Root hair length determines beneficial effect of a Glomus species on shoot growth of some pasture species

Transcription

1 Neu'PhvtoL (1995), 131, Root hair length determines beneficial effect of a Glomus species on shoot growth of some pasture species BY P. F. SCHWEIGERi*, A. D. ROBSON^ AND N. J. ^ Soil Science & Plant Nutrition, Faculty of Agriculture, The University of Western Australia, Nedlands, Western Australia 6009 '^Division of Soils, CSIRO, Wembley, Western Australia 6014 {Received 1 February 1995 ; accepted 28 June 1995) SUMMARY Differences hetween plant species in the benefit derived from arhuscular mycorrhizal colonization have often been attributed to differences in physical properties of their roots, especially in root hair development. To test this hypothesis, the growth response to phosphate of Ji\-e pasture species which differed markedly in the length of their root hairs was measured. Plants in the mycorrhizal treatments were inoculated with a Gtomus sp. (isolate WUM 10(1)) while non-mycorrhizal plants received control inoculum. Benefit was described as the relative effectiveness of phosphorus (P) for the mycorrhizal plants compared with non-mycorrhizal plants. The beneficial effect of Glomus sp. was inversely related to root hair length of the host plant but it was not well related to root diameter, root length per plant or root/shoot ratio. It is suggested that root hairs and external hyphae of Glomus sp. act as alternatne, similar ways of shortening the distance for the diffusion of phosphate in soils. Key words: Glomus sp., mycorrhizal benefit, pasture species, root hair iength. T N T R O D I" C T 1 O N Arbuscular mycorrhizal (AM) fungi colonize the roots of most plant species (Harley & Harley, 1987) and t\'pically increase plant phosphorus(p) uptake in P deficient soils (Harley & Smith, 1983). However, plant species differ in their growth response to mycorrhizal inoculation. The extent of this is often referred to as mycorrhizal dependence, which has often been related to morphological properties of the roots of the host plant (Hayman, 1983). Baylis(1970) suggested that plants with few and short root hairs were highly dependent on VA mycorrhizas. His hypothesis was based on results of a comparison of dry matter yields of a range of plants grown in steamed or unsteamed soil. Similarly, Crush (1974) reported a decrease in mycorrhizal dependence with increasing root hair length in four leguminous plants, and Manjunath & Habte (1991) also found root hair length to be one of the most important determinants of mycorrhizal dependence. ln all these studies, mycorrhizal dependence was assessed by comparing differences in growth of mycorrhizal and non-mycorrhizal plants at a single rate of P application. However, comparing different Current address: lnstitut fiir Mikrobiologie, Universitat Innsbruck, TechnikerstraUe 25, A Innsbruck. Austria. plant species is complicated by differences in the soil P concentration required to achieve their maximum growth, and by differences in their responsiveness to applied P (see, for example. Arias et al., 1991). Depending on the nutritional status of the soil, estimates of mycorrhizal dependence of different plant species will vary and can result in m^isleading conclusions. By describing quantitatively the growth response of mycorrhizal and non-mycorrhizal plants to applied P, these complications can be avoided. Benefit derived from mycorrhizal colonization is then described as the relative effectiveness of P for mycorrhizal plants compared with non-mycorrhizal plants (Abbott & Robson, 1984). Because of the slow" diffusion of phosphate through soil, a depletion zone forms around the absorbing root (Nye & Tinker, 1977). The w-idth of the depletion zone is mainly determined by the phosphate sorption capacity of the soil (Nye & Tinker, 1977) and the length of the root hairs (Lewis & Quirk, 1967). External hyphae produced by AM fungi have been shown to infiuence the depletion profile around the root in a similar way to root hairs (Viebrock, 1988). This leads to the hypothesis that root hairs and external AM hyphae might be alternative, similar ways of bypassing the phosphate depletion zone immediately around the root.

