Impact of phosphate-solubilizing fungi on the yield and phosphorus-uptake by wheat and faba bean plants

Microbio!' Res. (2000) 155,221-227 http://www.urbanfischer.de/journals/microbiolres © Urban & Fischer Verlag Impact of phosphate-solubilizing fungi on the yield and phosphorus-uptake by wheat and faba bean plants Omar A. Abdul Wahid1, Taha A. Mehana2 I Department of Botany, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt 2 Department of Soil & Water, Faculty of Agriculture, Suez Canal University, Ismailia. 41522, Egypt Accepted: March 30, 2000 Abstract Three fungal isolates (phosphate-dissolvers), Aspergillus niger, A. fumigatus and Penicillium pinophilum were isolated from the rhizosphere of different plants grown in Ismailia and South Sinai Governorates. They effectively solubilized rock phosphate or tricalcium phosphate in Pikovskaya's liquid medium. In pot and column experiments, they significantly reduced pH and increased available phosphorus in the soil treated with either rock phosphate or superphosphate. The yield components of wheat and faba bean plants increased as a result of soil inoculation with the isolated fungi. Penicillium pinophilum was the most efficient isolate. It increased the yield of wheat grains by 28.9 and 32.8% in the soil treated with rock phosphate and superphosphate, respectively. Similarly, it in- creased the production of faba bean seeds by 14.7 and 29.4% with the same treatments. The uptake of phosphorus by both crops significantly increased due to inoculation of the soil with the tested fungi. Key words: Phosphate-dissolving fungi - P-uptake - wheat- faba bean Introduction Phosphorus is one of the most vital elements for living organisms. It is a limiting factor in plant production. Soils contain about 0.05% (wjw) phosphorus; plants only make use of about 0.1 % of this phosphorus (Schef- fer and Schachtschabel 1992). Most of the newly re- claimed areas in Egypt are sandy and calcareous soils with alkaline pH. The soluble phosphate introduced into most of these soils, either by microbial degradation of Corresponding author: O. A. Abdul Wahid organic matters or by application of phosphatic ferti- lizers such as superphosphate, is rapidly converted to an unavailable form, mainly as tricalcium phosphate (Lewis and Racz 1969; Boutros et al. 1987). In such case, the only possible way to increase plant-available P is the use of phosphate-solubilizing microorganisms. Many investigators have shown that a wide range of microorganisms can solubilize water-insoluble phos- phate (Badr EI-Din et al. 1986; Alagawadi and Gaur 1988; Laheurte and Berthelin 1988; Gaind and Gaur 1991; Ibrahim et al. 1995). Although more attention has been paid to phosphate-dissolving bacteria than phos- phate-dissolving fungi (Barthakur 1978; Saber et al. 1983; Boutros et al. 1987), Venkateswarlu et al. (1984) reported that fungi were more efficient than bacteria in solubilizing insoluble phosphate. The present work aimed to study the effect of three isolates of phosphate- dissolving fungi on the yield and P-uptake by wheat and faba bean plants grown in a sandy soil treated with rock phosphate or superphosphate. Materials and methods Isolation, identification and counting of phosphate- dissolving fungi (PDF). The PDF used were isolated from soil samples collected from the rhizosphere of faba bean (Vicia faba L.), kidney bean (Phaseolus vulgaris L.), peas (Pisum sativum L.) and wheat (Triticum aesti- vum L.) grown in Ismailia and South Sinai Gover- norates. A mixture of Martin's medium (100 ml) (Allen, 1959), 10% CaCI2 (10 ml) and 10% K2HP04 (5 ml) was poured into ll-cm Petri dishes containing 1 ml of an appropriate soil-water dilution. Dishes were incubated 0944-5013/00/155/03-221 $15.0010 Microbiol. Res. ISS (2000) 3 221 # In soil-water suspension I :2.5. @ In soil saturation extract. Table 1. Some physical and chemical properties of the in- vestigated soil. in each pot, then the seedlings were thinned to 6 uniform plants per pot after one week of planting. As for faba bean, 4 pre-soaked seeds were sown in each column. After seedling emergence the bean plants were thinned to 2 plants per column. The following treatments were replicated 3 times in a randomized complete block design for each crop: The RP