Functional compatibility in arbuscular mycorrhizas measured as hyphal P transport to the plant

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1 New Phytot. (1995), 129, Functional compatibility in arbuscular mycorrhizas measured as hyphal P traport to the plant BY S. R A V N S K O V AND I. JAKOB SEN Plant Nutrition, Environmental Science and Technology Department, Riso National Laboratory, DK-4 Roskilde, Denmark {Received 3 August 1994; accepted 2 December 1994) SUMMARY The functional compatibility of symbioses between host plants and arbuscular mycorrhizal fungi was measured as hyphal P traport to plants. (Ciicimiis sativus L.), wheat (Triticum aestivum L.) and flax (Liiiiun iisitatissimum L.) were inoculated with Glomiis caledoiiium (Nicol. & Gerd.) Trappe & Gerdemann (isolate RIS42, BEG15) and Glomus invermaium Hall (isolate WUMIO) or left uninoculated and grown for 28 days in soil divided into three compartments. One side compartment was separated from the main compartment by a 2 /im nylon mesh, and radioactive labelling with ''-P was used to determine hyphal P uptake. The other side compartment was separated from the main compartment by a 7 //m nylon mesh and '''P was used to determine the combined P uptake by roots and hyphae. All plant-fungus combinatio were compatible with respect to mycorrhiza formation, measured both as root colonization and growth of external hyphae. In contrast, the symbioses differed markedly with respect to functional compatibility as phosphorus uptake by each fungus depended on the species of host plant. The hyphal traport of '' 'P was high in G. caledoiiium in symbioses with all three plant species, whereas G. invermaitim traported significant amounts of'"'p only when associated with flax. Coequently, to determine the P-traport effectiveness of a mycorrhizal fungus is meaningful only in the context of its associated host plant species. Key words: Arbuscular mycorrhizal fungi, functional compatibility, hyphal P traport, ''-^P, ''"'P. INTRODUCTION Plant growth respoes to arbuscular mycorrhizas vary with the fungal genotype (Plenchette, Furlan & Fortin, 1982; Haas & Krikun, 1985) the mycorrhizal dependency of the host plant species or cultivar (Plenchette, Furlan & Fortin, 1983; Hetriek, Wilson & Cox, 1992) and the environmental conditio. The overall symbiotic eflleetiveness is influenced by the functional or physiological compatibility between the two partners (Gianinazzi-Pearson, 1984). The reciprocal exchange of nutrients across the interface between the tvv'o partners is coidered as the key to functional compatibility (Gianinazzi, 1991). Quantification of this nutrient exchange on the basis of direct hyphal measurements is therefore superior to the traditional measurements of growth respoes and overall P nutrition of the plant (Jakobsen, 1995). This was emphasized by a recent investigation, where the P traport from a root-free compartment differed markedly between three fungi; these differences were not revealed by measuring the combined P uptake by hyphae and roots (Pearson & Jakobsen, 1993/)). The potential importance of interface processes ^^^^_, ^j^^ indicated by a substantial variation in P traport by different fungi, which was not related to levels of root colonization or hyphal length deities in soil (Jakobsen, Abbott & Robson, 1992a). Furthermore, interfungal differences in capacity for hyphal P traport were not related to the fungal use of host carbon (Pearson & Jakobsen, 1993a). The objective of the present work was to study functional compatibility measured as hyphal P traport in two arbuscular mycorrhizal fungi associated with three host plant species. The P traport from a root-free hyphal compartment was measured by mea of ^^P, while the combined P uptake by roots and hyphae was measured by mea of the tracer isotope ^''P. ^^,,,,^,^^,j g ^^^ METHODS ^,^^^^^^ ^^^^ mycorrhizal fungi The species of plants were cucumber {Cucumis.sa^/wM^-L.) cv. Amitiex (Fl hybrid), wheat (7)//«HW aestivum L.) cv. Cawain and flax {Linum usitatissimuiit L.) cv. Tardora, while the arbuscular

