Increased Sporulation of Vesicular-Arbuscular Mycorrhizal Fungi by Manipulation of Nutrient Regimenst

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 199, p /9/2413-6$2./ Copyright 199, American Society for Microbiology Vol. 56, No. 2 Increased Sporulation of Vesicular-Arbuscular Mycorrhizal Fungi by Manipulation of Nutrient Regimenst DAVID D. DOUDS, JR.,t AND N. C. SCHENCK* Plant Pathology Department, University of Florida, Gainesville, Florida Received 21 March 1989/Accepted 11 November 1989 Adjustment of pot culture nutrient solutions increased root colonization and sporulation of vesiculararbuscular mycorrhizal (VAM) fungi. Paspalum notatum Flugge and VAM fungi were grown in a sandy soil low in N and available P. Hoagland nutrient solution without P enhanced sporulation in soil and root colonization of Acaulospora longula, Scutellospora heterogama, Gigaspora margarita, and a wide range of other VAM fungi over levels produced by a tap water control or nutrient solutions containing P. However, Glomus intraradices produced significantly more spores in plant roots in the tap water control treatment. The effect of the nutrient solutions was not due solely to N nutrition, because the addition of NH4NO3 decreased both colonization and sporulation by G. margarita relative to levels produced by Hoagland solution without P. Large numbers of spores are frequently necessary to conduct research with vesicular-arbuscular mycorrhizal (VAM) fungi. Large populations of homogeneous spores are required for spore germination and storage research. More important, application of glasshouse-conducted research to the field depends upon the production of large amounts of inoculum of VAM fungi, including spores. These factors demonstrate the need for a broadly applicable method to enhance sporulation of VAM fungi in pot culture. Experiments were conducted to test the hypothesis that sporulation of VAM fungi would be increased if nutrient regimens were manipulated to provide conditions conducive to the bilateral transfer of nutrients (P from fungus to host and C from host to fungus). The ratio and amount of N and P in nutrient solutions were varied. Low levels of available P maintain high levels of colonization and enhance the efficacy of fungal transfer of P to the host. High levels of other nutrients increase the amount of photosynthate available to meet the metabolic demand for sporulation. Application of nutrient solution without P consistently yielded the greatest colonization of roots and production of soilborne spores by VAM fungi. MATERIALS AND METHODS Experimental material. Two-week-old seedlings of Paspalum notatum Flugge were transplanted into plastic pots (21 by 4 cm; Conetainers; Ray Leach Conetainer Nursery, Canby, Oreg.) containing pasteurized Arredondo fine sand soil (loamy, siliceous, hyperthermic Grossarenic Paleudult) previously inoculated with 2 to 4 spores of a VAM fungus placed on a cellulose filter buried under 5 cm of soil. P. notatum is a good host for the production of VAM fungal spores (13). The selected VAM fungi were Acaulospora longula Spain & Schenck (International Culture Collection of VA Mycorrhizal Fungi [INVAM] isolate 316), Scutellospora heterogama (Nicol. & Gerd.) Walker & Sanders * Corresponding author. t Florida Experiment Station Journal Series no t Present address: Plant and Soil Biophysics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, PA (INVAM 117), Glomus intraradices Schenck & Smith (IN- VAM 28), and four isolates of Gigaspora margarita Becker & Hall (INVAM 15, 185, 597, and 68). Plants were grown initially in a growth room with 14-h photoperiods (photosynthetic photon flux density, 8,mol m-2 min-'). Water only was applied during this period to allow for equal establishment of mycorrhizae across treatments. After 8 weeks, three plants were assayed for the percentage of root length colonized and VAM fungus spore populations (see below). The remaining plants were moved to a greenhouse, where they were grown with natural photoperiods for 1 to 12 weeks. Nutrient regimens were applied while the plants were in the greenhouse. Flowering shoots were removed as they appeared in order to avoid competition for photosynthates between root systems and reproductive structures. Experiments with A. longula and S. heterogama were conducted from 14 April