Scientia Horticulturae 84 (2000) 151±162

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1 Scientia Horticulturae 84 (2000) 151±162 Growth and owering in Petunia hybrida, Callistephus chinensis and Impatiens balsamina inoculated with mixed AM inocula or chemical fertilizers in a soil of low P fertility Anupama Gaur a, Atimanav Gaur b, Alok Adholeya c,* a Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Biological and Agricultural Sciences, University of Pretoria, Pretoria 0002, South Africa b Mycology Lab, Department of Botany, University of Pretoria, Pretoria 0002, South Africa c Microbial Biotechnology, Tata Energy Research Institute, India Habitat Centre, Lodhi Road, New Delhi , India Accepted 13 August 1999 Abstract Three seasonal ornamental plants, namely Petunia hybrida, Callistephus chinensis and Impatiens balsamina, were tested for their response to inoculation with mixed indigenous AM culture when grown on a marginal wasteland amended with organic matter. Subsequently, the treatments consisting of AM inoculations were compared with those consisting of recommended chemical fertilizers in terms of growth response and cost economics. In all the three plant species, mycorrhizal inoculation led to marked improvement in both reproductive (number of owers) and vegetative (dry matter) phase of the plants. P. hybrida showed a threefold increase over uninoculated plants in the reproductive growth as compared to twofold in C. chinensis and I. balsamina. Application of the recommended dose of chemical fertilizers produced a comparable response. The inoculated plants produced greater dry matter, grew taller, owered at least 15 days earlier and produced more owers when compared to uninoculated plants. In addition there was a signi cant increase in P and K uptake in shoots of all the three ornamentals. AM inoculation could be at least 30% cost economic as compared to the chemical fertilizers. Therefore, mycorrhizal inoculation is recommended at the nursery level for nutrient-de cient soil conditions because it is a cost-effective substitute for chemical fertilizers, either partly or fully, which makes the approach * Corresponding author. Tel.: ; fax: address: aloka@teri.res.in (A. Adholeya) /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (99)

2 152 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162 particularly suitable to marginal farmers with their low-input farming system. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Petunia hybrida; Impatiens balsamina; Callistephus chinensis; Indigenous AM consortium; Organic amendment; Chemical fertilizers; Cost economics 1. Introduction AM fungal inocula hold tremendous potential in increasing crop production as an integral component of sustainable crop production systems (Bagyaraj, 1992). They have been shown to confer a variety of bene ts on their hosts including growth and yield enhancement (Furlan, 1993). AM fungi are found in a wide variety of habitats (Brundrett, 1991). Wasteland are large stretches of land de cient in nutrients and bene cial microorganisms and account for approximately 20% of India's total geographical area (Sharma et al., 1996). Mycorrhizal fungi are likely to be bene cial in bringing these habitats under cultivation. The current study was designed to assess, in economic and physical terms, the bene t of cultivating economically important ornamental and oil seed plants on such wastelands using bene cial microorganisms and to see how it compares with the cultivation based on applying chemical fertilizers in terms of costs. Ornamental plants are often grown from seedlings and cuttings grown in disinfected soils or on inert substrates, mainly to lower the risk of contamination and to ensure controlled conditions to obtain homogenous material. Such activities and other horticultural practices that tend to eliminate AM fungi create the conditions best suited to using mycorrhizal biotechnology (Johnson et al., 1980). In addition, production of ornamental species in nutrient-de cient arid soils is more dif cult because these plants generally have a high fertilizer requirement. Adopting management practices to increase plant production with low fertilizer input will minimize adverse effects on the environment and keep production costs low, making it suitable for marginal farmers with low incomes. AM fungi are known to increase plant growth in arid and semi-arid regions (Hirrel and Gerdemann, 1980). Although many studies of host plant responses to mycorrhizal infection have been performed using a single AM fungal isolate, the present study used a mixed inoculum of AM fungi so as to simulate the natural conditions more closely, where an assemblage of many species is more common (Brundrett, 1991). The current experiment was planned with the following objectives: (1) to study the ef cacy of an indigenous, mixed AM inoculum in infecting and promoting the growth of selected ornamental plants; (2) to study the effect of mycorrhizal