2 612 S. Ravkov and I. jfakobsen mycorrhizal fungi were Glomus invermaium Hall (isolate WUMIO, obtained from L. K. Abbott, University of Western Australia) and Glomus caledonium (Nicol. & Gerd.) Trappe & Gerd (isolate RIS42, BEG15). The fungi had been multiplied on maize in the same soil as used in the experiment and fungal inoculum coisting of a mixture of soil, roots and spores was stored dry until use. Soil Plants were grown in a 1:1 mixture (w/w) of Pdeficient elay soil (Jakobsen & Nielsen, 1983) and quartz sand. The mixture was partially sterilized by irradiation (1 kgy, 1 MeV electron beam) in order to eliminate the native arbuscular mycorrhizal fungi. Nutrients were mixed into the soil at the following levels (mg kg"' dry soil): K2SO4, 71-; CaCl.,. 5H2O, 71-; C u S O,. 5 H, O, 2 ; ZnSO,,.7H2O, 5 ; MnSO^.H^O, 1-; COSO4.7H2O, -35; Na2MoO4.2H2O, -18; MgSO,,.7H,O, 2-. Experimental procedures Pots were cotructed from 5 mm PVC tubing and cross-shaped PVC fittings (Fig. 1). A central root compartment (RC) of 33 cm length had the bottom closed with a 21//m nylon mesh. A lateral compartment for both roots and hyphae (RHC) was separated from RC by a 7 /tm nylon mesh, while an opposite hyphal compartment (HC) was separated from RC by a 2//m nylon mesh. The central root compartment contained 72 g soil made up of 3 and 14 g inoculum-free soil in the bottom and top parts of the pots, respectively, with a mixture of 24 g soil and 4 g inoculum in the central part of the pot. The RHC contained a mixture of 5 g soil and 1 g inoculum, while the HC compartment contained 6 g soil and no inoculum. Non-mycorrhizal controls received no inoculum. The length of the soil column in RHC and in HC was 25 mm and both were closed by mea of capped plastic vials fitting iide the PVC tubes. Four replicate units of eaeh treatment were prepared as described. Twenty additional pots with cucumber were used to measure root colonization by each fungus after 14 d and to check for isotope discrimination in the uptake of ^^P and '^^P by nonmycorrhizal plants. All pots received 5 ml filtrate (Whatman No. 4 filter) of an aqueous suspeion of 25 g of eaeh inoculum as an attempt to establish a similar microfiora in all pots. The soil was watered to 5% of field capacity and incubated for one week. Two pregerminated seeds were sown in each pot and plants were later thinned to one per pot. Plants were plaeed in a growth chamber with a 16/8 h light/dark cycle, a photosynthetic photon flux deity of 5 //mol m"'' s"' (Osram H Q I - T 25 Figure 1. Three-compartment growth system used for the dual labelling experiment. Hatched areas in side compartments represent isotope-labelled soil sectio. HC, Hyphal compartment; RHC, root hypha) compartment. W/D) and day/night temperatures of 2/15 C. Pots were watered daily to a weight equal to 65 % of field capacity of the soil. All plants received 25 mg N kg"^ dry soil as NH^NO^ solution 7, 14, 17 and 2 d after sowing. The plastic vials were removed from side compartments 21 d after sowing, and RHC compartments and HC compartments were labelled with carrier-free solutio of 185 kbq ^''P and 185 kbq '^P, respectively. The isotope solutio were added to the exposed soil surface of each compartment as four parts of 1 ml separated by 15 min intervals. The plastic vials were put back into the side compartments after labelling.

