370 Techniques in Mycorrhizal Studies
synthetic medium supplemented with tryptophan solution. The indole compounds extracted from the culture medium were purified by thin layer chromatography and identified by, high petfonnanceliquid chromatography - HPLC (41).
Plant communities with Ericoid mycorrhizae occurs mainly in deep organic soils where due to low pH ferric (Fe3+) ions availability governs siderophores production (29,50). Mycorrhizal roots ofeal/una absorbs significantly more iron per unit time compared to uninfected Calluna roots.
This is attributed to the potential of ericoid mycorrhizal fungi to produce siderophores (96). Schaler and Haselwandter (95) demonstrated production of hydroxamate type of siderophores under pure culture conditions. Pure culture studies with ericoid mycorrhizal fungi like Hymenoscyphus ericae (87). Oidiodendron griseum (25) and Rhodothamnus chamaecistus (49) using Bioassay technique showed that H. cricae and O. griseum produced ferricrocin where as R.
chamaecistus produced fusarinine -C siderophore and pH of the culture medium strongly influenced the siderophore production (29).
4. Molecular Specificity and Compatibility
Specificity between symbiotic partners involves elicitors and receptor molecules. (38). An exocellular fibrillar sheath (76) is associated with the cell wall of ERM fungi and a strong correlation exists between the production offibrillar sheath and the ability ofERM fungal isolate to infect a host root (43,44). Cytochemical and affinity techniques show that mannose sugar residues are abundant in the fibriller sheath ofinfective strains. The hyphal cell wall and the fibrillar sheath are the site of active acid phosphatase and acid invertase activity (52,103,104,105).
Monoclonal antibodies, raised against hyphae of H. ericae have shown the presence of sugar molecules (81) specific for high infective strains.
The roots cell surface mainly consists of a layer of epidermal cells surrounded by a thin cortex of one or two cell layers and a narrow stele.
The walls show copious mucilage easily detectable with Alcian blue fixation or periodic acid Schiff reaction which is specific for the glycoproteins.
FITC (fluoresceinisothiocyanate)-labelled lectins are able to recognise soluble sugar and mannose along the roots surface, especially the cap region. The relative precision in the distribution of the surface molecules of
Geeta Singh and K. G. Mukerji 371
roots and hyphal fibrillar sheath of mycobiont has been implicated for the specificity in ericoid mycorrhizas (76,78).
Only hair roots of ericaceous plants with their low pectin walls and helicoidal cellulosic structure are colonized by ERM fimgi. In vitro, ericoid isolates have been induced to produce polygalacturonase (pG) enzyme in the presence of pectin substrate (75) as well as ~-1,4-glucanase in the presence of carboxymethyl cellulose substrate (11 0). Yet, there is little evidence of extensive enzymatic degradation at the sites of wall penetration at the time of establishment of symbiosis (78). Using antibody raised against PG enzyme of Fusarium spp. which cross reacted with an ericoid isolate. Peretto et al. (75) demonstrated complete absence oflabelling of intracellular hyphae but a strong labelling of extraradical hyphae. These workers showed complete repression ofPG activity inside the roots when they used monoclonal immunogenic probes to non esterified and methyl esterified pectins. The results showed pectins may be absent from tangential walls of differentiated epidermal cells of Calluna vulgaris, the normal site of penetration.
In compatible hosts the fibrillar sheath around external hyphae and the associated acid phosphatase activity disappears once the hyphae enters the host cortical cell indicating the regulatory control of host in symbiosis (42,44). Confirmation of host regulation of fimgal metabolism was given by Lemoine et al (64) by the use ofa probe in the form of poly clonal antiserum to a low molecular weight acid phosphatase of H. ericae.
Immunogold and cytochemical labelling demonstrated that the decline in intraradical fungal acid phosphatase was associated with an inhibition of activity rather than enzyme synthesis. This is achieved by conformational modification of the glycosidic portions of enzyme molecule; the glycosides have been shown to be essential for the enzyme's biological activity (64).
No evidence exists as to whether this control is exerted through transcription of new host gene products or through biochemical factors.