2 248 P. F. Schweiger, A. D. Robson and N. J. Barrozv The aim of this study was to quantify the relationship between mycorrhizal benefit and a measure of plant root morphology for some pasture species, with special emphasis on root hair length and incidence. M.^TERIALS AND METHODS Experimental design Five pasture species with marked differences in the length of their root hairs were grown in a sandy loam at 12 rates of applied P. The plants were either colonized or not by Glomus sp. There were no replicates. Experimental procedure For the experiment, a sandy loam (Non-calcic Brown Soil, 69% sand, 16% silt, 15 o clay, ph (0-01 M CaCl.^) 5-36, 2 /jg g"^ bicarbonate-extractable P (Colwell, 1963), 8//.gg-' NH,, 4/.gg-^ NO3, 63 //g g"' P sorbed after 24 h at 25 C at 1 fig m^^ P in solution (for method see Barrow & Shaw, 1979)) from near Bakers Hill (south western Western Australia) was collected and sieved to < 2 mm. After being steamed for 1 h on two consecutive days, the soil was weighed into 3 1 pots lined with plastic bags. Based on the results of a preliminary' experiment comprising 16 pasture legumes and nine pasture grasses (results not sbown), the follow-ing five species were chosen: suckling clover (Trifolium dubium Sibth.), subterranean clover {Trifolium subterraneum L.), burr medic {Aledicago polymorpha L.), yellow serradella {Ornithopus compressus L.) and annual ryegrass {Lolium rigidum Gaud.). Taking into account the individual requirements of the species for soil P concentration to achieve their maximum growth and the phosphate sorption capacity of the soil, phosphate was added as KH,2PO4 at 12 levels to define the response curves (see Fig. 1 a). A complete set of basal nutrients less nitrogen (mg kg"': KgSO^, 71; CaCl^, 71; CuSO^.SH^O, 2; ZnSO^.TH^O, 10; MnSO,.H2O,10; " CoSO,. 7H,O,0 35; NaMoO4.2H2O, 018; MgSO^.7H2O, 20) was applied in solution to the soil surface of every pot. After tbe soil had dried, tbe nutrients were mixed tbrougbout tbe soil. Dry soil inoculum (90 g) of Glomus sp. (isolate WUM 10(1)) was thoroughly mixed tbrougbout the soil of tbe mycorrbizal treatments. pots received 90 g of dry soil inoculum containing no mycorrbizal propagules (see Gazey, Abbott & Robson, 1992). The seeds of all species were sieved to supply seed of a narrow size range. Seeds of Medicago polymorpha and Ornithopus compressus were scarified. Germinated seeds were sown and tbe legumes inoculated with appropriate root nodule bacteria. Lolium rigidum was supplied with 63 mg NH^NOg kg"^ soil every fortnight; legumes received no added N. After emergence, seedlings w-ere tbinned to four per pot (eigbt for Trifolium dubium). Pots were maintained at 2O±1 C in a water batb and watered to field capacity by weigbt every second day witb deionized water. Plants were grown in a glassbouse during June and July at ambient air temperature (average day/nigbt temperature 22/17 C) and light conditions. All pots were bar\'ested at 46 d from sowing. Tbe tops were cut at tbe soil surface and weighed. Wbole tops were dried and digested in nitric/percbloric acid (Johnson & Ulrich, 1959). Tbe P concentrations in tbese digests were determined by tbe molybdovanado-pbospboric acid metbod (Boltz & Lueck, 1958). Tbe roots were wasbed free of soil and their fresb weight was recorded. Samples of root were cleared and stained for arbuscular mycorrbizal colonization as described by Gazey et al. (1992). Total length of root was assessed using a line intercept metbod (Newman, 1966). Presence or absence of mycorrhizal colonization was recorded at the same time at x 25 magnification (except for Lolium rigidum for wbich mycorrbizal colonization was assessed under a compound microscope at x 100 magnification using tbe metbod of McGonigle et al. (1990)). Root hair length was measured on 50 root segment intercepts at x 100 magnification using a compound microscope fitted witb an eyepiece scale. To estimate total root hair length mm"^ root length, slides were made witb root pieces preser\'ed in etbanol. All root bair intersects witb a grid line on one side of the root were counted at x 200 magnification. Root hair length was calculated using Tennant's (1975) metbod. Multiplying