Scientific Papers. Series A. Agronomy, Vol. LVII, 2014 ISSN 2285-5785; ISSN CD-ROM 2285-5793; ISSN Online 2285-5807; ISSN-L 2285-5785

THE EFFECTS OF PHOSPHORUS DEFICIENCY AND RHIZOBACTERIA ON PHOSPHORUS CONTENTS OF TWO SOYBEAN (Glycine max L.) CULTIVARS GROWN AT LOW WATER SUPPLY

Vladimir ROTARU1, Simion TOMA1, Ana BIRSAN2

1Institute of Genetics, Physiology and Plant Protection, Moldavian Academy of Sciences 20 Padurii str., MD 2002, Chisinau, Republic of Moldova 2State University of Moldova, 60 Mateevici str., MD 2009, Chisinau, Republic of Moldova

Corresponding author email: [email protected]

Abstract

Phosphorus (P) deficiency and low water supply are major environmental constraints for agricultural production in many regions. Compared to cereal crops, the soybean (Glycine max.L.) is more susceptible to phosphorus insufficiency and drought. A soil pot experiment was conducted in the greenhouse to investigate the effects of phosphorus deficiency and pseudomonas florescence and azotobacter chroococcum strains on phosphorus uptake by soybean grown under temporary drought conditions. Soybean cultivars Zodiac and Horboveanca were grown on soil-sand mixture with P deficiency at two water regimes - 70% water holding capacity of soil (WHC) and 35% WHC. Plants were harvested and analyzed for P contents in each organ after 12 days of water deficit at the setting pod stage. Phosphorus deficiency significantly decreased nutrient uptake by all plant parts especially in drought conditions. Leaves have shown a higher sensitivity to P deficiency than roots. Application of rhizobacteria induced P uptake in both cultivars but their effects were more pronounced in Horboveanca under both well watered and dry soil conditions. Phosphorus fertilization alone of plants significantly increased P accumulation irrespective of soil moisture regime and Horboveanca displayed a higher response than Zodiac cultivar. The P accumulation in the roots of both cultivars and in the pods of Zodiac enhanced after the application of bacteria or P under temporary drought. Interactive effects of applied bacterial strains and water soil regime depend on the cultivar used. From the results of the present experiment it can be concluded that seem to be particularly beneficial to P uptake in normal as well as in water limiting environment.

Key words: drought, phosphorus, rhizobacteria, soybean.

