Effect of phosphorus nutrition on morphological characteristics of vesicular arbuscular mycorrhizal colonization of maize

New Phytol. (99), 9. 07- Effect of phosphorus nutrition on morphological characteristics of vesicular arbuscular mycorrhizal colonization of maize By P. G. BRAUNBERGER\ M. H. MILLERS AND R. L. PETERSON^ ' Department of Land Resource Science, University of Guelph, Guelph, Ontario, NIG 2W, Canada '^Department of Botany, University of Guelph, Guelph, Ontario, NIG 2WI, Canada (Received October 990; accepted 0 May 99) SUMMARY The morphology of the colonization of maize {Zea mays L.) by the vesicular arbuscular mycorrhizal fungus Glomus versiforme (Daniels and Trappe) Berch was determined quantitatively to establish the stage at which colonization was limited in response to increased P nutrition. Harvests were taken 4, 9, 24 and d after planting maize in a calcined clay medium to whicb nutrient solution containing 0'6, 0-8 or 2-4 mm P was added daily. Tbe fraction of tbe root lengtb containing arbuscules (FAC) 9 d after planting was 04 witb 0'6 mm P, 0-, witb 0'8 mm P and 0'7 witb 2^4 mm P. Corresponding sboot P concentrations were -5, 2-0 and 2 mg g '. Increased shoot P concentration reduced the number of appressoria per unit root lengtb, but tbis accounted for only a part of tbe reduction in FAC. Increased root length did not account for tbe effect of sboot P concentration on FAC. Tbe lengtb of arbuscular colonized root lengtb (total root lengtb x FAC) per appressorium was strongly reduced in response to increased sboot P concentration. It was concluded tbat tbe reduction m FAC was mediated primarily tbrough an inbibition of intra-radical development. Key words; Zea mays, Glomus versiforme, mycorrbiza, appressoria, arbuscule, pbospborus. the number of entrv points of the fungus into the TNTRODUCTION, -, ".> i i- root. Increased plant r concentration may also limit Increasing phosphorus fertilization reduces the pro- the growth of the fungus once internal colonization is portion of root length colonized by vesicular- established. arbuscular (VA) mycorrhizal fungi in susceptible Several studies show a negative correlation beplant species (Mosse, 97 ; Sanders, 975 ; Schwab, tween the number of fungal entry points per unit of Menge & Leonard, 98a; Thomson, Robson & root length and P amendment (Jasper et al., 979; Abbott, 986; Amijee, Tinker & Stribley, 989). Same, Robson & Abbott, 98; Thomson et al.. Although several hypotheses have been proposed, 986). Amijee et al. (989) reported differences in the mechanism of reduced colonization is not well the number of entry points that were, however, understood. much more variable than differences in internal There is strong evidence that the reduction is due colonization. Schwab et a!. (98 a) indicated that a to increased plant P rather than root-medium P decrease in fractional colonization with increased P concentration (Sanders, 975; Menge et al., 978; fertilization was better correlated with a decrease in Jasper, Robson & Abbott, 979). Increased tissue P the number of infection units than with the mean concentrations following fertilization could influence length of infection units. the effectiveness of inoculum, either spores or hyphal There are contradictory reports on the rate of pieces, possibly due to changes in the quantity or internal spread of the fungus in response to variable quality of root exudates (Ratnayake, Leonard & P nutrition. Amijee e/a/. (989) reported a decrease Menge, 978; Graham, Leonard & Menge, 98; in the growth rate of internal colonization fronts. Schwab, Menge & Leonard, 98^; Thomson et al., Increased P fertilization has been reported to cause 986). These effects would be most likely to decrease increases (Buwalda et al., 982), decreases (Schwab

