Symbiotic Fungi: Principles and Practice (Soil Biology)

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Chapter 11 Isolation of Metabolically Active Arbuscules and Intraradical Hyphae from Mycorrhizal Roots Zakaria M. Solaiman 11.1 Introduction Arbuscular mycorrhizal (AM) fungi are obligate symbionts and colonize more than 80% terrestrial plant roots and fungi belonging to the Glomales. The mutualistic nutrient exchange in this symbiosis is characterized by the transfer of phosphorus from fungi to host plant in exchange for carbon compounds derived from photosynthesis (Smith and Read 1997; Solaiman and Saito 1997). It is now well-reported that phosphate present in soil is taken up into the extraradical hyphae by a phosphate transporter, subsequently condensed into polyphosphate, and translocated by protoplasmic streaming into the intraradical hyphae (Saito 2000). The arbuscule is supposed to be the main site for the nutrient exchange. Alkaline phosphatase activity has been expressed in arbuscules, and it is suggested that this relates to the efficiency of phosphorous uptake (Tisserant et al. 1992) and sugar metabolism (Ezawa et al. 1999; Solaiman and Saito 1997) of AM. However, the carbon and phosphorus metabolism involved in the nutrient exchange at the arbuscular interface remain to be investigated. The isolation of arbuscules from the host tissue and subsequent examination of biochemical activities are required to clarify the mechanism occurring in nutrient exchange in this symbiotic system. The isolation of arbuscules from host tissue is a powerful process, especially because this endo-symbiont cannot be independently cultured in vitro. However, it is not easy to isolate the arbuscules from host tissue because of the complex penetration of hyphae into cortical cells. Intraradical Z.M. Solaiman (*) Soil Science and Plant Nutrition, School of Earth and Environment, M087, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, WA 6009, Australia e‐mail: solaiman@cyllene.uwa.edu.au A. Varma and A.C. Kharkwal (eds.), Symbiotic Fungi, Soil Biology 18, 189 DOI: 10.1007/978‐3‐540‐95894‐9_11, # Springer‐Verlag Berlin Heidelberg 2009
190 Z.M. Solaiman hyphae have been isolated from the host by enzymic digestion of root tissue with cellulase and pectinase, followed by hand-sorting of the hyphae under a dissecting microscope (Capaccio and Callow 1982; Smith et al. 1985; Hepper et al. 1986). But this method was laborious, and enzymic digestion of the mycorrhizal roots for more than 12 h reduced the metabolic activity of the hyphae, as evaluated by histochemical staining for succinate dehydrogenase (SDH) activity (McGee and Smith 1990). Later on, Saito (1995) reported a method for isolation of metabolically active intraradical hyphae from AM onion roots after 1–2 h of enzymic digestion. The metabolic activity of the isolated hyphae, based upon the evaluation of SDH staining, was not significantly affected by this enzymic separation procedure. In addition, a mass of intraradical hyphae which are almost free from plant debris can be collected after Percoll gradient centrifugation. But this method is still laborious, and 2–3 h are needed to complete the isolation of the intraradical hyphae or arbuscules. A simple and rapid method is now available for the isolation of arbuscules (Senoo et al. 2007). But it needs highly colonized plant roots from which we can isolate arbuscules without intensive labor. We now have an increased arbuscule-forming mutant har1 (previously known as Ljsym78) ofLotus japonicus L. (Nishimura et al. 2002; Solaiman et al. 2000); this has shown increased colonization by arbuscules on its roots compared to the wildtype ‘Gifu’. Most of the arbuscules on the mutant root were SDH-active, welldeveloped and tough in morphology. The isolation procedure of arbuscules from the mutant roots, and the quantity and quality of the isolated arbuscule fraction have been described elsewhere (Senoo et al. 2007). 11.2 Isolation 11.2.1 Isolation of Arbuscules and Intraradical Hyphae with Enzymatic Digestion 11.2.1.1 Materials Waring blender, microscope, slides, reciprocal shaker, centrifuge machine, tubes. 11.2.1.2 Chemicals (a) Enzymic digestion solution Mix 1% cellulase, 0.2% pectolase, 0.1% bobin serum albumin, 1 mM dithiothreitol (DTT), 0.01 M Mesh-NaOH buffer of pH 5.5, 0.3 M mannitol
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Soil Biology Volume 18 Series Edito
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Ajit Varma l Amit C. Kharkwal Edito
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Foreword So old, so new... More tha
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Foreword vii Little AE, Robinson CJ
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x Preface work in this challenging
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xii Contents 9 Role of Root Exudate
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Contributors L.K. Abbott School of
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Contributors xvii Falko Feldmann Ju
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Contributors xix K. Nara Asian Natu
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Contributors xxi Marc St-Arnaud Ins
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2 A. Das and A. Varma Fig. 1.1 Type
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4 A. Das and A. Varma the scientifi
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6 A. Das and A. Varma 1.3.2 Symbiot
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8 A. Das and A. Varma all the root
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10 A. Das and A. Varma 1. Such poly
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12 A. Das and A. Varma 1.3.3.9 Nitr
