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 rhizosphere 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 microorganisms under temperate nursery conditions of Kashmir. Ann. For. Res. 55(2): 217-227, 2012.

Abstract. Microbial inoculants (Trichoderma harzianum, Pseudomonas fluorescens, 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 significantly higher plant growth, followed by Pseudomonas fluorescens and Trichoderma harzianum. The combined inoculation of rhizosphere microorganisms showed synergistic growth promoting action and proved superior in enhancing the growth of blue pine than individual inoculation. Co-inoculation of L. laccata with P. fluorescens resulted in higher ectomycorrhizal 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. laccata 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. (1986) was followed. Seed- by Rudresh et al. (2005) with inoculum load of lings were gently uprooted and their roots

219 Ann. For. Res. 55(2): 217-227, 2012 Research article washed under running tap water to remove by spectrophotometer at 460 nm wave length. adhering soil debris. The roots were later The amount of p-nitrophenol released was stained for 15 minutes in 10 per cent solution calculated with reference to a standard curve of glacial acetic acid (v/v) which contained 0.1 prepared by using different concentrations of per cent Ponceau S (w/v). Upon removal, the p-nitrophenol. The enzyme activity was ex- roots were immediately rinsed in 10 per cent pressed as μM p-nitrophenol (PNP) released acetic acid to remove excessive stain and then g-1 soil hr-1. washed with distilled water. To evaluate the accuracy of Ponceau S staining technique, the roots were stained for 5 minutes with trypan Results blue in lactophenol. The root systems were examined under a stereomicroscope to count The study on the impact of microbial inocu- the number of mycorrhizal short roots as sug- lants on growth and nutrient uptake of blue gested by Beckjord et al. (1984). pine seedlings revealed that microbial inocu-

No. of ectomycorrhizal lateral rootlets observed Degree of mycorrhization() %=⋅ 100 Total number of lateral rootlets examined

Nutrient content (NPK) of pine seedlings col- lants (Trichoderma harzianum, Pseudomonas lected at different growth stages was estimated ﬂ uorescens, Laccaria laccata) inoculated ei- as per standard procedures of Jackson (1973). ther individually or in combination signiﬁ cant- The nutrient uptake of plant was worked out on ly improved plant growth (seedling height and dry weight basis by using following formula: root length) and biomass (fresh and dry plant

Nutrient content (%) x dry matter (g) Nutrient uptake (g/plant) = 100

The acid phosphatase activity of rhizosphere weight) as compared to uninoculated control. soil was measured by the method of Tabata- Among the microbial inoculants tested indi- bai & Bremner (1969). One g of soil sample, vidually, the ectomycorrhizal fungus L. laccata drawn from the rhizosphere of plant in each showed signifi cantly higher plant growth fol- treatment, was put in 50 ml volumetric fl asks. lowed by P. fl uorescens and T. harzianum. The Then 0.2 ml toluene, 4 ml modifi ed universal blue pine seedlings inoculated with L.laccata buffer (prepared by dissolving 2.42 g tris, 2.32 showed 24.6, 50.6, 45.4 and 30.11 per cent g malic acid, 2.8 g citric acid and 1.26 g bo- increase in shoot height, root length, fresh and ric acid in 100 ml IN NaOH and volume made dry plant weight, respectively, as compared to upto 1 litre with distilled water) and 1 ml of uninoculated control (Table 1). The highest 0.025 M P-nitrophenyl phosphate was added. increase in growth and biomass of blue pine The fl asks were incubated at 37 ± 1o C for 1 seedlings was observed due to combined in- hour. Enzymatic reaction was stopped by add- oculation of all the three microbial inoculants ing 4 ml of 0.5 M NaOH and 1 ml 0.5 M CaCl2. (L. laccata + T. harzianum + P. fl uorescens). The contents were fi ltered through Whatman`s The seedling height, root length, fresh weight fi lter paper No. 42, and the absorbance of yel- and dry weight of blue pine seedlings were low colour (p-nitrophenol released) estimated increased by 61.0, 95.8, 105.8 and 61.33 per 220 Ahangar et al. Growth response and nutrient uptake of blue pine (Pinus wallichiana) ...

