Molecular diversity within and between ericoid endophytes from the Ericaceae and Epacridaceae

This study investigated the relationships between ericoid mycorrhizal endophytes of the Ericaceae (Northern Hemisphere) and the Epacridaceae (Australia). Over 200 fungi were isolated from the roots of two species of Epacridaceae from Victoria, Australia. The isolates were divided into 12 groups by morphology on quarter-strength potato dextrose agar. All were slow-growing and most were dematiaceous, but groups varied from white through pink to dark olive. The ITS1–5.8S–ITS2 ribosomal DNA was amplified and sequenced from eight isolates, forming typical ericoid mycorrhizal morphology in Epacris impressa and one nonmycorrhizal isolate. Sequences were compared, by using similarities and maximum-parsimony analysis, with those of Hymenoscyphus ericae (Leotiales) and Oidiodendron species (Hyphomycetes), the most common endophytes of the Ericaceae. Maximum-parsimony analysis produced four clusters: (1) all Oidiodendron species (at least 90% similarity); (2) all five Victorian dark grey-olive isolates (at least 96% similarity); (3) one Victorian isolate and Cistella grevillei (88% similarity); (4) two light-coloured Victorian isolates and H. ericae (81% similarity). This suggests that these isolates from the Epacridaceae do not belong to the same species as those forming ericoid mycorrhiza in the Ericaceae.     

Genetic diversity of root-associated fungal endophytes from Calluna vulgaris at contrasting field sites

A total of 107 putative ericoid mycorrhizal endophytes were isolated from hair roots of Calluna vulgaris from two abandoned arsenic/copper mine sites and a natural heathland site in southwest England. The endophytes were initially grouped as 14 RFLP types, based on the results of ITS-RFLP analysis using the restriction endonucleases Hin f I, Rsa I and Hae III. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with sequences for known ericoid mycorrhizal endophytes and selected ascomycetes. The majority of endophyte isolates (62–92%) from each site were identified as Hymenoscyphus ericae , but a number of other less common mycorrhizal RFLP types were also identified, all of which appear to have strong affinities with the order Leotiales. None of the less common RFLP types was isolated from C. vulgaris at more than one field site. Neighbour-joining analysis indicated similarities between the endophytes from C. vulgaris and mycorrhizal endophytes isolated from other Ericaceae and Epacridaceae hosts in North America and Australia.     

Phylogenetic Relationships of Empetraceae, Epacridaceae, Ericaceae, Monotropaceae, and Pyrolaceae: Evidence from Nuclear Ribosomal 18s Sequence Data

Recent studies of phylogenetic relationships have indicatedthat the traditional recognition of Epacridaceae and Empetraceaeas distinct from Ericaceae should be reevaluated. These studiesused morphological data and nucleotide sequence from the chloroplastencodedrbc L (rubisco, large subunit) gene. They indicated thatEricaceae as presently recognized are paraphyletic and shouldinclude Epacridaceae and Empetraceae, as well as Pyrolaceaeand Monotropaceae. A study of nuclear ribosomal 18s gene sequenceswas undertaken to test the hypothesis that Epacridaceae forma monophyletic derived group out of Ericaceae. The problematictaxaPrionotesandLebetanthuswere included because these taxahave been alternatively placed in Ericaceae and Epacridaceae.Representatives of the herbaceous (Pyrolaceae) and mycoparasitictaxa (non-chlorophyllous, Monotropaceae) were also includedin the study. Taxa that represented lineages peripherally relatedto Ericaceae and Epacridaceae were included in order to developa better understanding of the relationships and limits of Ericales.Parsimony analyses of 18s sequences and a combined analysisof 18s+rbcL sequences were performed. Results of these analysesindicate strong support for the recognition of a monophyleticEricaceae that includes Empetraceae, Epacridaceae, Pyrolaceae,and Monotropaceae. nr18s; Empetraceae; Epacridaceae; Ericaceae; Monotropaceae; Pyrolaceae; rbc L     

Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots

Ericoid mycorrhizal fungal endophytes form mycorrhizal associations with Ericaceae plant taxa and are regarded as essential to the ecological fitness of the plants in extremely nutrient-poor soils worldwide. We isolated fungi from roots of Epacris pulchella (Ericaceae) in a south-eastern Australian sclerophyll forest and compared rDNA internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs) and sequences for the cultured isolate assemblage with fungi identified in DNA extracted directly from the same root systems by cloning or denaturing gradient gel electrophoresis (DGGE). The most abundant RFLP types in the cultured isolate assemblage were identified as putative ericoid mycorrhizal ascomycete endophytes, and these also represented the most abundant RFLP types in the cloned assemblage and the most intense bands in DGGE profiles. Each method identified unique taxa, notably putative basidiomycetes in the DNA extracted directly from E. pulchella roots. However, the relative abundance of these was low.     

