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Development of a Nutrient Film Technique Culture System for Arbuscular Mycorrhizal Plants

Development of a Nutrient Film Technique Culture System for Arbuscular Mycorrhizal Plants

HORTSCIENCE 40(2):378–380. 2005. establishment of a fi lm technique (NFT) culture system for the AM , that is, G. mosseae plants. It was considered Development of a Nutrient Film that this system might enable the direct investi- gation of metal cations uptake by mycorrhizal Technique Culture System for hyphae and their transport to lettuce. Also, the possibility of using this system for mass Arbuscular Mycorrhizal Plants production of mycorrhizal horticultural under greenhouse conditions was evaluated. Yun-Jeong Lee1 Institute of (330), Hohenheim University, Fruwirthstr. 20, Materials and Methods 70593 Stuttgart, Germany & National Institute of and Two experiments were conducted. The fi rst Technology, RDA, Suwon, 441-707, Republic of Korea experiment was a trial to determine in Eckhard George2 substrate the optimum P concentration of the nutrient solution to avoid reduction of plant Leibniz Institute of Vegetable and Ornamental Crops, 14979 Grossbeeren, growth while maintaining an adequate Germany & Institute of Sciences, Humboldt University colonization rate. The second experiment tested Berlin, 10115 Berlin, Germany whether the modifi ed NFT could be used for the commercial production of mycorrhizal crop Additional index words. arbuscular , lettuce, inoculum production, mycorrhizal plants under greenhouse conditions. plant production, NFT system Plant and culture: Expt. 1. Abstract. A nutrient fi lm technique (NFT) culture system was developed to allow nursery were pregerminated overnight in saturated CaSO and sowed directly into 150-mL pots production of arbuscular mycorrhizal horticultural crops. This would benefi t horticultural 4 production and allow for uncomplicated production of mycorrhizal hyphae. of lettuce (thinned to one plant per pot after emergence) (Lactuca sativa var. capitata) plants were highly colonized by the arbuscular mycorrhizal containing Perlite and -based BEG 107 fungus, Glomus mosseae (BEG 107) after 4 weeks in the NFT system, following an initial inoculum (10% of pot volume) or Perlite and phase of fi ve weeks in inoculated in Perlite substrate. In the NFT system, a thin layer of inoculum fi ltrate (Blue Ribbon fi lter paper no. glass beads was used to provide solid support for plant and fungus growth and nutrient 5893; Schleicher & Schüll, Dassel, Germany) solution was supplied intermittently (15 min, six times per day). A modifi ed nutrient solu- and grown for 5 weeks. Perlite (1 to 3 mm) tion (80 µM P) was used and was replaced with fresh solution every 3 days. A signifi cantly was prepared by washing with tap water over higher dry weight was found for the mycorrhizal versus the nonmycorrhizal lettuce plants a 1-mm mesh sieve, sun-drying, and then in Perlite during the precolonization period. The root colonization rate was also high at heat treating for 24 h at 110 °C. Perlite was rates up to 80 µM P supply. On the NFT system, growth differences between mycorrhizal mixed uniformly either with the inoculum (for and nonmycorrhizal plants were less than in Perlite. However, root colonization rate was mycorrhizal plants) or the inoculum fi ltrate not reduced during the NFT culture period. In this system, high amounts of fungal and same amount of sterilized inoculum (for were produced. This would allow the determination of metal and other nutrient concentra- nonmycorrhizal plants). Plants in pots were ir- tions in fungal hyphae. Furthermore, we found large amounts of external fungal hyphae rigated daily with 40 mL nutrient solution with surrounding the root surface. As much as 130 mg fungal biomass were collected per culture different P concentration 10 µM, 40 µM, and 80 plate (three plants). Therefore, we suggest that this modifi ed NFT culture system would be µM one or two times per day (see below for the suitable for fungal biomass production on a large scale with a view to additional aeration by composition of nutrient solution), respectively. intermittent nutrient supply, optimum P supply, and a use of glass beads as support materials. After fi ve weeks, the dry weight and percentage Furthermore, bulk inoculum composition with a mixture of , colonized roots, and colonized root was determined. hyphae grown in soilless media by the modifi ed NFT system might be a useful way to mass- Plant and fungus culture: Expt. 2. Plants produce mycorrhizal crops and inoculum for commercial horticultural purposes. in Perlite, which had been colonized by AM after 5 weeks using a rate of 80 µM P, were Most studies of mycorrhizal plants have 3) Substrate-free