Mycoheterotrophic growth of Cephalanthera falcata (Orchidaceae) in tripartite symbioses with Thelephoraceae fungi and Quercus serrata (Fagaceae) in pot culture condition

Mixotrophy, obtaining carbon by mycoheterotrophy and photosynthesis, has been suggested in Cephalanthera species (Orchidaceae) by analyses on stable isotopes of carbon. In this study, we examined the growth of Cephalanthera falcata in pot cultured tripartite symbioses with Thelephoraceae fungi and Quercus serrata. Mycorrhizal fungi were isolated from roots of C. falcata in natural habitats. Two fungal isolates identified as Thelephoraceae were cultured and inoculated to fine roots of non-mycorrhizal seedlings of Q. serrata (Fagaceae). After the ectomycorrhizal formation, non-mycorrhizal seedlings of C. falcata were co-planted. The pots with tripartite symbioses were cultured in greenhouse for 30 months, and growth of C. falcata seedlings was examined. Fresh weight of C. falcata seedlings was significantly increased by the tripartite symbioses even in those with no shoot, thus providing further evidence for the mycoheterotrophic nature of this orchid. The achievement of seedling culture in tripartite symbioses would be valuable for conserving many forest orchids and for conducting experiments to understand their physiology and ecology.     

Distribution of ectomycorrhizal fungi in a Chamaecyparis obtusa stand at different distances from a mature Quercus serrata tree

Ectomycorrhizal fungal distributions at different distances from one mature Quercus serrata tree were demonstrated in a Japanese cypress stand. After 4 months for field-planted and 3 months for laboratory-incubated Q. serrata seedlings, they were evaluated ectomycorrhiza formations. For the field-planted seedlings, molecular discrimination was conducted to clarify the species diversity of ectomycorrhizal fungi. Ectomycorrhiza formation was confirmed at all distances in both methods. However, the formation and associating fungal species were decreased with increasing distance. These results indicate that the density and diversity of ectomycorrhizal fungi decrease rapidly within several tens of meters from arbuscular mycorrhizal forest edges.     

Dynamics of fruit-body production and mycorrhiza formation of ectomycorrhizal ammonia fungi in warm temperate forests in Japan

To investigate the quantitative correlation between dynamics of ectomycorrhiza (ECM) formation and fruit-body production of the ammonia fungi, we treated forest soils in two ECM forests, a Castanopsis cuspidata forest and a Quercus serrata forest, in warm temperate Japan with urea to induce fruiting of ammonia fungi. We identified the ectomycorrhizae of two known species of ammonia fungi, Alnicola lactariolens and Hebeloma vinosophyllum, using morphological typing and PCR-RFLP. ECM initiation, increase, and subsequent decrease preceded the start, increase, and decrease of fruit-body production for each species. We also found many kinds of ECM fungi that did not develop fruit bodies after urea treatment during the observation period, which suggests that it is necessary to expand the definition of ammonia fungi from one that refers solely to the species reproducing on the ground to one which includes those in somatic forms in the soil.     

Soil burrowing Muscina angustifrons (Diptera: Muscidae) larvae excrete spores capable of forming mycorrhizae underground

Muscina angustifrons (Diptera: Muscidae) is a mycophagous species that exploits a variety of fungi, including ectomycorrhizal fungi. Larvae of this species have been shown to feed on sporocarps (including spores), and full-grown larvae leave sporocarps and pupate 0–6?cm below the soil surface. In this study, we examined whether M. angustifrons larvae are capable of transporting ectomycorrhizal fungal spores and enhancing ectomycorrhiza growth on host-plant roots. Full-grown larvae were found to move horizontally 10–20?cm from their feeding sites and burrow underground. These wandering larvae retained ectomycorrhizal fungal spores in their intestines, which were excreted following relocation to underground pupation sites. Excreted spores retained germination and infection capacities to form ectomycorrhiza on host-plant roots. In the infection experiments, ectomycorrhizal fungal spores applied in the vicinity of underground host-plant roots were more effective in forming ectomycorrhiza than those applied to the ground surface, suggesting that belowground transportation of spores by M. angustifrons larvae could enhance ectomycorrhizal formation. These results suggested that M. angustifrons larvae act as a short-distance spore transporter of ectomycorrhizal fungi.     