3 Fmtctiotial compatibility in arbuscidar mycorrhizas 613 Table 1. Plant dry weight and root length in different combinatio of plant species and arbuscular mycorrhizal fungus Treatments Glomus inveriitmum Glomus caledonium ' G. inverninutin G. caledonium G. invermaiiim G. caledoniuin Two-way analysis of variance Plant species Fungus Interactio Plant dry weight (g plant"' ) Root length (m plant"') Shoot Root Total RHC *#* 11B 11S ** *««** ### p _ -1; **P = -1; * P = -5., non-significant. Root length measured in labelled area in RHC. Harvest Plants were harvested 28 d after sowing. Side compartments with soil, hyphae and/or roots were removed and kept at 5 C until extraction of roots and hyphae after 5 and 14 d, respectively. Shoots were weighed, dried at 7 C for 24 h, weighed again and ground for further analysis. Roots were washed, weighed and separated in two parts. One part was stored in 2-5 % acetic acid, the other dried at 7 C for 24 h, weighed and ground for further analysis. Three soil colum from side compartments of non-mycorrhizal plants were divided into -5 cm sectio, which were checked for radioactivity by mea of a table monitor (Radiation motiitor. Model RM-14, Eberline Itrument Corporation, Santa Fe, New Mexico). At least 9 5 % of the radioactive isotope was retained in the outermost 1 cm section and all soil colum from HC and RHC were then separated into this outermost labelled 1 cm section (HC, RHC ) and the 1-5 crn section closest to RC. Sectio from the HC compartments and 6 g of the outer RHC section were dried at 35 C for 24 h for hyphal counts. Roots from RHC sectio were washed and stored in 2-5 % acetic acid. Analyses Samples were taken from shoots and RC roots for wet digestion in nitric acid:perchloric acid (4:1). The digests were used to determine the activity of 32p^ :i.ip yj^j ^Qjjjj content of P. Radioactivity in 3 ml sample mixed with 1 ml scintillation fluid (Ecoscint) was measured in a Packard TR19 liquid scintillation counter by Dual Spectrum Analysis. Total P content was determined by the molybdate blue method (Murphy & Riley, 1962) on a Technicon Autoanalyzer II. Roots were stairied in lacto-glycerol (Kormanik & AicGraw, 1982), but with the acid fuchsin replaced by trypati blue, and colonized and total root lengths in RC and RHC were measured (Newman, 1966). Hyphal length in labelled soil from HC and RHC was measured after Abbott, Robson & de Boer (1984) by a grid-intersection method modified as described by Jakobsen et al. (1992o). Hyphal length was calculated according to Tennant (1975). Calculatio and statistics Levels of significarice of main treatments and their interactio were calculated by two-way analysis of variance and mea were compared by LSD^.^j. RESULTS Plant growth and inycorrhiza formation The three plant species differed in their growth respoe to mycorrhizas, which slightly depressed grov\'th of cucumber, generally had no effect on wheat, but markedly increased growth of flax. responded more to G. invermaiiim than to G. caledonium (Table 1). Total root length and the root length in the '''^Psoil section (RHC) was shorter in cucumber plants colonized by G. caledonium than in non-mycorrhizal plants, while flax with G. caledonium had less root length iti the RHC than when with. Roots of the non-mycorrhizal flax did not grow into

4 614 S. Ravkov and I. Jakobsen Table 2. Root colonization and hyphal length of different fungi associated with three host plants, 28 days following inoculation Root RC colonization RHC Hyphal length (m g"' soil) Treatments (%) (cm plant"') (%) (cm plant ') RHC HC Two-way analysis Plant species Fungus Interactio Glomus invermaium Glomus ealedonium G. catedonium G. ealedonium of variance ### #*# **# *## ## ### **# * **» # ## #* ## #* p = -1; compartments. **P = -1; P = -5. non-significant. Hyphal length measured in labelled area in side Table 3. Phosphorus concentratio and contents in three plant species combined with three arbuscular mycorrhizal treatments P concentration (mg g"' d. wt) P content (mg plant"') Treatments Shoot Root Shoot: Root Root/shoot ratio LSD., Glomus invermaium Glomus ealedonium G. ealedonium G. ealedonium Two-way analysis of variance Plant species Fungus