The Structure and Fungal Associates of Mycorrhizas in Leucopogon parviflorus (Andr.) Lindl.

Mycorrhizal root systems ofLeucopogon parviflorus (Andr.) Lindl.were collected from wild populations at three sites on the coastof New South Wales, Australia and examined by light and electronmicroscopy. The structure of the hair roots is typical of thefamily, there being an epidermal layer in which ericoid mycorrhizasare formed, two cortical layers (an exodermis and endodermis)and a very small stele. The colour, size and coil structureof the fungal symbionts indicate that there were at least twodifferent fungi that consistently formed ericoid mycorrhizalstructures at these sites. Transmission electron microscopyof the endophytes showed only ascomycete fungi. Plants fromtwo of the populations were used for fungal isolations. Fungiwere isolated by incubating surface sterilized hair-root piecesin a solution of bovine serum albumin with penicillin and streptomycin.Twenty-one different culture types were obtained, four of whichwere common to both sites. Two of the common culture types weredark, sterile, slow-growing cultures similar to the ericoidendophyteHymenoscyphus ericae(Read) Korf & Kernan. Leucopogon parviflorus ; bearded heath; Epacridaceae; Ericaceae; mycorrhiza; ultrastructure; endophytes; callose     

Somatic Embryogenesis for Mass Propagation of Ericaceae – A Case Study with Leucopogon verticillatus

An efficient three-phase culture has been developed for plant regeneration of Leucopogon verticillatus (R. Br.) (Ericaceae formerly Epacridaceae [Ann. Missouri Bot. Gard. 85 (1998): 531–553]) via somatic embryogenesis as indicative of likely culture scenarios for other Ericaceae. The Ericaceae, particularly many Australian species, are often difficult to propagate by conventional forms of nursery propagation. Initiation of somatic embryos was best achieved using Gamborgs B₅ medium, pH 6, 4% maltose, 0.7% agar with the plant growth regulators 10 µM TDZ and 5 µM IAA. Somatic embryos were removed from the parent tissue and transferred to half strength basal GB₅ medium for elongation. Root development did not occur unless specific treatments were used, a 2–5 day pulse treatment of 100 µM IBA significantly increased root production. All roots produced in agar-medium were fine and easily damaged when removed from culture. The most successful rooting medium (>60%) was sand on oat medium, which facilitated easy removal from the substrate and improved the survival of plants when transferred to soil.     

Co-occurrence of three fungal root symbionts in Gaultheria poeppiggi DC in central Argentina

The roots of Gaultheria poeppiggi (Ericaceae) were examined for fungal symbiont colonization. Typical structures of ericoid mycorrhizas (hyphae and intracellular coil hyphae complexes), dark septate fungal endophytes (hyphae and sclerotia), and arbuscular mycorrhizas (hyphae, coils, vesicles and arbuscules) were found in the roots of all the individuals examined. The evolutionarily derived position of Gaultheria within the Ericales may suggest that G. poeppiggi recently acquired the ability to form arbuscular mycorrhizas rather than having retained it from ancestral lines.     

Anthocyanins in the genus Erica

3-Glucosides, 3-galactosides and 3-arabinosides of cyanidin, delphinidin, malvidin, peonidin and pelargonidin have been identified as major floral pigments in Erica (Ericaceae). Unidentified 3-biosides are present as minor pigments in some species. A comparison is made with floral anthocyanins occurring in the related family Epacridaceae.     

Cytoevolution, Phylogeny and Taxonomy in Epacridaceae

Chromosome numbers in Epacridaceae were collated from variousauthors and superimposed on to recent cladograms for the family.The results strongly indicate that reduction in chromosome numberoccurs with evolutionary advancement. Thus Epacridaceae, incommon with ‘Ericaceaesens. strict .’ and ‘Vacciniaceaesens.strict. ’, has evolved from a primitive karyotype of atleastx =12 and probablyx =13. This conclusion dispels earlierpaleopolyploid models based on a primitivex =6 in the family.Other aspects of cytoevolution, such as chromosome number asa generic character especially in cytologically advanced generaof the Styphelieae, are discussed. Cytoevolution; cladistic models; primitive character states for chromosome number; dysploid falls; cytotaxonomy; Epacridaceae; Richeoideae; Epacridoideae; Epacrideae; Styphelieae; Ericaceae     