Importance of the ectomycorrhizal network for seedling survival and ectomycorrhiza formation in rain forests of south Cameroon

An investigation was undertaken at two sites south Cameroon to assess the importance of living roots of adult trees as sources of inoculum for survival, ectomycorrhizal colonisation and growth of seedlings of Paraberlinia bifoliolata. One-month-old seedlings of Paraberlinia bifoliolata, isolated from or in contact with roots of adult target trees, were transplanted in concentric circles 5, 10, 15, and 30 m away from the stem bases of four adult tree species (Afzelia bipindensis, Brachystegia cynometroides, Paraberlinia bifoliolata and Tetraberlinia bifoliolata). After 4 and 8 months, survival, ectomycorrhiza formation and seedling height were observed; plant biomass was determined after 8 months. After 4 months, there was no difference in seedling survival between seedlings in contact with roots of adult trees and isolated seedlings. The number of ectomycorrhizal seedlings was higher among seedlings in contact with adult trees than among isolated seedlings. After 8 months, both seedling survival and ectomycorrhiza formation were higher in seedlings in contact with roots than in isolated seedlings. Seedling survival and ectomycorrhiza formation were highest under Brachystegia. The fraction of surviving seedlings that had formed ectomycorrhizas was lowest under Afzelia. The fraction of surviving seedlings that were ectomycorrhizal declined monotonically towards the edge of the crown projection. Biomass of seedlings in contact with adult trees was significantly higher than that of isolated seedlings. This difference was consistent for all four tree species. The height of seedlings did not vary with the treatment.     

Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza

Fungus-derived indole-3-acetic acid (IAA), which is involved in development of ectomycorrhiza, affects both partners, i.e., the tree and the fungus. The biosynthesis pathway, excretion from fungal hyphae, the induction of branching in fungal cultures, and enhanced Hartig net formation in mycorrhiza were shown. Gene expression studies, incorporation of labeled compounds into IAA, heterologous expression of a transporter, and bioinformatics were applied to study the effect of IAA on fungal morphogenesis and on ectomycorrhiza. Tricholoma vaccinum produces IAA from tryptophan via indole-3-pyruvate, with the last step of this biosynthetic pathway being catalyzed by an aldehyde dehydrogenase. The gene ald1 was found to be highly expressed in ectomycorrhiza and induced by indole-3-acetaldehyde. The export of IAA from fungal cells is supported by the multidrug and toxic extrusion (MATE) transporter Mte1 found in T. vaccinum. The addition of IAA and its precursors induced elongated cells and hyphal ramification of mycorrhizal fungi; in contrast, in saprobic fungi such as Schizophyllum commune, IAA did not induce morphogenetic changes. Mycorrhiza responded by increasing its Hartig net formation. The IAA of fungal origin acts as a diffusible signal, influencing root colonization and increasing Hartig net formation in ectomycorrhiza.     

Recognition mechanisms during the pre-contact state of lichens: I. Mycobiont-photobiont interactions of the mycobiont of Fulgensia bracteata

