3.1. Acaulospora Gerdemann and Trappe emend. Berch.
Spores appear to form laterally on the neck of a small thin walled saccule called variously as a mother spore, a vesicle, a hyphal terminus, a sporiferous saccule, or a sporogenous saccule. At maturity the spore does not show a subtending hypha (Fig. 10), the vesicular structure that gives rise to it loses cytoplasm and collapses during development of the spore. The spores are globose or ellipsoid, range from 40 to 400 J,lm in diameter, hyaline, yellow or reddish brown. The surface of the spore wall may be ornamented with pits, projections of various shapes, folds, spines or reticulations. The wall is upto 12 J,lm thick, of two distinct, separable wall groups, part of the outer wall continuous, may be pigmented, laminated or composed of distinct walls. Inner wall composed of one or more walls that are usually membranous, hyaline, may be laminated, ornamented, stain pink, red or purple with Melzer's reagent.
3.2. Entrophospora Ames and Schneider
Azygospores or cblamydospores produced singly in soil by expansion within the stalk of the mother vesicle. Mother vesicle thin walled, dense, white, becoming empty, walls of the vesicular stalk expand to accommodate spore, forming an outer membranous spore wall, continuous, with two scars on either end (Fig. 9).
3.3. Gigaspora Gerdemann and Trappe
Spores produced singly in soil, large, variable in shape usually globose to sub globose, ovoid or obovoid, pyriform or irregular, borne on bulbous suspensor like cell, usually with narrow hyphae extending as one or more peg like projections towards the spore. Spore wall is of single wall group lacking flexible walls. One or more germ tubes are produced directly from spore wall near the base. Thin walled, echinulate, finely papillate, knobby auxiliary cells are produced on the hyphae in soil (Fig. 5,11).
C. Manoharachary, I.K. Kunwar and K. G. Mukerj; 261
3.4. Glomus Tulsane and Tulsane
Glomus is the most common genus, has over 85 species with globose, ellipsoid or rather irregularly shaped spores that range from 1 0-400 ~m.
Spores thick walled (upto 30 ~m)hyaline, yellow, brown, black. Spores are attached usually to a single or exceptionally to more subtending hyphae;
loose sporocarps, rarely compact sporocarps may be formed outside the root cortex or exceptionally in root cortical tissues (Figs. 6-8). Spores germinate mostly by emergence of a germ tube through the subtending hypha or directly through the spore wall. At maturity spore contents of the spore are separated from attached hyphae by a septum or occluded by spore wall thickenings.
3.5. Sclerocystis Berk. and Broome
Chlamydospores or spores are similar to Glomus, except that they tend to be clavate rather than globose. Spores always form in compact sporocarps which are upto 700 ~m in diameter, sporocarp normally covered with peridium, spores upto 205 ~m long, develop in a single layer, radially arranged, around a central plexus of sterile and sporogenous hyphae (Figs. 14, 15).
3.6. Scutellospora Walker and Sanders
Spores usually formed singly, size ranges from 55 - 600 ~m or more, spores develop terminally on a bulbous hypha! suspensor, which remains attached at maturity and may bear short lateral projections, spores globose, ovoid, obovoid, pyriform or irregular, hyaline, white, yellow, pinkish, grey or brown, wall of two layers with one or more flexible membranous or coriaceous walls in the inner group or groups. Germination by means of one or more germ tubes produced near the spore base, from a germination shield formed within or upon flexible inner wall (Figs. 12, 13).
Thin walled knobby or broadly papillate auxiliary cells are formed.
262 Techniques in Mycorrhizal Studies
4. Biochemical and molecular approach in the identification of VAM/AM fungi.
Besides conventional morphological characters isozyme analysis (16,33,34,37), ELISA technique and use of monoclonal antibodies (2,30) are currently being developed as more authentic taxonomic tools. By using flourescent antibody technique only, V AMI AM fungal genera could be distinguished.
Serological detection with light or electron microscopy represents a potential tool of differentiation between individual species or strains of endomycorrhizal fungi. Till date it has been applied only in few studies on ericoid or V AMI AM fungi using antibodies raised against antigen either from hyphae or spores (19,32). Aldwell et al. (2) have produced antisera
to four species ofV AMI AM fungi and presented results from the application of an indirect enzyme linked immunosorbent assay to detect differences between species. Wright et al. (46) raised monoclonal antibodies against Glomus occultum Walker and used them in ELISA to discriminate between geographically distint strains. A number of commercially available probes such as fluorescent gold or enzyme conjugated lectins and enzymes can be used to determine presence or absence of non-specific cell components ofthe fungus (30).
Molecular studies also include nucleotide sequence or RFLP of DNA regions, isoenzymes, glycoproteins and fatty acids. Nuclear r- DNA gene regions have been used most widely in systematic studies.
Simon et al (38) have found that nucleotide sequences of highly conserved region of the 185 ribosomal genes have been compared among isolates of glomalean fungi.