or SP at the rate of 31 mg PPs kg- I soil to- gether with 50 mg K20 kg-I soil as K2S04 (50% K20) were mixed with the top 15 cm soil in the pots or columns before cultivation of either crop. Ammonium sulphate (20.6% N) at a rate of 100 mg N kg-I soil was applied in three equal splits for wheat (before sowing, 55 and 80 d after sowing) and at a rate of 30 mg N kg-I soil in one split after 30 d of sowing of faba bean. The pots and columns were regularly irrigated up to the field capacity. The fungal suspension (2 x 109 spore ml- I ) was added with the irrigating water after plant thinning. After maturity, wheat and faba bean plants were har- vested, dried at 65°C and the dry weights of shoots and grains or seeds were recorded. Plant materials were ground and duplicate 0.5-g samples were acid digested and analyzed for P (Jackson 1958). Following the harvesting, soil of 3 columns and 3 pots (of each treat- ment) were sampled (0-20 cm depth). Sodium bicarbo- nate-soluble phosphorus was determined by the method of Olsen et al. (1954). The pH was measured in soil suspension (I: 2.5). Phosphate-dissolving fungi were isolated and enumerated as mentioned above. Control RP + Asp. n. RP + Asp. f. RP+Pen. Control SP+Asp. n. SP + Asp. f. SP+ Pen. value 94.5 2.5 3.0 Sandy 8.04# 0.55 1.40@ 1.05 0.09 6.50 4.08 parameter Sand (0/0) Silt (0/0) Clay (0/0) Texture pH CaC03 (0/0) ECe (dSm- l ) Organic C (g kg-1 soil) Total N (g kg-I soil) Available N (mg kg-I soil) Available P (mg kg-1 soil) RP RP + Aspergillus niger RP + A.fumigatus RP + Penicillium pinophilum SP SP + Aspergillus niger SP + A.fumigatus SP + Penicillium pinophilum for 5 d at 28°C in the dark. Colonies which produced clear halo zones indicated phosphate solubilization (Salih et al. 1989). From these colonies, three active iso- lates were identified as Aspergillus niger, A. fumigatus and Penicillium pinophilum. The total fungi were count- ed on Martin's medium containing rose bengal and streptomycin (Allen 1959). Preparation of inoculum. The three fungal species were grown on Martin's medium at 28°C for 10 d in the dark. The spores were harvested by flooding the cultures with sterile distilled water containing 0.05 % Tween 80. Each spore suspension was adjusted to the desired concen- tration and used for the inoculation of the flasks in the laboratory experiment and the soil in pot and column experiments. Laboratory experiment. In order to determine the solu- bilizing ability of the selected fungal isolates, rock phos- phate (RP, 29.6% Pps) of 125-500 J.l or powdered tricalcium phosphate (TCP, 45.1 % PPs) were used as substrates. The RP and TCP were autoclaved separately; a weight equivalent to 150 mg PPs of either RP or TCP was transferred aseptically to 250 ml Erlenmeyer flasks containing 100 ml of sterilized Pikovskaya's liquid me- dium (1948). The pH was adjusted to 7. Each flask was inoculated with 0.5 ml of either spore suspension. The flasks were incubated at 28°C in the dark. Each treat- ment was replicated twice. After 28 d of incubation, the fungal mat was removed and allowed to dry at 60°C for 3 d to determine the dry weight of each fungal biomass. The amount of soluble P20S was determined in the super- natant after sedimentation of the insoluble RP and TCP (Jackson 1958). This was done at three intervals: 5, 10 and 28 d. These two parameters were taken as a measure for the ability of the tested fungi to dissolve RP and TCP. The pH was also determined at these three intervals. Pot and column experiments. Two experiments, using wheat and faba bean plants, were carried out in the Botanical Garden of the Botany Department, Faculty of Science, Suez Canal University, Ismailia, Egypt. These experiments aimed to study the impact of the isolated fungi on the yield and P-uptake by plants grown on a sandy soil treated with either RP or superphosphate (SP, 15.5% P20 S)' The soil used in these experiments was collected from the Botanical Garden, grounded and sieved through a 2-mm screen before use. Some physi- cal and chemical properties of the used soil are given in Table 1. Soil was uniformly packed in earthenware pots (30 cm top diameter, 14 cm bottom diameter and 30 cm height) and plastic columns (15 cm diameter and 60 cm height) at rates of 7 kg pori and 15 kg