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Pinus Wallichiana) Seedlings Inoculated with Rhizo- Sphere Microorganisms Under Temperate Nursery Conditions

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Pinus Wallichiana) Seedlings Inoculated with Rhizo- Sphere Microorganisms Under Temperate Nursery Conditions Ann. For. Res. 55(2): 217-227, 2012 ANNALS OF FOREST RESEARCH www.e-afr.org Growth response and nutrient uptake of blue pine (Pinus wallichiana) seedlings inoculated with rhizo- sphere microorganisms under temperate nursery conditions M.A. Ahangar, G.H. Dar, Z.A. Bhat Ahangar M.A., Dar G.H., Bhat Z.A., 2012. Growth response and nutrient uptake of blue pine (Pinus wallichiana) seedlings inoculated with rhizosphere microorgan- isms under temperate nursery conditions of Kashmir. Ann. For. Res. 55(2): 217-227, 2012. Abstract. Microbial inoculants (Trichoderma harzianum, Pseudomonas fluo- rescens, Laccaria laccata) inoculated either individually or in combination significantly improved the growth and biomass of blue pine seedlings. The ECM fungus Laccaria laccata, when inoculated individually, showed sig- nificantly higher plant growth, followed by Pseudomonas fluorescens and Trichoderma harzianum. The combined inoculation of rhizosphere microor- ganisms showed synergistic growth promoting action and proved superior in enhancing the growth of blue pine than individual inoculation. Co-in- oculation of L. laccata with P. fluorescens resulted in higher ectomycor- rhizal root colonization. Uptake of nutrients (N, P, K) was significantly improved by microbial inoculants, tested individually or in combination. Combined inoculation of L. laccata with T. harzianum and P. fluorescens significantly increased in N, P and K contents in blue pine seedlings as compared to control. Acid phosphatase activity in the rhizosphere of blue pine seedlings was also enhanced by these microorganisms. L. lac- cata exhibited higher acid phosphatase activity followed by P. fluorescens. Keywords Pinus wallichiana, rhizosphere microorganisms, nutrient uptake, growth. Authors. M.A. Ahangar ([email protected]), G.H. Dar, Z.A. Bhat - Divi- sion of Plant Pathology, S. K. University of Agricultural Sciences and Technol- ogy Kashmir, India. Manuscript received April 10, 2012; revised September 04, 2012; accepted Sep- tember 14, 2012; online fi rst October 15, 2012. Introduction is propagated mainly through nursery-raised seedlings, which like most of the forest trees Blue pine ( Pinus wallichiana A. B. Jackson), face severe problems in successful regenera- 217 Ann. For. Res. 55(2): 217-227, 2012 Research article tion under existing natural conditions in soils et al. 2004). having low nutrient content. Pines are myco- Mycorrhizae improve plant growth through trophic in nature. Symbionts such as mycor- increased availability of phosphorus from non- rhizal fungi and free living organisms form in- labile sources (Jayachandran et al. 1989, Dar et tegral components of pine rhizosphere, an area al. 2007). The ectomycorrhizal fungal hyphae showing all kinds of antagonistic, parasitic and by growing away from the roots may detect growth promoting interactions (Zhang et al. zones of higher phosphorus availability and 1991). The ectomycorrhizal symbiosis alters hasten root proliferation in these zones (Har- the physicochemical and biological conditions ley & Smith 1983, Christophe et al. 2006). The in the surrounding soil and creates a highly other factors which contribute to enhance up- specialized environment called ‘ectomycor- take of phosphorus by mycorrhizal plants in- rhizosphere’, wherein selected microbial com- clude: (i) the possibility of fungi having a lower munities in association with mycorrhiza play threshold concentration for uptake of phospho- vital role in higher biochemical activities and rus, (ii) the production of organic acids and en- nutrient cycling (Christophe et al. 2007, Mey- zymes which increase the availability of phos- er et al. 2010). Microbial activity in the plant phorus and (iii) the ability of fungi to store and rhizosphere has substantial effect on plant effi ciently translocate phosphorus into the host productivity (Schroth & Weinhold 1986).The (Grove & LeTacon 1993). A major portion of free-living rhizosphere microorganisms such phosphorus in forest soils is in inorganic form. as Trichoderma, Gliocladium, Penicillium, Ectomycorrhizal fungi catalyze hydrolysis of Pseudomonas, Bacillus, Azotobacter, Azos- inorganic phosphorus and thereby enhance pirillum etc. favourably infl uence the plant phosphate uptake by host roots (Gerlitz & growth directly or indirectly. The direct pro- Werk 1994). It has been suggested that organic motion of plant growth by these organisms acids secreted by ectomycorrhizal fungi may occurs either through release of plant growth release phosphorus from organic and sparingly promoting compounds or by facilitating the soluble inorganic forms either by lowering the uptake of certain nutrients from the environ- soil pH or by chelation of metal ions (Malajc- ment. Further, they indirectly promote plant zuk & Cromack 1982, Gray & Dighton 2009). growth by reducing or preventing the delete- Ectomycorrhizal symbiosis also facilitates the rious effect of one or more phytopathogenic access of plant partner to soil nitrogen. This