SUMMARY

Incorporation of inoculum of arbuscular-mycorrhizal fungi (AMF) into containers filled with the sand/peat medium recommended for putting greens resulted in more rapid growth and establishment of creeping bentgrass (Agrostis palustris 'Penncross') and velvet bentgrass (A. canina 'Kingstown'). Two species of AMF, Gigaspora gigantea and Glomus intraradices, were tested and found to be effective. At low phosphorus concentrations, inoculated turf produced 40-75% more leaf matter than did non-mycorrhizal turf. At higher P concentrations, mycorrhizal-induced growth responses varied from no effect to a 63% increase in foliar biomass.

INTRODUCTION

Until recently, very little was known of the relationship between the symbiotic arbuscular mycorrhizal fungi (AMF) and amenity turf species (Rhodes & Larsen 1981, Petrovic 1984, Koske et al. 1997). AMF enter into association with the roots of most species of vascular plants (Harley & Smith 1983, Harley & Harley 1987), including the majority of grasses (Newman & Reddel 1987, Trappe 1987). While the mycorrhizal association often is reported as being beneficial to the plant (e.g. improved P nutrition and increased tolerance to drought, disease and pests), some instances of growth depression have been noted (Buwalda & Goh 1982, Kiernan et al. 1983, Modjo & Hendrix 1986, Thompson & Wildermuth 1989, Newsham et al. 1995).

Putting greens constructed according to the U.S. Golf Association (USGA) specification (Bengeyfield 1989) lack AMF at the time of sowing (Koske et al. unpubl. observ.) and populations of the fungi are slow to increase in the green (Koske et al. 1997). Thus, any beneficial effects that AMF may confer during the early establishment of turfgrass in a newly constructed putting green cannot be realised. Based upon earlier reports of the beneficial effects of AMF on the growth of various non-turfgrass species in the Poaceae (Hayman 1983, Hall et al. 1984, Sylvia & Burks 1988, Gemma & Koske 1989), it was postulated that incorporation of AMF inoculum into putting greens at the time of construction might result in the more rapid estalishment of the turf. The premise was tested on two common turf species used in putting greens: creeping bentgrass and velvet bentgrass. In addition, preliminary investigations were made into the effect of phosphorus fertilisation on the resultant growth.

MATERIALS AND METHODS

Growth medium, seeding and inoculation
Three greenhouse experiments were perform-ed, two involving creeping bentgrass (Agrostis palustris L. 'Penncross') and one using velvet bentgrass (A. canina L. 'Kingstown'). Experi-mental parameters are summarised in Table 1. The growing medium was a soil-less mixture consisting of four volumes of quartz sand to one volume of milled Canadian Sphagnum peat following the specification for use in putting greens recommended by the USGA (Bengeyfield 1989). Sand was pasteurised before use and its particle size distribution was: 1-2 mm = 2.01%, 0.5-1 mm = 36.6%, 240-500 µm = 52.8%, 106-200 µm = 8.4% and 106 µm = 0.14%. The pH of the medium was adjusted to 6.3 with lime after the fungal inoculum was added.

The velvet bent test was performed in tapered plastic containers of 165 ml capacity ("Cone-tainers"® [Steuwe and Sons, Corvallis, OR 97333]) measuring 20.7 cm ht. x 3.8 cm diam. and the creeping bent tests were carried out in 560 ml capacity tapered plastic containers ("Deepots"® [Stuewe and Sons, Corvallis, OR 97333]) measuring 25 cm x 6 cm. A piece of plastic window screen was inserted into each Cone-tainer and Deepot before filling to keep the contents from emptying out.

Seeds of both species were surface-sterilised by soaking for 20 mininutes in a 1.05% NaOCl solution containing a drop of Tween 80, followed by several rinses with sterile distilled water. After the final rinse, seeds were air-dried on filter paper. In the velvet bent study, fifteen seeds were sown in each Cone-tainer. After ten days, seedlings were thinned to ten per Cone-tainer. For the creeping bent, 0.14 g of the seed was surface sown in each Deepot.