through 24 September. Experiments with G. intraradices and Gigaspora margarita were conducted from 1 August through 15 December. Nutrient experiments. Twenty milliliters of one of seven nutrient solutions was applied three times per week (Table 1). Different N:P indices relative to that in the balanced, complete Hoagland nutrient solution (4) (solution 7) were achieved through manipulation of KH2PO4, Ca(NO3)2, and KNO3 concentrations. All N was supplied as NO3, the inorganic form of N that yields the greatest colonization of TABLE 1. Compositions of nutrient solutionsa Solution N:P Concn (mg kg-') of: no. index Ca(NO3)2 KNO3 KH2PO4 MgSO4 1 : 2 1: : : : : :2 1, asolutions also contained micronutrients as described by Hoagland and Arnon (4). One unit of N or P in the N:P index equals the approximate concentration of NO3 or P4, respectively, in half-strength Hoagland solution (7.5 and.5 mm, respectively). Downloaded from on November 3, 218 by guest 413

2 414 DOUDS AND SCHENCK APPL. ENVIRON. MICROBIOL. TABLE 2. Results of soil analyses conducted on pooled samples from the rhizospheres of plants colonized by A. longula Nutrient N:P Concn (mg kg-') of: soluion ndex ph solution index p P N (in NH4) N (in NO3) 1 : : : : : : : V,) tn (I en u Vl) 1o A I_ I+ Acculospora longula plants by VAM fungi (7). All nutrient solutions were applied to plants colonized by A. longula and G. intraradices; solutions 1, 2, and 4 were applied to plants colonized by S. heterogama; and solutions 1 through 5 were applied to plants colonized by Gigaspora margarita. Five replicates per nutrient solution were used for each fungal species except Gigaspora margarita, for which three replicates were used. A second experiment was a survey to test the results of the initial nutrient addition experiments. A variety of hostfungus associations was grown in soils conducive to the fungal isolates. Cultures were prepared as described above and divided into two groups. One received the routine cultural practices of INVAM (monthly addition of 2 ml of [N-P-K] fertilizer; Peter's Fertilizer Products, W. R. Grace & Co., Allentown, Pa.), and the other received 2 ml of full-strength Hoagland solution minus P three times per week for the final 4 of 6 months of growth. Only one or two cultures per symbiont combination per nutrient regimen were used, ruling out certain statistical analyses. Plants were grown from January through June. Cultures were analyzed for spore numbers and percentage of root length colonized by VAM fungi (see below). A third experiment was conducted to determine if the effect of nutrient addition on sporulation was due solely to nitrogen. Experimental material was prepared as described above. One of the following three nutrient solutions was applied to P. notatum colonized with one of two isolates of Gigaspora margarita (INVAM 185 or 68): (i) tap water; (ii) solution 3 (Table 1); or (iii) NH4NO3, which yielded the same concentration of N as solution 3 (14 mm N). No P was added to plants in this experiment. The ph of each solution was adjusted to 6.8 with.5 N H2SO4. Five replicates per isolate per nutrient level were prepared. This experiment was conducted from 26 May through 16 October. Solutions were added three times per week for the final 11 weeks. The percentages of root length colonized by the VAM fungi, spore numbers, and root morphology were studied (see below). Data collection. Soilborne VAM fungal spores were counted in a 45- or 53-cm3 section of soil in the middle of the pot, 5 cm below the soil surface. Spores were isolated by wet sieving (2) and centrifugation (5). Since spores of G. intraradices may be found in roots and soil, intraradical spores produced by G. intraradices were counted in all roots in 1-cm sections above and below the soil sampled for soilborne spores. Roots were cleared in 1% KOH, which allowed for quantification of G. intraradices spores with the dissecting microscope. The gridline-intersect method (11) was used to estimate the percentage of root length colonized by the VAM fungus and was quantified for all species except G. intraradices with roots from the soil section used to sample soilborne spores (12). Roots used for the enumera- I (a ~ U) 1) tn U) 1k Nutrient Solution Number FIG. 1. Sporulation of A. longula (A), S. heterogama (B), and G. intraradices (C) grown in