Effectiveness of Endomycorrhizal Fungi and Pseudomonas fluorescens Under Different Phosphorus Levels on Capsicum annuum L.

Transcription

1 Kasetsart J. (Nat. Sci.) 46 : (2012) Effectiveness of Endomycorrhizal Fungi and Pseudomonas fluorescens Under Different Phosphorus Levels on Capsicum annuum L. Anju Tanwar*, Ashok Aggarwal, Karishma and Neetu ABSTRACT The contribution of two indigenous arbuscular mycorrhizal fungi Glomus mosseae (G) and Acaulospora laevis (A) along with Pseudomonas fl uorescens (P) was examined at three levels of superphosphate fertilization (F1, g per pot; F2, g per pot; and F3, g per pot) for Capsicum annuum L. plants in a pot culture experiment in sterilized soil. After 120 d of inoculation, plants were harvested and analyzed for arbuscular mycorrhizal (AM) spore number, AM root colonization, alkaline phosphatase, acid phosphate, and root and shoot phosphorus. The lowest dose of phosphorus fertilization best promoted the growth of mycorrhizal fungi in terms of increasing the AM colonization and spore number compared with the two higher doses, as F1 had the maximum root colonization of 97.62%, which decreased to 76.2% and 23.4% with applications of F2 and F3, respectively. Of the two AM fungi, G. mosseae was found to be the better strain when applied along with P. fl uorescens. Increasing the phosphorus fertility decreased all the above measured parameters. The results showed that the activity of both acid and alkaline phosphatases increased with root colonization and was highest in the treatment with triple inoculation. There was a significant increase in the phosphorus content in F1 plants due to enhanced mycorrhization of roots which increases the secretion of phosphatases that helps increase phosphorus uptake in the plant and ultimately increases the fruit yield. On the basis of the results found in this study, G+P and G+A+P treatments in F1-fertilized plants were found to produce the largest increases of AM root colonization, phosphatase activity and phosphorus content and thereby increasing the yield of bell pepper plants. Keywords: Arbuscular mycorrhizal fungi, mycorrhization, phosphorus fertilizer, phosphorus uptake, Pseudomonas fl uorescens, enzyme activity INTRODUCTION Vegetables occupy an indispensable position in the dietary habits of the vast majority of the human population in the Indian subcontinent. Besides being nutritionally important, vegetables also contribute to the maintenance of health and the prevention of diseases. The primary limitation of vegetable crop production is the failure of establishment of the transplanted crops from the nursery to the field. Any technique that would enable the plants to become successfully established in the field would definitely help improve vegetable crop production. Mycology and Plant Pathology Laboratory, Department of Botany, Kurukshetra University, Kurukshetra , Haryana, India. * Corresponding author, anjutanwarbotany@gmail.com Received date : 27/03/12 Accepted date : 11/05/12

2 770 Kasetsart J. (Nat. Sci.) 46(5) Bell pepper (Capsicum annuum L.) is one of the most valuable crops of India (Kumar and Verma, 2009). It has nutritive and medicinal value and is a good source of vitamin A, C, E and also has antioxidant properties (Lee et al., 1995). The nutritional quality of vegetable crops is largely affected by any applied fertilizer (Schuphan, 1974). Among the various factors affecting the yield, the most important one is the supply of an adequate amount of chemical fertilizers (Singh and Srivastava, 1988). Phosphorus is one of the key macronutrients required for plant growth and metabolism; it plays an important role in the transfer of energy through the formation of energy-rich phosphate esters and is also an essential component of macromolecules such as nucleotides, phospholipids and sugar phosphates (Marschner, 1995). Plants obtain phosphorus from the soil (Mengel and Kirkby, 1987), from phytine (Bartnik and Szafranskia, 1987) or from other phosphorylated molecules and then store it in their vacuoles (Bieleski and Ferguson, 1983). When phosphorus fertilizers are applied