08 P. G. Braunberger, M. H. Miller and R. L. Peterson et ai, 98fl; Thomson et al., 986; Amijee et al., 989) or no changes (De Miranda, Harris & Wild, 989; Smith. 982) in the total length of colonized root. The objective of this study was to quantitatively analyze the morphology of VA mycorrhizal fungal colonization of maize {Zea mays L.) in response to variable P fertilization. MATERIALS AND METHODS Pot culture Cultures of Glomus versiforme (Daniels and Trappe) Berch were established on maize (cv. Pioneer 949) in a calcined montmorillonitc clay rooting medium (Turface-Internationa] Minerals and Chemical Corp., Blue Mountain, Miss., USA). Mycorrhizal fungal inocula were introduced into - pots of Turface from established Glomus versiforme-allium porrum L. pot cultures. Three maize plants were grown for five 2-d cycles without disturbance of the root medium between cycles. Following the five cycles, four groups of 2 pots were mixed in bulk and repotted in four blocks which were used as replicates. To account for an expected decrease in inoculum potential due to the vigorous Turface disturbance from mixing (Evans & Miller, 988), maize plants were grown for two further 2-d cycles. For al] cycles, maize seeds were surface sterilized by soaking for 5 min in 5-25 % sodium hypochlorite. The seeds were then rinsed for 5 min and germinated in aerated distilled U'ater for 48 h. For the final pre-cycle and the experimental cycle, seeds weighing between 0-27 and 0-0 g were selected. Four seeds were planted per pot and then thinned to two plants which were grown for the experimental cycle. The nutrient solutions applied are described by Clark (982) except for modifications of P concentrations. All seven pre-experimental cyc]es received solutions containing 0-6 mm P. Three P concentrations, 2-4, 0-8 and 0-6 mm P, designated high. medium and low respectively, were used in the experimental cycle. Each - pot was watered to excess daily with 600 to 700 ml of nutrient solution. The ph of the nutrient solutions ranged from 5-0 to 5-6. The first five cycles (pre-blocking) were in a glasshouse with a diurnal temperature cyc]e ranging from 20 to 25 C. The two fina] pre-cycles (post blocking), and the experimental cycle were run in a controlled environment growth chamber set at a 6/h, 24/8 C day/night cycle. The photosynthetically active radiation of the days was set at 600//mol m-2 s-\ Hari'ests were taken in the experimental cycle 4, 9, 24 and d after planting (DAP). Measurements Leaf number and plant height were determined immediately prior to each harvest. Dry weight of shoots was measured, then the material was ground and acid digested for P and N analyses by the method of Thomas, Sheard & Moyer (967). Roots were washed gently free of Turface. cut into approximately 2-cm lengths and stored in a preserving and fixing solution of 7% formaldehyde, glacial acetic acid, and 95% ethanol (O-O5/O-O5/ 0-90 v/v/v). The whole root sample was cleared and stained as described by Brundrett, Piche & Peterson (984), and then stored in glycerine. Root lengths were determined by a modified line intersect method (Tennant, 975), Root diameter was assessed on a subsample of approximately one hundred intercepts using a calibrated eyepiece micrometer of a binocular microscope set at 40 times magnification. Assessment of colonization by VA mycorrhiza! fungi followed McGonigle et al. (990). This method determines, at 200 x magnification, the fraction of the root length containing arbuscules (FAC), the fraction containing vesicles (FVC) and the fraction containing either arbuscules, vesicles or hyphae (FHC). The latter is equivalent to the often used percent root length infected. In this study the fractional arbuscular colonization is denoted by the acronym FAC instead of AC as used by McGonigle et al. (990) in order to clearly differentiate fractional arbuscular colonization fronn the total arbuscular colonized root length which is denoted as ACL. To obtain a representative root sample for assessment, a subsample of roots was randomly selected by grid coordinates from the tray used for root length determination, and mounted on glass