Indian Journal of Experimental Biology Vol. 38, April 2000, pp. 396-398

X-ray microanalysis of elements of VA mycorrhizal and non-mycorrhizal pedicellatum roots

C Ramesh , P Chellappan & A Mahadevan* Centre for Advanced Studies in Botany, University of Madras, Guindy Campus,Chennai 600 025. Indi a

Received 23 September 1999; revised 20 January 2000

Chemical composition of inter and intracellul ar hyphae was elucid ated in Pennisetum pedicellatwn roots colonized by Gigaspora marga rita by employing energy di spersi ve X-ray mi croanalysis. Maximum amount uf phosphate was estimated · in pol yphosphate gran ul es present in the roots. Amount of phosphate was low in fungal hyphae, arbuscular trunks and degenerated arbu scul es. El ements S, K and Ca were present in th e hyph ae, arbuscul ar trunk and degenerated arbuscules.

Vesicular arbuscular mycorrhizal (VAM) fun ga l on some of the cel lul ar component s has been association or arbuscular mycorrhiza (AM) is an reported . integral part of almost all vascular rootsl.2. Seeds of P. pedicellatum were sterilized in VAM fungi are of particular interest in tropics mercuric chloride (0.2%) and sown in earthen pots because they are found associated with a large ( 16x l0x l0 em) contai nin g sterilized so il , inocu lated number of agri cultural crops. Species of V AM fun gi with Gi. margarita (SO spores). After 15 days of seed either directly or indirectly increase plant growth by gerrnination, roots were collected and screened for improvi ng soil conditi ons. Direct benefits are usuall y AM fungal infection. Pl ants were sc reened at 15 days related to enh ancement of ph osphate uptake by the interval till maturity. Infected roots were washed in pl ants, but in some so il s, enhanced uptake of zinc and tap water and fixed in 2.5% of glutaraldehyde in 3 copper is known 0.0 l M of phosphate bu ffer (pH 7 .2) at 4 oc for 4 hr. In recent years, energy di spersive ana lysis X-ray Roots were th oroughl y was hed in 0.0 1 M ph osphate (EDAX) is used to elucidate the chem ical buffer (pH 7.2) twi ce and post fixed in 2% of os mi um compositi on of fun gi. Chemical composition .of inter tetroxide (Os0 4). Samples were dehydrated through and intracellui:J.r hyphae and ve icle has not been we ll acetone seri es, infiltrated wi th epoxy resin overni ght established but it is known that they accumulate lipid and po lymeri zed at 70°C for 24 hr. Sections (90- droplets during earl y developing stages and degene­ 4 5 140 nm) were cut with a glass kn ife mounted on 300 rating hyph ae are fill ed with lipid , P, Zn an d S . mesh gri ds and used fo r energy dispersive X-ray Occurrence of polyphosphate is confirmed by . I . to 6 mtc roa na ysts . X-ray mi croanalysis . Electron dense bodi es contain hi gh concentration of P and Ca. Highly vacuolated Energy dispersive X-ray mi croanalys is was intercellular hyphae, arbu scul es and vesicles contain performed in EDAX 700 series interface with a Data large number of polyphosphate gran ules. The General NOV A-2 compute r and a Texas In strument Silnent 700 ASR. EDAX was in terfaced with a granules are present in compl etel y co llapsed 7 Philips CM 12 TEM with a high til t (80) goniometer arbuscul es . Arbuscul es of Glomus aggregatum stage. Under STEM mode, secti ons for analyses were contain "large amount of P. Elements Mg, S, Ca and K ca 130 nm thick. Analys is time was 80 sec at 8 to are present in both mycorrhizal and non-mycorrhizal roots with margin al increase over non-mycorrhi za l 16 KV with 18 tilt I spot size of I ~un and emi ss ion 8 of 25 ~A . Data were co ll ected from 9 channel s with sugarcane roots . Ves icles contain hi gh concentration of Na, Ca, Cl , B and Si, but are not detected in roots of 12600 as the central channel and back ground was 9 white . stripped in all instances. In the present communication, effect of infection Results of X-ray microanalyse of P. pedicellatum of Pennisetum pedicellaturn by Gigaspora margarita roots co loni zed by Ci. margarita are presented in Tab le I. Zones selected in myco rrhizal colonized *Correspondent author roots included intercellul ar hyp hae, intracellular NOTES 397