to replenish soil fertility, a major portion gets bound in oxisols by Fe and Al oxides, reducing the effectiveness of P fertilization. Soluble phosphorus can be released from insoluble phosphates by a solubilization reaction involving rhizospheric microorganisms (Kapoor et al., 1989). Similarly, phosphorus uptake is also regulated by enzymes called phosphatases (Yan et al., 2001). Mycorrhizal fungi, especially arbuscular mycorrhizal fungi (AMF) and some phosphorus solubilizing bacteria like Pseudomonads, can be instrumental in obtaining phosphorus from organic matter and mineral forms by increasing the diffusion zone around roots and by the production of phosphatase enzymes (Bhromsiri and Bhromsiri, 2010). Phosphatases are generally classified as being acidic or alkaline depending upon their optimum ph for enzyme activity (Barret-Lennard et al., 1982). Phosphatases are believed to be important for phosphorus scavenging and remobilization in plants (Rodrígues and Fraga,1999). The recommended dose of phosphorus fertilizer for bell pepper is kg.ha -1 (Bajaj et al., 1979; Naeem et al., 2002) and should be applied in its soluble form as superphosphate to obtain the maximum increase in growth (Sreenivasa et al., 1993). Most farmers apply fertilizers in excess of the recommended dose. An abundance of phosphorus is critical for rapid and uniform growth that is required for most vegetable crops (Lorenz and Vittum, 1980). Even the adverse effect of high phosphorus on AMF is well documented (Mosse, 1973; Olsson et al., 2002). Under conditions of high exogenous phosphate supply, when a plant can meet its own P requirement without the fungus, AMF are suppressed (Peng et al., 1993). Thus, it is very important to compare the phosphatase activity of Glomus mosseae, Acaulospora laevis and Pseudomonas fluorescens alone or in combination against a control at different superphosphate fertilization levels and to relate their roles in supplying phosphorus to the host plant, for example, bell pepper. Therefore, this experiment investigated the effect of different doses of phosphorus fertilization incorporated in its soluble form as superphosphate (recommended level, above recommended level and below recommended level) with and without bio-inoculants (Glomus mosseae, Acaulospora laevis and Pseudomonas fluorescens) on the phosphatase activity, phosphorus uptake and yield of bell pepper. MATERIALS AND METHODS Source of arbuscular mycorrhizal spores and their isolation The native predominant AM fungi Glomus mosseae and Acaulospora laevis were isolated from the rhizosphere of bell pepper plants by the wet sieving and decanting technique of Gerdemann and Nicolson (1963).

3 Kasetsart J. (Nat. Sci.) 46(5) 771 Identification of arbuscular mycorrhizal fungal spores The isolated spores were given a thorough microscopic examination to record their morphological features namely, color, size, shape, wall structure, surface ornamentation, nature and size of subtending hyphae, bulbous suspensor and the number and arrangement on spores in sporocarp. These AM spores were identified using the keys of Schenck and Perez (1990). Pure culture preparation of selected arbuscular mycorrhizal spores The dominant AM fungi obtained were purified following the funnel technique (Menge and Timmer, 1982), by which a single spore was isolated and multiplied using maize as the host. Mass multiplication and maintenance of arbuscular mycorrhizal fungi The pure cultures of G. mosseae and A. laevis obtained were further mass multiplied in pots using soil and sand (3:1) and Lemongrass as the host. During multiplication, host plants were nourished by Hoagland s nutrient solution (excluding phosphorus) up to the age of 3 mth. These plants were then used as the inoculum. Maintenance and multiplication of Pseudomonas fluorescens P. fl uorescens (MTCC no. B103) was procured from IMTECH, Chandigarh, India and multiplied by culture in nutrient broth medium. Experimental design The experiment was conducted in a 3 6 factorial completely randomized design with five replications employing three levels of superphosphate fertilizer (F1, g per pot, the below recommended application; F2, g per pot, the recommended application; and F3, g per pot, the above recommended application) and six types of different microbial inoculations (C (control), G (Glomus mosseae), A (Acaulospora laevis), G+P (Glomus mosseae + Pseudomonas fl uorescens), A+P (Acaulospora laevis + Pseudomonas fluorescens), G+A+P (Glomus mosseae + Acaulospora laevis + Pseudomonas fl uorescens)). There was one plant in each pot. Experimental setup An experiment was designed to determine the effects of different fertilization levels of superphosphate in conjunction with mycorrhizal fungi (G. mosseae and A. laevis) and P. fluorescens on the AM spore number, colonization, phosphatase activity (alkaline as well as acid) and phosphorus content (root, shoot and total) of bell pepper. Soil from the Botanical Garden, Department of Botany, Kurukshetra University, Kurukshetra, Haryana, India was sieved and mixed with sand and soil (1:3) and autoclaved for 20 min for two consecutive days. The soil used in the experiment contained sand (64.2%), silt (21.81%) and clay (3.90%), had a ph of 8.08 and had total N and available P contents of and 0.017%, respectively. Separate earthen pots ( cm) each having a soil capacity of 2 kg were used for the different treatments. Initially, the sand and soil mixture was passed through a 2 mm sieve and sterilized twice at 1.5 atmospheres for 30 min and then added to the pots. A basal dressing of phosphorus as superphosphate was applied with the three levels of phosphate fertilizer. For the single inoculation (G, A), 10% of the soil (200 g) of air dried AM inoculum containing around 865 spores and 80% colonized root segments of trap host maize was added. For the double inoculations (G+P, A+P), 100 g of inoculum and for the triple inoculation (G+A+P), 65 g of AM inoculum was used. P. fluorescens was cultured in nutrient broth medium. P. fl uorescens was applied to each pot directly in amounts of approximately (colony forming units (cfu) ml -1 ). The soil was mixed thoroughly, and finally the sand and soil mixture was added to

4 772 Kasetsart J. (Nat. Sci.) 46(5) make the final volume 2 kg. Control pots without any inoculation were also maintained. There were five replications of the experiment under polyhouse conditions. Bell pepper seedlings (Capsicum annuum cultivar California Wonder) were procured from a local nursery in Kurukshetra, India and a single seedling was planted in each pot. The seedlings were watered regularly and Hoagland s solution (excluding the phosphorus source) was also provided every 15 d. Harvest and analysis After 120 d, plants were harvested and separated into the root, shoot and fruit components. A soil sample (10 g) was used to determine the AM spore number by the wet sieving and decanting technique. Small root pieces were washed and prepared for root colonization by the rapid clearing and staining technique of Phillips and Hayman (1970). Fresh roots were used for extraction of alkaline and acid phosphatases, assayed by using p-nitrophenyl phosphate as the substrate which is hydrolyzed by the enzyme to p-nitrophenol. For this procedure, 1 g of fresh, washed roots was homogenized in 5 ml of ice-cold sodium acetate buffer (0.05 M with ph 4.8) for acid phosphatase and sodium carbonate-bicarbonate buffer (0.05 M with ph 10) for alkaline phosphatase activity using a pre-chilled mortar and pestle. The resulting homogenate was centrifuged at 10,000 revolutions min -1 for 15 min and the supernatant thus obtained was referred to as crude enzyme extract and was used for the assay of acid phosphatase activity, measured in international units per gram of fresh weight (IU per gram FW). For yield parameters, the number of fruits per plant and their fresh weight was recorded. The phosphorus content in the shoots and roots was determined by the vanadomolybdophosphoric acid yellow color method in a nitric acid system outlined by Jackson (1973), which is actually based on the yellow color of the unreduced vanadomolybdophosphoric heteropoly complex. Statistics All results were analyzed using analysis