Table 1 Inﬂ uence of microbial inoculants on growth and biomass of blue pine seedlings Seedling Root length Plant fresh weight Plant dry weight Treatments height (cm) (cm) (mg/plant) (mg/plant) Laccaria laccata (LL) 9.60 11.0 791.00 350.00 Trichoderma harzianum (TH) 8.30 9.2 690.00 322.00 Pseudomonas ﬂ uorescens (PF) 8.80 10.2 740.00 330.00 LL + TH 9.80 11.5 885.00 361.00 LL + PF 11.00 12.6 954.00 383.00 TH + PF 9.00 10.4 854.00 358.00 LL + TH + PF 12.40 14.3 1120.00 434.00 Control 7.70 7.3 544.00 269.00 Mean 9.50 10.8 822.20 250.80 CD (p = 0.05) 0.48 0.4 8.02 6.04

D = Days after seedling emergence Figure 1 Mycorrhizal root colonization of blue pine seedlings by L. laccata, also combined with P. fl uorescens and T. harzianum cent, respectively as compared to uninoculated ally or in combination as compared to unin- control. oculated control. The combined inoculation Roots of pine seedlings were colonized by proved superior over individual inoculants. L. the ectomycorrhizal fungus L. laccata, inocu- laccata proved most effi cient in improving the lated as vegetative inoculum. Generally co-in- uptake of nutrients followed by P. fl uorescens oculation of L. laccata with P. fl uorescens in- and T. harzianum. L. laccata and caused 99.2, creased the total number of mycorrhizal short 241.9 and 145.2 per cent increase in nitrogen, roots as compared to individual inoculation phosphorus and potassium uptake respective- (Fig. 1). ly, as compared to uninoculated control (Table The higher uptake of macronutrients (N, P, 2). The combined inoculation of L. laccata + K) was recorded in blue pine seedlings inocu- T. harzianum + P. fl uorescens resulted in sig- lated with microbial inoculants (L. laccata, P. nifi cantly higher uptake of N, P and K by fl uorescens and T. harzianum) either individu- blue pine seedlings as compared to individual

221 Ann. For. Res. 55(2): 217-227, 2012 Research article

Table 2 Inﬂ uence of microbial inoculants on nutrient uptake ( NPK) of blue pine seedlings Nitrogen uptake Phosphorus uptake Potassium uptake Treatments (mg/plant) (mg/plant) (mg/plant) Laccaria laccata (LL) 6.475 0.465 3.759 Trichoderma harzianum (TH) 4.733 0.332 2.737 Pseudomonas ﬂ uorescens (PF) 5.214 0.363 3.003 LL + TH 6.931 0.504 3.971 LL + PF 7.660 0.612 4.289 TH + PF 6.479 0.455 3.710 LL + TH + PF 9.461 0.824 5.685 Control 3.250 0.136 1.533 Mean 6.200 0.461 3.585 CD (p = 0.05) 0.557 0.026 0.477

Figure 2 Impact of microbial inoculants on rhizosphere acid phosphatase activity of blue pine seedlings inoculation and control. Discussion Acid phosphatase is solely of extra cel- lular enzymatic origin and is involved in the The impact of ectomycorrhizal fungi or rhizo- mineralization of organic phosphates. Among sphere bacteria on seedling growth and nutri- the rhizosphere microorganisms inoculated ent uptake is well known. However, few stud- individually, L. laccata exhibited higher acid ies have combined these microorganisms in phosphatase activity followed by P. fl oures- one experiment to clarify their relative contri- cens and T. harzianum (Fig. 2). The combined bution and interactions in nutrient acquisition. inoculation of L. laccata + T. harzianum + P. In the present study ectomycorrhizal fungus L. fl uorescens caused highest increase in acid laccata , mycorrhizosphere bacterium P. fl uo- phosphatase activity in the rhizosphere of blue rescens and mycorrhizosphere fungus T. har- pine seedlings as compared to uninoculated zianum were monitored for the respective con- control. This was followed by the coinocula- tributions, on nutrient uptake and plant growth tion of L. laccata + P. fl uorescens. of pine seedlings. L. laccata, P. fl uorescens and T. harzianum are considered to be opportunistic avirulent plant symbionts, benefi ting