3 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151± inoculation on owering, and nutrient uptake and compare; (3) the cost effectiveness of using mycorrhizal inoculation and chemical fertilizers. 2. Materials and methods 2.1. Preparation of AM inocula A mixed indigenous culture (containing native populations of Glomus, Gigaspora and Scutellospora spp.) collected from the experimental site was used as the AM inoculum and multiplied for 1 year in clay pots (5 kg capacity) lled with soil similar to the poting mixture, with Sorghum bicolor as the host plant. At maturity, the tops of the sorghum plants were removed and substrate was allowed to dry for a week at 25 58C. The roots were nely chopped and the dried root/soil mixture was thoroughly mixed to obtain a homogeneous inoculum. Spores were isolated by wet sieving and decanting (Gerdemann and Nicolson, 1963) and counted on a lter paper (Gaur and Adholeya, 1994). The percentage of root colonization by mycorrhizae was assessed as described by Biermann and Lindermann (1981) after staining the roots with acid fuchsin (Phillips and Hayman, 1970). The total number of infectious propagules (IPs) per gram of inoculum was also counted (Sharma et al., 1996) and the value was found to be 15 IPs/g. The colonization percentage was 62 and the spore density was 20 spores/g inoculum Preparation of seedlings Seeds of Petunia hybrida cv. blue bird, Callistephus chinensis cv. dwarf chrysanthemum and Impatiens balsamina were surface sterilized with 10% H 2 O 2 for 5 min. Subsequently, the seeds were washed repeatedly with sterile water and kept for germination on moist sand in sterile petriplates at 308C in dark for 48 h. On germination, the seedlings were given half-strength Hoagland solution (Hoagland and Arnon, 1950) for even 15 days Preparation of growth substrate The experimental site is located at Gwal Pahari in Haryana state, India ( E and latitude N) 255 m above the mean sea level and receives a mean annual rainfall of 500 mm. Two experiments were conducted. In Experiment 1, the poting mixture was prepared by mixing two parts of soil (sandy loam Hyperthermic Typic Haplustalf, ph 7.5, NO 3 N ˆ 124 mg/kg, available P ˆ 0.53 mg/kg, available K ˆ 124 mg/ kg) from the experimental site and one part of compost (made from leaves of

4 154 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162 Table 1 Soil characteristics of the substrate used in both the experiments Substrate ph Phosphorus (ppm) Nitrogen (ppm) With chemical fertilizers With organic manure only Potassium (%) Albizzia and Poplars, ph ˆ 7.2, available P ˆ 12 ppm, organic C ˆ 11.40% and N ˆ 0.75%). The compost amended substrate showed a ph of 7.3, available P ˆ 2.5 ppm (Olsen et al., 1954), and organic C ˆ 3.46% (Datta et al., 1962). The substrate was autoclaved before inoculation (1218C for 1 h at 15 psi). Clay pots, lled with 5 kg of substrate, were used in the experiment. In Experiment 2, the same potting mixture was used and one set of pots was given chemical fertilizers in the form of SSP (single super phosphate), micronutrients (commercially available), ammonium nitrate and potassium chloride at the recommended levels (Table 1) Experiment layout and mycorrhizal inoculation Experiment 1 followed a completely randomized design consisting of a 3 2 structure three types of ornamental plants and two treatments (inoculated and uninoculated). Each treatment was replicated six times. The substrate was inoculated by thoroughly mixing the crude inoculum with soil in each pot (at 2000 IP per pot). The inoculum consisted of spores, hyphae and infected root bits. Five kg of soil containing the inoculum mixture was transferred to earthen pots (17 cm diameter). Uninoculated plants served as controls. Experiment 2 also followed a completely randomized design consisting of a 3 3 structure (having an additional treatment, inoculated, uninoculated and uninoculated with chemical fertilizer) each treatment was replicated six times. Six 15-day-old germinated seedlings of each host were selected for uniformity and transplanted to the pots. All the plants were grown under 70% daylight in a greenhouse and watered to maintain the soil at 60% of its water holding capacity. The pots were rotated regularly to avoid any positional effect. All the parameters were analysed at harvest except the ower count, which was carried out as described below Observation, harvest and analysis Flowers were counted (non-destructively) at 10 days intervals for all the six replicates in each treatment for 120 days (Experiment 1 and 2). Shoots were severed just above the crown, weighed while fresh, rinsed in distilled water, dried