of variance, followed by a post hoc test using the computer software SPSS version 11.5 (SPSS Inc. Chicago, IL, USA). Means were then ranked at the P < 0.05 level of significance using Duncan s multiple range test for comparison. RESULTS AND DISCUSSION The response of the host plant to the indigenous AM fungi in terms of mycorrhization was evaluated by measuring the AM spore number and root colonization. The acid and alkaline phosphatase activity was studied to gain further insight into the physiological changes occurring as a result of applying different levels of phosphorus fertilization. Similarly, the amounts of shoots, roots and total phosphorus were measured to correlate with the role of mycorrhiza in the production of phosphatases, which then help in solubilization of soil phosphorus and are then absorbed by plants. AM colonization is known to alter the inherent phosphorus supply by increasing the phosphatase activity in the plant rhizosphere (Allen et al., 1995). Finally, yield parameters, which are of utmost importance, were observed to see the combined effect of phosphatase activity and phosphorus uptake on fruit formation. AM root colonization and spore number Bell pepper plants grown under different superphosphate doses inoculated with AM fungi and Pseudomonas fluorescens showed mycorrhization in their roots (Table 1). The extent of colonization and the AM spore number varied with different treatments. The maximum intensity of root colonization (mean ± SD) was observed in treatments of F1, G+A+P (97.62 ± 3.3), A (94.29 ± 6.82), G (93.98 ± 6.13), A+P (86.44 ± 2.5), G+P (85.68 ± 7.2), followed by F2, G (89.28 ± 2.7), G+A+P (79.1 ± 4.4), A (77.3 ± 3.1) and least in F3, G+P (45.02 ± 6.34), G (30.79 ± 9.2), A+P (30.81 ± 4.6). The spore number

5 Kasetsart J. (Nat. Sci.) 46(5) 773 also varied with different treatments. It was found that in treatments with a high concentration (above the recommended dose) of fertilizer, the AM spore number as well as root colonization decreased, which was in accordance with the findings of Aguilera-Gomez et al. (1999) and Linderman and Davis (2004). In the F3 treatment, the maximum spore number was produced by G+A+P (23.4 ± 4.87), followed by A (21.8 ± 1.9), C (19.3 ± 2.4), G (19 ± 1.87), G+P (17.6 ± 2.1), and the minimum spore number was produced in A+P (16.8 ± 2.6), but were found to be less in comparison to the recommended and below recommended doses. The AM colonization in the above recommended dose was maximized in G+P (45.02 ± 6.34), followed by A+P (30.81 ± 4.6), G (30.79 ± 9.2), A (27 ± 4.41), G+A+P (26.66 ± 3.3) and lowest in C (14.45 ± 4.3), which were again less than the values for the recommended and the below recommended doses. It was found Table 1 Arbuscular mycorrhizal (AM) spore number, AM root colonization and pattern of colonization of C. annuum under different doses of superphosphate. AM spore AM root Superphosphate Treatment number per 10 g colonization concentration soil (%) F1 Control 24.0±1.58 f 11.62±2.42 f Below recommended G 93.8±6 a 93.98±6.13 a (0.200 g per pot) A 76.8±3.4 b 94.29±6.82 a G+P 62.6±4.5 c 85.68±7.2 b A+P 51.4±4.3 d 86.44±2.5 b G+A+P 80.2±3.5 ab 97.62±3.3 a F2 Control 20.0±4 f 22.14±2.2 e Recommended G 76.2±3.7 b 89.28±2.7 b (0.400 g per pot) A 62.4±3.2 c 77.30±3.1 bc G+P 44.0±2.9 e 73.52±4.7 bc A+P 33.0±3.67 e 69.32±6.9 c G+A+P 56.4±2.7 d 79.10±4.4 bc F3 Control 19.3±2.4 f 14.45±4.3 f Above recommended G 19.0±1.87 f 30.79±9.2 de (0.800 g per pot) A 21.8±1.9 f 27.00±4.41 e G+P 17.6±2.1 f 45.02±6.34 d A+P 16.8±2.6 f 30.81±4.6 de G+A+P 23.4±4.87 f 26.66±3.3 e ANOVA (F 5, 72 ) Treatment 372.6* 331.8* (F 2, 72 ) Superphosphate 569.6* 358* concentration (F 10, 72 ) Treatment 78.0* 43.9* superphosphate conconcentration Each value is a mean of five replicates ± standard deviation; F = Fertilization; G = Glomus mosseae; A = Acaulospora laevis; P = Pseudomonas fl uorescens. Mean values followed by same lowercase superscript letter within a column are not significantly different by Duncan s multiple range test, P < 0.05). * = Significance at P < 0.05 level. ANOVA = Analysis of Variance.