Two types of inocula were used. For the velvet bent (VB) and one of the creeping bent trials (CB-1), an attapulgite-based inoculum of Glomus intraradices Schenck & Smith ("Nutri-Link"® [NPI, Salt Lake City, UT 84108]) containing 1000 spores g^-1 was dispersed throughout the medium. Each Cone-tainer and each Deepot received 8.2 ml (12.5 g) and 30 ml (46 g) of inoculum respectively. The size of the Nutri-Link particles ranged from 1.5-4.5 mm (avg. 3 mm). The control treatment contained the same volume of attapulgite carrier particles (also provided by NPI) without spores. A different type of inoculum was employed in the CB-2 trial. This consisted of chopped roots of Asparagus officinalis L., spores and 2-4 mm diameter particles of calcined clay (Terra-Green® [Oil Dri Corp., Chicago, IL 60611]) from a four-month-old pot culture of the AMF Gigaspora gigantea (Nicol. & Gerd.) Gerd. & Trappe. The soil and roots were air dried and refrig-erated for one month prior to use. The control treatment for this study was prepared from Asparagus plants that had been grown in Terra-Green in the absence of the fungus and consisted of chopped roots and particles of Terra-Green. The controls had been exposed to the same conditions (duration of growth, fertilisation schedule, light regime, drying and refrigeration) as had the plants inoculated with G. gigantea. Six g of inoculum or control material was dispersed throughout the contents of each Deepot as it was filled.

The Gi. gigantea was originally isolated from a P-deficient sand dune in Rhode Island. The site of origin of Gl. intraradices is not known, but is not thought to have come from a sand dune (NPI, pers. comm.).

Growth conditions
Plants that were grown in a heated glasshouse (expts. VB and CB-1) received supplemental lighting from high pressure sodium vapour lamps giving an intensity at the leaf surface of 350-1375 µE m^-2 sec^-1 on a 16/8 L/D cycle. The mean minimum (night) and mean maximum (day) temperatures were 21 and 27°C respectively. Plants in expt. CB-2 were grown on a laboratory bench under fluorescent lighting (using a combination of Gro-Lux® and Vitalite® full spectrum, high-output lamps) suspended 26.5 cm above the edge of the Deepots, giving a light intensity of 260-285 µE m^-2 sec^-1 on a 16/8 L/D cycle. The temperature in the growth room ranged from 20-34°C (avg. 26.5°C). Plants were watered as needed (ca. every other day) with tap water.

Containers in each experiment were fertilised with a modified Hoagland's solution at the beginning of the experiment and every two weeks after. The Hoagland & Arnon (1950) macronutrient solution was applied at 1/2-strength, Fe-EDTA at 46 mg/L, Epstein's (1972) micronutrient solution at 1/4-strength, and phosphorus at 7.5, 15.0 or 30 mg l^-1 supplied as KH₂PO₄. The pH of the fertiliser solution was 6.0-6.2. Deepots received 50 ml of this solution and Cone-tainers received 20 ml at each fertilisation. All three P concentrations were tested on velvet bent turf, and 7.5 and 30 mg l^-1 P were used for the creeping bent trials.

Assessment of response
The effects of inoculation and P level were assessed by measuring the dry weight of the leaves at the end of the experiment. Leaves were clipped at the level of the top of the container and dried at 50 C for 24 hr before weighing.

At the completion of each experiment, roots of the control and inoculated plants in each experiment were collected, cleared and stained (Koske & Gemma 1989). Stained roots were examined at X40-60 with a dissecting microscope, and portions of each root system were examined at X400 with a compound microscope. The presence of vesicles, arbuscules, hyphal coils and internal hyphae was noted for each specimen. Only those specimens in which arbuscules were found were considered to have formed functional mycorrhizas.

Data were analysed by ANOVA, means were separated using Duncan's Multiple Range Test and significance was assigned at P<0.05. Because the experiments were performed at different times and under different growth conditions, results from one experiment were not compared with the other experiments.

RESULTS

In all three trials, turf inoculated with AM fungi produced significantly more leaf matter (40-75% more) than did non-mycorrhizal turf when fertilised with the lowest concentration of P (7.5 mg l^-1) (Fig. 1). In creeping bent inoculated with Gl. intraradices (CB-1), significant growth increases (41 and 63%) occurred in mycorrhizal turf at both P concentrations. When creeping bent was inoculated with Gi. gigantea, there were no significant differences at 30 mg l^-1 P. Similarly, velvet bent showed no enhancement by Gl. intraradices at 15 or 30 mg l^-1 P. A 75% increase in growth resulted when Gi. gigantea was used with creeping bent at 7.5 mg l^-1 P.

In the creeping bent trials, the mycorrhizal plants fertilised with 7.5 mg l^-1 P produced more leaf matter than did the non-mycorrhizal plants grown with four times as much P (30 mg l^-1) (Fig. 1). The mycorrhizal growth benefit in velvet bent at low P could be achieved in non-mycorrhizal turf by applying additional P.

At the conclusion of the experiments, roots of all inoculated turf were mycorrhizal, and non-inoculated turf was free of mycorrhizas.

FIGURE 1. Leaf dry matter production by creeping bent and velvet bent in response to inoculation with arbuscular mycorrhizal fungi and fertilisation with nutrient solutions containing different amounts of phosphorus. Creeping bent was inoculated with Glomus intraradices (CB-1) or Gigaspora gigantea (CB-2), and velvet bent (VB) was inoculated with Gl. intraradices. White bars indicate growth of non-mycorrhizal turf, shaded bars represent mycorrhizal turf. Bars in each experiement containing the same letter did not differ significantly (Duncan's Multiple RangeTest, P<0.05).