is organisms (Glick 1995, Hayat et al. 2010). of paramount importance in ecosystems where Many plant growth promoting rhizobacteria primary production appears to be restricted are free living diazotrophs and can convert by nitrogen availability (Linder 1989). In ad- molecular nitrogen into ammonia by virtue of dition to provide access to organic nitrogen nitrogenase enzyme complex (Postgate 1982). resources, ectomycorrhizae through improved Plant growth promoting rhizobacteria (PGPR) root systems help to counteract the effects of stimulate plant growth by facilitating the min- nitrogen depletion zones around roots (Finlay eral uptake particularly phosphates in plants et al. 1988). The dominant form of nitrogen + (Kloepper et al. 1989, Rashmi Awasthi et al. in forest soils is NH 4-N and ectomycorrhiaze 2011 ). Trichoderma species are opportunistic through their mycelial network increase the + avirulent plant symbionts as well as myco- uptake of NH 4-N (Reid et al. 1983). Inorganic parasites. Root colonization by Trichoderma nitrogen absorbed into hyphae is assimilated species also frequently enhances root growth and translocated as amides and amino acids in and development, improves crop productiv- the fungus utilizing metabolic pathways (Mar- ity, induces resistance to abiotic stress and in- tin & Botton 1993). creases uptake and use of nutrients (Harman The present investigation was under taken 218 Ahangar et al. Growth response and nutrient uptake of blue pine (Pinus wallichiana) ... to assess the impact of microbial inoculants 1 x 109 c fu/g. on nutrient uptake and growth of blue pine in Potting mixture was sterilized at 1.4 kg/cm2 nursery under temperate conditions of Kash- for one hour for three successive days. One kg mir. of sterilized potting mixture [organic carbon (%) = 0.7, pH = 6.8, available N = 281.2 kg/h, available P = 15.8 kg/ha, available K = 243.0 Materials and methods kg/ha] was put in each plastic bag of 1.5 kg capacity. The growth promoting microorgan- Ectomycorrhizal fungus (Laccaria laccata isms were inoculated separately and in com- Broome & Berkely) was isolated from sporo- bination in desired quantities into the upper 8- carps collected from the canopy of blue pine 10 cm of potting mixture and mixed properly. plantations in Lidder Forest Division, Kash- Ectomycorrhizal fungal inoculum prepared in mir on Modifi ed Melin-Norkran’s medium a vermiculite based carrier was added 15 days (Marx 1969). Trichoderma harzianum Rifai. before sowing at the rate of 15 ml/kg. Prepared and Pseudomonas fl uorescens Migulla were talc based formulation of Trichoderma har- isolated from the soil collected from the rhizo- zianum with inoculum load of 1 x 109 cfu/g, sphere of blue pine seedlings. The fungal an- was added at 5 g/kg of potting mixture, while tagonist was isolated by dilution plate method as Pseudomonas fl uorescens multiplied on talc on potato dextrose agar medium and the culture formulation with inoculum load of 2.5 x 108 was purifi ed by single spore/hyphal tip method cfu/g, was added at 5 g/kg of potting mixture and maintained for further studies. Rhizobac- fi ve days before seed sowing. teria were isolated by dilution plate method on Healthy seeds of blue pine were surface King`s B medium (King et al. 1954) and main- sterilized in 30% hydrogen peroxide for 30 tained in culture tubes at 4oC. Mycelial inocu- minutes, washed thoroughly with sterile dis- lum of ectomycorrhizal fungus was prepared tilled water and stratifi ed for 48 hours at 40 in a vermiculite-based carrier according to the C in dark. Five seeds were sown per bag and method of Marx & Bryan (1975). Ectomycor- after germination seedlings were thinned out rhizal fungus was grown aseptically in 1 litre to one per bag. The inoculated and uninocu- Erlenmeyer fl asks containing 750 ml of ver- lated seedlings were arranged in a completely miculite moistened with 375 ml of MMN liq- randomised design, with each treatment repli- uid medium. The fl asks were incubated at 26 ± cated 12 times. The seedlings were grown un- 2oC in the dark for 10 weeks. After incubation, der greenhouse conditions at 25 ± 3oC and ir- inoculum was removed from fl asks just before rigated with sterile distilled water as and when inoculation and leached with distilled water to required. No fertilizers or protective chemicals remove unused nutrients. were applied throughout the study. The seed- Pseudomonas fl uorescens was multiplied in lings were out planted gently and observations talc formulation with prior growth on King’s on plant biomass, seedling height, root length, ‘B’ broth medium using the procedure given ectomycorrhizal root colonization and nutrient by Vidhyasekaran & Muthamilan (1995). The uptakes were recorded at 30, 60, 90 and 120 product with fi nal bacterial population of 3 x days after seedling emergence. Dry weight of 108 cfu/g was shade-dried to reduce the mois- seedlings was determined by drying the plants ture content to 20 per cent, packed in poly- at 60o C for 48 hours in hot air oven. propylene bags, sealed and stored at 4o C for For the estimation of per cent ectomycor- further study. A talc based formulation of Tri- rhizal infection in roots, standard method of choderma harzianum was prepared as given Daughtridge et al.
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