6 774 Kasetsart J. (Nat. Sci.) 46(5) that the spore number and root colonization did not respond similarly, as the number of spores in the rhizosphere was frequently unrelated to the intensity of AM root colonization. All the main AMF structures (intraradical hyphae, vesicles and arbuscules) observed in the colonized roots were found at all dose levels but the number varied. As was reported above, in F1, followed by F2 and F3, it was found that mycorrhization of roots and the AM spore number decreased with an increase in the fertilizer dose. The results presented in Table 1 show that plants inoculated with G. mosseae alone had the maximum spore number whereas the triple inoculation of both the AMF and P. fl uorescens resulted in the highest mycorrhizal colonization that may have been due to a synergistic interaction between the AM fungi and P. fl uorescens. Higher sporulation and root colonization helps increase fungal host contact and the exchange of nutrients. Several earlier reports also noted the positive influence of AMF along with rhizobacteria on AM root colonization (Gamalero et al., 2004; Das et al., 2007) Phosphatase activity In addition to changes in the mycorrhization of the plants, there were also changes in the functioning of the systems, as evaluated by measuring the plant enzyme activity. The measurement of the phosphatase activity provided a good index of mycorrhizal effect on the host plant growth and nutrient uptake, especially phosphorus. Data presented in Table 2 indicate that phosphatase activity (alkaline as well as acid) was higher in the AM-colonized plants compared to the non-mycorrhizal plants. A similar trend was observed in the effect of different fertilizer doses on enzyme activity. The enzyme activity decreased with an increase in the fertilizer dose. The maximum activity was observed in F1, followed by F2, then F3. The higher fertilizer dose suppresses the mycorrhization of roots, which in turn decreases the phophatase activity as observed in this experiment. In bell pepper plants, acid phosphatase activity was greater than alkaline phosphatase activity. The inoculation with P. fl uorescens along with two AM fungi significantly increased the phosphatase activity, as the maximum activity (international units per gram FW) was observed in G+A+P (alkaline, 0.08 ± 0.005, acid, 0.53 ± 0), followed by G+P (alkaline, 0.06 ± 0, acid, 0.38 ± 0.004), A+P (alkaline, 0.05 ± 0, acid, 0.34 ± 0.005). Nonmycorrhizal plants and plants with a high dose of fertilizer showed minimum phosphatase activity (Table 2). Several previous studies have also shown increased acid and alkaline phosphatase activity in mycorrhizal plants (Krishna et al., 1983; Tarafdar and Marschner, 1994). The increased phosphatase activity found in the mycorrhizal roots indicated an increase in the P content. The ability of mycorrhizae to produce phosphatase enzymes actually depends upon the availability of phosphorus in the soil. In the P cycle, enzyme activities are inversely related to P availability (Tadano et al., 1993). According to Sumana (1998) and Kumar et al. (2008), the acid phosphatase activity actually increases with increased root colonization by AM fungi. Those treatments which decrease the available phosphate, cause an overall increase in the phosphatase activity (Azcón and Barea, 1997). With low phosphorus availability, P demand increases, resulting in an increase in the phosphatase activity, as was found in this experiment in the AM-colonized roots. Phosphorus content The phosphorus concentration in the shoots, roots and whole plant was found to be influenced by the AM inoculation and varied among the different doses of fertilizer (Table 3). The P content was greater in roots than shoots. After 120 d of inoculation, a higher P content (%) in roots was recorded in the F1 treatment (below recommended dose) G+P (0.79 ± 0.005), followed by F1, G+A+P (0.77 ± 0.005), F2 (recommended dose) G+A+P (0.67 ± 0.012) and F1, A+P (0.66 ± 0.011). The increased uptake of