Table !-Compari son of energy intensities of nutrient elements in Pennisetum pedicellatum and in different components of Gi. margarita

Component Count per second (CPS ) Mg p s K Ca Resin (Cont rol) 01.09 01.09 00.80 00.72 00.35 Host cells 02 .18 01.21 01.74 02.79 0 1.72 without infecti on (Control) Host cell s wi th infection 03 .19 13.02 22.02 07.54 12.19 Hyphae 02.87 17.08 03.87 05.99 0.41 Arbuscular trunks 04.36 12.35 06.27 14.34 11.28 Arbuscules 03 .87 23 .R5 37.70 34.78 20.26 Degenerating arbusculcs 02.14 09 .2 1 02.18 03. 19 07.29 Polyphosphate granules 00.66 49.64 05 .13 14.12 2 1.62 hyphae, arbuscular trunk, mature arbuscules, Presence of P in arbuscules has been demonstrated degenerating arbuscules and polyphosphate granules. in Allium cepa colonized by G. fasciculatum using X­ To compare the results, epoxy resin and randomly ray microanalyses12. Metachromatic granules which selected non-mycorrhizal cortical cell s were chosen accumulate P are identified in both ecto- and endo­ and EDAX spectra were taken as control. To find out mycorrhizae6·13-16. In mycorrhizzal fungi, high amount the fluxes , both count per second (CPS) value and of Pis stored in mycelium as orthophosphate. apparent relative atomic per cent of a particular Capacity to accumulate P as polyphosphate varied mineral in different components were made. X-ray among YAM fungi . Polyphosphate granules found in microanalyses of thi ck sections ( 120 to 150 nm) of hyphae and arbuscules of Gi. margarita colonized P. P. pedicellatum colonized by Gi. margarita roots pedicellaltum roots showed maximum amount of P. were destained, since lead and uranyl acetate used for Presence of P was an indication of its transfer from P. staining might interfere with EDAX analyses . The rich arbuscule to host cell cytoplasm. Vacuole in destained sections could reduce the possibility of arbuscule containing polyphosphate granule showed di spl acement of ions and allowed X-ray maximum P flux indicating that P was stored in the microanalyses of elemental composition within the form of metac hromatic granule (polyphosphate components of Gi. margarita. granules) in arbuscule. Elements present in mycorrhizal cortical cells with Ca flux rate exhibited variation. Occurrence of and · without arbuscules have been presented in calcium was high in polyphosphate granules. This Table I . Spectra from control res in revealed no observation was in contrast to the finding that Ca is measurable concentration of elements. not present in Saccharum officinarum roots colonized X-ray microanalyses of phosphate granules showed 8 17 by G. aggregatum • However, Strullu's finding higher fluxes th an control. Per cent (5 1%) of P in lends credence to our observation. X-ray analyses of hyphae, arbuscules and polyphosphate granules also roots reveal that calcium is a common constituent of increased over control. CPS was also more in 7 polyphosphate granules than control. Flux rate of P in most granules . fungal components ranged from 9.21 to 49.64 which Flux rate of Mg was low in non-mycorrhizal was higher than control. The control had the flux rate ti ssues, hyphae, arbuscules, infected host cells and 1.09 and host cells without infection had CPS 1.2 1. polyphosphate granules. Flux rate of S also differed. X-ray microanalyses of phosphate granules revealed Degenerating arbuscules recorded CPS value of 38, that Ca was a common secondary constituent of most while cortical cells without infection had CPS value granules. Other elements such as S, Mn and K were of 1.74. In other fungal components, CPS values also detected as components of the granules. ranged from 2.18 to 22. Flux rate of K exhibited Polyphosphate granules were present in small much variation. Maximum CPS was 34.78 in arbuscular branches. P rich inclusions are referred to arbuscules. According to Schoknecht and Hattingh12· as volutin granules, metachromatic granules and K occurs in higher concentration in control cells of A. polyphosphate granules 11 . Fungal component devoid cepa than in colonized by G. fasciculatum, however of phosphate granules was the degenerated arbuscule. our observation was in contrast to these findings. 398 INDIAN J EXP BIOL, APRIL 2000