Interactio *» ** * ** Ml «** P = -1, **P=-1; *P = -5., non-significant. the RHC. Otherwise, length and dry weight of roots of each plant species were only little influenced by mycorrhizas (Table 1). Both mycorrhizal fungi had colonized about 4 % of the root length in 14-d-old cucumber plants (data not shown) and mycorrhizas were well established in all treatments after 28 d, except of the uninoculated controls which remained non-mycorrhizal (Table 2). Percentage root colonization in RC decreased in the order: cucumber, wheat and flax. Colonized root length of wheat was lower with G. ealedonium than with, whereas colonized root lengths of cucumber and flax were similar with the two fungi. Treatment effects on root colonization in RHC were similar to those observed for RC (Table 2)- Average hyphal lengths in RHC and HC soil of non-mycorrhizal plants were 2'71 and 1-99 m g"' dry soil, respectively. Hyphal measurements, corrected for these background counts, were in the range 4'5 16'4 m g~' dry soil in the mycorrhizal treatments (Table 2). The RHC soil of cucumber and wheat

5 Functional compatibility in arbuscular mycorrhizas 615 Figure 2. Total phosphorus content in three plant species combined with three mycorrhizal treatments. Bars represent standard errors of the mean. Empty colum, Nonmycorrhizal; hatched colum, Glomus invermainm; dotted colum, Glomus caledoniwn. plants contained more hyphae of G. invermatum than of G. caledonium, whereas the corresponding HC soil contained more hyphae of G. caledonixtm than of G. invermainm. Hyphal lengths associated with flax varied much less hetween fungi and hetween side compartments. Concentration and content of P Shoot and root P concentratio of flax were markedly increased hy hoth fungi over levels present in non-mycorrhizal flax (Table 3). The P concentration was higher in shoots of wheat in association with than in non-mycorrhizal wheat, and root P concentratio were increased by G. caledoitium and G. invermaiutn in cucumher and wheat, respectively. Only shoot P concentratio of cucumber were not significantly affected hy the mycorrhizal treatments. Phosphorus contents in shoots of cucumber and wheat colonized by G. iiwermaium were similar to those of non-mycorrhizal plants, while cucumher and wheat associated with G. caledoniiim had significantly lower P content in shoots than non-mycorrhizal plants (Table 3). Total P content in wheat and flax was higher with than with G. caledortium (Fig. 2). Root/shoot ratios of P contents in cucumber and wheat were higher in mycorrhizal than in non-mycorrhizal plants, especially in wheat associated with G. caledonium. Root/shoot ratio of P contents in flax associated with G. caledonium was lower than in the other treatments (Table 3). External hyphae of G. caledonium traported large amounts of ^' ^P from H C to all host plants. The total *^P content in plants (Fig. 3a) cotituted 15, 21 and 23 % of the ''"P applied in flax, cucumber and wheat, respectively. associated with flax traported 13 % of the ^^P applied, whereas its traport to cucumher and wheat was negligible (Fig. 3 a). The ^^P uptake per unit hyphal length Figure 3. Total radioactivity in three plant species combined with three mycorrhizal treatments (a) Radioactivity of ^^P in plants; b, radioactivity of ''^P in plants. Bars represent standard errors of the mean. Empty colum, non-mycorrhizal; hatched colum, Glomus invernmium; dotted colum, Glomus caledonium. difl^ered between treatments in a similar way: High levels were measured with G. caledonium associated with all plant species, whereas had a high specific uptake only in association with flax (Table 4). The root/shoot ratios of "'"P in cucumher, wheat and flax associated with G. inverntaiunj ^vere 1-29, 2-65 and -58, respectively. Root/shoot ratios of ^^P in cucumber, wheat and flax colonized by G. caledonium were -59, 1-12 and -42, respectively. Uptake of ''''P by roots and hyphae from the R H C of cucumber and wheat was lower in mycorrhizal than in non-mycorrhizal plants and decreased in the following order of mycorrhizal treatments: Nonmycorrhizal,, G. caledonium (Fig. 2b). No uptake of ''''P was measured in non-mycorrhizal flax. Uptake of ''^P per cm root length was significantly influenced by mycorrhizas in wheat, where the length-specific uptake in associatio with G. invermaium and G. caledonium was only 5 or 4 4 %, respectively, of the length-specific uptake of nonmycorrhizal plants (Table 4). In all three associatio with G. caledonitmi a higher quantity of P was taken up by hyphae (^^P) from HC than by roots and hyphae (''''P) from RHC. The P uptake from HC was 1-3, 1-6 and 2-6 times as

