In vitro GROWTH OF ECTOMYCORRHIZAL FUNGI ASSOCIATED

Transcription

1 Scientific rticle Rev. Fitotec. Mex. Vol. 4 (4): , 217 In vitro GROWTH OF ECTOMYCORRHIZAL FUNGI ASSOCIATED WITH Pinus rdit PLANTATIONS IN CHILE CRECIMIENTO in vitro DE HONGOS ECTOMICORRÍCICOS ASOCIADOS CON PLANTACIONES DE Pinus rdit EN CHILE José-Leonrdo Grcí-Rodríguez 1, 4 *, Jesús Pérez-Moreno 2, Drcy Ríos-Lel 1, Ptrici Sez- Delgdo 1, Cristin Atl-Binchi 3, Mnuel Sánchez-Olte 1 nd Guillermo Pereir-Cncino 1, 4 1 Progrm de Postgrdo en Ciencis Forestles, Fcultd de Ciencis Forestles, Universidd de Concepción. Victori 631, Brrio Universitrio, Concepción Región del Bioío, Chile. Tel. (56-41) Colegio de Postgrdudos, Postgrdo en Edfologí, Lortorio de Microiologí, Colegio de Postgrdudos, Cmpus Montecillo. Crreter México-Texcoco km , Montecillo, Texcoco, Estdo de México Tel. (595) 9522 Ext Lortorio de Antomí y Ecologí Funcionl de Plnts, Instituto de Biologí, Fcultd de Ciencis, Pontifici Universidd Ctólic de Vlpríso, Cmpus Curum. Avenid Universidd 33, Vlpríso, Chile. 4 Lortorio de Biotecnologí de Hongos. Cmpus Los Ángeles, Universidd de Concepción. Jun Antonio Colom 21, Los Ángeles, Chile. Teléfono: (56-43) *Corresponding utor (jgrcir@udec.cl) SUMMARY A comprtive study of in vitro growth of three species of ectomycorrhizl fungi (ECMF) (Rhizopogon luteolus, Suillus grnultus nd Suillus luteus) ws performed. Fungl mteril ws collected in dult Pinus rdit plnttions. Isoltion nd purifiction of the strins were performed on potto-dextrosegr medium nd the evlution of the rdil growth rte nd the increse in mycelil iomss, under different culture conditions, ws performed on the Modified Melin Norkrns growth medium. The effects of temperture (24, 28 nd 32 ºC) nd ph (4.8, 5.3, 5.8, 6.3 nd 6.8) of the growth medium were tested for the three fungl species in two independent ssys. The results indicte tht the temperture hd significnt effect on the rdil growth rte (RG) nd mycelil iomss increse (MB) in ll of the evluted fungl species. The highest RGR nd MBI were recorded in R. luteolus, nd the lowest vlues for these vriles were registered in S. luteus. Rhizopogon luteolus hd the highest sensitivity to ph chnges. Menwhile, there ws no pttern in S. grnultus nd S. luteus growth response under different ph conditions. When cultivted in vitro, the three studied species of ECMF presented dpttion, exponentil, declining nd sttionry growth phses. The in vitro growth responses recorded in the present study showed the gret potentil of R. luteolus to e used in future progrms using mycorrhizl inocultion in the production of Pinus rdit trees in nurseries in Chile. Index words: Rhizopogon, Suillus, iomss, pure culture, rdil growth rte. RESUMEN Se hizo un estudio comprtivo del crecimiento in vitro de tres especies de hongos ectomicorrícicos (ECMF) (Rhizopogon luteolus, Suillus grnulus y Suillus luteus). El islmiento y purificción de ls ceps se hizo en medio de cultivo pp-dextros-gr (PDA), y l evlución de l velocidd de crecimiento rdil (RG) y del incremento en ioms micelil (MB), jo diferentes condiciones de cultivo, se hizo en el medio de crecimiento Melin Norkrns Modificdo (MMN). Los efectos de l tempertur (24, 28 y 32 ºC) y ph (4,8, 5,3, 5,8, 6,3 y 6,8) del medio de crecimiento fueron prodos pr ls tres especies fúngics en dos ensyos independientes. Los resultdos indicn que l tempertur tuvo un efecto significtivo en l ts de crecimiento rdil (RG) y en el ncremento de ioms micelil (MB), en tods ls especies fúngics evluds. Los más ltos vlores de GR y MB fueron registrdos en R. luteolus, y los vlores más jos fueron registrdos en S. luteus. Rhizopogon luteolus presentó l myor sensiilidd los cmios de ph del medio de cultivo. En cmio, no existió un ptrón en l respuest de crecimiento de S. grnultus y