DISCUSSION

Inoculation of two frequently used species of greens turf with AMF was shown to significantly improve establishment in the sand/peat putting green mix under certain conditions of P fertilisation. These results are in agreement with numerous reports documenting the influence of soil P on the extent of growth enhancement by AMF (Mosse 1973, Biermann & Linderman 1983, Harley & Smith 1983, Kiernan et al. 1983, Miller et al. 1987, Habte & Fox 1993) in a variety of non-turf species.

Fine-rooted cool-season (C3) grasses inoculated with AMF have been reported to produce greater shoot biomass than did non-mycorrhizal plants at low P concentrations (Hall et al. 1984, Petrovic 1984, Hetrick et al. 1986, 1988). As in our study, growth benefits were not always apparent at higher P levels in the studies cited above, and in some cases mycorrhiza caused a growth depression (Crush 1976, Biermann & Linderman 1983, Kiernan et al. 1983, Petrovic 1984).

This experiment and others cited above clearly demonstrate that amount of P applied is an important factor that determines the ability of AMF to promote growth of turf species in the sand/peat putting green mix. Species of AMF differ in their ability to improve plant growth and in their behavior at different P con-centrations (Harley & Smith 1983, Sylvia & Schenck 1983, Davis et al. 1984, Hetrick et al. 1986). Species or isolates that promote plant growth over a broad range of soil P levels may be better suited for use in putting greens than were the fungi used in the trial reported in our study. No such isolates are com-mercially available at present, but there are no scientific reasons preventing the production of inoculum with greater soil P tolerance.

Application of the results from these indoor experiments to preparation of new putting greens will require further study to determine appropriate inoculation rates as well as optimal levels of soil nutrients and pesticide effects. The size of the inoculum particles (1.5-4.5 mm) may need to be reduced to conform to the USGA specification for particle size to maintain the drainage properties of the medium.

In addition to growth enhancement, AMF may provide other benefits to greens turf. Mycorrhizal fungi can confer significant protection to creeping bentgrass against drought stress even in the apparent absence of growth enhancement (Koske et al. 1995, Gemma et al. 1997). Further, AMF may confer some protection against nematodes and fungi that attack roots (Thompson & Wildermuth 1989, Newsham et al. 1995, Little & Maun 1996). Management of greens turf may require new strategies to accommodate AMF populations and maximise their benefits.

ACKNOWLEDGEMENTS

This study was supported by the Greens Research Section of the U.S. Golf Association to whom we are grateful for assistance, interest and guidance. For technical assistance we thank Tom Bellohusen, Peter Newcomb and Lisa Rowley. We thank the reviewers for their excellent suggestions for improving the manuscript.

REFERENCES

Bengeyfield, W.H. (Ed.) (1989). Specifications for a Method of Putting Green Construction. United States Golf Association, Far Hills, New Jersey. R=14838

Biermann, B.J. & Linderman, R.G. (1983). Effect of container plant growth medium and fertilizer phosphorus on establishment and host growth response to vesicular-arbuscular mycorrhizae. J. Amer. Soc. Hort. Sci. 108, 962-971.

Buwalda, J.G. & Goh, K.M. (1982). Host-fungus competition for carbon as a cause of growth depressions in vesicular-arbuscular mycorrhizal ryegrass. Soil Biol. Biochem. 14, 103-106. R=4380

Crush, J.R. (1976). Endomycorrhizas and legume growth in some soils of the Mackenzie Basin, Canterbury, New Zealand. NZ J. Agric. Res. 19, 473-476.

Davis, E.A., Young, J.L. & Rose, S.L. (1984). Detection of high-phosphorus tolerant VAM-fungi colonizing hops and peppermint. Pl. Soil 81, 29-36.

Epstein, E. (1972). Mineral Nutrition of Plants: Principles and Perspectives. Wiley and Sons, New York.

Gemma, J.N. & Koske, R.E. (1989). Field inoculation of American beachgrass (Ammophila breviligulata) with VA. mycorrhizal fungi. J. Environ. Manag. 29, 173-182.

Gemma, J.N., Koske, R.E., Roberts, E.M., Jackson, N. & De Antonis, K.M. (1997). Mycorrhizal fungi enhance drought resistance in creeping bentgrass. J. Turfgrass Science 73, in this volume. R=44658

Habte, M. & Fox, R.L. (1993). Effectiveness of VAM fungi in nonsterile soils before and after optimization of P in soil solution. Pl. Soil 151, 219-226.