INTRODUCTION these and many farmers don’t have the capacity to buy them. In addition, the long- Phosphorus (P) is an essential nutrient required term sustainability of fertilizers application is for plant growth, development and questionable because according to some productivity, but its low availability in soils estimates the world reserves of rock phosphates makes it one of the least mobile mineral are expected to be exhausted in the next 50 elements. Phosphorus deficiency is worldwide years at current rates of consumption (Gilbert, spread and it affects agriculture sustainability 2009). in many agricultural regions. In field Under low levels of soil moisture, not only conditions, low frequently is water but also nutrient availability may be accompanied by droughts. Drought and low P severely decreased when compared with well availability in soil presents a serious problem to watered conditions (Gahoonia et al., 1994). agriculture production on global and national There is need to identify suitable biological levels. Chemical P fertilizers are the main approach to improve under poor source of plant available phosphates alleviation soil resources such as scarce availability of in agroecosystems, but the majority phosphates and water. (approximately 75-80%) of applied phosphates Plant growth promotion rhizobacteria (PGPR) are fixed in soil and they became less available due to their ability to increase nutrient uptake for crops (Vance et al., 2003). Nowadays, the and thus to promote plant growth, have often use of P fertilizer is reduced substantially in been suggested as a means to improve crop many countries because of the higher costs of production (Adesemoye et al., 2009). There are 316 evidences that the application of P-mobilizing deficiency and pseudomonas florescence and microorganisms is regarded to be an alternative azotobacter chroococcum suspension strategy to improve the supply of P to application on P accumulation within soybean agricultural crops (Rodrigues and Fraga, 1999). organs of two cultivars were either cultivated Whitelaw (2000) stated that the bacterial under normal or suboptimal soil water regime. genera Pseudomonas are the most powerful P solubilizer. Simulative effects on plant P MATERIALS AND METHODS uptake under P deficient conditions were found after the application of Pseudomonas strains in To accomplish the objective of this study it was a pot experiment with cotton and pea conducted out a pot experiment in a greenhouse (Egamberdiyeva and Höflich, 2004). under controlled humidity conditions. The Plant growth promoting bacteria could research included two soybean (Glycine max., effectively colonize plant roots and provide L.) cultivars: Zodiac and Horboveanca that plant growth through different mechanisms that differ by potential productivity and include enhancing mobilization of less responsiveness to phosphorus fertilization. The available nutrients and subsequent increase in soil was chernoziom carbonated with low level nutrient uptake (Dey et al., 2004). There are of available phosphates, basic pH (7,7), which many experimental results which demonstrated was mixed with sand in a 3:1 ratio (by volume). the beneficial effects of PGPR on nutrient Seed inoculation with bacterial preparation was acquisition and plant growth. Their analyses carried out on the basis of rhizobium have shown that the application of PGPR is a japonicum. Phosphorus as monocalcium suitable approach for reducing the doses of phosphate dose of 100 mg kg-1 was chemical fertilizers and their pollution hazards administered to the soil (P100), being regarded that could also support eco-friendly crop as sufficiently supplied with phosphorus and production (Dey et al., 2004). control treatment (without chemical and Legumes are well known to be an essential biofertilizers, P0) - deficient in phosphorus. component of various agroecosystems in order The bacterial suspension of azotobacter to develop organic agriculture. Legumes, chroococcum and pseudomonas fluorescens particularly soybean, have a higher demand of strains (equivalent to 108 cells ml-1) was P nutrition because nitrogen fixation required applied to the soil before sowing. Soil moisture considerable sources of energy. This species is regime was achieved by watering to 70% of the sensitive to low P supply and drought. water holding capacity of the soil (WHC) in the We hypothesized that particularly under control as optimal value and 35% WHC unfavorable conditions P deficiency and low treatment option, being considered as water water supply the application of rhizosphere stress for soybean. The pots were distributed in microorganisms would improve plant growth a complete randomized block design with 3 by increasing phosphorus absorption in plant replications and three plants per pot. parts. Temporary drought regime began in the full- Moreover, the analyses of available literature flowering stage and lasted 12 days. The plants data revealed that the effects of PGPR on were harvested at the end of water stress and nutrient uptake were examined as a rule under separated into roots, leaves, stems, nodules and normal water conditions and there is scarce pods and dried at 60ºC for 3 days. Dry weight information regarding their impact on P uptake of each fraction was measured. The by legumes under combined abiotic stresses the concentration of P was measured in samples of low P and water deficit. Suspension of ground tissues following wet digestion, using nitrogen-fixing (Azotobacter chroococcum) and the phosphovanado-molybdate method of phosphate solubilizing (Pseudomonas Murthy and Riley (1962). The phosphorus florescence) bacteria (Rh - in Figures) was used contents in plant parts were calculated by as tested strains in the study and soybean plants multiplying dry weight of each organ with P which have a higher demand of P nutrition. concentration. Thus, the main objective of the research was to Data in figures represent the average value of investigate the influence of phosphorus the results of chemical analysis of plants in

317 three replications. The experimental results droughts on plant growth and development. As were analyzed statistically, determining we expected, the phosphorus sufficient significant differences at the level of P = 0.05. treatment (P100) had positive impact on P In figures are presented means of 3 replicates uptake in all parts of soybean plants. The P with standard error. concentrations of roots in the control treatment and with biofertilizers administration were RESULTS AND DISCUSSIONS similar in Zodiac cultivar under drought but rhizobacteria increased this trait by 14.5% in Plant growth usually benefits from application normal soil moisture level. of rhizobacteria in nutrient-deficient soils. It is well known that PGPR can improve the 60 nutrient status of their host plants (Rodrigues P0 P0+Rh P100 and Fraga, 1999). 50 One of the main objectives of our research was to explore the beneficial effects of potential 40