S. luteus jo diferentes condiciones de ph. Cundo se cultivron in vitro, ls tres especies de ECMF estudids presentron en su crecimiento micelil ls fses de dptción, crecimiento exponencil, declinción y estcionri. Ls respuests de crecimiento in vitro registrds en el presente estudio mostrron el grn potencil de R. luteolus pr ser utilizdo en futuros progrms de micorrizción controld pr l producción en vivero de ároles de Pinus rdit en Chile. Plrs clve: Rhizopogon, Suillus, ioms, cultivo puro, ts de crecimiento rdil. INTRODUCTION Around 95 % of higher plnts nturlly estlish mutulistic symiotic reltionships with mycorrhizl fungi whose externl mycelium cn explore up to 1 times more soil thn the roots (Brundrett et l., 1996; Honrui et l., 1992). Therefore, this symiosis improves the nutrition of the ssocited plnts y moilizing nutrients nd wter (Honrui et l., 1992; Quoreshi, 28; Skinner nd Bowen, 1974). Qulity of plnts successfully mycorrhized y ectomycorrhizl fungi (ECMF) is improved in terms of vigor, drought tolernce, increse in nutrient uptke nd resistnce to post-trnsplnt stress in the field (Bücking et l., 212; Dulmer et l., 214). The inocultion with ECMF is recommended prctice to produce plnts of high qulity in nurseries (Díz et l., 29). The selection of the pproprite ectomycorrhizl fungl species s symionts nd their susequent mnipultion, oth in the lortory nd in the nursery, cn e key fctor for the successful estlishment of mny tree species in the field (Chávez et l., 27; Honrui et l., 1992; Mrx et l., 1991). One criteri of gret relevnce in the selection of the ECMF is the use of species which Reciido: 23 de Ferero de 217 Aceptdo: 24 de Octure de 217

2 ECTOMYCORRHIZAL MUSHROOMS FROM CHILE Rev. Fitotec. Mex. Vol. 4 (4) 217 re growing nturlly ssocited with the selected plnts in the sites of interest (Honrui et l., 1992; Pereir et l., 27). Different types of mycorrhize hve een descried depending on the structures tht they form, lthough in the cse of tree species relevnt in forestry in temperte climtes, the ectomycorrhiz is the most importnt type of mycorrhize (Hrley nd Smith, 1983; Mrks nd Kozlowski, 1973; Smith nd Red, 28). The ECMF is n importnt component of microil soil communities in orel, temperte, mediterrnen nd some tropicl forests, where they ply n importnt role in nutrient recycling processes (Allen et l., 1995). In nture, ECMF growth depends on fctors such s temperture, ph, humidity, physicl nd chemicl soil properties nd nutrient vilility, minly cron nd nitrogen sources (Bowen, 1973; Hrley nd Smith, 1983). The ECMF re usully mesophilic; the temperture directly influences their growth ecuse it ffects the enzymtic production; nd thus, cn cuse the denturtion of proteins nd other mcromolecules of the cell memrnes (Frioni, 1999). Additionlly, the soil ph ffects the microil ctivity through the soluility nd the ioniztion of the inorgnic nd orgnic compounds in the soil solution (Voroney, 27). In vitro pure culture studies contriute to the study of the ECMF in order to understnd their ehvior in different cultivtion conditions (Pirt, 1975). Growth differences of ECMF under in vitro conditions hve een considered guide for prescreening potentil fungl species (strins or ecotypes) to e used in nursery mycorrhiztion progrms. In the present study, the iomss production in different in vitro conditions ws nlyzed in order to understnd the optiml temperture nd ph conditions for the evluted ECMF (Eng et l., 23; Lzrević et l., 216; Pereir et l., 27; Sánchez et l., 21). The ojective of the present study ws to nlyze the effect of three tempertures nd five different ph levels on the in vitro growth of the species of ECMF R. luteolus, S. grnultus nd S. luteus nturlly ssocited with Pinus rdit plnttions growing t low productivity sites in