association with P. notatum. Means of five observations are shown, with error bars representing the standard error of the mean. See Table 1 for compositions of nutrient solutions. Spore populations 2 months after inoculation and before the addition of nutrient solutions were 24.3 and 5.1 spores cm-3 for A. longula and S. heterogama, respectively. tion of intraradical spores were used to measure colonization of plants by G. intraradices. Phosphorus contents in leaf and root tissues of plants colonized by A. longula and Gigaspora margarita (INVAM 597 and 68) were analyzed by combustion in a muffle furnace and quantified by the ascorbic acid-molybdenum blue method (1). At the end of the experiment with A. Iongula, soils were analyzed by ph (in water), P (Mehlich I extractable), and NH4' and N3 (water extractable) by the Downloaded from on November 3, 218 by guest

3 VOL. 56, 199 SPORULATION OF VAM FUNGI 415 Florida Institute of Food and Agricultural Sciences Extension Soil Testing Laboratory. Data were analyzed by analysis of variance and least squares linear regression. Significant treatment effects were characterized further with the Duncan multiple-range test. Percent colonization data were analyzed after arcsine transformation. Data for the survey of host-fungus-soil combinations were analyzed after differences in sporulation and colonization between paired cultures from the two treatments were calculated. Sporulation data (normalized [Shapiro-Wilk W test] by a log1o transformation) and colonization data were then analyzed with a t test (ot =.5). RESULTS Soil characteristics, plant growth, and P nutrition. Addition of nutrient solutions containing KH2PO4 (solutions 4 through 7) resulted in an increase in the ph of Arredondo fine sand (Table 2). P concentrations in soil reflected the nutrient additions. N3 concentrations in soil-appeared to be greater for the high-n addition than for the'low-n addition only for treatments in which no P was added (solutions 2 and 3). Data for plant growth and P contents were presented only for plants colonized by A. longula. These data are complete for all nutrient solutions studied and reflect data for plants colonized by other VAM fungi. Addition of nutrient solution without P (solutions 2 and 3) resulted in an increase in shoot biomass of P. notatum over that of the control (water only added) (Table 3). Addition of N and P (solutions 4 through 7) caused a further increase in shoot mass, a trend toward less root mass, and a corresponding decrease in root/shoot ratio relative to the addition of nutrient solution without P. P concentrations in shoots and shoot contents reflected nutrient additions and plant growth. The growth stimulation caused by solutions 2 and 3 resulted in a dilution of tissue P (Table 3). At the lowest level of P addition, P concentrations in shoots increased to the levels obtained when only water was added (solutions 4 and 5; N:P = 1:1 and 2:1 [Table 3]). P concentrations in roots exhibited responses different from those in shoots. P concentrations in roots remained constant as growth increased with the addition of nutrient solution without P and increased with P addition (Table 3). The P concentrations in shoots and roots decreased because of increased plant growth with increasing levels of N at a given level of P addition. Spore numbers and colonization of roots of P. notatum. The addition of nutrient solutions without P (solutions 2 and 3) significantly increased sporulation of A. longula, S. heterogama, and three isolates of Gigaspora margarita (INVAM TABLE 3. 15, 597, and 68) over sporulation in water controls and nutrient solutions with P (Fig. 1A and B and 2a, c, and d, respectively). Sporulation of A. Ionggula exhibited a patterned response to N:P indices of the nutrient solutions. Sporulation decreased as P increased. At a given P level, sporulation was greater at the higher N level, e.g., N:P ratios of 1:1 versus 2:1 and 1:2 versus 2:2 (Fig. 1A; solutions 4 versus 5 and 6 versus 7, respectively). This tendency was not seen for Gigaspora margarita isolates (Fig. 2) or for soilborne spores of G. intraradices (data not shown). G. intraradices differs from other species used in this experiment because it sporulates primarily in the host root rather than in the soil. Spore densities of this fungus averaged less