Molecular diversity of ericoid mycorrhizal endophytes isolated from Woollsia pungens

One hundred and sixty-eight sterile endophytic mycelia were isolated from roots of four Woollsia pungens (Cav.) F. Muell. (Epacridaceae) plants collected from a field site in New South Wales, Australia. All isolates formed typical ericoid mycorrhizal structures when inoculated onto roots of Vaccinium macrocarpon Ait. (Ericaceae). Microsatellite-primed PCR fingerprints generated using the primers (GTG)₅ and (GACA)₄ indicated that considerable genetic diversity exists within the endophyte population. It was estimated that a minimum of 43 genetically distinct mycelial genets were present in the root systems of the sampled W. pungens population, with most genets confined to individual plants. Two genets, however, were present within the root systems of two adjacent plants. While most genets were represented by less than eight isolates, three genets contained up to 41 isolates, suggesting that root system colonization by some endophytic mycelia might be extensive.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Decomposition of organic matter by the ericoid mycorrhizal endophytes of Formosan rhododendron (Rhododendron formosanum Hemsl.)

Ericoid mycorrhizas are associated with a number of host plants in the Ericaceae in high-elevation regions of Taiwan. The ability of these microorganisms to thrive in harsh environmental conditions in the regions implies their capability of decomposing plant organic matter (raw humus). The objective of this study was to investigate the decomposition characteristics of three ericoid mycorrhizal endophytes isolated from the roots of Formosan rhododendron (Rhododendron formosanum Hemsl.). Molecular analysis indicated that strains Rf9 and Rf32 belong to the genus Cryptosporiopsis while strain Rf28 is a member of the genus Phialocephala. Mycorrhizal synthesis experiment showed that the roots of synthesized seedlings produced hyphal coils, a characteristic of ericoid mycorrhiza. Decomposition ability analysis revealed that strains Rf28 and Rf32 had the highest rates of decomposition of organic matter (up to 10.4% after 70 days) while the value for strain Rf9 was about 6.8%. Consistently, these strains secreted extracellular oxidases when cultured on tannic acid medium. Enzyme assay revealed that strains Rf28 and Rf32 secreted peroxidase, laccase, tyrosinase, and cellulase, but strain Rf9 secreted mainly peroxidase and tyrosinase. Apparently, the differences in secreted hydrolytic enzymes among the three endophytes are related to their ability to decompose organic matter. In the mycorrhizal synthesis experiment, all inoculated seedlings survived in the organic matter substrate for 70 days and exhibited a stronger vigor than the control. This study demonstrated that these three isolated endophytes, Rf9, Rf28, and Rf32, are ericoid mycorrhizal fungi, capable of forming ericoid mycorrhiza with Formosan rhododendron. Meanwhile, all three endophytes can secrete hydrolytic enzymes to decompose organic matter for growth, presumably a prerequisite for the adaptation of Formosan rhododendron to the harsh environments of high elevation.     

VESICULAR-ARBUSCULAR MYCORRHIZAE IN HAWAIIAN ERICALES

Vesicular-arbuscular mycorrhizae (VAM) are reported for the first time in four species of Hawaiian Ericales, Vaccinium calycinum, V. dentatum, and V. reticulatum of the Ericaceae and Styphelia tameiameiae of the Epacridaceae. The coarse roots (> 1.5 mm diam) of many specimens were densely colonized by VAM fungi, with up to 90% of the length of roots containing arbuscules, vesicles, coils, and internal hyphae. Spores of an undescribed Glomus sp. were associated with two species of Vaccinium. The hair roots of all species bore the ericoid mycorrhizae typical of certain families of this order. The high frequency of VAM in Hawaiian populations of Ericales suggests that ancestral Ericales possessed the capacity to form both VA and ericoid mycorrhizae. An evolutionary sequence of mycorrhizal dependency in the Ericales is presented.     