Chile, ssuming tht ech strin or ecotype hs its own ecologicl optiml conditions. MATERIALS AND METHODS Fungl mteril. The ectomycorrhizl fungi (ECMF) Rhizopogon luteolus Fr., Suillus grnultus (L.) Roussel nd Suillus luteus L. Gry were hrvested in n dult plnttion of Pinus rdit D. Don growing on representtive site of lrge forest re with sndy soils of low productivity (ph 5.6; orgnic mtter: 2.39 %; N:1.3 %; P:6.7 %; K:47.9 %) in the province of Bioío, Region VIII, Chile (37º 2'42.58 ''S, 72º 17'55.15 ''W) during June to Septemer 215 (Figures 1A, B, C). The species identifiction ws performed y using mcro- nd micro-morphologicl chrcteriztion of their sporomes (Clonge, 29; Gerhrdt et l., 2; Honrui et l., 21; Lzo, 216). In lortory, under septic conditions in lminr flow cinet (ESCO, USA) the sporomes were dissected nd smll prts of the pile context locted immeditely ove the hymenium in epigeous mushrooms; nd in the centrl re of the gle in hypogel fungi, were respectively extrcted (Brundrett et l., 1996; Honrui et l., 1995; Kumr y Stynryn, 22). The tissue frgments were plced in 5 mm dimeter Petri dishes with 1 ml of potto-dextrose-gr medium (HiMedi, Indi) with ph djusted to 5.8. For ech species, five strins were collected from the collection site, nd from them, the strin with the est initil growth ws studied in detil. The pltes were incuted t 24 ± 1 ºC (BINDER incutor, Germny) until n ctive growth of the myceli (stock cultures) ws otined. Lter, grmycelil discs of 5 mm in dimeter of the mrgin of the isolted strins (Díz et l., 29), were trnsferred to new Petri pltes with 2 ml of potto-dextrose-gr medium nd ph djusted to 5.8, in order to complete the processes of isoltion nd purifiction. These pltes were incuted t 24 ± 1 ºC for 18 d, otining pure strins of the three ECMF species of R. luteolus, S. grnultus, nd S. luteus (Figures 1A, B, C). Experimentl pproch. Two independent experiments were set up. In the first experiment, the effect of the temperture ws evluted. The three ECMF strins were cultivted t ph 5.8 nd the effect of three different tempertures ws evluted. In the second experiment, the effect of ph on mycelil growth ws evluted for the three isolted strins. The strins were cultivted t 24 ºC nd the effect of five different ph vlues ws evluted. Culture conditions tested. In the first experiment, the evluted tempertures were 24, 28 nd 32 ºC (Andrino et l., 211; Arn-Griel et l., 214; Brn et l., 215; Lzrević et l., 216). From growing ctive colonies (stock cultures), 5 mm dimeter gr-mycelil discs were trnsferred to 9 mm dimeter Petri dishes contining 2 ml of gr Modified Melin Norkrns (MMN) s culture medium, with ph djusted to 5.8. This ph vlue ws used ecuse it hs een successfully employed in the cultivtion of other ECMF. Five pltes per species nd three temperture vlues were set up (n = 45). Cultures were grown in three seprte incutors (Memmert, Germny). In the second experiment, five different ph vlues were evluted: 4.8, 5.3, 5.8, 6.3 nd 6.8 (González et l., 215; Pereir et l., 27; Sundri nd Adholey, 23; Vázquez-Grcí et l., 22). Prior to steriliztion, the ph djustment of the culture medium ws performed with PH 21 Hnn Instruments electrode y pplying HCl to cidify the medium nd KOH to sify 416