5 at 708C for 48 h and weighed again, which were then ground ne enough to pass through a 0.5 mm screen and digested in H 2 SO 4. The P and K content in the digest was determined chemically (Jackson, 1973) and using ame photometry (Chapman and Pratt, 1978). The harvested roots were washed clean or cut into 1 cm segments, and homogenized thoroughly. Samples of root segments were analysed for mycorrhizal colonization. Colonization percentage was determined on 100 root segments from each sample. Roots were stained using the method of Phillips and Hayman (1970). Root pieces were mounted between the glass slides and examined under a microscope at X40 for AM hyphae, arbuscules, vesicles and spores. The extent of colonization was assessed by using the method of Biermann and Lindermann (1981) and expressed as the percent of root segment colonized for each root piece (Experiment 1 and 2). Spores of AM fungi were extracted (Gerdemann and Nicolson, 1963) from 50 ml samples of the homogenized substrate from three replicates for each treatment (Experiment 1). The spores retained on different sieves were collected in a beaker and recovered by sucrose density centrifugation. Only visually intact spores were counted under a stereoscopic microscope (Gaur and Adholeya, 1994). The average number of spores in 50 ml of the substrate soil was used to estimate the spores per gram soil (g 1 soil) in each of the treatments. Sporocarps were gently crushed to count the number of spores in each sporocarp Statistical analysis The data were analysed using one-way ANOVA using the least signi cant difference (Duncan's multiple range test at 5% signi cant level). The data were also analysed for observing the standard deviation within the treatments using Costat software (Cohort, Berkeley, CA, USA). 3. Results 3.1. Flowering A. Gaur et al. / Scientia Horticulturae 84 (2000) 151± The inoculated plants owered signi cantly earlier than uninoculated controls in both the experiments though the difference was not signi cant in Experiment 2. The inoculated plants of C. chinesis owered 27 DAT (days after transplanting), whereas the uninoculated plants took 22 days longer. Those of I. balamina owered 37 DAT, which was 16 days earlier than uninoculated plants and those of P. hybrida owered 29 DAT, 12 days before the uninoculated plants (Experiment 1, Table 2). The inoculated host plants not only owered earlier but also produced signi cantly more owers, 190%, 106% and 75% more in P. hybrida, I. balsamina and C. chinensis, respectively (Fig. 1).