7 Kasetsart J. (Nat. Sci.) 46(5) 775 phosphorus from the soil could have been due to an increase in the uptake of P facilitated by the increased colonization by AM fungi (Gaur and Adholeya, 1999) or it could have been due to an increase in the number of uptake sites per unit area and a greater ability of these roots to exploit the soil nutrient (Bolan, 1991). Furthermore, in the current experiment, the highest AM colonization and maximum P content were found in the F1 treatments. Similarly, the maximum P content (%) in shoots was observed in F1, G+A+P (0.59 ± 0.031), followed by F1, A+P (0.51 ± 0.025), F2, G+A+P (0.5 ± 0.029) and F2, G+P (0.43 ± 0.016). Mycorrhizal fungi and P. fl uorescens can offer considerable benefits in terms of growth as AMF Table 2 Effect of different doses of superphosphate along with arbuscular mycorrhizal fungi (G. mosseae and A. laevis) and Pseudomonas fl uorescens on alkaline phosphatase and acid phosphatase activity of bell pepper. Alkaline phosphatase Acid phosphatase Superphosphate activity (international activity (international Treatment concentration units per gram fresh units per gram fresh weight) weight) F1 Control 0.02±0 f 0.08±0 f Below G 0.04±0 d 0.37±0 b recommended A 0.03±0 e 0.27±0 c (0.200 g per pot) G+P 0.06±0 b 0.38±0.004 b A+P 0.05±0 c 0.34±0.005 b G+A+P 0.08±0.005 a 0.53±0 a F2 Control 0.02±0 f 0.15±0 de Recommended G 0.02±0.005 f 0.27±0.005 c (0.400 g per pot A 0.03±0 e 0.22±0 d G+P 0.04±0 d 0.26±0 c A+P 0.03±0 e 0.23±0 d G+A+P 0.04±0 d 0.33±0 b F3 Control 0.01±0 g 0.15±0 de Above G 0.01±0 g 0.12±0.005 e recommended A 0.01±0 g 0.07±0 f (0.800 g per pot) G+P 0.02±0 f 0.19±0.004 de A+P 0.02±0 f 0.05±0 f G+A+P 0.02±0 f 0.17±0 de ANOVA (F 5, 72 ) Treatment 16.3* * (F 2, 72 ) Superphosphate 48.0* * concentration (F 10, 72 ) Treatment 9.7* * superphosphate concentration Each value is a mean of five replicates ± standard deviation. Note that some standard deviation amounts shown as zero as the values were very small. F = Fertilization; G = Glomus mosseae; A = Acaulospora laevis; P = Pseudomonas fl uorescens. Mean values followed by same lowercase superscript letter within a column are not significantly different by Duncan s multiple range test, P < 0.05). * = Significance at P < 0.05 level. ANOVA = Analysis of Variance.

8 776 Kasetsart J. (Nat. Sci.) 46(5) are potential substitutes for chemical fertilizers. The increase in the P content of shoots may have been due to the ability of the AM fungi to form extrametrical hyphae in the roots that ultimately help in better absorption of water and in solubilization of phosphorus and other nutrients (Gosling et al., 2005). Another possibility is that the P. fluorescens promoted the germination of AM fungal spores and thereby increased the rate and extent of mycorrhizal root colonization (Johansson et al., 2004). The data revealed that the total P content (%) was maximized in the dual inoculation of G+P (1.39 ± 0.036) and in the triple inoculation of G+A+P (1.3 ± 0.089) in the F1 treatment followed by the treatments of F2 and F3. In earlier reports, Akhtar and Siddique (2008) and Basu and Santhaguru (2009) observed a synergistic interaction between AM fungi and Pseudomonads with regard to the total phosphorus content of plants. The total P content increase may have been due to the enhanced activities of the acid Table 3 Shoot, root and total plant phosphorus percentage uptake in C. annuum under different doses of superphosphate. Total Superphosphate Shoot Root phosphorus Treatment phosphorus in concentration phosphorus (%) (%) whole plant (%) F1 