3 GARCÍA-RODRÍGUEZ et l. Rev. Fitotec. Mex. Vol. 4 (4) 217 A) B) C) RI Sg SI D) E) F) 24 ºC 28 ºC 32 ºC 24 ºC 28 ºC 32 ºC 24 ºC 28 ºC 32 ºC G) H) I) Figure 1. Sporomes of the ectomycorrhizl fungi Rhizopogon luteolus (A), Suillus grnultus (B), S. luteus (C) nd in vitro mycelil growth t three tempertures (D, E nd F) nd five ph vlues (G, H nd I) in Modified Melin Norkrns culture medium. it. In this experiment, 5 mm dimeter gr-mycelil discs were trnsferred to 9 mm dimeter Petri dishes contining 2 ml of gr MMN culture medium, with ech level of ph tested. Five pltes for ech ph vlue were set up (n = 75), which were incuted in drkness t 24 ± 1 ºC sed on the fct tht this ws the optiml temperture in terms of fungl growth in the first experiment. Growth trits. The verge rdil growth (RG) of the evluted ECMF strins ws recorded for ech of the evluted tempertures nd ph vlues. The pltes were incuted for 21 d nd the rdil growth of the colonies ws mesured every 3 d on the ck of the pltes (in four directions tht pssed through the center of the colonies) with digitl cliper (Mitutoyo, Mod. CD-6, Jpn) (Murriet-Hernández et l., 214). The non-ccumulted rdil growth (NARG) ws the verge of the growth distnce of four rdil points for ech mesurement, to which the verge growth of previous mesurements ws not dded. These dt were djusted y liner regression eqution to clculte the slope of the growth curve corresponding to the verge growth rte of ech fungl species nd expressed in mm d -1 (Sntigo-Mrtínez et l., 1995; Vázquez- Grcí et l., 22). After the culture period, the increse in mycelil iomss (MB) ws evluted for oth experiments y recovering the iomss produced in Petri dishes with vcuum filtrtion (Filtering Pump K, model SU 417

4 ECTOMYCORRHIZAL MUSHROOMS FROM CHILE Rev. Fitotec. Mex. Vol. 4 (4) , Germny). The filtering of the growth medium previously melted in microwve ws crried out y using filter pper, previously oven dried nd weighed in n nlyticl lnce (RADWAG, USA). The mycelil mss retined on the filter pper ws oven dried (Memmert, model BE-4, Germny) t 6 ºC during 48 h in order to determine the constnt dry weight (Duñeiti et l., 24; Pereir et l., 214). Sttisticl nlysis. In every experiment, completely rndomly sttisticl designs with fctoril rrngements, 3x3 in the first experiment nd 3 x 5 in the second experiment, ws used. In the first experiment the fctors were: ECMF species nd culture tempertures, giving totl of 45 experimentl units. In the second experiment, the fctors were the ECMF species nd the ph of the culture medium, giving totl of 75 experimentl units. In order to determine the effect of the studied fctors on the RGR nd MB, the tests of homogeneity of vrinces nd normlity through the Shpiro-Wilk nd Levene Test were previously performed. When the distriutionl ssumptions were not verified, Box-Cox powers for dt normliztion (Olivier nd Norerg, 21) were pplied. In oth experiments two-wy nlysis of vrince ws performed with 95 % confidence level nd ecuse the interction ws significnt, contrsts were performed y LSD Fisher (α =.5) for the nlysis of significnt differences. Tukey (P <.5) men comprison tests (Steel nd Torrie, 1989) were crried out mong tretments. Sttisticl nlysis of the dt ws performed y using the sttisticl progrm STAT- GRAPHICS. RESULTS Experiment I (temperture) Rdil growth (RG). The results showed tht the temperture hd significnt effect on the RG for the three ECMF evluted species, with negtive effect when this vrile incresed (Figures 1D, E nd F; Figure 2A). The highest RG ws otined t 24 ºC nd the lowest t 32 ºC, regrdless of the ectomycorrhizl fungi (ECMF), hving differences (P <.5) mong species. It ws oserved tht R. luteolus nd S. luteus hd greter sensitivity to the temperture chnge, expressed in the differences etween their tretments (P <.5). Rhizopogon luteolus hd the highest RG in the three evluted tempertures eing 2.49, 1.81, 1.1 mm d -1, respectively. These RG were different (P <.5) to those recorded for S. luteus nd S. grnultus in the three evluted tempertures. The lst two species did not show differences (P <.5) in RG etween them, independently of the tested tempertures. Mycelil iomss (MB). The MB of the three evluted ECMF ws lso ffected y the tested tempertures (Figure 2B). The highest (P <.5) MB ws registered t 24 ºC nd the lowest t 32 ºC for ll species. Rhizopogon