spores and colonized roots transplanted into the NFT system to be culti- been conducted in soil. However, the collec- can be collected for various uses such as taxo- vated for another 4 weeks. In the NFT system, tion of clear fungal hyphae for determination nomic identifi cation of the fungus, production P was supplied at a concentration of 80 µM of nutrient concentration is impossible in soil of inoculum and molecular studies (Hawkins P. To transplant, the pots were submerged in culture since hyphae of AM cannot be fully and George, 1998). deionized water, the roots carefully separated separated from the soil. It has been reported that Mycorrhizal plants have been cultured in from the Perlite, and the plants placed into the some cultures in nutrient solution, such as hy- (Dugassa, 1995; Hawkins and NFT system plate (Plexiglas material, 15 × 45 droponics, and nutrient fl ow culture George, 1999; Peuss, 1958), aeroponics (Hung cm, three plants per plate). In the NFT plate, have various advantages in this respect. and Sylvia, 1988; Mohammad et al., 2000; glass beads were used as thin layer (about 1 1) Soilless culture provides soilless prepara- Sylvia and Hubbel, 1986) and in a nutrient fl ow cm thickness) to provide solid support for plant tions of mycorrhizal material. system (Mosse and Thompson, 1984). Aero- and fungus growth. The roots were placed on 2) Rates of nutrient supply at the root surface ponic culture of mycorrhizal plants has been the glass bead layer and fi xed in black plastic can be manipulated easily. experimentally used in nurseries (Jarstfer and foil, which was carefully laid to cover the Sylvia, 1995). Nutrient fi lm technique, which entire plate. The percentage colonized root was Received for publication 16 Apr. 2004. Accepted for is now widely used in commercial horticultural determined again at the time of harvest. The publication 22 Aug. 2004. This research was partly crop production, was pioneered by Cooper fungal hyphae were collected from the glass fi nanced by the Ministry of Education & Human (1975) and more fully described by Winsor beads in NFT plate for the determination of Resources Development of Republic of Korea and fungal biomass. The extraradical hyphal mats the Deutsche Forschungsgemeinschaft (DFG), grant et al. (1979). The plant roots lie in a shallow no. GE 920/4. layer of rapidly fl owing nutrient solution and surrounded with lettuce roots were excluded 1Plant nutritionist and corresponding author. Current as root mats develop, the upper layers above for the determintion of fungal biomass because address: Division of Technology, the liquid retain a fi lm of moisture around it could not be separated completely from National Institute of Agricultural Science and them. Therefore, this system may provide the the root. After separation of plant root from Technology, RDA, Suwon, 441-707, Republic of fungus with enough air and nutrient solution the glass beads layer, all of grass beads were Korea; e-mail [email protected]. without disturbing hyphal growth. collected in a beaker (5 L) and then rinsed 2Professor. The aim of these experiments was to with deionized water four times. The fungal

378 HORTSCIENCE VOL. 40(2) APRIL 2005

AAprilHSBook.indbprilHSBook.indb 337878 22/9/05/9/05 44:02:59:02:59 PPMM Table 1. Total root colonization and fresh weights of mycorrhizal lettuce plant after 5 weeks of age remained high (about 85.3%) compared precolonization in Perlite. Different letters indicate statistical difference due to P supply (P < 0.05, to the initial percentage root colonized in one-way ANOVA). Data are means of four replications ± SE. Perlite. The fungal biomass per culture plate P supply (µM) 10 40 80 (three plants) was as much as 130 mg of dry Colonization rate (%) 58.7 ± 2.1 a 86.3 ± 4.2 b 77.5 ± 3.9 b weight. Furthermore, external hyphae of AM Shoot fresh weight (g) 13.94 ± 0.61 a 19.00 ± 2.12 b 23.95 ± 1.80 b were well developed in this system. After 4 weeks in this system, we found large amounts of external hyphae surrounding the root (Fig. 1). Better developed root mats were formed in mycorrhizal plants compared to nonmycor- rhizal plants due to aggregation with fungal hyphae (Fig. 1). Noninoculated plants showed no colonization in this system. The shoot dry weights of mycorrhizal and nonmycorrhizal plants grown in the NFT sys- tem were signifi cantly different 4 weeks after transplanting into the NFT system (Table 2). Dry weight of mycorrhizal lettuce shoot and root were higher than those of nonmycor- rhizal lettuce. The P concentrations in roots were also higher in mycorrhizal plants compared to nonmycorrhizal plants (Table 3). However, no signifi cant difference in shoot P concentra- tion between mycorrhizal and nonmycorrhizal plants was observed.