bacterial inoculants (nitrogen fixer and organ / 30 phosphate solubilizer) that can enhance mineral nutrition of plants. In our study, phosphorus mg P 20 fertilization and PGPR application generally increased plant growth and P uptake of both 10 soybean cultivars in soil with initial P deficiency. The data in Figures 1-4 show that 0 Leaves Stems Roots Pods the accumulation of phosphorus in plant parts varied among cultivars and were significantly Figure 1. Effects of P deficiency and rhizobacteria (Rh) altered by rhizobactria (Rh) application and soil application on P content of cultivar Zodiac grown in water regime. normal water conditions (70% WHC) In P deficiency treatment the total plant uptake of P was generally higher for Horboveanca 45 P0 compared with Zodiac under optimal moisture 40 level (70% WHC). However, there was no P0+Rh 35 significant difference between cultivars when P100 plants were subjected to limited water 30 conditions (35% WHC). Consequently, the 25

water suboptimal regime reduced substantially organ / 20 P accumulation in Horboveanca than in Zodiac.

mgP 15 Phosphorus accumulation in leaves of the P deficiency and bacteria application treatments 10 did not differ significantly while the P 5 sufficiency (P100) treatment increased 0 evidently the uptake of nutrient in Zodiac Leaves Stems Roots Pods (Figure 1 and 2). The analysis of experimental results revealed that the administration of Figure 2. Effects of P deficiency and rhizobacteria (Rh) biofertilizers improved nutrient contents in the application on P content of cultivar Zodiac grown in stems of Zodiac. Drought is considered one of suboptimal water conditions (35% WHC) the major environmental abiotic factors with a This was linked with more biomass considerable adverse influence on the plant accumulation by roots in no water stress performance, in particular on the nutrient status conditions (data are not presented). Compared of crops. In our study it was revealed that low to the P-deficiency plants the P content of pods water supply significantly reduced P contents in the treatments with biofertilizers and in both cultivars irrespective of treatments. chemical P fertilizer was approximately twice There is evidence that improving P nutrition of as high at low water supply (Figure 2). Wani plants could attenuate the negative impact of and collaborators (2007) revealed a beneficial 318 impact of PSB and N2-fixing bacteria on and 32.1% in comparison to control plants chickpea plants with a significant increase in (without rhizobia supply), when plants were productivity and uptake of phosphorus and not subjected to low water regime. The highest nitrogen. increase in phosphorus accumulation was Likewise, Li and coworkers (2013) observed in the pods of plants grown with demonstrated an increase of N2 fixation in pseudomonas florescence and azotobacter inoculated alfalfa with phosphate solubilizing chroococcum biofertilization under well rhizobium and nitrogen fixing bacteria when watered conditions. However, the application both nitrogen and phosphorus are limited. It of these strains had no significant effects on the may be concluded that the application of accumulation of nutrient in the pods under rhizobacteria increases significantly P uptake in water limited conditions (Figure 4). the stems of Zodiac in normal water conditions but their influence was not registered in roots in 90 water deficit treatment. Probably, these results P0 could be explained by the decreased transport 80 P0+Rh of nutrients from roots to above ground plant 70 P100 parts at low water supply. Our results are 60 consistent with experimental data obtained in 50

cowpea (Franzini et al., 2010; Neuman and organ / George, 2009). 40

In normal water soil conditions the differences mg P 30 between treatments with P deficiency and the 20 application of PGPR were not so pronounced in terms of P contents in leaves. In contrast, under 10 dry soil conditions P deficiency and 0 biofertilizers administration treatments were Leaves Stems Roots Pods significantly different in biomass production (data not presented) and total plant P content.

Thus, soil dryness limited P availability in the P deficiency-plants, compared with the Figure 3. Effects of P deficiency and rhizobacteria (Rh) application on P content of cultivar Horboveanca grown biofertilizers treatment. The similar effects in normal water conditions (70% WHC) were registered in maize (Krey et al., 2011) and rice (Panhwar et al., 2011). Interesting data 60 were obtained in Horboveanca cultivar which is P0 P0+Rh P100 considered more responsive to supplemental P 50 nutrition than Zodiac. The results registered by this cultivar indicate that the suspension 40 application of pseudomonas florescence and organ azotobacter chroococum contributed / 30 significantly to increase plant P uptake and plant biomass production (Figure 3 and 4). mg P 20 At the optimal soil moisture level, as shown in Figure 3, there were significantly higher P 10 contents in stems, roots and pods of treatments with P and biofertilizers application compared 0 with those of the control plants. However, there Leaves Stems Roots Pods was poor difference in terms of P accumulation in leaves between P deficiency and microorganism treatments. Experimental Figure 4. Effects of P deficiency and rhizobacteria (Rh) results demonstrated that the administration of application on P content of cultivar Horboveanca bacterial suspension induced a significant grown in suboptimal water conditions (35% WHC) increase of P uptake in stems and roots of 7.5%