luteolus showed the highest sensitivity, expressed s differences (P <.5) etween ech of the tested tempertures. Suillus grnultus nd S. luteus only showed differences (P <.5) etween extreme tempertures (24 nd 32 ºC). Rhizopogon luteolus hd the highest MB with 143.5, 11.8 nd 6.7 mg, nd S. luteus hd the lowest MB with 28.5, 17.3 nd 1.9 mg for the three evluted tempertures, respectively. At the most extreme tempertures (28 nd 32 ºC) there were greter MB differences (P <.5) in the three ECMF. Experiment II (ph) Rdil growth (RG). The ph hd different effect on the RG of the evluted fungl species, nd there ws not defined pttern (Figures 1G, H, I, nd Figure 3A). Rhizopogon luteolus reched its mximum (P <.5) RG t ph 4.8 (1.52 mm d -1 ) nd the lowest t ph 6.3 (1. mm d -1 ), with significnt differences etween ph 4.8 compred to 5.8, 6.3 nd 6.8. This species showed the highest RG in ll of the evluted ph vlues compred with the other two fungl species. In the cse of S. grnultus nd S. luteus, the RG only differed (P <.5) t ph 4.8, 5.3 nd 6.8. Mycelil iomss (MB). Rhizopogon luteolus showed only MB differences (P <.5) etween the extreme ph tretments (4.8 nd 6.8) (Figure 3B). Suillus grnultus nd S. luteus did not show pttern in terms of MB when grown in the different evluted ph vlues. The ph of 4.8 yielded higher (P <.5) MB in R. luteolus (14.76 mg) compred to S. grnultus (63.26 mg) nd S. luteus (64.74 mg). Experiments I nd II (temperture nd ph) Accumulted rdil growth (ARG) nd non-ccumulted rdil growth (NARG). The three evluted ECMF showed the four cultivtion growth phses: culture medium dpttion, exponentil, declining nd sttionry phses. In generl, this trend ws oserved in ll of the evluted tempertures nd ph vlues (Figures 4B nd 4D). The dpttion phse ws longer (pproximtely 6 d) in the experiment where the influence of temperture ws evluted compred with tht of different ph vlues (pproximtely 3 d) (Figures 4A nd 4C). Rhizopogon luteolus showed the highest ARG in oth the temperture nd ph experiments (Figures 4A nd 4C). In R. luteolus, the period of the exponentil phse of NARG ws dependent on the temperture. This phse concluded on dy 12 t 24 ºC nd on dy 9 t oth 28 ºC nd 32 ºC (Figure 4B). After this phse, the decline of NARG in 418

5 GARCÍA-RODRÍGUEZ et l. Rev. Fitotec. Mex. Vol. 4 (4) 217 Rdil growth (mm dí -1 ) A B C A A Rl Sg Sl Species B A B C A) B) C 28 C 32 C Rl Sg Sl Species Figure 2. (A) Rdil growth (mm d -1 ) nd (B) mycelil iomss (mg) of three fungl ectomycorrhizl species t three culture tempertures. Vlues re mens ± stndrd error of the men (n = 5). Brs with the sme letter, for ech species, re not different ccording to the Tukey test (P <.5). Rl = Rhizopogon luteolus; Sg = Suillus grnultus; Sl = Suillus luteus. Mycelil iomss (mg) Rl Sg Sl Species ph 4.8 ph 5.3 ph 5.8 ph 6.3 ph 6.8 Figure 3. (A) Rdil growth (mm d -1 ) nd (B) mycelil iomss (mg) of three fungl ectomycorrhizl species t five ph. Vlues re mens ± stndrd error of the men (n = 5). Brs with the sme letter, for ech species, re not different ccording to the Tukey test (P <.5). Rl = Rhizopogon luteolus; Sg = Suillus grnultus; Sl = Suillus luteus. Mycelil iomss (mg) C 28 C 32 C A) 1.8 B) ph ph 5.3 ph ph ph Rdil growth (mm dí -1 ) Rl Sg Sl Species R. luteolus continued until dy 21, nd then the sttionry phse strted. In the cse of the experiment were different ph vlues were evluted, in generl the exponentil phse of the NARG ws shorter, finishing in etween dys 6 nd 9 depending on the ECMF species. Then, the exponentil period ws recorded etween dys 15 nd 18, nd fter tht the declining phse egn until dy 24, to conclude with the sttionry phse of NARG (Figure 4D). DISCUSSION The knowledge of the iologicl, physiologicl nd symiotic chrcteristics of ECMF, s well s the specificity tht they