Discussion

The results indicate that it would be Fig. 1. Root development of nonmycorrhizal (NAM) and mycorrhizal (AM) lettuce plants and external possible to produce arbuscular mycorrhizal hyphae developed in NFT system at harvest. plants with root colonization in a NFT culture system. Therefore, NFT would be one way to hyphae in supernatant were collected using of root length colonized by mycorrhizal fungi mass-produce inoculum and commercially sieve (30 µm pore size). After rinsing the was determined on roots stained in trypan produce mycorrhizal horticultural crop under fungal hyphae on the sieve carefully with blue (Koske and Gemma, 1989) using the greenhouse conditions. Growth or P uptake of double distilled water, fungal hyphae were gridline-intersect method (Giovannetti and mycorrhizal plants on NFT culture system was collected into Eppendorf tube and dried at 50 Mosse, 1980). not clearly superior to that of nonmycorrhizal °C in an oven for 2 d to determine dry weights. Plant P analysis. The P concentrations of plants (Tables 2 and 3). This result was expected Shoot and roots were harvested for dry weight the dried, pulverized shoot and roots material because are delivered to the root determination after sampling the fresh root (0.5 were determined using the molybdo-P-blue surface by the solution fl ow in NFT culture, g fresh weight per plant) for the determina- method (Murphy and Riley, 1962). so that the additional absorbing surface of the tion of mycorrhizal colonization. Shoot and Statistics. Four replications per treatment extraradical hyphae is of no benefi t to the plant roots sampled were dried at 70 °C in an oven were used in both experiments. Pots and NFT (George, 2000). Nevertheless, the NFT system for three days and thereafter determined dry plates were placed randomly in the growth can be used to raise mycorrhizal plants that may weights. The experiments were carried out in chamber and the position of the plates was be superior in outplanting success, nutritional a growth chamber with a PAR of 400 to 500 varied each time after replacing the nutrient composition, or selling price. µmol·m–2·s–1 (mercury halide lamps, Osram solution. The Student’s t test and one-way In the modifi ed NFT system, the AM fungus Powerstar HQI-T-2000 W/D), a day/night analysis of variance were applied to determine temperature of 25/20 °C and a relative humid- differences due to P concentration in the fi rst Table 2. Dry weights of nonmycorrhizal (NAM) ity of about 60%. experiment and mycorrhizal colonization in and mycorrhizal (AM) lettuce plants grown in Nutrient media. The plants in Perlite cul- the second experiment. the NFT system after 5 weeks of precolonization in Perlite. Different letters indicate statistical ture were supplied with 1/10 strength nutrient difference between NAM and AM within one solution for the fi rst 4 d in all experiments and Results P treatment (P < 0.05, Student’s t test). Data thereafter with a full strength of nutrient solu- are means of four plates with three plants per tion, which consisted of the following macro- Roots colonization of mycorrhizal plants in plate ± SE. nutrients (mM): Ca (NO ) 4H O (2), KH PO 3 2 2 2 4 Perlite culture was colonized to approximately Dry wt (g) NAM AM H O (0.0752), K HPO 2H O (0.0048), K SO M 2 2 4 2 2 4 60% when 10 µ P was supplied (Table 1). Shoot 8.09 ± 0.49a 10.79 ± 0.15b (0.7), KCl (0.1), MgSO4 7H2O (0.5) and mi- When more P was supplied, root colonization Root 1.84 ± 0.16a 3.16 ± 0.27b cronutrients (µM): H3BO3 (10), MnSO4 H2O rates further increased. However, there was no (3), ZnSO 7H O (0.5), CuSO 5H O (0.2), 4 2 4 2 signifi cant difference in root colonization rate Table 3. P concentrations of nonmycorrhizal (NAM) (NH4)6Mo7O24 4H2O (0.01), FeEDTA (10). A between 40 and 80 µM P supply (Table 1). and mycorrhizal (AM) lettuce plants grown in pH of 6.0 was maintained by 0.15 mM MES- After 5 weeks of colonization in Perlite, the NFT system after 5 weeks of precolonization KOH. The nutrient solutions was circulated and the shoot fresh weight of lettuce had increased in Perlite. Different letters indicate statistical replaced every 3 d. The nutrient solution was as P supply increased in the nutrient solution. difference between NAM and AM within one supplied by water