Lichens are an association of a photoautotrophic alga/cyanobacteria (photobiont) and a heterotrophic fungus (mycobiont) constituting the lichen thallus as a complex phenotype. Many mycobionts reproduce sexually and the ascospores are dispersed without the photobiont. For successful re-lichenization the specific photobiont must be recognized, contacted, and incorporated by the mycobiont. A so-called pre-contact stage has been postulated as the initial step of a gradual recognition process. In the present study, the effect of the specific Trebouxia photobiont, an unspecific Asterochloris photobiont and the non-lichenizing green alga Myrmecia bisecta on the development of the mycobiont Fulgensia bracteata was assessed by pre-contact assays. Three hypotheses were confirmed: (i) the pre-contact stage exists, (ii) it is characterized by morphological reactions in the development of the mycobiont, and (iii) the reactions depend on the interacting alga. Control conditions revealed a mycelial growth arrest but this effect was not observed in the presence of any of the three algae. Different algae induce distinct growth patterns with respect to hyphal length, morphological characteristics, and formation of mucilage. The specific Trebouxia photobiont had a positive impact on hyphal growth, branching frequency, and mucilage formation. These effects were less explicit with the non-specific Asterochloris photobiont. Myrmecia bisecta induced uncharacteristic growth patterns with pronounced hyphal growth and high numbers of aerial hyphae but less formation of mucilage. These results indicate that symbiont recognition mechanisms are established before physical contact. Pre-contact reactions may be an evolutionary advantage that supports the persistence of the mycobiont on newly colonized sites and improves the probability of re-lichenization.     

Indentification of huperzine A-producing endophytic fungi isolated from Huperzia serrata

This present study was designed to investigate the production of huperzine A (HupA), an acetylcholine inhibitor, which was produced by an endophytic fungi isolated from Huperzia serrata. Screening of 94 endophytic fungal isolates obtained from plant H. serrata was carried out for the production of HupA. Their morphological characteristics were studied and rDNA sequence analysis was carried out. The cultures were grown in liquid culture medium and the extracted metabolites were analyzed by thin layer chromatography and high performance liquid chromatograph for the presence of HupA. The DPPH scavenging ratio and inhibition ratio of acetylcholinesterase (AchE) of the same were determined. 3 out of 94 strains i.e. S29, L44 and S94 showed significant AchE-inhibitory activity and antioxidant activity. Strain L44 which exhibited maximum yield of HupA (37.63 μg/g on dry weight basis) was identified as Trichoderma species by ITS sequence analysis. In conclusion, endophytic fungi from H. serrata can be used as a new resource of HupA.     

Identification of a novel endophytic fungus from Huperzia serrata which produces huperzine A

Huperzine A is isolated from Huperzia serrata and is used for treatment of Alzheimer’s disease, due to its low toxicity and long effective period. The decrease in H. serrata sources means that natural huperzine A cannot meet the needs of clinical therapy. In this study, >200 endophytic fungal strains were isolated from H. serrata, and screened using high-performance liquid chromatography. Strain ES026 produced huperzine A. Production was identified and quantified by liquid chromatography–tandem mass spectrometry, and the yield of huperzine A was 1 μg/g dried mycelium. ES026 strain was identified as Colletotrichum gloeosporioides by morphology, polymerase chain reaction with species-specific primers and rDNA internal transcribed spacer sequence. ES026 contributes to the breeding of cultivated strains with high yield of huperzine A. Meanwhile, the strain was suitable for the study of biosynthesis of huperzine A.     

In vitro zygotic embryo germination and propagation of an endangered Boswellia serrata Roxb., a source of boswellic acid

This study aims to establish an efficient protocol for development of seedlings of an endangered medicinally important forest tree Boswellia serrata Roxb., for mass plantation and consistent supply of salai guggul. The green mature fruits served as source of seeds. The excised green zygotic embryos were cultured on Gamborg (B5), McCown and Loyd (WPM) and Schenk and Hildebrandt (SH) media fortified with different concentration of sucrose and on Murashige and Skoog (MS) medium containing 3 % sucrose, polyvinylpyrrolidone (PVP) (0–300 mg l^−l), Gibberellic acid (GA₃), Indoleacetic acid (IAA), Naphthaleneacetic acid (NAA), Indole-3-Butyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4 D) and 6-benzylaminopurine (BA) or kinetin (Kin) individually. The highest frequency of embryo germination (96 %) and conversion into seedling was obtained on MS medium containing 3 % sucrose together with 200 mg l^−l PVP; other media were either inferior or induced abnormalities in the seedlings including callus formation from the zygotic embryos. Fully developed seedlings could be successfully established in soil with about 94 % survival. The embryos from mature dry seeds did not respond for germination in any of the experiments. In conclusion, selection of zygotic embryo from green mature seeds and their in vitro germination is important for propagation of B. serrata.     