slides. Approximately 200 intersects were assessed from each root sample. Total root length colonized by arbuscules (ACL), by vesicles (VCL) and by any combination of arbuscules, vesicles or intra-radical hyphae (HCL) were calculated as the product of total root length and FAC, FVC and FHC respectively. For each of the 200 root intersects per sannple, the width of the field of view was scanned for total number of appressoria. All hyphal entry points observed in this study met the definition criteria for appressoria outlined by Garriock, Peterson and Ackerley (989). After a random subsample of the whole root system was selected, another random sample of only main axes was analyzed separately for FAC, FVC and FHC. Statistical analysis The experiment comprised a full factorial design of three phosphorus levels and four harvest times with four replicates arranged in randomized complete blocks. Analysis of variance was performed on all

Phosphorus and VA mycorrhizal morphology and colonization of maize data sets. Due to non-homogenity of variance, the dependant variables shoot dry weight, root length, hyphal-colonized root length (HCL), arbuscularcolonized root length (ACL) and vesicle-colonized root length (VCL) were log transformed for the analysis of variance. A separate analysis was also performed for each harvest on these parameters to provide an LSD applicable to each harvest. A further analysis was performed in which all dependent variables were regressed against the continuous independent variables of shoot tissue P concentration and days after planting. This provides a more powerful indicator of the effect of shoot P than does the class variable of phosphorus level in the rooting medium. The analysis of variance tables describe the regression model which provides tbe best fit of dependent variables against the shoot P concentration and days after planting. The type III sums of squares reported represent the variation unique to a given term in the model (Steel & Torrie, 980). Statistical effects were considered significant at the P > F 0'05 level unless otherwise stated. The FHC of the whole root system was inversely related to P amendment for the final three harvests (Table 2). The FHC of tbe main axes increased between the first and second harvest but w-as not related to P fertilization. The total length of colonized root (HCL) increased with time, but did not show any consistent relation with P fertilization (Table 2). The FAC was inversely related to P fertilization (Fig. 2). A harvest time by P fertilization interaction was significant (P < 0-05), with the FAC of the medium and high P treatment decreasing, and of the low P treatment increasing between the 4 and 24 d harvests (Fig. 2). The FAC of tbe main axes was RESULTS The three P treatments produced significantly different shoot phosphorus concentrations at each of the four harvest times (Fig. ). Phosphorus amendment increased leaf number at the final three harvests (Table ). Shoot dry mass and root length (Table ) increased at a tog linear rate with time, with improved P nutrition resulting in greater rates of increase. Mean root diameter decreased with time (Table ). 4 79 24 Figure. Shoot phosphotus concentration (mg g ^) of high, medium and low phosphorus treatmetits. The bar shown is the LSD at P = 0-05. Table. Effect of P rate and days after planting on shoot N concentration, shoot dry weight, leaf number, root length and root diameter Time (DAP) P rate Shoot N (mgg-') Shoot dnweight* (g pot"') Leaf number Root* length (m pot"^) Mean root diameter (mm) 4 9 24 Low Medium High Low Medium High Low Medium High Low Mediuni High LSD(P = 0-05) 42 4 46 42 42 40 9 42 4 40 4 4 40 0-0-46 0-46 (0-26) 0-75 0'99 98 (0-57) ]-05 2-66 4-95 (27) -84 5-8 9-5 (-5) 5-5-6 5-6 7'4 7'8 8 8 9-5 0-9' 0'5 8 08 7'8 9.4 8-7 (2-4) -0 5 9 (-) 8-24-0-0 (6-) 24-9-6 57-0 (2-6) 0-46 0-40 04 0-45 0'9 0-7 0-6 07 0 0-4 0'28 0-2 0-0 LSD {P = 0-05) for each harvest indicated in parentheses.