pump, which connected to However, there was no signifi cant difference P treatment (P < 0.05, Student’s t test). Data a time switch so as to supply nutrient solution between 40 and 80 µM P supply (Table 1). are means of four plates with three plants per for 15 min once every 4 h, i.e., six times per After colonization in Perlite for 5 weeks in plate ± SE. day, so that each plate received 2 L per day the second experiment, lettuce was transplanted Dry wt (mg·g–1) NAM AM in total. into NFT culture plates. After four weeks in Shoot 1.23 ± 0.13a 1.16 ± 0.05a Mycorrhizal root colonization. Percentage NFT culture, the total root colonization percent- Root 1.48 ± 0.11a 2.22 ± 0.15b

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AAprilHSBook.indbprilHSBook.indb 337979 22/9/05/9/05 44:03:01:03:01 PPMM G. mosseae (BEG 107) could colonize and Using a thin layer glass beads as substrate ent fl ow and hydroponic culture, p. 809–826. In: develop well with lettuce, high root coloniza- in this system might be helpful to support A. Varma (eds.). Microbes: For health, wealth tion remained or even slightly increased and, plant and fungal growth and additionally to and sustainable environment. Malhotra Publ. in addition, large amounts of fungal biomass avoid complete drying of the root during the House, New Delhi. were produced. This offers the potential of nonwatering period. However, it was somewhat Hawkins, H.-J., M.D. Cramer, and E. George. 1999. Root respiratory quotient and uptake determining nutrient concentration directly diffi cult to collect most of the external hyphae in hydroponically grown nonmycorrhizal and in external hyphae through plant-fungus from the root and glass beads because the mycorrhizal . Mycorrhiza 9:57–60. interaction. Interestingly, in this system the mycorrhizal root had developed well and ag- Hung, L.-L. and D.M. Sylvia. 1988. Production external hyphae of G. mosseae (BEG 107) gregated tightly to the glass beads together with of vesicular–arbuscular mycorrhizal fungus developed very well and even surrounded the the external hyphae. So it may be preferable in inoculum in aeroponic culture. Appl. Environ. lettuce root to form large mats of hyphae. It future experiments to use a membrane bag (30 Microbiol. 54:353–355. could give some advantages to commercial µm in diameter) to separate root and hyphae Jarstfer, A.G. and D.M. Sylvia. 1995. Aeroponic inoculum production not only for inoculum for the better collection of hyphae. culture of VAM fungi, p. 428–441. In: A. Varma quantity and richness but also inhibition of A NFT culture system may have some and B. Hock (eds.). Mycorrhiza—Structure, function, molecular biology and biotechnology. pathogenic occurrence (one advantages in comparison to other culture Springer-Verlag, Berlin, Germany. possible draw back of NFT). Many research- system. In aeroponics, wilting of plants is a Kahn, A.G. 1988. Inoculum density of Glomus ers reported that once arbuscular mycorrhiza possibility when the water supply is briefl y mosseae and growth of plants in was colonized and established well with host interrupted by some technical problem. In hy- unsterilized bituminous coal spoil. Soil Biol. plant, it could give an activation of host plant droponics, fungal growth may be inhibited by Biochem. 20:749–753. defense mechanisms against other pathogenic limited aeration. Therefore, it is suggested that Koske, R.E. and J.N. Gemma. 1989. A modifi ed organisms (Azcon-Aguilar and Barea, 1996; NFT is a better production system compared procedure for staining roots to detect VA mycor- Gianinazzi-Pearson et al., 1996; Pozo et al., to aeroponics and hydroponics due to not only rhizas. Mycol. Res. 92:486–505. 1999). However, further study should be made for inoculum and mycorrhizal crop production Lee, K.K, M.V. Reddy, S.P. Wani, and N. Trimurtulu. 1996. Vesicular-arbuscular mycorrhizal fungi in to test the inoculum potential of these hyphae but also for investigating activity of external earthworm casts and surrounding soil in relation by most probable number (MPN) (Kahn, 1988; and elemental composition of fungal hyphae to soil management of a semi-arid tropical Alfi sol. Lee et al., 1996) or other techniques (Lesueur to nutrient uptake. Appl. Soil Ecol. 3:177–181. et al., 2001) compared to those of other culture Lesueur D, K. Ingleby, D. Odee, J. Chamberlain, systems so as to be qualifi ed as commercial Literature Cited J. Wilson, T. T. Manga, J.