The results indicate a great potential establishing relationships at family leve1. Proteins or glycoproteins identified by immunological procedures appear to have some taxonomic value in seggregating isolates of species (46). Jabaji-Hare (18) evidenced different fatty acids in suborganismal parts of Gigaspora and Glomus species. The results indicated convergent evolution of these molecules. In order to construct genetic phylogenics the studies mentioned above will help immensely and only researchers know the difficulties in identifying isolates of different species. Morton (24) suggested that the similarities in lipid profiles between V AMI AM fungi and protoctisian fungi are convergent characters rather than indicators
C. Manoharachary, l.K. Kunwar and K.G. Mukerji 263
of phylogenetic relationships. Protoctisian fungi revealed the presence of glucans in their cell walls rather than chitin which is present in the cell walls ofVAM/ AM fungi. The nature of nuclei ofVAM/ AM fungi is similar to zygomycetous fungi.
5. Classification
Arbuscular mycorrhizal fungi and their taxonomy has been the subject of much debate. The identification of V AM/ AM fungal species is mainly based on mOlphological criteria and spore characteristics. Dangeard (11) was the first to name V AM/ AM fungi. Peyronel (31) was the first to recognize the VAM/AM fungi as Endogone species. Thaxter (40) described all the species known at that time. Mosse (27) was the first to demonstrate experimentally that Endogone species could produce vesicles and arbuscules. She was able to produce typical arbuscular mycorrhizae using Endogone species as inoculum. Recently Berch (7), Hall (15), Schenck and Perez (35) and Trappe and Schenck (41) have compiled additional information to the taxonomic knowledge. Mosse and Bowen (28) have worked on cytoplasamic structures of VAM/AM fungi.
Gerdemann and Trappe (13) reported different types of spore walls. Hall (14) listed 115 characteristics that can be used for separating V AM/ AM fungal species. Walker (42) reported standardized terminology to describe the wall layers. Different monographs have been used to illustrate the interrelation of these spore walls. Morton (22,23,24) emphasized that mOlphological characters are important in identifYing endomycorrhizal fungi besides elaborating their evolutionary relationships. Almeida (4) has proposed critical evaluation of V AM/ AM fungi and discussed scientific names in Endogonales. Berch (7) has detailed out the taxonomy, specificity, fossil record and phylogeny of endogonaceae. Bonfante - Fasolo (8) proposed that anatomy and morphology ofVAM/ AM fungi are important.
Hall (15) has made valid contributions in understanding the taxonomy of AM fungi based on spore morphology. Abbott and Robson (1) provided evidence that morphological, architectural and histochemical properties ofintraradical hyphae may be taxonomically informative at the genus level and above. Morton (24) used arbuscules as the only character that unites Glomineae and Gigasporineae under Glomales. Brundrett and Kendrick
264 Techniques in Mycorrhizal Studies
(9) have revealed critical differences in arbuscuIar structures ofGigaspora and Glomus. Morton and Benny (25) have reported that structural differences of spores and sub-cellular structures ofV AMI AM fungi form important criteria in the identification and classification of V AMI AM fungi.
Various types of walls (42) form a stable and reliable criterion in the identification and classification offungi. Morton and Bentivenga (26) have emphasized that the level of diversity in endomycorrhizal fungi with reference to vegetative characters, morphological characters, spore formation etc.
have an important role in defining taxonomic and non taxonomic groups.
It is concluded by them that any taxonomic character that relate to mycorrhizal functions will come only from comparative studies involving organisms from shared habitats rather than those having shared spore morphologies.
Arbuscu1ar mycorrhizal fungi have been placed in zygomycotina as the cell walls ofthis group of fungi possess chitin, coenocytic hyphae, aplanospore or chlamydospore formation and nature of nuclei being similar to zygomycetous fungi. Morton and Benney (25) have proposed a classification ofV AMI AM fungi based on morphological characters, spore ontogeny and germination. Table 2 presents a comparative account of the old and new classification ofVAMI AM fungi (29).
At present there are about 164 species of V AMI AM fungi reported from allover the world. The following is the dichotomous key for the seggregation ofVAMI AM fungal genera.
TABLE 2.