Control 0.28±0.019 ef 0.34±0.056 f 0.62±0.016 f Below recommended G 0.40±0.011 c 0.46±0.005 cd 0.86±0.016 cd (0.200 g per pot) A 0.37±0.027 d 0.44±0.004 cd 0.81±0.03 d G+P 0.31±0.03 e 0.79±0.005 a 1.39±0.036 a A+P 0.51±0.025 b 0.66±0.011 b 1.16±0.028 b G+A+P 0.59±0.031 a 0.77±0.005 a 1.30±0.089 a F2 Control 0.31±0.005 e 0.37±0.030 e 0.67±0.033 ef Recommended G 0.38±0.023 d 0.45±0.007 cd 0.83±0.029 cd (0.400 g per pot) A 0.36±0.012 d 0.38±0.005 e 0.74±0.014 e G+P 0.43±0.016 c 0.48±0.009 cd 0.91±0.022 c A+P 0.41±0.016 c 0.53±0.004 c 0.95±0.015 c G+A+P 0.50±0.029 b 0.67±0.012 b 1.17±.035 b F3 Control 0.23±0.01 f 0.35±0.013 f 0.58±0.02 f Above recommended G 0.37±0.013 d 0.37±0.01 e 0.59±0.019 f (0.800 g per pot) A 0.28±0.022 ef 0.34±0.013 f 0.62±0.034 f G+P 0.37±0.036 d 0.48±0.065 cd 0.84±0.03 cd A+P 0.30±0.016 e 0.41±0.015 d 0.71±0.018 e G+A+P 0.41±0.026 c 0.51±0.013 c 0.91±0.027 c ANOVA (F 5, 72 ) Treatment 193.5* * 247.1* (F 2, 72 ) Superphosphate 793.9* * 458.3* concentration (F 10, 72 ) Treatment 20.4* 274.5* 18.6* superphosphate concentration Each value is a mean of five replicates ± standard deviation; F = Fertilization; G = Glomus mosseae; A = Acaulospora laevis; P = Pseudomonas fl uorescens. Mean values followed by same lowercase superscript letter within a column are not significantly different by Duncan s multiple range test, P < 0.05). * = Significance at P < 0.05 level. ANOVA = Analysis of Variance.

9 Kasetsart J. (Nat. Sci.) 46(5) 777 and alkaline phosphatases (Feng et al., 2002). In the present investigation, maximum phosphatase activities (acid and alkaline) were observed in G+P and G+A+P of the F1 treatment. These phosphatases produced by extraradical hyphae of AM fungi could hydrolyze extracellular phosphate ester bonds and ultimately made P available to the plants (Joner et al., 2000). Further solubilization of soil P is also achieved by the release of organic acids and phosphates (Bolan, 1991). The responses to the range of fertilizer doses was different with the bio-inoculants. In the F3 treatment, the P uptake was comparatively lower than in F1 and F2, possibly because the higher dose of superphosphate was detrimental to the growth of mycorrhizal stains and P. fl uorescens, which in turn decreased colonization and ultimately decreased the P uptake from the soil, thus supporting the well documented fact that higher fertilizer doses inhibit mycorrhizal growth (Treseder and Allen, 2002; Rotor and Delima, 2010) directly by reducing spore germination and hyphal growth from the germinating spores (Nagahashi et al., 1996). Sreenivasa et al. (1993) also reported G. macrocarpum caused maximum nutrient enhancement at 50% of the recommended dose of phosphorus in its soluble form as superphosphate. Among the two AM fungal strains, G. mosseae was found to be a much more compatible strain than A. laevis. AM symbiosis is known to substantially increase the amount of roots, shoots and total P content. Also, in the present investigation, all AM-inoculated seedlings of F1 and F2 had a greater P content compared to the control. These results were in accordance with the findings of Khare and Rodrigues (2009) and Rakshit and Bhadoria (2008) who reported an increased P content in mycorrhizal Carica papaya, Arachis hypogea and tomato over nonmycorrhizal plants following inoculation with different AM fungi. Ortas (2008) reported under field and greenhouse conditions that several plant species inoculated with mycorrhizae have higher P availability than non-inoculated plants. Fruit yield Bio-inoculants (AM fungi, P. fluorescens) and superphosphate had significant effects on fruit yield. A comparison of the yields from bioinoculated pots with uninoculated ones revealed that fruit formation did not occur in the latter (Table 4). The application of superphosphate