-M. Sarrailh, inoculum. and A. Pottinger. 2001. Improvement of Azcón-Aguilar, C. and J.M. Barea. 1996. Arbuscular Mosse and Thompson (1984) also found forage production in Calliandra calothyrsus: and biological control of soilborne methodology for the identifi cation of an effective large amounts of external mycelium fl oating plant pathogens- An overview of the mechanisms as a gray fi lm on the water surface in a nutri- inoculum containing Rhizobium strains and involved. Mycorrhiza 6:457–464. arbuscular mycorrhizal isolates. J. Biotechnol. ent fl ow culture system (bean colonized with Cooper, A.J. 1975. Crop production in re-circulating 91:269–282. a G. mosseae isolate). In comparison with our nutrient solutions. Sci. Hort. 3:251–258. Mohammad, A., A.G. Kahn, and C. Kuec. 2000. experiments, they supplied nutrient solution Dugassa, D.G., G. Grunewaldt-Stöcker, and F. Improved aeroponic culture technique for continuously at a rate of 1 L·min–1. However, Schönbeck. 1995. Growth of Glomus intrara- production of inocular of arbuscular mycorrhizal the intermittent supply of nutrient solution dices and its effect on linseed (Linum usitatis- fungi. Mycorrhiza 9:337–339. in this study could be helpful for providing simum L.) in hydroponics culture. Mycorrhiza Mosse, B. and J.P. Thompson. 1984. Vesicular-arbus- 5:279–282. adequate aeration while avoiding disturbance cular endomycorrhizal inoculum production. I. George, E. 2000. Nutrient uptake. Contributions of Exploratory experiments with beans (Phaseolus of fungal growth. Intermittent nutrient sup- arbuscular mycorrhizal fungi to plant ply also reduces costs for horticultural crop vulgaris) in nutrient fl ow culture. Can. J. Bot. nutrition, p. 307–343. In: Y. Kapulnik and D. 62:1523-1530. production. Our system provided about 22 h Douds, Jr. (eds.). Arbuscular mycorrhizas: Murphy, J. and J.P. Riley. 1962. A modifi ed single so- aeration time. and function. Kluwer Acad.Pub., lution method for the determination of phosphate The P concentration in the nutrient solution Dordrecht. in natural waters. Anal. Chem. Acta 27:31–36. could be an important factor because it could Giovannetti, M. and B. Mosse. 1980. An evaluation Peuss, H. 1958. Untersuchungen zur Ökologie und reduce fungal inoculation at high level of sup- of techniques for measuring vesicular–arbuscular Bedeutung der Tabakmycorrhiza. Arch. Mikro- ply. Hawkins and George (1997) have reported mycorrhizal infection in roots. New Phytol. biol. 29:112–142. 84:489–500. that modifi ed Knop and Hoagland nutrient Pozo, M.J., C. Azcón-Aguilar, E. Dumas-Gaudot, Gianinazzi-Pearson, V., E. Dumas-Gaudot, A. and J. M. Barea. 1999. ß-1,3-glucanase activities solution with 0.9 mM P concentration reduced Gollotte, A. Tahiri-Alaoui, and S. Gianinazzi. the colonization rate in hydroponics with Linum in roots inoculated with arbuscular 1996. Cellular and molecular defense-related root mycorrhizal fungi and-or Phytophthora usitatissimum colonized with G. mosseae. In responses to invasion by arbuscular mycorrhizal parasitica and their possible involvement in the present study, 80 µM P concentration was fungi. New Phytol. 133:45–57. bioprotection. Plant Sci. 141:149–157. selected for the pre colonization of lettuce. In the Hawkins, H.-J. and E. George. 1997. Hydroponic Sylvia, D.M. and D.H. Hubbel. 1986. Growth and NFT system, both 80 µM (Table 3) and 160 µM culture of the mycorrhizal fungus Glomus mos- sporulation of vesicular–arbuscular mycorrhizal P (data not shown) used in nutrient solution did seae with Linum usitatissimum L., Sorghum fungi in aeroponic and membrane systems. not reduce the root colonization rate. The host bicolor L. and Triticum aestivum L. Plant Soil 1:259–267. 196:143–149. Winsor, G.W., R.G. Hurd, and D. Price. 1979. Nutri- plant species may certainly have an effect, but it Hawkins, H.-J. and E. George. 1998. Substrate-free is possible that the useful range for colonization ent fi lm technique. Glasshouse Crops. Res. Inst. culture of mycorrhizal plants: Aeroponic, nutri- Grower’s Bul. 5. in NFT is 200 to 1.0 mM P concentration.

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