column-I. The pots were planted with wheat grains, the columns with faba bean seeds. Five grams of wheat grains were sown 222 Microbiol. Res. 155 (2000) 3 Results Isolation and counting ofphosphate-dissolving fungi Counts (colony fonning units, CFU) of phosphate-dis- solving fungi (PDF), as detected by the clear zone around the colony on agar plates, ranged from 2-29 X 103 CFU per gram dry soil depending on the soil location and cultivated crop (Table 2). They represent about 3.51 to 25% of the total fungal biota recorded in the rhizosphere of wheat and faba bean plants grown in South Sinai Governorate respectively. In vitro phosphate solubilization ability ofisolatedfungi The 3 tested fungi showed different abilities to dissolve either RP or TCP in Pikovskaya's liquid medium (PLM). They can be arranged in a decreasing order, according to their ability, as follows: P. pinophilum > A. fumigatus > A. niger (Table 3). P. pinophilum re- leased 13.7 and 21.8 mg P20s/lOO ml PLM containing ISO mg P20 S as RPorTCP, respectively. The amount of solubilized P increased with the incubation period. The pH values of PLM inoculated with either fungal isolate were lowered during the incubation period. The pH reached its minimum value after 28 d. P. pinophilum showed the highest biomass value among the three fungal isolates used. This could be attributed to the greatest solubilization ability exhibited by this fungus. Soil pH and available P. Data in Table 4 indicate that the three fungal isolates significantly decreased the pH and increased the value of available P in the soil treated with RP or SP. 3.4. Population ofPDF in the tested soil Table 4 indicates that the total count of PDF in the soil was elevated as a result of inoculating the soil with either one of the three fungal isolates compared to the control. The counts ranged from 1.61-30.1 X 103 CFU g-l dry soil depending on the inorganic phosphate source, the inoculated fungus and the cultivated plant. The max- imum population was recorded in the soil treated with Table 2. Counts* of total fungi (TF) and phosphate-dissolving fungi (PDF) in the rhizosphere of different plants grown in Ismailia and South Sinai Governorates. Location Plant TF PDF PDFrrF % Ismailia Faba bean 312 29 9.29 Kidney bean 160 13 8.13 South Sinai Faba beans 24 6 25.00 Peas 31 3 9.68 Wheat 57 2 3.51 * count X 103 g-J dry soil Table 3. Effect of phosphate-dissolving fungi on the pH, soluble PPs (mg/l 00 ml medium) and fungal biomass (FBM, mg dry matter/l 00 ml medium) in Pikovskaya liquid medium containing rock phosphate (RP) or tricalcium phosphate (TCP). Treatments Incubation period (d) 0 5 10 28 pH P20 S pH PPs pH P20 S pH P20 S FBM RP (Control) 7.00 0.02 6.99 0.05 6.91 0.06 6.90 0.11 0 RP+Asp n 7.00 0.02 5.82 0.19 5.51 0.43 5.02 3.34 190 RP+Asp f 7.00 0.02 5.33 0.29 3.85 6.53 3.28 11.30 295 RP+ Pen 7.00 0.02 5.13 0.35 3.10 7.91 3.02 13.74 285 r -0.9844* -0.9528* -0.9687* TCP (Control) 7.00 0.10 6.95 1.19 6.90 3.24 6.85 7.80 0 TCP+Asp n 7.00 0.10 5.10 2.45 4.65 5.78 4.58 13.51 295 TCP+Asp f 7.00 0.10 4.62 2.70 3.50 8.90 3.14 19.47 370 TCP+ Pen 7.00 0.10 4.24 2.73 2.80 9.13 2.78 21.75 380 r -0.9891 * -0.9785* -0.9876* * Significant at P < 0.05; r = correlation coefficient between pH and Pps; Asp n: Aspergillus niger; Asp f: A. fumigatus; Pen: Penicillium pinophilum. Microbiol. Res. 155 (2000) 3 223 Table 4. Effect of the inoculation of soil with phosphate-dissolving fungi (PDF) on pH, available P (mg kg-I) and counts of PDF (count x 103 g-I dry soil) in a sandy soil treated with either rock phosphate (RP) or superphosphate (SP) and cultivated with wheat or faba bean plants. Treatments Wheat Faba bean pH Available P PDF pH Available P PDF RP (Control) 7.88 8.8 1.6 7.87 9.1 2.2 RP + Asp n 7.77 11.9 6.2 7.81 12.8 10.6 RP+Asp f 7.76 11.8 4.9 782 1l.5 5.9 RP+ Pen 7.73 14.2 8.8 7.80 14.0 16.1 LSD 0.05 0.10 1.21 0.05 1.76 SP (Control) 7.86 16.0 1.8 7.85 15.5 204 SP+Asp n 7.8 21.0 6.8 7.76 20.3 11.6 SP+Asp f 7.77 21.6 6.8 7.78 21.1 17.1 SP+ Pen 7.74 23.1 lOA 7.73 24.2 30.1 LSD 0.05 0.06 2.79 0.06 2.63 Asp n: Aspergillus niger; Asp f: A. fumigatus; Pen: Penicillium pinophilum. Table 5. Correlation coefficient (r) between levels of avail- able P and soil pH values, counts of phosphate-dissolving fungi (PDF) and values of the yield components of wheat and faba plants grown on a sandy soil treated with either rock phos- phate (RP) or superphosphate (SP). RP SP Soil pH Wheat -0.9608* -0.9745* Faba bean -0.9707* -0.9641 * Counts of PDF Wheat 0.9870* 0.9726* Faba bean 0.9661 * 0.9679* Yield components Wheat Straw 0.9904** 0.9209 Grains 0.9673* 0.8956 Total 0.9860* 0.9413 Faba bean Straw 0.9975** 0.9587* Grains 0.9729* 0.8668 Total 0.9936** 0.9408 * significant at P < 0.05 ** significant at P < 0.01 SP, inoculated with Penicillium pinophiLum and culti- vated with faba bean. It was about 25.1 times that of the initial count of the untreated soil. In general, the fungal count was always higher in the soil treated with SP compared to RP. The same picture was found in the soil cultivated with faba bean and wheat. This is supported by the data of Table 2. Data of Table 5 reveal a positive and significant cor- relation between levels of available P and counts of PDF in soil treated with either inorganic phosphate sources and cultivated with faba bean or wheat plants. 224 Microbiol. Res. 155 (2000) 3 YieLd components Table 6 indicates that the yield components of the two tested crops increased as a result of soil inoculation with PDF. However, this effect was not always statistically significant (Table 6). Grain and biological yields of wheat plants grown on soil treated with RP or SP were significantly affected by the inoculation with P. pino- phi/um and A. fumigatus compared to the corresponding control (uninoculated soil). In the case of faba bean plants, straw, seed and biological yields significantly in- creased when the soil was treated with RP plus A. niger or P. pinophiLum relative to the control. However, no significant differences were observed in the yield com- ponent values of both crops due to the inoculation with either one of the three fungi when compared to each other. In RP treated soil, the highest yields of wheat grains and faba bean seeds were recorded when the soil was inoculated with P. pinophiLum, where the increases over the control were 28.9 and 14.7%, respectively. In SP-treated soil, P. pinophiLum was also found to be the most efficient fungal isolate. It significantly increased wheat grains and faba bean seeds over the control by 32.8 and 29.4%, respectively. Phosphorus concentration and uptake Data in Table 7 indicate that the concentration of P in wheat straw significantly increased in the soil treated with either phosphate compound and inoculated with P. pinophiLum or A.fumigatus relative to the correspond- ing control. On the other hand, concentration of P in the other yield components of both crops was not signif- icantly affected under the same conditions (Table 7) though the P uptake significantly increased. Table 6. Effect of the inoculation of the soil with phosphate-dissolving fungi on some yield components of wheat and faba bean plants grown on a sandy soil treated with either rock phosphate (RP) or superphosphate (SP). Treatments Wheat yield (g column-I) Faba bean yield (g porI) Straw Grain Biological Straw Seed Biological RP (Control) 23.2 12.8 36.0 31.7 29.9 61.6 RP + Asp n 25.7 14.5 40.2 36.6 33.0 69.6 RP+Asp f 26.1 15.4 41.5 35.0 31.1 66.1 RP+ Pen 28.7 16.5 45.2 38.8 34.3 73.1 LSD 0.05 ns! 2.37 5.40 4.61 2.64 7.45 SP (Control) 25.4 13.7 39.1 34.2 32.0 66.2 SP+Asp n 29.6 15.1 44.7 37.8 34.7 72.5 SP+Aspf 29.5 17.5 47.0 41.2 33.8 75.0 SP+ Pen 34.4 18.2 52.6 46.4 41.4 87.8 LSD 0.05 2.75 1.98 5.70 5.97 4.05 5.91 Asp n: Aspergillus niger; Asp f: A.fumigatus; Pen: Penicillium pinophilum. ns!: not significant. Table 7. Effect of inoculation with phosphate-dissolving fungi on the concentration and uptake of P by wheat and faba bean plants grown on a sandy soil treated with rock phosphate (RP) or superphosphate (SP). Treatments Wheat Faba bean Straw Grains Total Straw Seeds Total --- Cone. Uptake Cone. Uptake Uptake Cone. Uptake Cone. Uptake Uptake % mg/pot % mg/pot mg/pot % mg/col % mg/col mg/col RP (Control) 0.17 39.4 0.53 67.8 107 0.36 114 0.50 150 264 RP+Asp n 0.20 51.4 0.55 79.8 131 0.36 132 0.57 188 320 RP+Asp f 0.34 88.7 0.54 83.2 172 0.45 158 0.53 165 323 RP+ Pen 0.41 117.7 0.56 92.4 210 0.37 144 0.51 175 319 LSD 0.05 0.05 11.7 ns! 10.0 20 ns! 16 ns! 14 33 SP (Control) 0.22 55.9 