The genetic control mechanism determining compatibility and specificity in ericoid mycorrhiza is an area to be explored in future.
Fungi can be identified in the roots from species specific isozyme profiles (108) and by their reactions with antibodies (21) but these techniques are difficult to use when more than one or two fungi are present in the same roots as in case of hair roots of ericoid endomycorrhiza.
Morphological discrimination may be possible at the genus level when
372 Techniques in Mycorrhizal Studies
hyphal mOlphology is sufficiently different between fungi. (68). ERM fungi are taxonomically diverse and are difficult to be identified in pure culture as they often grow as sterile mycelium. PCR technique has greatly benefitted in morphological-phylogenetical identification ofERM fungal-symbiotic system (55).
According to Gardes and Bruns (39) the genetic material for PCR amplification should be (i) present in all fungi of interest (ii) easily amplifiable and variable enough to enable probes to be designed for several taxonomic hierarchies (iii) preferentially amplified from fungi when plant and fungal DNA are mixed. Genes coding for ribosomal RNA (r DNA) satisfy many of these criteria. The ribosomal coding regions are conserved, the internal transcribed spacers (ITS) and intergenetic spacers are highly polymorphic non coding regions which provides a useful tool to examine taxonomic and phylogenetic diversity existing in the ERM fungi (39). ITS regions are generally constant within a species but vary between species in a genus.
Amplification of these regions is normally followed by restriction fragment length polymorphism (RFLP) analysis on the PCR generated fragments for identification of the fungal species (33), and to construct species specific primers (13,97).
RAPD (Random amplified polymorphic DNA) : Finger printing method do not require any nucleotide sequence information for primers design and allow amplification of DNA fragment which are of undefined length and sequence. This is performed under low stringency conditions (i.e. low annealing temperature) with non specific primers. RAPD analysis has enabled greater resolution in investigation of the genetic polymorphism of isolates sharing the same ITS-RFLP pattern (79, 111).
Species-specific primers can only be used to amplify genes from known fungi in root tissue by PCR (1,69,109,118). RAPD-PCR is of limited use for filed studies as several mycorrhizal fungi may colonize the roots (58). SEAD-PCR (20) which selects for fungal DNA before amplification using 18s r DNA genus specific primers only discriminates at the family level (37). In contrast, systematic sequencing ofPCR products from 18 s r DNA of endomycorrhiza (AMF) has shown how complex genetic variability can be within communities colonizing roots in natural ecosystems (51). Similar observations were made using RFLP analysis of the ITS (r DNA) regions amplified from hyphae colonizing Calluna vulgaris roots. Four putative endophyte taxa were observed in the root system of a single Calluna vulgaris plant (79). ERM fungi Scytalidium
Geeta Singh and K. G. Mukerji 373
vaccinii and o. maius were found to co-occur from roots of several specimens of 19 ericaceous genera.
Dalpe et al. (261989) isolated a slow growing dematiaceous hyphomycete from roots of Vaccinium angustifolium with cultural and mycorrhizae-forming characteristics similar to Hymenosyphus ericae. But it differed because of presence of aseptate arthroconidia. Egger and Sigler (34) used polymerase chain reaction (PCR) amplification, restriction fragment analysis (RFLP) and nucleotide sequencing to show that there was a very low divergence of insertions' deletions (1.2-3. 5%) distinguishing isolates of H. ericae and S. vaccinii which indicated that the taxa are closely related and conspecific. This work highlights the potential of molecular techniques to establish the relationship between H. ericae and other ascomycetes of similar ultrastructure.