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14 A. Das and A. Varma about 1-2 mm
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16 A. Das and A. Varma fixed nitrog
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18 A. Das and A. Varma Mycorrhizae
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20 A. Das and A. Varma Fig. 1.6 Dia
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22 A. Das and A. Varma a b Root hai
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24 A. Das and A. Varma intracellula
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26 A. Das and A. Varma Becker A, Ni
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28 A. Das and A. Varma Trappe JM, B
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30 A. Jumpponen were once active in
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32 A. Jumpponen GA, USA) to avoid d
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34 A. Jumpponen 2.2.3.6 Cloning, Se
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36 A. Jumpponen Table 2.1 Fungi det
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38 A. Jumpponen of the community st
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40 A. Jumpponen Girvan MS, Bullimor
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42 I.A.F. Djuuna et al. 1991). Crop
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44 I.A.F. Djuuna et al. 3.2.2 Indir
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46 I.A.F. Djuuna et al. Mycorrhiza
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48 I.A.F. Djuuna et al. Boomsma CR,
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50 I.A.F. Djuuna et al. Saito M (19
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52 M. Giovannetti et al. evidenced
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54 M. Giovannetti et al. a c e Ster
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56 M. Giovannetti et al. 4.2.4 Rema
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58 M. Giovannetti et al. a b c Fig.
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60 M. Giovannetti et al. to 4.1 mm
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62 M. Giovannetti et al. The detect
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64 M. Giovannetti et al. Jones MD,
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66 A. Gobert and C. Plassard NO3 fl
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68 A. Gobert and C. Plassard After
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70 A. Gobert and C. Plassard with k
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72 A. Gobert and C. Plassard 10 9 2
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74 A. Gobert and C. Plassard Fig. 5
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76 A. Gobert and C. Plassard connec
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78 A. Gobert and C. Plassard any ti
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80 A. Gobert and C. Plassard Net fl
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82 A. Gobert and C. Plassard a Rati
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84 A. Gobert and C. Plassard - upta
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86 A. Gobert and C. Plassard Table
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88 A. Gobert and C. Plassard Newman
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90 I.M. van Aarle as mycorrhizal hy
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92 I.M. van Aarle a wash buffer. Th
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94 I.M. van Aarle l Usually 20-50 m
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96 I.M. van Aarle Fig. 6.1 Extramat
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98 I.M. van Aarle A disadvantage of
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Chapter 7 In Vitro Compartmented Sy
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7 In Vitro Compartmented Systems to
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Chapter 8 Use of the Autofluorescen
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8 Use of the Autofluorescence Prope
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242 K. Vogel-Mikusˇ et al. Frey B,
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244 E. Dumas-Gaudot et al. TEMED N,
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246 E. Dumas-Gaudot et al. system i
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248 E. Dumas-Gaudot et al. Bromophe
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250 E. Dumas-Gaudot et al. taken to
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252 E. Dumas-Gaudot et al. Note 6:
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254 E. Dumas-Gaudot et al. by Bradf
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256 E. Dumas-Gaudot et al. 3. Take
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258 E. Dumas-Gaudot et al. solubili
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260 E. Dumas-Gaudot et al. Table 15
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262 E. Dumas-Gaudot et al. Table 15
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264 E. Dumas-Gaudot et al. Followin
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266 E. Dumas-Gaudot et al. Once hom
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268 E. Dumas-Gaudot et al. to both
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270 E. Dumas-Gaudot et al. were the
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272 E. Dumas-Gaudot et al. Dumas-Ga
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274 E. Dumas-Gaudot et al. Valot B,
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276 P.A. Olsson Fig. 16.1 Schematic