In the present study, mycorrhizal fungus 8 Bojan C R, Studies on SO II'Ie aspects of vesicular arbuscular accumulated substantial amount of P, S, K and Ca in mycorrhizal fungi on sugarcane (Sacchamm officinarum), Ph,D. Thesis, University of Madras, Madras, 1995, hyphae, arbusculars trunk, arbuscul es and they are 9 Walker G & Powell C L, Vesicul ar arbuscul ar mycorrhiza in made available to host ce ll s during degeneration. whi te clover: a scanning electron microsco pe and X-ray The author (CR) is grateful to CSIR for awarding microanalytical st ud y. N Z J Bot, 17 ( 1979) 55, I 0 Hayat M A, Basic techniques for transmission electron fellowship. Thanks are also due to DBT and Coal microscopy, (Academic Press, London) 1986, Ltd for financial assistance. We th ank RSIC, IlT II Strullu D G, Golll-ret J P, Garrec J P & Nancy A, Chennai for providing EDAX facility. Ultrastructure and electron-probe mi cro-analysis of th e metachromati c vacuolar granul es occ urring in Taxus References mycorrhizas. New Phytol, 87 ( 1981) 537. 12 Schoknecht J D & Hattingh M J, X-ray microanalysis of I Mahadevan A, Raman & Natarajan K, Mycorrhizae for elements in cells of VA mycorrhizal and non-myco rrhizal green As ia, Proceedings I Asian Conferr'nce Mycorrhizae oni on , Mycologia, 68( 1976) 297. (University of Madras, Mad ras) 1988, 35 1 13 Ashford A E, Ling-Lee M & Chi Ivers G A, Polyphosphate in 2 Mukerji KG, in Advances in Botany, edited by K G Mukerji , eucalypt myco rrhizas- Cyrochemical demonstration, New 8 Mathur, B P Chamola & P Chitralekha (As hi sh, New Phytol,74 ( 1975) 447, Delhi ) 1996, 211. 14 Ashford A E, Peterson R L, Dwarte D & Chilvers G A, 3 Raman N & Mahadevan A, Mycorrhizal research-a priority Po lyphosphate granu les in eucalypt mycorrhizas, in agricu lture. in Concepts in mycorrhizal research, ed ited determination by energy dispersive X-ray microanalyses, by KG Mukerji (Kluwer, Dordrecht) 1996, 41. Can J Bot 64 ( 1986)70 4 Cooper K M & Lose! D M, Lipid physiology of ves icular 15 Scannerini S & Bonfante-Fasolo P, Comparative arbuscular mycorrhiza. New Phytol, 80 ( 1978) 145. ultrastructural analyses of mycorrhiza associati ons. Can 1 5 Cooper K M & Tinker P B, Translocatio n and transfer of Bot, 61(1983)917. nutrients in vesicu lar arbuscular mycorrhiza. II . Uptake and 16 Strullu D G, Grellier 13 , Garrec J P. Mac Cready C C & translocation of phosphorus, zinc and sulphur. New Phytol, Harley J L, An experimental study of the effect of 81 (1978) 43. monovalent and divalent cations on phosphate abso rpti on by 6 Straker C J & Mitchell D T, The characterizat ion and beech mycorrhizas, New Phytol, I03 ( 1986) 403. estimation of polyphosphates in endomycorrhi za of the 17 Stru llu D G, Chamel A, Eloy J F & Gourret J P. Eri caceae. Nell' Phytol, 99( 1985) 431 . Ultrastructure and analyses by laser probe mass 7 White J A & Brown M F. Ul trastructure·and X-ray analyses spectrography of the mineral composition of the vesicles of of phosphorous granules in a vesicular arbu scu lar Trifolium pratense endomycorrhizas. New Phytol, 94 (1983) mycorrhizal fungus. Can J Bot, 57 ( 1979) 2812. 8 1.