222 Ahangar et al. Growth response and nutrient uptake of blue pine (Pinus wallichiana) ... their host by frequently enhanced root growth, is generally assumed that these developmen- resistance to abiotic stress and the uptake and tal responses are triggered by phytohormones use of nutrients. These organisms have the produced by bacteria (Bloemberg & Lugten- ability to increase plant growth and productiv- berg 2001, Persello-Cartieaux et al. 2003). Sig- ity by solubilization of otherwise unavailable nifi cant increase in plant height, basal area and mineral nutrients. crown breadth was observed in Bacillus and ECM fungus L. laccata showed signifi cantly Pseudomonas inoculated Pinus massoniana higher plant growth as compared to uninocu- plants ten months after their inoculation (Yang lated control. The seedlings with considerable et al. 2002). Pseudomonas fulva signifi cantly ectomycorrhizal colonization rapidly regener- stimulated the growth of Pinus sylvestris seed- ate new lateral roots, create more new sites for lings in a pasteurised soil and increased the ectomycorrhizae and thereby utilize available number of mycorrhizal roots in seedlings in- nutrients more effi ciently than non-mycorrhiz- oculated with Suillus luteus (Pokojeska et al. al seedlings (Marx & Hatchell 1986). Positive 2004). effects on seedling growth and survival in the In present study T. harzianum showed stim- fi eld environment after nursery inovulation ulatory effect on growth and biomass of pine with different mycorrhizal fungi have reported seedlings. Trichoderma species reportedly by various researchers (Diaz et al. 2009, Alves produce hormone like metabolites and release et al. 2010, Dalong et al. 2011). The favourable nutrients from soil or organic matter thereby infl uence of ectomycorrhizal fungus on plant facilitate better plant growth (Windham 1986). growth and health may be attributed to the Pinus sylvestris seedlings inoculated with T. excretion of growth promoting substances by virens produced a signifi cantly higher biomass mycorrhizae (Duchesne et al. 1987, Strzelezyk of needles, trunks and root than uninoculated et al. 1985) or indirectly, by alteration in root plants (Werner 2002). physiology, uptake of minerals and pattern of In combined inoculation of all the three mi- exudation into the mycorrhizosphere (Leyval crobial inoculants (L. laccata + T. harzianum & Berthelin 1990). Sudhakara & Natarajan + P. fl uorescens) steep increase in plant growth (1997) reported that 38.0 and 186.5 per cent may be ascribed to the synergistic growth increase in Pinus patula seedling height and promoting action of microbial inoculants as shoot dry weight, respectively due to L. lac- well as more solubilization of mineral nutri- cata after 8 months of inoculation. ents. P. fl uorescens seems to be a good can- Application of plant growth promoting didate for optimizing the effi ciency of ecto- rhizobacterium Pseudomonas fl uorescens mycorrhizal mycelium inoculum. Garbay & signifi cantly improved seedling height and Duponnois (1992) reported that P. fl uorescens root length as well as fresh and dry biomass promoted mycorrhizal formation of L. laccata as compared to uninoculated control. The im- with Douglas-fi r and oak seedlings, thereby provement in plant growth and biomass seems increased the growth of seedlings. Co-inocu- to be a consequence of intensive root coloni- lation of ectomycorrhizal fungus, S. citrinum zation by bacteria which may have promoted with the Collimonas sp. bacterial strain signifi - the synthesis of growth promoting compounds cantly improved the Pinus sylvestris biomass as well as facilitated more nutrient supply as compared to non-inoculated pine plants in rhizosphere. The positive effects of plant (Koele et al., 2009). Werner (2002) observed growth promoting bacteria on plant growth that mycorrhizal Pinus sylvestris seedlings in- are always correlated with remarkable changes oculated with T. virens produced signifi cantly in root morphology (Pacovsky 1990, Okon & higher plant growth and biomass. Vanderleyden 1997, Bertrand et al. 2000). It Roots of pine seedlings were colonized by