Hall, I.R., Johnstone, P.D. & Dolby, R. (1984). Interactions between endomycorrhizas and soil nitrogen and phosphorus on the growth of ryegrass. New Phytol. 97, 447-453. R=4261

Harley, J.L. & Harley, E.L. (1987). A check-list of mycorrhiza in the British flora. New Phytol. 105, 1-102.

Harley, J.L. & Smith, S. E. (1983). Mycorrhizal Symbiosis. Academic Press, London.

Hayman, D.S. (1983). The physiology of vesicular-arbuscular endomycorrhizal symbiosis. Can. J. Bot. 61, 944-963. R=6761

Hetrick, B.A.D., Kitt, D.G. & Wilson, G.W.T. (1986). The influence of phosphorus fertilization, fungal species, and nonsterile soil on mycorrhizal growth response in tall grass prairie species. Can. J. Bot. 64, 1199-1203.

Hetrick, B.A.D., Kitt, D.G. & Wilson, G.W.T. (1988). Mycorrhizal dependency and growth habit of warm-season and cool-season tallgrass prairie plants. Can. J. Bot. 66, 1376-1380. R=12635

Hetrick, B.A.D., Wilson, G.W.T. & Todd, T.C. (1990). Differential response of C3 and C4 grasses to mycorrhizal symbiosis, P fertilization, and soil microorganisms. Can. J. Bot. 68, 461-467. R=17874

Hoagland, D.R. & Arnon, D.I. (1950). The Water Culture Method of Growing Plants Without Soil. Calif. Agric. Expt. Sta. Circ. 347.

Kiernan, J.M., Hendrix, J.W. & Maronek, D.M. (1983). Fertilizer-induced pathogen-icity of mycorrhizal fungi to sweetgum seedlings. Soil Biol. Biochem. 15, 257-262.

Koske, R.E & Gemma, J.N. (1989). A modified procedure for staining roots to detect V-A mycorrhizas. Mycol. Res. 92, 486-488. R=36353

Koske, R.E., Gemma, J.N. & Jackson, N. (1995). Mycorrhizal fungi benefit putting greens. USGA Green Section Record 33(6), 12-14. R=44635

Koske, R.E., Gemma, J.N. & Jackson, N. (1997). A preliminary survey of mycorrhizal fungi in putting greens. J. Turfgrass Science 73, in this volume.

Little, L.R. & Maun, M.A. (1996). The "Ammophila problem" revisited: a role for mycorrhizal fungi. J. Ecol. 84, 1-7.

Miller, R.M., Jarstfer, A.G. & Pillai, J.K. (1987). Biomass allocation in an Agropyron smithii-Glomus symbiosis. Amer. J. Bot. 74, 114-122.

Modjo, H.S. & Hendrix, J.W. (1986). The mycorrhizal fungus Glomus macrocarpum as a cause of tobacco stunt disease. Phytopathol. 76, 688-691.

Mosse, B. (1973). Plant growth responses to vesicular-arbuscular mycorrhiza. New Phytol. 72,127-136.

Newman, E.I. & Reddel, P. (1987). The distribution of mycorrhizas among families of vascular plants. New Phytol. 106, 745-751.

Newsham, K.K, Fitter, A.H. & Watkinson, A.R. (1995). Arbuscular mycorrhiza protect an annual grass from root pathogenic fungi in the field. J. Ecol. 83, 991-1000.

Petrovic, A.M. (1984). Endomycorrhizal fungi: Friend or foe. In: Proceedings of the 53rd Annual Northeastern Turfgrass Conference, Univ. of Massachusetts, Amherst, Massachusetts, pp. 18-20. R=9891

Rhodes, L.H. & Larsen, P.O. (1981). Effects of fungicides on mycorrhizal development of creeping bentgrass. Plant Dis. 65, 145-147. R=1397

Sylvia, D.M. & Burks, J.N. (1988). Selection of a vesicular-arbuscular mycorrhizal fungus for practical inoculation of Uniola paniculata. Mycologia 80, 565-568.

Sylvia, D.M. & Schenck, N.C. (1983). Application of superphosphate to mycorrhizal plants stimulates sporulation of phosphorus-tolerant vesicular-arbuscular mycorrhizal fungi. New Phytol. 95, 655-661.

Thompson, J.P. & Wildermuth, G.B. (1989). Colonization of crop and pasture species with vesicular-arbuscular mycorrhizal fungi and a negative correlation with root infection by Bipolaris sorokiniana. Can. J. Bot. 69, 687-693.

Trappe, J.M. (1987). Phylogenetic and ecologic aspects of mycotrophy in the angiosperms from an evolutionary standpoint. In: Ecophysiology of VA Mycorrhizal Plants (Ed. G.R. Safir), CRC Press, Boca Raton, Florida, pp. 5-26.

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