6 156 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162 Table 2 In uence of inoculation with an indigenous arbuscular mycorrhizal (AM) consortium on growth and nutrient uptake of the three ornamentals (Experiment 1) a Hosts MCP (%) AM propagules Spores (g 1 soil) IP d (g 1 soil) Flower initiation (DAT e ) Nutrient uptake Shoot P (mg g 1 ) Shoot K (%) Shoot height (cm) Dry matter (g/plant) P. hybrida AM b a 6.5a a 6.21a 70.57a 0.84a NM c ± ± ± b 5.32b 64.53b 0.64b I. balsamina AM b 6.0b a 5.0a 53.06a 0.72a NM ± ± ± b 4.02b 33.12b 0.62b C. chinensis AM b 5.3c a 5.33a 16.01a 0.34a NM ± ± ± b 3.72b 11.50b 0.27b a Means followed by the same letters are not signi cantly different (p < 0.05). b AM Ð mycorrhizal. c NM Ð non-mycorrhizal. d IP Ð infectious propagule. e DAT Ð days after transplanting. In Experiment 2, the number of owers in AM inoculated P. hybrida was signi cantly higher, though the plants that had received chemical fertilizers also produced more owers, as compared to control plants (Fig. 2) Plant dry matter In both the experiments, dry matter was signi cantly higher in the inoculated plants than in their uninoculated counterparts (Tables 2 and 3) Nutrient uptake In Experiment 1, the P and K content of the three hosts tested was signi cantly higher in the inoculated plants (Table 2). In Experiment 2, both the AM fungal inoculation and addition of chemical fertilizer increased P and K uptake over the uninoculated controls, though the increase was greater in the later plants (Table 3) Mycorrhizal parameter In both the experiments, AM consortia produced the maximum colonization in P. hybrida followed by I. balsamina and C. chinensis (Tables 2 and 3). In terms of

7 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151± Fig. 1. Effect of mycorrhizal inoculation on the number of owers produced by: (a) P. hybrida; (b) I. balsamina; (c) C. Chinensis in Experiment 1. Error bars denote standard errors of mean.

8 158 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162 Fig. 2. Effect of mycorrhizal inoculation on the number of owers produced by: (a) P. hybrida; (b) I. balsamina; (c) C. chinensis in Experiment 2. Error bars denote standard errors of mean.

9 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151± Table 3 In uence of inoculation with an indigenous arbuscular mycorrhizal (AM) consortium on the growth and nutrient uptake of three ornamentals (Experiment 2) a Hosts MCP (%) Nutrient uptake Shoot height (cm) Shoot P Shoot (mg g 1 ) K (%) Dry matter (g/plant) P. hybrida AM b 6.18b 70.43b 0.86b CF b ± 5.52a 6.52a 75.62a 0.91a NM ± 3.21c 5.27c 65.3c 0.63c I. balsamina AM b 5.08b 54.96b 0.72a CF a ± 3.15a 5.21a 58.32a 0.72a NM ± 2.58c 4.11c 35.16c 0.62b C. chinensis AM b 5.37b 16.8b 0.34a CF ± 3.1a 5.52a 17.32a 0.33b NM ± 2c 3.68c 11.8c 0.28c a Means followed by the same letters are not signi cantly different (p < 0.05). b CF Ð chemical fertilizer. AM multiplication (number of spores and infectious propagules), the trend was the same, with maximum multiplication in P. hybrida followed by I. balsamina and C. chinensis (Table 2) Cost analysis Application of chemical fertilizers proved to be 30% more expensive than inoculation. The total cost of growing a chemically fertilized plant was $4.15, compared to $2.93 in the case of a mycorrhizal plant, a saving of 30%. 4. Discussion The result of the present study demonstrated that indigenous mycorrhizal endophytes established well in the targeted plants and the occurrence of these endophytes in plant roots and soil varied with host plant. The data also indicate the potential role of AM fungi in the growth and mineral nutrition of the host plants tested. In both the experiments, the soil used as a substrate was low in P (except in the treatment with chemical fertilizers). However, mycorrhization by the indigenous AM consortia enhanced vegetative growth, nutrient uptake (P and K) and ower production in all the three ornamental spp. tested. Several workers, namely