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278 P.A. Olsson Furthermore, C tran
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280 P.A. Olsson in AM fungi and a h
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282 P.A. Olsson (Graham et al. 1995
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284 P.A. Olsson Nakano A, Takahashi
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286 X.H. He et al. In many cases, N
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288 X.H. He et al. Table 17.1 Exper
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290 X.H. He et al. 17.3.1.1 Comment
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Chapter 18 Analyses of Ecophysiolog
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18 Analyses of Ecophysiological Tra
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308 I. Brito et al. fungi, little i
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310 I. Brito et al. 60% of moisture
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312 I. Brito et al. Thompson 1990;
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314 I. Brito et al. 19.8 Crop Rotat
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316 I. Brito et al. References Abbo
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318 I. Brito et al. Smith SE, Read
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320 F. Feldmann et al. inoculum of
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322 F. Feldmann et al. Table 20.1 B
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324 F. Feldmann et al. transferred
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326 F. Feldmann et al. 20.3.3 The A
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328 F. Feldmann et al. Inoculum is
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330 F. Feldmann et al. Fig. 20.5 Pr
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332 F. Feldmann et al. value for th
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334 F. Feldmann et al. of abiotic a
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336 F. Feldmann et al. Lackie SM, B
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338 M. Vestberg and A.C. Cassells b
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340 M. Vestberg and A.C. Cassells M
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342 M. Vestberg and A.C. Cassells a
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344 M. Vestberg and A.C. Cassells T
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348 M. Vestberg and A.C. Cassells M
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350 M. Vestberg and A.C. Cassells 2
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352 M. Vestberg and A.C. Cassells 2
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354 M. Vestberg and A.C. Cassells C
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356 M. Vestberg and A.C. Cassells G
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358 M. Vestberg and A.C. Cassells P
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360 M. Vestberg and A.C. Cassells V
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362 A. Baldi et al. the capabilitie
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364 A. Baldi et al. 22.2.3 Initiati
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366 A. Baldi et al. 2. Place inocul
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368 A. Baldi et al. 22.5 Analysis 2
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370 A. Baldi et al. 22.5.4 Phenylal
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372 A. Baldi et al. Nicholas JB, Jo
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374 A. Baldi et al. Among these, fu
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376 A. Baldi et al. 3. Place one ex
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378 A. Baldi et al. 23.4 Analysis F
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380 A. Baldi et al. Chattopadhyay S
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382 A. Sirrenberg et al. physical c
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384 A. Sirrenberg et al. Fig. 24.1
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390 A. Sirrenberg et al. in Fig. 24
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392 A. Sirrenberg et al. 24.5.2 Tru
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394 K. Haselwandter and G. Winkelma
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404 E. Mohammadi Goltapeh et al. Fi
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406 E. Mohammadi Goltapeh et al. Th
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408 E. Mohammadi Goltapeh et al. 26
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410 E. Mohammadi Goltapeh et al. co
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412 E. Mohammadi Goltapeh et al. Tw
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414 E. Mohammadi Goltapeh et al. Co
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416 E. Mohammadi Goltapeh et al. qu
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418 E. Mohammadi Goltapeh et al. 26
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420 E. Mohammadi Goltapeh et al. Ch
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Index A A. bisporus var. burnettii,
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Index 425 Dark septate root endophy
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Index 427 Leghemoglobin, 11 Lentinu
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Index 429 Proteomics, 244 Protoplas
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Symbiotic Fungi: Principles and Practice (Soil Biology)

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