A comparative account of the old and new classification of endomycorrhizal fungi (29)
Old classification New classification
Class Order Family Genera
Zygomycetes Endogonales Endogonaceae
Acaulospora, Endogone Gigaspora, Glaziella, Glomus, Modicella Sclerocystis
Class Zygomycetes
Order Glomales
Type family: Glomaceae Suborder Glomineae Family
Genera Family Genera Suborder Family Genera
Glomaceae
Glomus, Sclerocystis Acaulosporaceae
Acaulospora, Entrophospora Gigasporineae
Gigasporaceae
Gigaspora, Scutellospora
C. Manoharachary, l.K. Kunwar and K.G. Mukerji 265
Key to genera in Glomales
A "AzygosporeslCblamydospores" produced on the apex of a sporogenous cell of a fertile hypha; auxiliary cell fonned ... ... ... Gigasporineae With a single family ... Gigasporaceae (B) B. Genn tubes produced directly through the spore wall; inner flexible wall group absent, auxiliary cells finely papillate or echinulate ... . ... Gigaspora BB. Genn tubes funned from gennination shield; inner flexible wall group
present; auxiliaIy cells knobby, broadly papillate or smooth ... Scutellospora AA Intercalary, lateral or terminal chlamydospores fonned singly and/or in
sporocarps; auxiliary cells not produced ... Glomineae (C)
C. Chlamydospores borne on fertile hyphae ... Glomaceae ( D ) D. Fruiting body, the sporocarp composed of spores with lateral walls
adherent to one another; connecting hyphae embedded in a central hypha! plexus; chlamydospores in a single layer except at the base;
base composed of sterile hyphae ... Sclerocystis
D.D. Spores produced singly or in loose to tight aggregates in soil, less commonly in roots; sporocarp not fonned as in D above
... Glomus C. C. Cblamydospores fonned from or within the "neck" of a sporogenous saccule
... Acaulosporaceae ( E)
E Spores arise laterally from the neck of a sporogenous saccule
... Acaulospora E.E. Spores fonned in the neck of the sporogenous saccule
... Entrophospora
Table 3 lists all the V AMI AM fungal species reported from India.
The association of spores and sporocarps ofVAMlAM fungi with the plant tissues is presented in Table-4.
TABLE 3.
Vesicular-Arbuscular mycorrhizal fungi reported from India
1. Acaulospora appendiculata 2. A. bireticulata
3. A. delicata 4. A. denticulata 5. A. dilata 6. A. elegans.
7. A.foveata 8. A. lacunosa 9. A.laevis 10. A. longula 11. A. mellea 12. A. morrowiae 13. A. myriocarpa
266 Techniques in Mycorrhizal Studies 14. A. nicolsonii
15. A. rehmii 16. A. rugosa 17. A. scrobiculata 18. A. spinosa 19. A. sporocarpia 20. A. tuberculata 21. Endogone aggregata 22. E. incrassata
23. Entrophospora colombiana 24. E. infrequens
25. E. schenckii 26. Gigaspora albida 27. G. candida 28. G. coralloidea 29. G. decipiens 30. G. gigantea 31. G. margarita 32. G. ram is porum 33. G. rosea 34. G. tuberculata 35. Glomus aggregatum 36. G. albidum
37. G. ambisporum 38. G. botryoides 39. G. caledonium 40. G. canadense 41. G. citricola 42. G. claroideum 43. G. clarum 44. G. clavispoum
= Sclerocystis
= S. microcarpus 45. G. constrictum 46. G. convolutum 47. G. delhiense 48. G. deserticola 49. G. diaphanum 50. G. dimorphicum 51. G. epigaeum 52. G. etunicatum 53. G.fasciculatum 54. G.fecundisporum 55. G. j1avisporum 56. G. formosanum 57. G.fragalis 58. G.fuegianum 59. G.fulvum 60. G. geosporum 61. G. globiferum 62. C. halonatum 63. G. heterosporum 64. G. hoi
65. G. intraradices
C. Manoharachary, 1.K. Kunwar and K.G. Mukerji 267 66. G. invermaium
67. G. lacteum 68. G. leptotichum 69. G. macrocarpum 70. G. maculosum 71. G. magnicaule 72. G. manihotis 73. G. melanosporum 74. G. microaggregatum 75. G. microcarpum 76. G. monos porum 77. G. mosseae 78. G. multicaule 79. G. multisubstensum 80. G. occultum 81. G. pallidum 82. G. pubescens 83. G. pulvinatum 84. G. pustulatum 85. G. radiatum 86. G. reticulatum 87. G. rubiforme
= Sclerocystis rubriformis
=S. indicus
= S. pachycaulis 88. G. scintil/ians 89. G. sinuosum
= Sclerocystis sinuosa
= S. pakistanica 90. G. tenebrossum 91. G. tenue 92. G. tortuosum 93. G. tubiforme 94. G. versiforme 95. G. warcupii
96. Sclerocystis coremioides
= S. coccogena
=S. dussii
97. Scutellospora alborosea 98. S. arinicola
99. S. aurigloba 100. S. calospora
= G. calospora 10 1. S. dipurpurascens 102. S. erythropa 103. S.julgida 104. S. gilmorei 105. S. gregaria 106. S. heterogama 107. S. nigra 108. S. pellucida 109. s. percica 110. S. reticulata 111. S. savamicola
268 Techniques in Mycorrhizal Studies
TABLE 4.
Association of spores and sporocarps of vesicular - arbuscular (VAMP) and arbuscular mycorrhizal fungi (AMF) with plant tissues
Spores I Sporocarps Aggregation of spores
Sporocarp Spores
Obligately sporocarpic Loose sporocarps
Spores only