the count by 2 (for two sides of the root) and l/(widtb of grid in mm) gave total root hair length mm'^ root. Twenty measurements were made for eacb species. Tbe proportion of the total length of root covered by root bairs was assessed by recording absence or presence of root hairs on more than 100 root segment intercepts at x 25 magnification. Diameter of roots was calculated from the relation: Diameter = 2iW/L7T)'*'\ where W is root fresb weigbt and L root length. This relationship assumes a specific root weight of 1, which is close to the value of 1-03 that was found in tbe preliminary experiment. Linear relations and polynomials were fitted to tbe root colonization data in relation to relative dr>' weight of sboots. Significant differences in this relationship between the plant species were assessed by fitting relations at first to all data, then to the data from eacb plant species separately. An improvement in description was tested using the principle of extra sum of squares (Mead & Pike, 1975). Two phase linear relations (for shape see Fig. ] b) were fitted to the dry weight data plotted against internal P concentration by the method of least squares using a computer program written in

3 Root hair lengths and effects of a Glomus sp. on pasture species r Trifolium dubium Trifolium dubium Trifolium subterranean ' Medicago polymorpha 0.5 Medicago polymorptia Ornithopus compressus Ornithopus compressus ^ o Lolium rigidum " Lolium rigidum 0 05' Phosphate applied (mg P kg"^ soil) Shoot P concentration (mg P g"^) Figure 1. The relationship between dry weight of shoots of (D) non-mycorrhizal and ( ) mycorrhizal plants and (a) P application rate (left column) and (b) internal phosphorus concentration of shoots (right column). GENSTAT (Scott, 1990). Tbe two lines were found by tbe following process: the data were ranked based on the values for internal P concentration and divided into two sets at the second lowest value. Subsequently, separate lines were fitted to these sets. This procedure was repeated by dividing the data stepwise into two sets at each successive, higher value for internal P concentration. The two lines. where R^ calculated from the residual and total sums of squares of both relations was maximum, were chosen. The intercept of the two lines was taken as an estimate for critical tissue P concentration. Mitscherlich equations of increasing complexity (ranging from a common equation for both mycorrhizal and non-mycorrhiza! plants to two completely separate equations) were fitted to the dry matter

4 250 P. F. Schweiger, A. D. Robson and N.jf. Barrow yields of the mycorrhizal and non-mycorrhizal plants. The significance of improvement in description for equations of greater complexity was tested using the principle of extra sum of squares (Mead & Pike, 1975). The data were best described by the following equation: where Y is log^, yield. A' is the P applied (mg P kg"^ soil), A is the estimated maximum value for log^ Y, B is the range betw-een A and the estimated value for logp Y when no P was added, C is the slope coefficient, and the subscript i indicates either mycorrhizal (m) or non-mycorrhizal (nm). All curves were fitted using the simplex method for function minin:iization (Nelder & Mead, 1965). Benefit due to mycorrhizal colonization was calculated as the ratio of the slopes of the mycorrhizal and non-mycorrhizal curves at their intercept w^ith each other. This approach is slightly different from tbe one Abbott & Robson (1984) suggested, in that it also allows comparison between mycorrhizal and non-mycorrhizal treatments when they give different intercepts on the yield axis. In this case, the x-coordinate of the intercept of the mycorrhizal with the non-mycorrhizal curve refiects the P supplied by the inoculum and the unfertilized soil. The y-coordinate of the intercept refiects the growth of the plants relying entirely on the P supplied by the seeds. RESULTS Growth response Maximum total dry matter production was similar for the species, except for the very small-seeded Trifolium dubium, which yielded only half as much with P not limiting, with twice as many plants as the other species. For all species, the