than 1.3 spores cm-3 of soil. It also responded differently to nutrient addition. Sporulation in roots receiving tap water (solution 1) was much greater than that in roots receiving other treatments (Fig. 1C). There were no significant differences in sporulation among the other treatments. The total number of spores per root system was also greater for plants receiving only water (mean standard error of the mean, 81,6 + 2,2 for water, 8,35 3,35 for solution 3, and 1, for solution 7). Nutrient addition had a significant effect on the percentage of root length colonized by the VAM fungi. For all species studied, the addition of nutrient solution without P (solution 2 or 3; N:P = 1: or 2:) significantly increased the percentage of root length colonized compared with the addition of water (Table 4). Colonization by A. Iongula, G. intraradices, and most of the Gigaspora margarita isolates increased with increasing N addition at a given level of P. Although solution 3 (N:P = 2:) produced the greatest sporulation by A. longula, the number of spores per unit of root weight was not significantly different from that of cultures receiving water only (mean + standard error of the mean, 54.5 x x 13 versus 75.7 x x 13 spores g [dry weight] of root-', respectively). Expressing sporulation on the basis of colonized-root weight showed that the water treatment (N:P = :) yielded significantly more spores than other treatments (mean + standard error of the mean, x x 13, x x 13, and 72.6 x x 13 spores g of dry colonized-root weight-' for N:P ratios of :, 2:, and 2:2, respectively). Spore populations in soil were all significantly (P <.1) and positively correlated to the percentage of root length colonized (r2 =.688,.531, and.696 for A. longula, Gigaspora margarita (INVAM 15), and S. heterogama, respectively). Sporulation of G. intraradices within the roots of P. notatum, however, was not correlated to colonization (r2 =.25). Physical characteristics and P concentration of P. notatum seedlings colonized by A. longula and fertilized with nutrient solutiona Nutrient N:P Dry wt (g) of: Dry wt ratio P concn (% [dry wt]) in: Solution index Shoot Root (root/shoot) Shoot Root 1 :.923 (d).989 (d) 1.2 (d).13 (b).46 (c) 2 1: 4.14 (c) 2.32 (a, b).58 (b).37 (c).45 (c) 3 2: (c) 2.62 (a).69 (b).44 (c).42 (c) 4 1: (b) (c).3 (c).135 (b).8 (a) 5 2: (a, b) (b, c).32 (c).11 (b).68 (b) 6 1: (a) (a, b).35 (c).189 (a).87 (a) 7 2: (a) 2.96 (b, c).33 (c).141 (b).81 (a) a Values are means of five observations. Values followed by the same letter in a column are not significantly different (a =.5; Duncan multiple-range test). Physical characteristics of plants after 2 months of growth, before the beginning of nutrient additions: dry weight of shoot,.16 g; dry weight of root,.287 g; and dry weight ratio (root/shoot), 1.85 (n = 3). Downloaded from on November 3, 218 by guest

4 416 DOUDS AND SCHENCK APPL. ENVIRON. MICROBIOL. to.5_ 1 E 31. u} o 21 a!o ri o f6 C II _ K 6 Ln I 4- E uin 4 IIV Q 11~ 2 _-_ U) 2 D I~~T T F I Nutrient Solution Number Nutrient Solution Number FIG. 2. Sporulation of four isolates of Gigaspora margarita (15 [a], 185 [b], 597 [c], and 68 [d]) grown in association with P. notatum. Means of three observations are shown, with error bars representing the standard error of the mean. See Table 1 for compositions of nutrient solutions. The populations 2 months after inoculation and before the addition of nutrient solutions were 9., 5., 2.8, and 2.1 spores cm-3 for isolates 15, 185, 597, and 68, respectively. The survey experiment that tested the addition of nutrient solution 3 minus P (N:P = 2:) on a variety of VAM fungi, hosts, and soils confirmed the results shown above. The only VAM fungus isolate tested that showed less sporulation with the addition of Hoagland solution minus P was Scutellospora dipapillosa (INVAM 678) (Table 5). The percentage of root length colonized was greater for Hoagland solution without P in all plant-fungus-soil combinations studied. Analysis of paired observations showed significantly greater sporulation TABLE 4. Percentage of root length of P. notatum colonized by VAM fungi as affected by nutrient additiona % Root length colonized by: Nutrient N:P Gigaspora solution index A. longula S. hetero- G. intra- margarita gama radices INVAM 68 1 : 24 (d) 41 (c) 5 (b) 17 (c) 2 1: 61 (a) 71 (a) 55 (b) 27 (b) 3 2: 59 (a) 65 (a) 5 (a) 4 1:1 38 (b, c) 6 (b) 53 (b) 22 (b, c) 5 2:1 46 (b) 64 (a) 42 (a) 6 1:2 18 (d) 44 (c) 7 2:2 34 (c) 56 (b) a Values are means of five observations except for Gigaspora margarita (means of three observations). Values followed by the same letter in a column are not significantly different (a =.5; Duncan multiple-range test). Colonization of plants 2 months after inoculation and before addition of nutrients: A. longula, 2%; S. heterogama, 21%; G. intraradices, 41%; and Gigaspora margarita, 23%. 