Purification and part characterization of a novel antibacterial protein Scygonadin, isolated from the seminal plasma of mud crab, Scylla serrata (Forskål, 1775)

A novel protein was isolated from the seminal plasma of the mud crab, Scylla serrata (Forskål, 1775). It exhibited an antibacterial activity against the Gram-positive bacterium Micrococcus leteus with IC90 of 0.125 mg/ml. The extraction procedure for the protein included techniques of acid extraction, ion-exchange chromatography on SP-Sepharose Fast Flow and reverse-phase liquid chromatography on Source 5R RPC. It showed a molecular mass of 10.8 kDa by SDS-PAGE. A partial 20 residue NH₂-terminal sequence was determined by Edman degradation and MS-fingerprint of the protein was conducted. Similarity search in protein databases (BLAST) revealed that the protein exhibited no significant homology to any other reported antimicrobial peptides. We propose the name Scygonadin (from the gonad of S. serrata) for this antibacterial protein.     

Symbiotic performance,nitrogen flux and growth of lima bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L.) varieties inoculated with different indigenous strains of rhizobia

Nitrate uptake and nitrogen inclusion into amino acids were studied in the intact thallus and isolated bionts of the lichen Parmelia sulcata with the aid of mass spectroscopic tracing of heavy isotope ¹⁵N. The isolated photobiont, the green algae Trebouxia sp. did not take up nitrate, whereas the mycobiont and intact thalli were enriched in ¹⁵N when incubated with Na¹⁵NO₃. Pulse feeding experiments with intact thalli followed by separation of photobiont showed that the labelled nitrate was originally assimilated by the mycobiont and only after that was detected in the photobiont. The isolated mycobiont after pulse labeling excreted labeled compounds into the incubation medium. Amino acids were detected in the exudate. The quantities of two amino acids considerably exceeded those of the others. One was identified as alanine, the other could not yet be identified with certainty. Both of these high-quantity compounds were also much more enriched in ¹⁵N than the others. These two compounds are proposed to be the transport forms of nitrogen within the Parmelia sulcata thallus.     

Morphological characterization of mycorrhizae formed between three Terfezia species (desert truffles) and several Cistaceae and Aleppo pine

Six Cistaceae species, Helianthemum ledifolium, Helianthemum lippii, Fumana procumbens, Cistus albidus, Cistus incanus, Cistus salvifolius, and Pinus halepensis (Aleppo pine) were inoculated with three mycorrhizal desert truffles, Terfezia leptoderma, Terfezia boudieri, and Terfezia claveryi under greenhouse conditions, on soil originating from desert truffle natural habitat in Algeria. The syntheses have led to the formation of typical endomycorrhizae in annual Cistaceae (H. ledifolium) and perennial ones (H. lippii and F. procumbens) and an ectomycorrhiza with a less developed sheath in Cistus species and Aleppo pine. These results demonstrate the plasticity of Terfezia species to form different mycorrhizal types. The formation of an endomycorrhiza with H. ledifolium and F. procumbens and a sheathing ectomycorrhiza with P. halepensis inoculated by T. leptoderma in in vivo culture conditions was obtained for the first time.     