response of mycorrbizal and non-mycorrhizal plants to added P was well described by the Mitscherlich equation (Eig. 1 a, Table 1). Eitting Mitscherlich equations of greater complexity than a common equation to the dry matter yields of mycorrhizal and non-mycorrhizal Lolium rigidum plants did not significantly improve the description of their response to added P. Ornithopus compressus and Lolium rigidum plants reached a slightly higher maximum yield than their non-mycorrhizal counterparts, but the difference was not significant. Values for benefit due to mycorrhizal colonization ranged from I-() for Lolium rigidum to 2319 for Trifolium dubium (Table 1). The relation of dry matter yields of mycorrhizal plants converted to percentage values of maximum yield to applied P was similar for all five species and was fairly well described by a common Mitscherlich equation (i?^ = 0-92; relation not shown). The responsiveness of non-mycorrhizal plants to added P differed between the species and increased in the same order as did root hair length. and non-mycorrhiza! plants produced about the same amount of dry matter with the same internal P concentration (Eig. \b). Values for critical tissue concentration ranged from 3-13 mgpg"' in Trifolium subterraneum to 4-2 mg P g~^ in Trifolium dubium. The other three species had critical tissue concentrations of about 41 mg P g~*. Below that concentration, however, Medicago polymorpha was more efficient in its P utilization for growth than was Ornithopus compressus, and even more than was Lolium rigidum. ln all pasture species tested, inoculation with Glomus sp. resulted in 70"^'o or more of total root Table 1. Parameters of the regression equations which describe the relationship between dry weight of shoots and phosphate applied Plant species A B C R^ benefit Trifolium dubium Trifolium subterraneum Medicago polymorpha Ornithopus compressus Lolium rigidum Mycorrhizai O'39O ' ' ' A = estimated maximum dry weight (log,. Y), B = difference between minimum and maximum dry weight (log F), and C = the slope coefficient.

5 Root hair lengths and effects of a Glomus sp. on pasture species r? E T3 0) rsi o "o u JZ lengi Root A * % Maximum dry weight of shoots 125 Figure 2. Relationship between % root length colonized and "o maximum dry weight of shoots (A as estimated by the Mitscherlith equation) for all plants in inoculated treatments., TrifoLium dubium;, Trifolium subterraneum; #, Medicago polymorpha; /\, Ornithopus compressus; D, Lolium rigidum. length colonized at low levels of P added (Fig. 2). Levels of colonization in Trifolium dubium. Trifolium subterraneum and Ornithopus compressus showed similar distributions and were not significantly different from each other. For these species, values ranged from c o of total root length colonized in plants yielding between 5 and 75 "^ of the estimated maximum yield. Increases in dry matter yield above 75 'o were associated with a sharp decline in "^ root length colonized. Levels of colonization in Lolium rigidum declined linearly from c "^o at the lowest yields to reach about 5 o at the estimated maximum yield. Levels of colonization in Medicago polymorpha showed an intermediate distribution, hetween the other three legumes and Lolium rigidum. In this relationship, Medicago polymorpha and Lolium rigidum differed significantly from all the other plant species. No colonization occurred in any of the non-mycorrhizal treatments. Root hairs plants of the five species differed markedly in root hair development (Table 2). For Trifolium dubium, the mean root hair length was 127 //m whereas Lohum rigidum produced root hairs with an average length of 1155 ftm. The means of the estimates for total root hair length mm"^ root ranged from 5-1 mm for Trifolium dubium to 60 mm for Lolium rigidum. Except for Trifolium subterraneum and Medicago polymorpha, for which very similar estimates were obtained, estimates for total root hair length mm"' root increased in the same order as root hair length, so that numbers of root hairs mm"' root varied little between the species, with the exception of Ornithopus compressus which produced nearly twice as many root hairs as the other species.