0 P.G. Braunberger, M. H. Miller and R. L. Peterson Table 2. Effect of P rate and harvest time on several colonization parameters. For abbreviations, see text. Time (DAP) P Rate FHC FHC (axes) HCL* (m pot"') FAC (axes) ACL* (m pot"^) FVC FVC (axes) VCL* ACL/VCL (cm pot~^) (axes) 4 9 24 Low- 0-54 Medium 0-54 High 0-42 Low 0'76 Medium 0-49 High 0-5 Low 0-84 Medium 0-46 High 0-40 Low 0'85 Medium 0'46 High 0-6 LSD(P = 0-05) 0-6 0-22 0-45 0-27 0-57 0'54 0-40 0-66 0-66 0-47 049 0-5 0-64 0-2 0-42 0-49 0-7 (0-) 0-0 7-6-6 (-9) 5'6 0'9 2-8 (4-) 7-4 2-4 20-5 (5-2) 0-0-27 0-4 0-26 0-28 0-9 0'2! 0'0 0-2 0-0 0-7 0-24 0-20 2-8 2-8 2-2 (0-7) 5-4 4'2-9 (0) 8-5 5-4-8 (2-i) 9-8 0-8-8 (6-) 0-09 0-0 0-08 0-2 0-0-09 0-0-8 0'7 0-28 0-6 0-2 0-0 0-06 0-4 0-07 0'27 0-2 0-9 0-42 0'4 0' 0' 0-2 0-4 0-8 66 9 79 (45) 02 78 7 (97) 56 420 59 (26 J) 682 750 624 (440) -9-9 2- -0-2 -5 0-5 0-8 0-5 0-6 0-7 l'o -6 LSD (P = 0-05) for each har\'est indicated in parentheses. 2.00-5-00-4.00- -00- voo- High P Medium P * Low P 2A Figure 2. Fractional arbuscular colonization of roots of high, medium and low phosphorus treatments. The bar shown is the LSD at P = 0-05. highly variable and not related to either P fertihzation or harvest time (Table 2). The ACL increased with time, and was inversely related to P fertilization for the 9 and 24 d harvests (Table 2). The FVC was inversely related to P fertilization at the second and subsequent harvests, and increased with time until the third harvest (Table 2). The FVC of the main axes was not affected by P fertilization but did increase with time similar to the FVC of the whole root system. Phosphorus fertilization did not significantly affect the ACL/VCL ratio, although the ratio declined with time in both the whole root system (Fig. ) and the main axes (Table 2). The decrease in ACL/VCL ratio in all treatments until the 24 d harvest reflects an increase in FVC with little change in FAC. Q.OO U 9 24 Figure. The arbuscule to vesicle colonization ratio (ACL/VCL) of high, medium and low P treatments. The bar shown is the LSD at P = 005. The number of appressoria per unit of root length (appressoria density) decreased with time (Fig. 4). There appeared to be a decrease in appressoria density with increasing P fertilization at the 9 and 24 d harvests but the main effect of P was significant only at P=0-4. The interaction between P and harvest was not significant even at P = 0-5. The appressoria density was, however, significantly related to shoot P concentration (Table a). The ratio of ACL to total number of appressoria was inversely related to shoot P concentration for the final three harvests (Fig. 5). The reduced regression models of appressoria density (Table a) and FAC (Table 4 a) against shoot P concentration and harvest time were modified

Phosphorus and VA mycorrhizal morphology and colonization of maize.-^ 0-80- 6 0-60- ^ 0-40 - Table 4. Analysis of variance of fractional arbuscular colonization as described by the (a) best-fit regression model against shoot P concentration {shoot P) and time and with (b) log of root length (T^^n^^J and {c) appressoria density included as covariates. F values test the significance of the type III sums of squares of each term in the model Source d.f. value P> F 0-20- O'OO Figure 4. Appressoria density for high, medium and law phosphorus treatments. The bar shown is the LSD at P = 0-05. {a) * *Time ib) T * «Time I! 4-97 4074-4 7'67-24 5-4-02 0-8 6-55 0-0050 O'OOOl 0-000 0-000 0-278 0-006 0-000 O'OOOl 0-045 Table. Analysis of variance of appressoria density as described by the {a) best-fit regression model against shoot P concentration [shoot P) and time and with (b) ^^S ^f ^oot length (Tig^gj^) include as a covariate. F values test the significance of the type III sums of squares of each term in the model { ) Appressoria (mm"') *»Time 6-56 0-45 5'46 22'59 47 0-044 0-7207 0-000 0-000 0-057 Source d.f. /value P>F - 0-7- E -0-0-4- Time * T ieugtu Time * 7'86 2-4 5-2 4-5 -28 6'87 0-9 -2-4 0-000 0-000 0-0276 0-095 0'264 0-000 0-544 0-0809 0-072 Low P Medium P with root length or appressoria density as covariates to indicate possible mechanisms of variation. When the log of root length (Ti^^j^,,^) is included in the reduced model for FAC, the effect of shoot P concentration and the shoot P concentration by harvest interaction remain highly significant (Table Ah). Thus T p,,jj,,, does not significantly account for variation in FAC. When the appressoria density is included in the model for FAC, the effect of shoot P concentration and shoot P concentration by harvest interaction remain significant (Table Ac). The appressoria density is, however, a significant source of variation, and Its inclusion in the model eliminates the block effect. The inclusion of Tj^^.