10 160 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162 Boerner (1990), Davies (1987), Gianinazzi et al. (1989), Gianinazzi et al. (1990), and Jeffries and Dodds (1991) have reported similar results in a number of ornamental plants. In Experiment 2, though the application of chemical fertilizers led to increased uptake of nutrients (non-signi cant), the plants produced fewer owers. This can perhaps be explained by the common observation that luxurious consumption does not help in better owering.the extent of colonization was substantially higher in all the three host species with P. hybrida showing the highest value. It is likely that P. hybrida relies more on AM fungi for the uptake of phosphorus than I. balsamina and C. chinensis. Also, it was observed that these horticultural plants have a fairly well-developed ne root system, offering a larger surface area for the AM to colonize. By contrast, our observation of high MCP values in all three hosts in both the experiments supports the hypothesis of Baylis (1970) that plant with poorly developed ne root system may be mycotrophs in P de cit soils. Mycorrhizal inoculation improves P (Sharma et al., 1996) and K (Johnson et al., 1980) uptake because roots are supplemented with the AM fungal hyphae in tapping soil resource (Abbot and Robson, 1982). This is shown in the present study by the increased P and K recovery in the inoculated plants as compared to the uninoculated controls. The inoculated plants were able to obtain greater quantities of soil phosphorus and produce more plant dry matter. In addition, increased K concentration in plants has been shown to increase the number of owers (Dufault et al., 1990) and plant yield (Albregts et al., 1991). Mycorrhizal inoculation had a pronounced in uence on the time required for ower initiation, as studied in Experiment 1. Also, the infection led to prolonged owering and produced signi cantly greater number of owers in the inoculated plants than in their uninoculated counterparts. Our ndings are consistent with those in many other species that the number of owers produced by a plant is proportional to plant size and nutrient content (Lee and Bazzaz, 1982). Thus, the two experiments showed that these ornamental species performed equally well with chemical fertilizers and mycorrhizal inoculation. Application of fertilizers accounts for 30% of the cash expenses in nurseries (Thakur and Panwar, 1997). This will continue to be high. With inoculation, the expenses on phosphorus fertilizers could be reduced to 70%. Current levels of N, K and micronutrients could also be reduced. Thus, there is a wide potential in exploiting mycorrhizal fungi for improved establishment, survival, nutrient uptake and growth of plants especially in nutrient de cient soils. Acknowledgements The present study was supported by the Department of Biotechnology, Government of India. Thanks are due to the Director of Tata Energy Research

11 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151± Institute for providing support. Copy editing by Yateen Joshi is thankfully acknowledged. References Abbot, L.K., Robson, A.D., The role of vesicular±arbuscular mycorrhizal fungi in agriculture and the selection of fungi for inoculation. Aust. J. Agric. Res. 33, 389±408. Albregts, E.E., Howard, C.M., Chandler, C.K., Strawberry responses to K rate on a ne sand soil. HortScience 26(2), 135±138. Bagyaraj, D.J., Vesicular±arbuscular mycorrhiza: application in agriculture. In: Norris, J.R., Read, D.J., Varma, A.K. (Eds.), Methods in Microbiology, vol. 24. Academic Press, London, pp. 359±373. Baylis, G.T.S., Root hairs and phycomycetous mycorrhizas in phosphorus-de cient soil. Plant Soil 33, 713±716. Biermann, B.J., Lindermann, R.G., Quantifying vesicular±arbuscular mycorrhizae; a proposed method towards standardization. New Phytol. 87, 63±67. Boerner, R.E.J., Role of mycorrhizal fungus origin in growth and nutrient uptake by Geranium robertianum. Am. J. Bot. 77(4), 483±489. Brundrett, M., Mycorrhizas in natural ecosystems. Adv. Ecol. Res. 21, 171±313. Chapman, H.D., Pratt, P.F., Methods of analysis for soils and waters. University of California, Division of Agriculture Science, CA, USA. Datta, N.P., Khera, M.S., Saini, T.R., A rapid calorimetric procedure for the determination of the organic carbon in soils. J. Ind. Soc. Soil Sci. 10, 67±74. Davies Jr., F.T., Effects of VA-mycorrhizal fungi on growth and nutrient uptake of cuttings of Rosa multi ora in two container media with three levels of fertilizer application. Plant Soil 104, 31±35. Dufault, R.J., Phillips, T.L., Kelly, J.W., Nitrogen and potassium fertility and plant populations in uence eld production of Gerbera. HortScience 25(12), 1599±1602. Furlan, V., Large scale application of endomycorrhizal fungi and technology transfer to the farmer. In: Abstracts of the Ninth North American Conference on Mycorrhizae. Guelph, Ont., 8±12 August, p. 77. Gaur, A., Adholeya, A., Estimation of AMf spores in soil: a modi ed method. Myco. News 6, 10±11. Gerdemann, J.W., Nicolson, T.H., Spores of mycorrhizal endogone species extracted from soil by wet sieving and decanting. Trans. Br. Mycol. Soc. 46, 235±244. Gianinazzi, S., Gianinazzi-Pearson, V., Trouvelot, A., Potentialities and procedures for the use of endomycorrhizas with emphasis on high value crops. In: Whips, J.M., Lumsden, B. (Eds.), Biotechnology of Fungi for Improving Plant Growth. Cambridge University Press, Cambridge, pp. 41±54. Gianinazzi, S., Trouvelot, A., Gianinazzi-Pearson, V., Conceptual approaches for the rational use of VA endomycorrhizae in agriculture: possibilities and limitations. Agric. Ecosyst. Environ. 29, 153±161. Hirrel, M.C., Gerdemann, J.W., Improved growth of onion and bell pepper in saline soils by two vesicular±arbuscular mycorrhizal fungi. Soil Sci. Soc. Am. J. 44, 654±655. Hoagland, D.R., Arnon, D.I., The water culture method for growing plants without soil. California Agricultural Experimental Station, circular no University of California, Berkeley, CA.