6 252 P. F. Schzveiger, A. D. Robson and N. J. Barrow 20 r % Maximum dry weight of shoots 125 Figure 3. Relationship between the root fresh weight/ shoot dry weight ratio and "f, tnaximum dry weight of shoots of non-mycorrhizal plants {A as estimated by the Mitscherlich equation). Fitted line: y = Y V; R^ = Symbols as in Figure B 10 a m Mean root hair length (mm) 1 25 Figure 4. Relationship between mycorrhizal benefit ( ) and root hair length ( : y = 2-7- Benefit of producing long root hairs compared with shorter root hairs following Itoh & Barber's (1983) calculation is also given ( ). Trifolium dubium produced tbe finest roots, followed by Lolium rigidum, witb the otber tbree species having slightly thicker roots. Tbe root/shoot ratio of the non-mycorrhizal plants was a similar declining function in the five species (Fig. 3). Tbere was a strong inverse relationship (R^ = 0-98) between mean root hair length of the host and benefit due to mycorrhizal colonization by Glomus sp. (Fig. 4). A similar relationship, although not as clear, was obtained by plotting mycorrhizal benefit against total root hair length per mm root. benefit was not well related to root diameter, root length per plant nor root/shoot ratio. Also values for critical tissue P concentrations were of poor predictive value for mycorrhizat benefit. DISCUSSION There was a strong inverse relationship between root hair length and the beneficial effect of Glomus sp. for some pasture species. Tbis result therefore supports the initial hypothesis. It has to be considered, however, that tbe mycorrbizal benefit for Trifolium dubium was probably overestimated since the value for root density was much lower (only c %) compared witb the values recorded for the other species. Baath & Hayman (1984) and Koide (1991) showed strong inverse relationships between growth response due to mycorrhizas and root density. Applying the relationship between sboot weight response and root density for Koide's data to tbis experiment, the benefit for Trifolium dubium at a root density similar to the other species is still expected to be markedly greater than for Trifolium subterraneum, the species with the second greatest benefit. These findings are thus in good agreement with Baylis's hypothesis (1970). He stated that 'root hairs and phycomycetous mycorrhizas are alternative means of obtaining phosphorus that is not readily available'. The response to added P of the mycorrbizal plants of all five species, whose yields had been converted to percentage values of the estimated maximum yield, was quite well described by a common equation. Since the responses of the mycorrhizal and nonmycorrhizal Lolium rigidum plants to increasing levels of added P were not significantly different, values for the benefit of having long root hairs compared with shorter root hairs were very close to values for mycorrhizal benefit. Plants with longer root hairs than Lolium rigidum are not expected to benefit from mycorrhizal colonization, because the inverse relation of mycorrhizal benefit to root hair length in tbis study approached a value that was not significantly different from 1. Similarly, producing root hairs longer than c mm is not expected to lead to a marked increase in plant P uptake. Based on tbese observations and the strong inverse relationship between root hair length and mycorrhizal benefit itself, it is suggested that root hairs and external byphae of Glomus sp. can be considered as alternative ways of shortening the distance for tbe diffusion of phosphate in soil to absorbing structures. Direct comparisons between root hairs and external mycorrhizal hyphae are difficult to make. hyphae are only slightly thinner than root hairs, and values for length of hyphae cm"' plant root of the same order of inagnitude as root hair length cm"* root have been reported (Sanders & Tinker, 1973). However, external hyphal length cm"^ root produced by Glomus sp. can range up to 450 cm (Abbott & Robson, 1985). These values are well above estimates for root hair length cm"' root obtained in this study. However, because of the limited soil volume, not only root hairs but also mycorrhizal hyphae would have started to com^pete for nutrient uptake. The limited soil volume might have reduced the uptake effectiveness of the otherwise more dispersed hyphae. Itoh & Barber (1983), using a model that predicts P uptake by roots and root hairs, calculated the effect