5 d and colonization with the addition of solution 3 three times per week than with the monthly addition of (N-P-K) fertilizer. The addition of NH4NO3 to P. notatum produced plants intermediate in size between those receiving water and those receiving the balanced nutrient solution minus P (data not shown). Spore populations and root colonization levels were not increased by the addition of NH4NO3 (Table 6). DISCUSSION We have found strong support for the hypothesis that sporulation of VAM fungi is enhanced when the pot culture nutrient regimen is manipulated to provide high amounts of nutrients other than P. Two processes may be at work here: the transport of fixed carbon from plant to fungus and the effect of nutrition on the level of colonization of the root system by the VAM fungus. The addition of nutrient solution without P greatly increased the growth of plants above ground under the conditions of these experiments. Nitrate addition has been shown to increase the rates of photosynthesis and assimilate export (9). This would make more C available for partitioning below ground, including intraradical structures of the fungus and transport to soilborne hyphae. Spores of VAM fungi contain much lipid, and their production would require much C. Transport of C into extraradical hyphae would work in concert with the low P concentrations in the soil and plant to enhance the spread of colonization. A highly colonized root Downloaded from on November 3, 218 by guest

5 VOL. 56, 199 SPORULATION OF VAM FUNGI 417 TABLE 5. Effect of three-times-weekly addition of nutrient solution without P (solution 3) or monthly addition of (N-P-K) fertilizer on sporulation and colonization for several fungus-host plant-soil combinations Sporulation Root length colonization INVAM isolate Host Soilb No. of (spores cm of soil-3) with: (%) with: plant(s) observations Fertilizer Solution 3 Fertilizer Solution 3 Gigaspora margarita 15A, S C Gigaspora margarita 15B, S C Gigaspora margarita 274 B, A A Gigaspora margarita 15 B, A A Gigaspora rosea 676 B, A A Scutellospora gregaria 336, S A Scutellospora heterogama 117 B, S A Scutellospora pellucida 37 B, S A Scutellospora pellucida 371B B, S B Scutellospora pellucida 371C B, S B Scutellospora dipapillosa 678 B, A A Glomus clarum 24 B, S A c Glomus etunicatum 452 B, A A Glomus etunicatum 67 B A Acaulospora sp. B, A A a A, Medicago sativum; B, P. notatum;, Allium cepa; S, Stylosanthes quianensis. b A, Arredondo fine sand (loamy, siliceous, hyperthermic Grossarenic Paleudult); B, Wauchula sand (sandy, siliceous, hyperthermic Ultic Haplaquods); C, Hague sand (loamy siliceous, hyperthermic Arenic Hapludalfs). c Spores mg of root (dry weight)-'. system would have more hyphal connections to the source of C for the production of soilborne spores. G. intraradices produced more spores when cultures received only water than when they received other treatments. Possibly because of its habit of sporulating in the root, Glomus clarum produced more spores in roots when plants received Hoagland nutrient solution without P than when they received a treatment similar to the tap water received by G. intraradices. Another experiment with G. intraradices showed colonization to be unaffected and sporulation to be enhanced after 26 weeks by applications of P (6). Therefore, G. intraradices may respond to stress differently from other VAM fungi. Literature on the effect of nutrient addition other than P on VAM fungus colonization is divided between reports of positive and negative effects on the percentage of root length colonized. Hepper (3) found that colonization levels increased with increasing NO3 application at all levels of P studied. Basal fertilizer without P improved colonization in soils left fallow for long periods when inoculated with