Xiao and Berch (115) identified four different mycorrhizal fungi associated with Gaultheria shallon roots two groups of isolates from which remained sterile in culture were identified by DNA sequence technique as members ofthe Leotiales distinct fromH. ericae (70). The other two known ericoid mycorrhizal fungi of salal are Oidiodendron maius Robak (46,47) and Acremonium strictum Gams (112). This showed that fungi with different ITS restriction patterns may co-exist in a thin root system ofEricales. Presence offungi with different ITS restriction patterns has been reported from Gaultheria shallon roots. (45,71). ITS DNA sequence comparisons have shown that H. ericae exist as an endophyte in the rhizoids of an Australian leafY liverwort (19). Chambers et. al. (18) found a parallel situation in Epacridaceae when they compared ITS regions, sequence of 14 isolates of endophytic fungi obtained from the root systems of four Woollsia pungens (Epacridaceae species) with the sequence of Hymenoscyphus ericae, Oidiodendron maius and to sequences from other fungal taxa available in the Gene bank and EMBL nucleotide databases. Sequence data suggested that six distinct taxa were present in the four plants and upto four of them existed in the root system of a single plant. One of the endophyte was identified as an Oidiodendron species. While most other isolates probably belonged to Leotiales. This showed that the root systems of individual Epacrldaceae plant also harbours a diverse population of ericoid mycorrhizal endophyte.
Comparison of rDNA ITS region sequences suggests that many Oidiodendron isolates were misidentified in previous studies, and that
374 Techniques in Mycorrhizal Studies
only 0. maius fonTIS mycorrhizal associations with Ericaceae in field (47).
Same technique was utilised and similar observation were made in ectomycorrhiza when Pritsch et al. (86) characterized 20 isolates of black alder ecto mycorrhizas into four groups; three were closely related; while one showed high sequence dissimilarity within the ITS 1 and ITS2 spacer regIOns.
Recently strain specific primers amplifying a part of gene coding for the large ribosomal subunit gene (LSu r DNA) were described (109) and were used in nested PCR. Systematic sequencing ofPCR products from 18S rDNA of AM fungi has shown a complex genetic variability within communities colonizing roots in natural ecosystem (51). The advantages of nested PCR is its utility in study of microcosm fungal community in plants and in soils. The technique is simple, rapid, and enhances the sensitivity ofPCR techniques. The method can be used to study spores, mycelium fresh or trypan blue stained mycorrhizal roots segments. The design of taxon specific primers from the 25S rDNA has opened the possibility of discriminating between fungi at the species level directly in a mixed community (109).
The method given by Van Tuinen et al. (109) using strain-specific primers in the nested PCR enables to detect the presence or absence of fungi depending on whether amplification is achieved or not. This method is applicable for microcosm experiment using a known fungi but is of no value ifunknown fungi colonised the roots. The limitation ofRFLP is that it is dependent on base differences in the specific restriction sites, and may not detect all polymorphic sites in a given sequence. On the other hand single stranded conformation polymorphisms (SSCPs) can detect single base substitutions any where in the amplified sequence (73). The use of mini gels followed by silver staining makes this technique suitable for screening a large number of samples (57).
Amplification of 18S and 28S ribosomal genes using specifically designed universal primers has yielded DNA fragments which were larger in size than expected (35,83). Sequencing of these fragments has revealed that this error was due to the insertion of group I interons. Group I interons have four relatively conserved regions involved in the formation of secondary structures that are important in splicing (56). The distribution and sequence of group I interon are highly variable and may account for the observed genetic diversity in ericoid fungi (77).
Geeta Singh and K. G. Mukerji 375 4. Conclusion
Ericoid mycorrhizas are complex organisms consisting offungal and plant components and their formation requires enzymatic synergisms and mutualist compatibilities resulting form cooperation of recognition and defence systems of both plant and fungus. Ericoid mycorrhizae shares fundamental features that are associated with interactions between symbionts. These features include chemotropism, recognition, compatibility and early alteration in morphogenesis of the symbionts. The use of in vitro methods for synthesis of mycorrhizas may help in dissecting the colonization process more precisely. The development ofPCR-based techniques has helped in identification of isolates through specific finger printing methods. This is particularly useful to study, ericoid fungi as these fungi lack identifiable morphological characters. Because ofPCR based techniques many crucial questions about phylogenesis, identification and polymorphisms of mycorrhizal fungi have begun to be answered. For instance many phylogenetically different symbiotic fungi have been located within the roots of Eric ales. Use ofmolecular techniques may improve our understanding of the factors that influence the success of establishment of a mycorrhizal association. This will help in optimum use and manipulation of the mycorrhizal association for plant or ecosystem benefit.
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