7 Root hair lengths and effects of a Glomus sp. on pasture species 253 of varying root hair density and length on predicted P uptake W'hile keeping all the otber parameters constant. The effect they suggested of crowding root hairs together is similar to the effect on plant growtb of increasing the amount of external byphae (Gazey, unpublished). Using Itoh & Barber's (1983) estimates for P uptake of roots w-ith root hairs of different lengths, the relative increase in P uptake of roots with l-2mm-long root hairs compared with roots with shorter root hairs was calculated. It was assumed that an increase in root bair length beyond 1-2 mm would not increase P uptake. Witb tbe exception of root hair lengths below about 0 2 mm, the estimates obtained w^itb Itoh & Barber's model (1983) were of tbe same order as the values for mycorrbizal benefit presented in this paper and followed a similar trend (Fig. 4). More knowledge about the physiology of the fungi has to be gained before comparisons between fungal species showing different spreading patterns and root hairs of varying lengths can be made. There are reports in tbe literature of grasses witb abundant and long root hairs showing marked growth responses following mycorrhizal colonization (Mosse, Hayman & Arnold, 1973; Hetrick, Kitt & Wilson, 1988). As Hetrick f? a/. (1988) indicated, tbe marked growth response of grasses seems mostly to be conhned to species with a C^ photosynthetic pathway. The underlying cause of this phenomenon is still unknown. Hayman (1983) suggested that tbe greater photosynthetic efficiency of C^ plants might cause this tendency. In contrast to Hetrick, Wilson & Leslie (1991) and other studies (e.g. Pope et al, 1983; Graham & Syvertsen, 1985). mycorrhizal benefit was not well related to root diameter in the present experiment. These slightly contrasting results might be explained in the case of the studies of Hetrick et al (1988, 1991) by physiological differences betw^een C3 and C4 plants. Pope et al. (1983) compared the mycorrhizal dependence of hardwood tree species and found American sycamore to be much less dependent than tbe other species. Apart from having a fibrous root system, sycamore has also been reported to produce abundant root hairs (Richardson, 1953). A species of ash different from that used in the experiment of Pope et al. (1983) was observed not to form any root hairs on tbe main root (Dittmer. 1949). Although no reports on root hair development could be found for the other two species tested by Pope et al. (1983), it cannot be discounted that root hairs also had an effect on the outcome of their study. Graham & Syvertsen (1985) found that mycorrbizal dependence was negatively correlated with length of fibrous roots (i.e. roots with a diameter < 2 mm). That observation could not be confirmed in a later study (Graham, Eissenstat & Drouillard, 1991). However, root diameter cannot be disregarded as a parameter infiuencing mycorrhizal dependence, especially when as in tbe study of Graham et al. (1991) comparisons are made between plants of similar root hair development. In agreement with Baylis' hypothesis, it is concluded that root hairs and external mycorrhizal hyphae act as alternative ways of bypassing the phosphate depletion zone immediately around the root. Because of limited knowledge of the physiology of mycorrhizal fungi and even more so about the physiology of root hairs, direct comparisons betw'een root hairs and external hyphae are at the moment difficult to make. ACKNOWLEDGEMENTS P.F.S. received a Universit>' of Western Australia Research Scholarship throughout the course of this study. We wish to thank Lyn Abbott for invaluable comments and discussion throughout the study. REFERENCES Abbott LK, Robson AD The effect of VA mycorrhizae on plant growth. In: Powell CL, Bagyaraj DJ, eds. VA Alycorrliisa. Boca Raton. FL, USA: C.R.C. Press, Abbott LK, Robson AD Formation of external hyphae in soil by four species of vesicular-iirbu.scular mycorrhizal fungi. SeiL Phytologisl 99; Arias I, koomen I, Dodd JC, White RP, Hayman DS Growth responses of