Phylogeographical structure in Zelkova serrata in Japan and phylogeny in the genus Zelkova using the polymorphisms of chloroplast DNA

The genetic differentiation inherent in Zelkova serrata in Japan and the southern portion of the Korean Peninsula was examined by comparing a chloroplast DNA (cpDNA) sequence over a 16?k baselength in 40 individual samples collected from an area covering the natural distribution range of Z. serrata in Japan and the southern portion of the Korean Peninsula. We detected over 50 single-nucleotide polymorphisms in the protein-coding and intergenic regions, and over 30 insertions/deletions in the intergenic region. From the polymorphisms detected in the cpDNA, 14 haplotypes were identified. These 14 haplotypes had cluster-like structures and genetic differentiation between the clusters was large. Closely related haplotypes existed in adjacent regions. One haplotype existed in both Japan and the Korean Peninsula. By comparison with other Zelkova species, Z. serrata is apparently distinct from European and East Asian Zelkova species and Z. serrata is closest to the Ulmus species in the genus Zelkova. The effects of the analyzed length of the cpDNA sequence on the detection of polymorphisms were analyzed by re-sampling simulation.     

Ascomycetes associated with ectomycorrhizas: molecular diversity and ecology with particular reference to the Helotiales

Mycorrhizosphere microbes enhance functioning of the plant–soil interface, but little is known of their ecology. This study aims to characterize the ascomycete communities associated with ectomycorrhizas in two Tasmanian wet sclerophyll forests. We hypothesize that both the phyto‐ and mycobiont, mantle type, soil microbiotope and geographical distance affect the diversity and occurrence of the associated ascomycetes. Using the culture‐independent rDNA sequence analysis, we demonstrate a high diversity of these fungi on different hosts and habitats. Plant host has the strongest effect on the occurrence of the dominant species and community composition of ectomycorrhiza‐associated fungi. Root endophytes, soil saprobes, myco‐, phyto‐ and entomopathogens contribute to the ectomycorrhiza‐associated ascomycete community. Taxonomically these Ascomycota mostly belong to the orders Helotiales, Hypocreales, Chaetothyriales and Sordariales. Members of Helotiales from both Tasmania and the Northern Hemisphere are phylogenetically closely related to root endophytes and ericoid mycorrhizal fungi, suggesting their strong ecological and evolutionary links. Ectomycorrhizal mycobionts from Australia and the Northern Hemisphere are taxonomically unrelated to each other and phylogenetically distant to other helotialean root‐associated fungi, indicating independent evolution. The ubiquity and diversity of the secondary root‐associated fungi should be considered in studies of mycorrhizal communities to avoid overestimating the richness of true symbionts.     

Recognition mechanisms during the pre-contact state of lichens: II. Influence of algal exudates and ribitol on the response of the mycobiont of Fulgensia bracteata

Successful re-lichenization between the two bionts of the lichen symbiosis, the fungal mycobiont and its specific photobiont, is a process that is not well understood yet. To assess potential signalling between the two bionts during initial pre-contact, exudates of the Trebouxia photobionts of Fulgensia bracteata, Fulgensia fulgens, and Xanthoria elegans, of the Asterochloris photobiont of Lecidea lurida, and of the non-lichenizing green alga Myrmecia bisecta were investigated. The compounds identified in these exudates were tested with respect to their influence on germination and early development of the Fulgensia bracteata mycobiont. Additionally, carbohydrates (glucose, sucrose, ribitol) were tested to appraise their effect on the mycobiont growth patterns. Three hypotheses were confirmed: (i) photobionts exude various substances, (ii) the photobiont exudation pattern varies with the identity of the photobiont, and (iii) a pre-contact influence induces changes in the early development of the mycobiont of F. bracteata. This study gives comparative insight to exudates of lichen photobionts. In vitro photobionts differentially release compounds belonging to several substance classes which include indole-3-carbaldehyde, two cyclic dipeptides, and rhamnose. Two compounds had inhibitory effects on germination and germ-tube growth of the mycobiont and one other enhanced spore germination. Additionally, ribitol was found to elicit a strong effect on the mycobiont’s growth. In general, photobiont-exudation, its effect on the mycobiont, and the response to ribitol suggest that complex pre-contact signalling has a crucial role in lichen biont recognition.