^^.^^ in the regression model for appressoria density eliminates the effect of harvest, with the F value changing from 2-4 to 09 (Table 6). Root length also changes tbe significance of the shoot P concentration effect by changing the P >F from < 005 to between 0-05 and 00. The effect of harvest on appressoria density is apparently mediated primarily by changes in root length, while only part of the effect of shoot P concentration on appressoria density is mediated by root length. 0 - I I I I I I I I I I I 4 9 24 T Figure S. Arbuscular colonized root length (ACL) per appressoriutt\ for high, medium and low phosphorus treatments. The bar shown is the LSD at P = 005. DISCUSSION Phosphorus amendment resulted in the expected decrease in FAC (Fig. 2). The usually observed laglog-plateau pattern of mycorrhizal development with

2 P.G. Braunberger, M. H. Miller and R. L. Peterson time was not apparent. The FHC and FAC were near maximum 9 d after planting. The FVC increased with time for all P fertilization levels until 24 d after planting (Table 2). The delay in maximum FVC is not inconsistent w-ith the report that vesicle formation follows arbuscule formation (Brundrett, Piche & Peterson, 985). The absence of an effect of shoot P concentration on ACL/VCL (Fig. ) does nor agree with Abbott, Robson & De Boer (984) who observed that vesicle development decreased to a greater extent than arbuscule development with increasing P fertilization, and suggested that a decrease in energy supply hmited the production of lipid-dense vesicles. Although appressoria density accounted for some of the effect of shoot P concentration on FAC, the effect of shoot P concentration remained highly significant. Shoot P concentration may affect appressoria density through a reduction in extra-radical hyphal development which would limit the formation of secondary colonization points. Appressorium formation from secondary colonization cannot be separated from formation from dispersed inoculum in this study. Elimination of the effect of block on FAC (Table 4a) with the inclusion of appressoria density as a covariate in the regression model indicates that variation of FAC due to blocks was caused by differences in inoculum potential. Each block was composed of 2 pots selected from the pot culture development system. Differences in inoculum potential among blocks may have occurred with pot selection and mixing, or after blocking while inoculum levels were being raised just prior to the experimental cycle. Phosphorus fertilization had a strong effect on FAC in addition to its effects on root length and appressoria density. It is evident that this effect is expressed as a reduction in the intra-radical development of the fungus. The length of root colonized per appressorium increased with decreasing P amendment for the last three harvest times (Fig. 5). This contrasts with Amijee et al. (989) who reported no differences in the total colonization to entry points ratio. It is important to note, however, that in their study the inoculum was layered below the seed so that spread of colonization was dependent on mycorrhizas of the same root system. Thus, inhibition of intra-radical growth could limit secondary infection and appressorium formation to the same extent. Our report also contrasts with Schwab et al. (9Sa) who showed that the decrease in the percent root infection in response to increased P fertilization was more closely correlated w ith a decrease in the number of infections per unit root length than with a decrease in the mean length of each infection unit. Amijee et al. 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