mycorrhizai and non-mycorrhizal tropical forage species to different levels of soil phosphate. Plant and Soil 132: Baath E, Hayman DS Effect of soil volume and plant density on mycorrhizal infection and growth response. Platit and Soil 11: Barrow NJ, Shaw TC Effects of solution: soil ratio and vigour of shaking on the rate of phosphate adsorption hy soil. Journal of Soil Science 30: bl-lh. Baylis GTS Root hairs and phycomycetous mycorrhizas in phosphorus-deficient soil. Plant and Soil 33: Boltz DF, Lueck CH Phosphorus. In: Boltz DF, ed. Colorimetric Determination of Non-metals. New York; Interscience Publishers, Colwell JD The estimation of phosphorus fertilizer requirements of wheat in southern New South Wales by soil analysis. Australian Journal of Experimental Agriculture and Animal Husbandry 3: Crush JR Plant growth responses to vesicular-arbuscular mycorrhiza. VIL Growth and nodulation of some herbage legumes. Se^i- Phyloiogist 73: Dittmer HJ Root hair variations in plant species. American Jotirnal of Botany 36: , Gazey C, Abbott LK, Robson AD The rate of development of mycorrhizas affects the onset of sporulation and production of external hyphae by two species of Acaulospora. Mviological Research 96; Graham JH, Syvertsen JP Host determinants of mycorrhizal dependency of citrus rootsiock seedlings. Neu' Phytologist 101: Graham JH. Eissenstat DM, Drouillard DL On the relationship between a plant's mycorrhizal dependency and rate of vesicular-arbuscular mycorrhizal colonization. Functional Ecology 5: Harley JL, Smith SE symbiosis. New York: Academic Press. Harley JL, Harley EL A check-list of mycorrhiza in the British rtora. Nev: Phytologist 105: Hayman DS The physiology of vesicular-arbuscular endomycorrhizal symbiosis. Canadian Journal of Botany 61: Hetrick BAD, Kitt DG, Wilson GT dependence and growth babit of warm-season and cool-season

8 254 P. F. Schzveiger, A. D. Robson and N. J. Barrow tallgrass prairie plants. Canadian Journal of Botany 66; Hetrick BAD, Wilson GWT, Leslie JF Root architetture ot warm- and cool-stason grasse.s: relationship to mycorrhizal dependence. Canadian Journal of Botany f>9: Itoh S, Barber SA Phosphorus uptake by six piant species as related ro roor hatrs. Agronomy yournal 75: ^ Johnson CM, Ulrich A Analytical methods for use in plant analysis. Bulletin of the Californian Agricultural Experimental Station. No. 7t»6. Koide RT Density-dependent re.'^pon.se to mycorrhiza! infection in Abutilon theophrasti Medic, Oecologia 85: Lewis DG, Quirk JP Phosphate diffusion in soil and uptake by plants. III. '"P movement and uptake by plants as indicated by ^'^P autoradiography. Plant and Soil 26: 445^53. Manjunatfa A, Hahte M Root morphological characteristics of host species having distinct mycorrhizal dependency. Canadian Journal of Botany 69; McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA A new method which gives an objective measure of colonization of roots by vesicular-arbuscular myccrrhizal fungi. New Phytologist 115; Mead R, Pike DJ, A review of response surface methodology from a biometric viewpoint. Biometrics 31; Mosse B, Hayman DS, Arnold DJ Plant growth responses to vesicular-arbuscular mycorrhiza. V. Phosphate uptake by tbree piant species from P-deficient soils labelled with *^P. New Phytologist 72; Nelder JA, Mead R A simplex method for function minimization. Computer Journal 7: Newman El A method for estimating the total length of root in a sample. Journal of.4ppl!ed Ecology 3; Nye PB, Tinker P. 1977, Solute movement in the soil-root system. Oxford: Btackwt'll Scientific. Pope PE, Chaney WR, Rhodes JD, Woodhead SH The mycorrhizal dependency of four hardwood tree species. Canadian Journal of Botany 61: 412-^17. Richardson SD A note on some differences in root-hair formation between seedlings of sycamore and American oak. NetL- Phytologist 52: Sanders FE, Tinker PB Phosphate flow into mycorrhizal roots. Pesticide Science 4: Scott BJ Diagnosis of magnesium deficiency by plant analysis. Ph.D. thesis. Perth, Australia. Tennant D A test of a modified line intersect method of estimating root length. Journal of Ecology 63: Viebrock H Ursachen der Erhohung des Phosphat- Aneignungs vermtigens von Pfiansen durch VA-Mykorrhiza. Ph.D. tbesis, Gottingen, Germany.

9