicinally important taxon

Crepidium acuminatum (Orchidaceae) is a threatened medicinal orchid that grows under shady and moist forest floor where light remains for a very short period of time. Mycorrhizal association is known to be essential for seed germination and seedling establishment in a majority of orchids. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant–fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of the cortex. Also, spiral-root hairs were found along with root hairs, which is an unusual observation. Spiral-root hairs provide more surface area for fluid absorption and entrance of colonizers. Further, total root genomic DNA was isolated and fungal internal-transcribed spacer (ITS) regions were polymerase chain reaction (PCR)-amplified using specific primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were obtained and analysed to know the closest sequence matche in the GenBank using BLASTn hosted by NLM-NCBI. Subject sequences were identified to be belonging to three main genera, namely, Tulasnella, Aspergillus and Penicillium. Results indicate that mycorrhizal association is necessary for the growth and development of the plant. In addition, this symbiosis influences the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to an enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future in vitro germination and seedling establishment programmes, because they rely on fungi for germination. Identification of mycorrhizal fungi can be used for orchid propagation and conservation programmes.     

A new method of propagation of arbuscular mycorrhizal fungi in field cropped sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.)

Though arbuscular mycorrhizal (AM) fungi are indigenous to agricultural soils, their beneficial effects to host plants could be further improved by inoculation with efficient species. The method of AM propagation described in the present study uses oil cake as a supporting medium for the simultaneous delivery of sesame seeds and AM inoculum to the field. Experiment was conducted in a farmer’s field located at Avoor, Kerala, India where sesame was cultivated as a winter crop in rice fallows. Oil cake entrapped with sesame seeds (var. Tilatara) and AM fungus (Funneliformis dimorphicus) inoculum was prepared by thoroughly mixing sterilized coconut cake and neem cake (5:1 v/v), surface sterilized sesame seeds and sterilized spore sieving of F. dimorphicus from a pot culture in a 10% solution of a polysaccharide gum obtained from the seeds of Strychnos potatorum L. Entire mix was moulded into 2.5 cm cubes (ca. 5g) containing approximately 25–30 seeds and 200–300 spores cube^?1 and shade dried before application. The cubes were broadcast @ 600 kg ha^?1 in inoculated treatments. In uninoculated treatments, the oil cake cubes devoid of the fungal component was used. Harvested root samples from the inoculated treatments showed a high frequency (%F) and intensity (%M) of colonization by AM fungi as well as frequency of vesicles (%V) and arbuscules (%A) compared to uninoculated control. The growth (root length, shoot length and leaf area) and yield characters (pod number, seed number, seed weight and oil content) of sesame plants were significantly (p=0.05) improved under the present method of AM propagation indicating its viability under field condition.     

Effect of inoculation of symbiotic fungi on the growth and antioxidant enzymes’ activities in the presence of <Emphasis Type="Italic">Fusarium subglutinans</Emphasis> f. sp. <Emphasis Type="Italic">ananas</Emphasis> in pineapple plantlets

The inoculation with symbiotic fungi, Arbuscular mycorrhizal fungi (AMF) and/or Piriformospora indica on the growth, nutrient absorption, and induction of antioxidant enzyme activities in plantlets of pineapple ‘Imperial’ (fusariosis-resistant) and ‘Pérola’ (fusariosis-susceptible) in the presence of Fusarium subglutinans f. sp. ananas was investigated. The experiment was comprised by two cultivars, with or without fungal inoculation (Claroideoglomus etunicatum, Rhizophagus clarus, and P. indica, a mixture of all the fungi, and the control—absence of fungal inoculation), with or without applying Fusarium conidia, and with four replicates. In both cultivars, nutrient absorption was higher in the AMF plantlets compared to those inoculated with P. indica or the control ones, although it was more efficient in ‘Imperial’ than in ‘Pérola’. Inoculation with AMF and/or P. indica as well as the pathogen influenced differently the activities of superoxide dismutase, catalase, glutathione reductase, peroxidase, and polyphenol oxidase, in the shoots or roots of pineapple plantlets in both cultivars. Inoculated plantlets with mixture of all the fungi also exhibited a better growth and nutrient absorption, and generally, the ‘Imperial’ responded better than ‘Pérola’. In addition, these plantlets developed better than the control even in the presence of pathogen, indicating that inoculation with AMF and/or P. indica may contribute to the production of more resistant propagative material. Increased antioxidant enzyme activity is a potential strategy for managing this plant for explore biological control as an alternative to reduce environmental and health impacts by reducing the use of fungicides.