hve with certin hosts, re fundmentl requirements in order to choose the most pproprite fungl species in the production of ectomycorrhizl tree seedlings. Environmentl fctors, including temperture nd ph, lrgely influence the formtion nd the development of mycorrhizl structures (Smith nd Red, 28). In generl, most ECMF re considered to grow well t moderte tempertures etween 11 ºC nd 28 ºC (Hutchison, 199). However, there re differences in the fungl growth mong isoltes of ECMF t different environmentl tempertures (Dmes et l., 1999; Hutchinson, 199). Some studies hve shown tht the ectomycorrhizl mycelium might e very sensitive to temperture vritions (Lzrević et l., 216; Mrx et l., 197; Sánchez et l., 21). If the soil drmticlly cools, the metolic ctivity of the fungi nd the roots decrese, with negtive consequences for mycelil growth nd nutrient vilility for oth the fungi nd the ssocited plnts (Lzrević et l., 419

6 ECTOMYCORRHIZAL MUSHROOMS FROM CHILE Rev. Fitotec. Mex. Vol. 4 (4) 217 Rdil growth (mm) Rdil growth (mm) Growth time (dys) Growth time (dys) A) Rl 24 ºC B) Rl 28 ºC Rl 32 ºC Sg 24 ºC Sg 28 ºC Sg 32 ºC Sl 24 ºC Sl 28 ºC Sl 32 ºC C) D) Rl ph 4.8 Rl ph 5.3 Rl ph 5.8 Rl ph 6.3 Rl ph 6.8 Sl ph 4.8 Sl ph 5.3 Sl ph 5.8 Sl ph 6.3 Sl ph 6.8 Sg ph 4.8 Sg ph 5.3 Sg ph 5.8 Sg ph 6.3 Sg ph Growth time (dys) Growth time (dys) Figure 4. Averge (A nd C) ccumulted nd (B nd D) non-ccumulted rdil growth of three fungl ectomycorrhizl species t three tempertures (t ph 5.8) nd under five ph vlues (t 24 ºC). Vlues re mens (n = 5). In order to hve more clrity in the grphs the ± error rs re not shown. Rl = Rhizopogon luteolus; Sg = Suillus grnultus; Sl = Suillus luteus Rdil growth (mm) Rdil growth (mm) 216). The results of the present study showed tht the culture temperture hd n effect on the in vitro growth of R. luteolus, S. grnultus nd S. luteus species. The est growth, oth in terms of RG nd MB, ws reched t 24 ºC for the three species in the evluted ECMF, nd when the temperture ws incresed to 28 nd 32 ºC, negtive effect ws oserved. Different uthors hve reported similr findings to those tht we found here. Jh et l. (26) reported optiml growth for R. luteolus in tempertures rnging from 2 to 25 ºC. Sánchez et l. (21) recorded the highest growth vlues for R. roseolus, Suillus collinitus, S. grnultus nd S. luteus t tempertures of 23 ºC; similrly to our own findings, these uthors registered significnt decreses with incresing tempertures. Dennis (1985) reported optiml growth for S. grnultus t 25 ºC nd for S. luteus in the intervl of 2 to 25 ºC. Lzrević et l. (216) recorded the est growth for S. collinitus nd S. grnultus t 22 ºC. Cline et l. (1987) reported the est growth for S. grnultus t 27 ºC, nd for Suillus spp. optiml mycelil growth etween 16 nd 32 ºC. In the present study, R. luteolus showed the est mycelil growth mong the three studied ECMF species independently of the tempertures tested. Sánchez et l. (21) found similr results for Rhizopogon nd Suillus species. The mximum rdil growth rte reched y R. luteolus (2.49 mm d -1 ) in the present work ws ove those previously reported 1.15, 1.98 nd 1.28 mm d -1 y Chávez et l. (27), Pereir et l. (27) nd Vázquez-Grcí et l. (22), respectively. However, the mximum vlues of RG otined here y S. grnultus nd S. luteus re elow those registered y Sntelices et l. (212) for S. luteus, under the sme culture conditions. The ph cn iologiclly determine the type of orgnism le to develop on soil or sustrte, due to its significnt influence on the nutrient vilility (Pereir et l., 27). The ph cn strongly ffect the microil ctivity through nutrient vilility, ioniztion of the constituents of the inorgnic nd orgnic soil solution, nd therefore cn hve prmount importnce in the enzymtic ctivity of the soil (Voroney, 27). In the present study, different ehvior of the evluted ECMF species ws recorded t the different 42