12 162 A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162 Jackson, M.L., Soil Chemical Analysis. Prentice-Hall, Englewood Cliffs, NJ, USA. Jeffries, P., Dodds, J.C., The use of mycorrhizal inoculants in forestry and agriculture. In: Arora, D.K., Rai, B., Mukerji, K.G., Knudsen, G.R. (Eds.), Handbook of Applied Mycology, vol. I, Soil and Plants. Marcel Dekker, New York, pp. 35±53. Johnson, C.R., Joiner, J.N., Crews, C.E., Effects of N, K, and Mg on growth and leaf nutrient composition of three container grown woody ornamentals inoculated with mycorrhizae. J. Am. Soc. Hort. Sci. 105(2), 286±288. Lee, T.D., Bazzaz, F.A., Regulation of fruit maturation pattern in annual legume, Cassia fasciculata. Ecology 63, 1374±1388. Olsen, S.R., Cole, C.V., Watanabe, F.S., Dean, L.A., Estimation of available phosphorus in soils by extraction with sodium bicarbonate. US Department of Agriculture, Washington, DC, Circular. Phillips, J.M., Hayman, D.S., Improved procedures for clearing roots and staining parasitic and vesicular±arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Myco. Soc. 55, 158±161. Sharma, M.P., Gaur, A., Bhatia, N.P., Adholeya, A., Growth responses and dependence of Acacia nilotica var. cupriciformis on the indigenous arbuscular mycorrhizal consortium of a marginal wasteland soil. Mycorrhiza 6, 441±446. Thakur, A.K., Panwar, D.S., Response of Rhizobium vesicular arbuscular mycorrhizal symbionts on photosynthesis, nitrogen metabolism and sucrose translocation in green gram (Phaseolus radatus). Ind. J. Agric. Sci. 67(6), 245±248.

COMPONENTS OF VA MYCORRHIZAL INOCULUM AND THEIR EFFECTS ON GROWTH OF ONION

COMPONENTS OF VA MYCORRHIZAL INOCULUM AND THEIR EFFECTS ON GROWTH OF ONION New Phytol. (1981) 87, 3 5 5.161 355 OMPONENTS OF VA MYORRHIZAL INOULUM AND THEIR EFFETS ON GROWTH OF ONION BY A. MANJUNATH AND D. J. BAGYARAJ Depart?nent of Agricultural Microbiology, University of Agricultural