0.54 74.0 130 0.32 109 0.50 160 269 SP + Asp n 0.24 71.0 0.58 87.6 159 0.33 125 0.50 174 299 SP + Asp f 0.54 159.3 0.57 99.8 259 0.32 132 0.52 176 308 SP+ Pen 0.43 147.9 0.59 107.4 255 0.35 162 0.53 219 381 LSD 0.05 0.05 6.1 ns! 11.1 18 ns! 12 ns! 13 24 Asp n: Aspergillus niger; Asp f: A.fumigatus; Pen: Penicillium pinophilum. ns!: not significant. Discussion It is obvious that the rhizosphere of leguminous crops is more appropriate than that of wheat for the isolation of PDF. This is coincident with the results of Alexander (1983) that legumes create a more pronounced rhizo- sphere than cereals. This stimulative effect is attributed to the root exudates which contain amino acids, carbo- hydrates, organic acids and growth-promoting sub- stances. In vitro, the three fungi showed different abilities to dissolve either RP or TCP. It was clearly evident that the greater the reduction of pH values the greater the amount of the solubilized P. Significant negative cor- relation coefficients were observed between values of pH and amounts of solubilized P20 S at different inter- vals of incubation. This was reported by some other scientists (Arora and Gaur 1979; Gaur et ai. 1979). Although the change of soil pH was slight, there was a significant negative correlation coefficient between values of soil pH and available P. It is obvious that the change in P availability could be related to the inocula- tion of the soil with anyone of the tested fungi. This phe- nomenon is attributed mainly to the release of organic acids by the fungi (Dighton and Boddy 1989; Yadav and Singh 1991; Bolan et ai. 1994). On the other hand, Salih et ai. (1989) found that the solubilization of P by some Aspergillus and Penicillium isolates was not related to changes in soil pH, but due to the nature of organic acids produced by these fungi. However, some authors be- lieved that some other factors may be involved in this process (Asea et ai. 1988; Cunningham and Kuiack Microbiol. Res. 155 (2000) 3 225 1992). Illmer and Schinner (1995) proposed that the release of protons accompanying respiration or NH4+ as- similation is the most probable mechanism involved in P solubilization in soil. Casida (1959) and Chhonkar and Tarafdar (1984) suggested that neutral phosphatases are also involved. It is more likely that a complex mecha- nism is involved in the P dissolution by the fungi Asper- gillus and Penicillium. This mechanism may include the production of organic acids, the release of protons during respiration and NH4+ assimilation as well as the secretion of phosphatase enzymes. Faba bean favored the development of larger popula- tions of PDF compared to wheat. Supplementing the soil with SP supported establishment and development of PDF in the rhizosphere of faba beans. Although RP was less effective than SP, both inorganic phosphates increased the PDF population significantly. The stimu- lating effect of SP in the presence of PDF may be direct- ly attributed to the increased supply of available P; indirectly, it could be due to the change in the plant growth rate and metabolic activity that in tum increases the root exudates. This will create a favorable habitat for development and growth of microorganisms (Gaind and Gaur 1991). It is clear that inoculating the soil with PDF to increase their population in the rhizosphere of dif- ferent plants may improve the efficiency of the added inorganic phosphatic fertilizers. P. pinophilum gave the highest yield components of both crops. This could be explained on the basis that values of the highest available P and lowest soil pH were recorded in soil inoculated with P. pinophilum. Partic- ularly in the soil treated with RP, the crop yield depends on the availability of P. The reduction in soil pH values due to inoculation with fungi may also increase the availability of some other micronutrients (Matloub and Mehana 1998; Mehana 1998), which in tum support plant growth. It could be concluded that the application of RP along with PDF may be considered to be a possible substitute for SP. Furthennore, PDF inoculation improves the efficiency of SP through increasing the P availability, and consequently, decreasing the amount of phosphatic fertilizers needed. 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