7 GARCÍA-RODRÍGUEZ et l. Rev. Fitotec. Mex. Vol. 4 (4) 217 ph vlues. Rhizopogon luteolus presented the gretest sensitivity of RG nd MB to chnges in ph, showing decrese in these vriles when the ph ws incresed. Jh et l. (26) nd Pereir et l. (27) found similr trends for R. luteolus. Vázquez-Grcí et l. (22) reported optiml growth for Rhizopogon sp. in BAF medium t ph 6., nd significnt growth decreses when ph ws either incresed or decresed. Duñeiti et l. (24) did not find chnges in terms of colony dimeter nd mycelil growth iomss of R. luteolus when the ph ws modified. In the present study, S. grnultus nd S. luteus did not show cler trend when the ph chnged. Similr responses were found for S. luteus y Sntelices et l. (212) nd for S. ellinii y Pereir et l. (27). In their studies with severl species of Suillus, Murriet-Hernández et l. (214) nd Vázquez-Grcí et l. (22) reported different ehviors in terms of growth when the ph of the culture medium ws modified. In contrst, Dennis (1985) found significnt differences in rdil growth when S. grnultus nd S. luteus were grown in rnges of ph from 3 to 6. Brros et l. (26), nd Pereir et l. (27) recorded differences in terms of RG for S. luteus when the ph ws modified. In our work, R. luteolus showed the highest vlues of RG t ph 4.8 (152 mm d -1 ) nd t ph 6.3 the lowest one (1. mm d -1 ). The results showed tht, in generl, R. luteolus tends to grow etter under cidic thn lkline conditions. This finding grees with the trends mentioned y Hung nd Trppe (1983), Lzrević et l. (216) nd Willenorg et l. (199), who mentioned tht ECMF hve in generl, n cidophilic nture when grown under pure culture conditions. However, it is importnt to mention tht there re some exceptions either specific or intrspecific, i.e. Sánchez et l. (21) found tht S. grnultus, S. luteus nd S. collinitus hd their est iomss increse responses t ph of 8.5. In vitro mycelil growth of fungus chnges ccording to the culture medium used nd the experimentl culture conditions (Pereir et l., 214; Sntelices et l., 212). The lck of significnt trends otined in the ph experiment could proly e explined ecuse no iologicl uffers were dded to growth medi. Such uffers re essentil to stilize the ph vrile. According to Grcí-Rodríguez et l. (26) nd Hung nd Trpe (1983) during the development of in vitro mycelium, ECMF produce series of orgnic cids, through the sorption of ions, cidifying the growth medium, nd negtively ffecting the growth nd development of the fungi. Additionlly, conventionl culture medi such s Modified Melin Norkrns hve low uffering cpcity nd therefore the effects of ph on fungl growth in such medi re difficult to evlute (Child et l., 1973, Hung nd Trppe, 1983; Ymnk, 23). Orgnic nd inorgnic cids, such s cettes, citrte, phthlte nd phosphte, hve een used s lterntive uffers to stilize ph. Guerin-Lguette et l. (2) showed the importnce of iologicl uffers in ph experiments with fungl growth, they found very strong evidence of the influence of ph on the in vitro mycelil development of two edile ectomycorrhizl mushrooms. The growth of Lctrius deliciosus nd L. snguifluus ws optiml t ph nd , respectively. They used 5X gr medium to which the iologicl uffer MES (2-(N-morpholino) ethnesulfonic cid ws dded nd the ph ws djusted with 4 M NOH prior to utoclving. Although some of these cids my inhiit the growth of fungi (Giltrp nd Lewis, 1981; Hilger et l., 1986; Inoue nd Ichitni, 199), MES nd other uffers such s piperzine-n nd N -is (2-ethnesulfonic cid) re considered to e physiologiclly inert (Good et l., 1966). It hs lso een shown tht these uffers cn stimulte the growth of some ectomycorrhizl fungi (Ymnk, 23). The fungi present growth kinetics tht