319 In contrast, at the suboptimal soil moisture mineral nutrients. Also, they could lead to regime, significant differences between increase photosynthesis, making a greater treatments were recorded, regarding proportion of photosynthates available to plant phosphorus contents in leaves, stems and roots. - rhizobium symbiosis (Mortimer et al., 2008). Thus, the biofertilizers and mineral phosphorus In summary, our results have evaluated the applications contributed to an increase in total effects of rhizobacteria application and P nutrient uptake of 19.4% and 47.9% in plants fertilizer on low soil fertility on the soybean for Horboveanca under suboptimal water phosphorus uptake under suboptimal soil conditions, respectively. As related in literature moisture regime. These investigations show a the rhizobacteria application improved P potential strategy of microorganisms used for availability in soil from hard compounds of agricultural development in marginal lands that organic form. In addition, they in association are often deficient in P. with plant roots could stimulate protons and Likewise, these findings point to a higher low weight organic acids release that in turn dependency of Horboveanca, cultivar improve the nutrient condition of plants responsive to supplemental P nutrition, (Rodrigues and Fraga, 1999). Thus, the lowest compared with Zodiac on the bacterial phosphorus accumulation in plant parts was application for growth and nutrient uptake obtained in control plants of both cultivars, under conditions of drought and/or low nutrient particularly in water limiting environment. availability. In this study we used non-sterile The highest phosphorus accumulation in the soil due to the fact that in many researches, present work was obtained in the plants plant growth promotion with PGPRs was fertilized with P for Zodiac and Horboveanca. observed usually under controlled conditions Hence, considering the influence of the PGPR where these bacteria do not have to compete application on this parameter, the effect was with a range of indigenous soil microbes. At lower than those of the industrial fertilizer. present, unfortunately, the use of There is a body of reports on plant growth microorganisms in the biotechnology of promotion by bacteria (Kery et al., 2011) that legume production is very modest. Although have the ability to enhance P availability for the effects of rhizobacteria symbiosis are more plants. The use of PGPR can improve difficult to reveal in the field there is a need to phosphorus uptake by the plant which in turn examine their functional properties in further would result in more energy available for studies. nitrogen fixation by rhizobium. In addition, inoculation with PGPR improves CONCLUSIONS plant growth parameters and nutrient uptake, but this effect was more pronounced in Phosphorus deficiency decreased the Horboveanca than in Zodiac, expressing that phosphorus contents in soybean organs in the colonization of rhizobacteria also particular under water-limited conditions. significantly reduced the negative effect of P In P-deficient soils, application of P. deficiency (Figures 1-4). The uptake of other fluorescens and A. chroococcum strains can essential micronutrients from the soil due to affect the P supply of soybean plants. However, biofertilizers could play a role in general plant the magnitude of this influence is affected by growth improvement as well as in more soil water regime. indirect effects upon the N2-fixing system. The The application of rhizobacteria biofertilizers obtained results indicated that bacteria led to a significant increase in total phosphorus application increased the phosphorus content in uptake by Zodiac and Horboveanca soybean roots and pods, compared with their controls cultivars. Cultivar Horboveanca displayed a which were not supplied with rhizobacteria. higher response to PGPR application than These findings are in agreement with that of Zodiac. Subsequent field experiments should Xiurong et al. (2011) and Li et al. (2013). be consider the agronomic effects of Increased mineral nutrient status in roots would microorganisms in combination with different contribute to enhance the acquisition of scarce doses of phosphorus fertilizer and organic resources from dry soil in our case of water and manure on crop production.

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