Pectic Zymograms and Water Stress Tolerance of Endophytic Fungi Isolated from Western Australian Heaths (Epacridaceae)

Forty-nine isolates of root-inhabiting fungi were obtained fromthirteen species of eleven genera of native Epacridaceae andcompared in relation to host taxonomy and habitat of origin.Pectic zymograms of extracts of the cultured endophytes showeda marked degree of homogeneity of banding patterns amongst isolatesfrom a mesic wetland site, whereas those from dryland habitatdisplayed more heterogenous banding. It is speculated that hostspecies can operate with only a limited number of fungal associatesunder mesic conditions but require a higher degree of endophytevariation when combating dry and impoverished environments.Considerable distinction between the geographically diverseendophytes isolated from the common hostLysinema ciliatum suggestedthat selection of endophytes was not driven primarily by hosttaxonomy. Ascribing functional significance to the observed differencesbetween endophytes was studied by examining responses of culturedisolates to polyethylene glycol induced-water stress coveringa range of potentials from -0.16 to -2.96MPa. Three responsetypes were observed: (a) the isolate produced minimal radialgrowth at all water potentials tested, (b) maximum growth ofthe isolate occurred under least water stress, with progressivesuppression of radial extension with decreasing water potentialand (c) maximum growth of the isolate occurred under a degreeof water stress. The broad range of responses to water stressobserved was suggested to reflect the diverse habitat tolerancedisplayed by epacrids and their endophytic partners in southwest Australia. ericoid mycorrhizas; pectic zymogram; water stress; Epacridaceae     

Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3–65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.     

The Co-occurrence and Morphological Continuum Between Ericoid Mycorrhiza and Dark Septate Endophytes in Roots of Six European Rhododendron Species

Ericaceae associate with a wide spectrum of root mycobionts, but the most common are ascomycetous ericoid mycorrhizal fungi and dark septate endophytes (DSE), followed by basidiomycetous fungi and glomeracean arbuscular mycorrhizal fungi. We investigated distribution and morphological diversity of ericoid mycorrhizae (ErM), DSE associations, ectomycorrhizae (EcM) and arbuscular mycorrhizae (AM) in hair roots of six European native Rhododendron species and found that i) while EcM and AM were absent, ErM and DSE associations were simultaneously present in all screened plants; ii) their levels were negatively correlated, suggesting Ericaceae preference for certain root-fungus association in certain habitats; iii) the highest ErM colonization occurred at sites in southern and central Europe, while the highest DSE colonization was found in a subarctic site in northern Finland and in a subalpine site in the Carpathians, suggesting a latitudinal/altitudinal shift in Ericaceae root-fungus associations; iv) some mycelia could simultaneously form structures corresponding to ErM and DSE association, which occasionally resulted in a unique ectendomycorrhizal colonization comprising an intercellular parenchymatous net and intracellular hyphal coils. These results indicate frequent interactions between ErM fungi and DSE in roots of European rhododendrons and a morphological continuum between ErM and DSE associations. The new ectendomycorrhizal type deserves further investigation.     

锦绣杜鹃菌根真菌rDNA ITS序列分析及接种效应研究

利用rDNA ITS序列对锦绣杜鹃菌根真菌的16个菌株进行了分类分析。根据菌株ITS序列全长计算各菌株间序列相似度和遗传距离,并与GenBank中最相似菌株序列构建系统发育树。结果表明：16个菌株在系统树上聚为3个大分支。其中7个菌株在支持率为100%的基础上与树粉孢属真菌Oidiodendron sp.聚为一类;2个菌株与未鉴定的杜鹃花科植物根系真菌unidentified root associated fungi聚为一类,支持率为100%;其他7个菌株在98%的支持率上与几种未命名的欧石楠类菌根真菌     

The Structure and Function of the Ericoid Mycorrhizal Root

The uniformity of structure of the anatomically simple ericoidmycorrhizal hair root across many plant families, includingEpacridaceae, that are diagnostic of heathland, and the characteristicrestriction of its occurrence to nutrient impoverished soils,are both emphasized. The extent to which the predominantly ascomycetousfungal endophytes of these roots are taxonomically related isdiscussed. In functional terms, the role of the mycorrhiza innutrient mobilization is evaluated on the basis of experimentswith ericaceous plants. The considerable saprotrophic potentialof endophytes such asHymenoscyphus ericae is demonstrated andthe significance of this for nitrogen (N) and phosphorus (P)nutrition of plants growing in sclerophyllous litter of highC:N and C:P ratios is discussed. The need to carry out experimentsusing epacrid hosts is stressed. It is considered that the selectiveprovision, by ericoid mycorrhizal fungi, of access to recalcitrantorganic sources of N and P facilitates niche differentiationand so contributes to the maintenance of species diversity whichis a feature of heaths with a significant component of epacridor ericaceous plants particularly in the southern hemisphere. Ericoid mycorrhiza; hair root; nitrogen mobilization; heathland; Epacridaceae