usully involves the dpttion, exponentil, declining nd sttionry phses (Mnero et l., 212; Sánchez nd Royse, 22). The three species of evluted ECMF in the present work showed these four well-defined phses in our in vitro culture conditions. It ws oserved tht the dpttion phse ws more conspicuously mrked in the first experiment where tempertures were evluted, compred to the second experiment where the effect of ph conditions ws studied. Sntelices et l. (212) reported only the dpttion, exponentil nd sttionry phses for S. luteus. Colemn et l. (1989) reported the sme growth pttern for S. luteus in Modified Melin Norkrns culture medium, with sence of the initil dpttion phse; however, when wter stress tretments were involved in their trils, the initil phse of dpttion ecme more conspicuous. In our study the exponentil phse of R. luteolus ws strongly dependent on the culture temperture. When the ph of the medium vried, the period of the exponentil phse ws in generl short, producing in some species second period of exponentil growth. In the kinetics study of the ECMF, the growth phses involved in their cultivtion re fundmentl to nlyze nd understnd ecuse they provide importnt informtion relted to the prticulr growth dynmics of ech ECMF species. These studies cn show the exct moment when the mycelium of prticulr fungus is physiologiclly ctive in culture, nd hs the mximum potentil to e used s n inoculum in possile progrms of inoculum production in order to produce mycorrhizl plnts. The understnding of the optiml in vitro conditions to grow ntive strins will llow the genertion of ectomycorrhizl inoculum in enough quntities to e pplied to P. rdit tree production under greenhouse conditions. Such work hs previously een crried 421

8 ECTOMYCORRHIZAL MUSHROOMS FROM CHILE Rev. Fitotec. Mex. Vol. 4 (4) 217 out in other pine species such s P. pine in Spin with Rhizopogon spp. (Rincón et l., 25). Afterwrds, if there is successful nd undnt ectomycorrhizl coloniztion the estlishment of plnttions with the locl plnt nd the studied fungl genotypes might e fvoured. CONCLUSIONS The results indicted tht the men rdil growth rte nd the iomss increse of strins of R. luteolus, S. grnultus nd S. luteus were significntly ffected y incresing the temperture. The optimum cultivtion temperture for ll of the evluted species ws 24 ºC. There ws significnt decrese with temperture increses. Under the present culture conditions without iologicl uffers, the ph vriility of the medium, in the rnge of 4.8 to 6.8, did not significntly ffect the mycelil growth of S. grnultus nd S. luteus species. However, R. luteolus decresed its growth when ph ws incresed. Among the three evluted fungl species, R. luteolus showed the gretest mycelil production. This species lso shows the gretest potentil for the lrge-scle production of mycelil inoculum t lrger scle, useful in the production of ectomycorrhizl Pinus rdit trees. ACKNOWLEDGMENTS The support of the Ntionl Institute of Forestry, Agriculture nd Livestock Reserch of México (INIFAP, CORECADE NC13/83), of The Council Ntionl of Science nd Technology of México (CONACYT 1727), nd of The Chilen Ntionl Council for Science nd Technology (CONICYT ) through doctorl scholrship grnted to J. L. Grcí-Rodríguez is cknowledged. The uthors lso thnk two nonymous referees nd the Editor whose vlule comments helped to sustntilly improve the originl mnuscript. BIBLIOGRAPHY Allen E. B., M. F. Allen, D. J. Helm, J. M. Trppe, R. Molin nd E. Rincon (1995) Ptterns nd regultion of mycorrhizl plnt nd fungl diversity. Plnt nd Soil 17:47-62, f Andrino A., M. A. Morte G. y M. Honrui G. (211) Crcterizción y cultivo de tres ceps de Pleurotus eryngii (Fries) Quélet sore sustrtos sdos en residuos grolimentrios. 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