223 Ann. For. Res. 55(2): 217-227, 2012 Research article the ectomycorrhizal fungus L. laccata, inocu- secreted by ectomycorrhizal fungi may release lated as vegetative inoculum. Generally co-in- phosphorus from organic and sparingly solu- oculation of L. laccata with P. fl uorescens in- ble inorganic forms either by lowering the soil creased the total number of mycorrhizal short pH or chelation of metal ions (Malajczuk & roots compared with individual inoculation Cromack 1982). Improved uptake of nutrients (Fig. 1). The degree of colonization of L. lac- in P. fl uorescens plants might have attribut- cata varied among the treatments with highest ed due to their mineralization activity in the root colonization observed due to co-inocula- rhizosphere. Plant growth promoting rhizobac- tion of L. laccata with P. fl uorescens. It seems teria are free living diazotrophs that can con- that P. fl uorescens might have stimulated the vert molecular nitrogen into ammonia in a free mycorrhizal development of L. laccata with state by virtue of nitrogenase enzyme complex the roots of pine seedlings. Our results are sup- (Postgate 1982). Brimecombe et al. (1998) ported by the observations of Garbaye et al. reported increased mineralization of organic

(1992) where as P. fl uorescens isolate BBC6 residues by the two strains of Pseudomonas promoted mycorrhiza formation with L. lac- fl uorescens. Several reports have suggested cata with four conifer species. The mycorrhiz- that PGPR stimulate plant growth by facilitat- al helper bacteria seem to have acted in early ing the mineral uptake particularly phosphates stage to improve the receptivity of the root to in plants (Kloepper et al. 1989). Trichoderma the fungus before the fi rst mycorrhizas were spp. also frequently enhances root growth and formed. development, and increase uptake and use of L. laccata proved most effi cient in improv- nutrients (Harman et al. 2004). T. harzianum ing the uptake of nutrients (N, P, K) followed (T-22) was found to solubilize phosphate and by P. fl uorescens and T. harzianum. Increased other micronutrients that could be made avail- nutrient uptake in L. laccata inoculation seed- able to provide plant growth (Altomare et al., lings could be attributed to the solubilization 1999). Combined inoculation of L. laccata + of unavailable nutrients and presence of more T. harzianum + P. fl uorescens resulted signifi - root absorbing surfaces. Ectomycorrhizal fun- cantly higher uptake of N, P and K by blue gi catalyse hydrolysis of inorganic phosphorus pine seedlings as compared to individual in- and thereby enhance phosphorus uptake by oculation and control. The inoculation of two roots (Gerlitz & Work 1994). Organic acids or more benefi cial microbes have synergistic secreted by ectomycorrhizal fungi may release effect on host plants. The signifi cant increase phosphorus from organic and inorganic forms in nutrient uptake is due to more nutrient avail- either by lowering the soil pH or by chelation ability in the soil, which ultimately enhanced of metal ions (Melajeuk & Cromack 1982). the plant growth and biomass. Present fi ndings Ectomycorrhizal symbiosis facilitated the are supported by the work of Singh & Singh access of plant partner to soil nitrogen. Ecto- (1998) & Singh et al. (2001). Deepa et al. mycorrhizae through improved root systems (2003) reported that L. laccata. T. harzianum help to counteract the effects of nitrogen de- and Pseudomonas corrugata infl uenced the pletion zones around roots (Finlay et al. 1988) soil microfl ora, nutrient status of rhizosphere and through their mycelial network increase soil and that of different parts of Cedrus deo- + the uptake of NH 4-N (Reid et al. 1983). The dara seedlings with increased uptake of N, P ectomycorrhizal fungal hyphae growing away and K. from the root may detect zones of higher phos- Among the inoculants, L. laccata exhibited phorus availability and hasten the prolifera- higher acid phosphatase activity followed by tion of roots in these zones (Harley & Smith P. fl ourescens (Fig. 2). The phosphatases pro- 1983). It has been suggested that organic acids duced by ectomycorrhizae, solubilize insolu-

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