positively influenced fruit formation in the presence of AM fungi and P. fluorescens but not at the higher concentration. Fruits appeared at all levels of phosphorus fertilizer, but the number varied with the different concentrations (Table 4). The maximum number of fruits and fruit weight, respectively, were recorded in F1, G+A+P (3.6 ± 0.55, 26.4 g ± 2.2), followed by G+P (3.6 ± 0.45, 14.2 g ± 1.12), A+P (2.4 ± 0.55, 11.6 g ± 0.48). This could have been due to the high uptake of nutrients and also the buildup of sufficient photosynthates by beneficiary inoculants, resulting in an increase in the fruit size and weight. Indeed, fruits appeared in the high fertilization pots (F3) also, but were fewer in number and also lower in weight at harvest on day 120 compared to the F1 and F2 treatments. AM fungi along with P. fluorescens showed prominent results regarding fruit yield at all levels of fertilizer application indicating the presence of a synergetic interaction among the AM fungi, bacteria and the phosphorus fertilizer. Tohidi-Moghaddam et al. (2004) reported that phosphorus-solubilizing bacteria increase the available nitrogen and phosphorus in the soil which could enhance the crop production. The current findings were in accordance with the work done by Davies and Linderman (1991), and Baar (2008) that bell pepper seedlings fertilized with the lowest concentration of phosphorus showed enhanced mycorrhizal colonization and spore numbers compared to a high concentration. Similarly, Soleimanzadeh et al. (2010) recommended 50% P application along with inoculation of VA mycorrhizae to increase

10 778 Kasetsart J. (Nat. Sci.) 46(5) the seed yield and oil production of sunflower; so this treatment could be considered as a suitable substitute for chemical phosphorus fertilizer. CONCLUSION The current study results support a recommendation for bell pepper growers in India to apply a lower dosage of superphosphate in combination with some bio-inoculants like G. mosseae, A. laevis and P. fluorescens for better growth, establishment, nutrient uptake and fruit yield. The current study showed that the recommended higher concentration of superphosphate fertilizer did not significantly enhance nutrient uptake and yield. ACKNOWLEDGEMENTS The authors are grateful to Kurukshetra University, Kurukshetra, India for providing the laboratory facilities and financial assistance. Special thanks are recorded to Dr. Vipin Parkash for giving valuable suggestions during the course of the research. Table 4 Efficacy of bioinoclulants and superphosphate doses on yield of bell pepper. Superphosphate Number of fruits Fresh weight of Treatment concentration per plant fruits (g) F1 Control 0 0 Below recommended G 1.8±0.45 c 9.24±0.39 bc (0.200 g per pot) A 1.8±0.45 c 4.10±0.48 c G+P 3.6±0.45 a 14.2±1.12 b A+P 2.4±0.55 b 11.6±0.48 bc G+A+P 3.6±0.55 a 26.4±2.2 a F2 Control 0 0 Recommended G 0.6±0.55 d 2.06±1.92 d (0.400 g per pot) A 0.4±0.55 e 0.84±1.16 e G+P 2.6±0.55 b 9.06±0.69 bc A+P 0.8±0.84 cd 1.99±1.81 d G+A+P 2.6±0.55 b 15.0±1.2 b F3 Control 0 0 Above recommended G 0.4±0.55 e 0.40±0.54 f (0.800 g per pot) A 0 0 G+P 0.8±0.84 cd 0.62±0.57 ef A+P 0.4±0.55 e 0.39±0.54 f G+A+P 1.4±0.55 c 1.83±0.50 d ANOVA (F 5, 72 ) Treatment 89.0* 731.4* (F 2, 72 ) Superphosphate 43.7* 388.8* concentration (F 10, 72 ) Treatment 6.6* 105.7* superphosphate concentration Each value is a mean of five replicates ± standard deviation; F = Fertilization; G = Glomus mosseae; A = Acaulospora laevis; P = Pseudomonas fl uorescens. Mean values followed by same lowercase superscript letter within a column are not significantly different by Duncan s multiple range test, P < 0.05). * = Significance at P < 0.05 level. ANOVA = Analysis of Variance.

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