soil from cropped fields (14). In addition, Verkade and Hamilton (15) found increased colonization with the addition of slow- TABLE 6. Spore populations and percentages of P. notatum root length colonized by one of two isolates of Gigaspora margarita Results with isolate': Nutrient solution No. of Colonization No. of Colonization spores cm-3 (%) spores cm-3 (%) Water 17.5 (b) 53 (b) 4.8 (b) 25 (b) Solution (a) 67 (a) 13.7 (a) 39 (a) NH4NO (b) 58 (b) 1.8 (b) 34 (a, b) a Values are means of five observations. Values followed by the same letter in a column are not significantly different (a =.5; Duncan multiple-range test). release fertilizer containing N, P, and K. Other researchers have seen lower percentages of root length colonized after the addition of N (1, 8). We conclude that the addition of nutrient solution without P increases the colonization of roots in soils low in N. A. longula produced significantly more spores per unit of colonized-root weight when receiving water than when receiving the different nutrient solutions. This may be an adaptation of this fungus to optimize reproduction under the nutrient-poor conditions commonly found in nature. We have presented a pot culture nutrient regimen that increases the sporulation of VAM fungi that produce spores in the soil. One of two fungi tested which produce spores in the roots of the host plant also responded favorably. Nutrient solution without P is added to P. notatum grown in sandy soil low in N and available P. This method promises to be quite beneficial in the production of VAM fungus inoculum because it increases spore populations and enhances colonization of roots. ACKNOWLEDGMENTS This work was supported by National Science Foundation grant BSR We thank D. M. Sylvia and J. H. Graham for their reviews of the manuscript. LITERATURE CITED 1. Buwalda, J. G., and K. M. Goh Host-fungus competition for carbon as a cause of growth depressions in vesiculararbuscular ryegrass. Soil Biol. Biochem. 14: Gerdemann, J. W., and T. H. Nicolson Spores of mycorrhizal Endogone species extracted by wet sieving and decanting. Trans. Br. Mycol. Soc. 46: Hepper, C. M Effect of nitrate and phosphate on the vesicular-arbuscular mycorrhizal infection of lettuce. New Phytol. 92: Hoagland, D. R., and D. I. Arnon The water-culture method for growing plants without soil. Agricultural Experi- Downloaded from on November 3, 218 by guest

6 418 DOUDS AND SCHENCK ment Station circular 347. University of California College of Agriculture, Berkeley. 5. Jenkins, W. R A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Dis. Rep. 48: Johnson, C. R Phosphorus nutrition on mycorrhizal colonization, photosynthesis, growth and nutrient composition of Citrus aurantium. Plant Soil 8: Johnson, C. R., W. M. Jarrell, and J. A. Menge Influence of ammonium:nitrate ratio and solution ph on mycorrhizal infection, growth and nutrient composition of Chrysantheinum morifolium var. Circus. Plant Soil 77: Johnson, C. R., J. N. Joiner, and C. E. Crews Effects of N, K, and Mg on growth and leaf nutrient composition of three container grown woody ornamentals inoculated with mycorrhizae. J. Am. Soc. Hortic. Sci. 15: Kerr, P. S., D. W. Israel, S. C. Huber, and T. W. Rufty, Jr Effect of supplemental N3- on plant growth and components of photosynthetic carbon metabolism in soybean (Glycine max). Can. J. Bot. 64: Murphy, J., and J. P. Riley A modified single solution APPL. ENVIRON. MICROBIOL. method for the determination of phosphate in natural waters. Anal. Chim. Acta 27: Newman, E. I A method of estimating total length of root in a sample. J. Appl. Ecol. 3: Phillips, J. M., and D. S. Hayman Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55: Struble, J. E., and H. D. Skipper Vesicular-arbuscular mycorrhizal fungal spore production as influenced by plant species. Plant Soil 19: Thompson, J. P Decline of vesicular-arbuscular mycorrhizae in long fallow disorder of field crops and its expression in phosphorus deficiency of sunflower. Aust. J. Agric. Res. 38: Verkade, S. D., and D. F. Hamilton Effects of soil fertility on growth, nutrient concentration and mycorrhizal development of Liriodendron tulipifera seedlings inoculated with the vesicular-arbuscular mycorrhizal fungus, Glomus fasciculatus. Sci. Hortic. (Amsterdam) 21: Downloaded from on November 3, 218 by guest