6 616 S. Ravkov and I. Jakobsen Table 4. Uptake of length, respectively and ^^P on the basis of hyphal length and root ''^P uptake (Bq cm"' hyphae) Treatments Glomus invermaiutn Glomus caledonium G. caledonium G. caledonium LSD Two-way analysis of variance Plant species Fungus Interactio p = -1; ' 'P u p t a k e (Bq cm"' root) M O'l '2 «* ## * n8-7 **p = -1; *P = -5., non-significant. association with these two hosts. Similar results were obtained with cucumber by Pearson & Jakobsen (1993i)). In contrast, a high P eflectiveness of both fungi was expressed in marked growth respoes in flax, where the root hair length was only one-fifth of the length in cucumber and wheat (data not presented). Interfungal differences in ability to improve P uptake by different host plants have been related to differences in rate of colonization (Al-Nahidh & DISCUSSION Sanders, 1987). This variable can be excluded in the present work, where the root colonization in cualthough arbuscular mycorrhizal fungi and their cumber was about 4% after 14 d, and where the host plants are highly compatible with respect to mycorrhiza formation, the present work confirms length deity of external hyphae was high in all that they are not always compatible at other plant-fungus combinatio at time of harvest. It is functional levels. Our measurements of hyphal P more likely that the functioning of hyphal P traport traport clearly showed that the effectiveness of one by was diflerently affected by fungus depended on the associated species of host different host plants. plant. The hyphal length deity diflered between fungi Direct measurement of hyphal P traport from a in previous studies (Jakobsen et al a; Pearson root-free compartment by mea of radiotracers is a & Jakobsen, 19936) and in the present study. novel approach for the assessment of fungal P However, no clear relatiohip existed between effectiveness (Jakobsen, Abbott & Robson, 19926). hyphal P traport and hyphal length deity. The method enables us to quantify the potential Although the hyphal length deity of G. caledonium fungal P traport to different host plants, as in HC varied with plant species the ^^P uptake was traport is studied in the absence of competing almost the same. The pattern was opposite for G. roots. The method is therefore superior to con- invermaium, where the hyphal length deity in HC ventional growth and P uptake studies based on pot was almost unaffected by host speeies, but where ''^P experiments, where the mycorrhizal potential uptake was recorded only in the combination with becomes obscured in plant species with high root flax. The interfungal ranking by hyphal length length deities (Baath & Hayman, 1984). This is deity was similar in HC and in RHC in previous exemplified by the absence of a mycorrhizal growth experiments (Pearson & Jakobsen, 19936; Olsson et respoe in cucumber and wheat in the present al., 1995), whereas the hyphal length deity of G. work, where both species had high root length caledonium was the highest in HC, but the lowest in deities (Table 1) and long root hairs (data not RHC in symbioses with cucumber and wheat in the presented). However, the direct studies of hyphal present work (Table 2). This discrepancy may be traport demotrated that G. caledonium, but not related to the supply of inoculum to RHC in the, had a high P traport capacity in present experiment and coequently a more unihigh as the uptake from R H C in cucumber, wheat and flax, respectively. External hyphae of G. invermaium contributed significantly to P uptake only in flax, where the traport of ^^P from H C was -7 times as high as the traport of ^'P by roots and hyphae from R H C. Non-mycorrhizal root systems of cucumber showed no discrimination in their uptake of ' ' P and ''"P.

7 Functional compatibility in arbuscular mycorrhizas form colonization of the roots in RHC. The pattern of hyphal spread differs between fungi, such that one fungus may be the most abundant near the root surface (e.g. in RHC), but least abundant further away (e.g. G. inverrnaimn in HC) (Jakobsen et al., 1992a). Non-mycorrhizal cucumber and wheat had a significantly higher ^^P uptake from RHC compartments than the corresponding uptake by roots of mycorrhizal plants even when the uptake was expressed on the basis of a root length unit. This indicates that the P uptake mechanisms are different in non-mycorrhizal and mycorrhizal root systems. Jungk et al. (199) found that plants raised under Pdeficient conditio had a higher P influx after trafer to a P solution than plants raised under Psufficient conditio. Such feedback regulatio could possibly explain the higher ^^P uptake in nonmycorrhizal plants, as their root-p concentratio were lower than those in corresponding mycorrhizal plants. The difference in root-p concentratio between mycorrhizal and non-mycorrhizal plants could even have been larger at the time of ^^P application, where root deities would have been lower. Plants in combination with G. caledonium all had a greater uptake from HC than RHC. This would seem to imply that the fungus alone depleted the soil P more effectively than the roots and fungus in combination. However, in a previous study bacterial growth and biomass was markedly higher in soil containing both roots and hyphae than in soil containing hyphae only (P. A. Olsson, personal communication). Therefore, competition by bacteria for phosphorus would have been higher in compartments with roots than in compartments with hyphae only, and this could possibly explain the lower phosphorus uptake from the compartments containing roots. The present demotration of differences in functional compatibility was obtained from measurements over a relatively short time interval in one experiment only; the results might have been different if measurements had been performed at another stage of plant growth, over a longer time interval or under different environmental conditio. So far we can speculate on the mechanisms which could be respoible for the observed host-dependency in P traport by. Arbuscules were well developed in the cortex cells of all three plant species, but processes at the arbuscular interface might have been respoible for the differences obtained. The high rate of fungal P efflux into the interfacial apoplast is coidered the most ' unusual' step in the fungal traport of P from soil to plant in arbuscular mycorrhizas (Smith et al., 1994). Coequently, it is possible that the measured differences in compatibility were related to the regulation of the fungal P efflux by the host plant. This hypothesis is 617 supported by the ohservation that the root/shoot ratios for ^'P distribution in cucumber and wheat associated with were more than twice the ratios in the same host plants associated with the P traport-effective G. caledonium. Future attempts to identify the regulating mechanisms could benefit from the functional differences demotrated in this work. The results of the present work imply that the P effectiveness of an arbuscular mycorrhizal fungus is a useful term only when a host plant is also defined. This mea that for practical purposes the selection of an effective inoculant fungus should be based on bioassays including the host plants with which it is going to be used. Our work also shows that root-free hyphal compartments are essential in such bioassays designed to assess the P traport capacity by mycorrhizal fungi: marked interfungal differences were revealed by direct measurements of the hyphal traport of ''^P, but not by measurements of the traport of '^^P by mycorrhizal root systems. Confounding interactive effects of a limited soil volume and high root deities can be avoided in future work on fungal effectiveness if this is measured directly as hyphal P traport. REFERENCES Abbott LK, Robson AD, De Boer G The effect of phosphorus on the formation of hyphae in soil by the vesicular-arbuscular mycorrhizal fungus, Glomus fasciculatum. New Phytologist 97: Al-Nahidh S, Sanders FE Compariso between vesicular-arbuscular mycorrhizal fungi with respect to the development of infection and coequent effects on plant growth. In: Pegg GF, Ayres PG, eds. Fungal infection of plants. 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