Impaginato 171 Adv. Hort. Sci., 2019 33(2): 171-177 DOI: 10.13128/ahs-23326 Relationship between chlorosis, photo- synthesis and the nutrient content of plane trees in the presence of chemical and organic fertilizers H. Aalipour (*), A. Nikbakht, N. Etemadi Department of Horticulture, College of Agriculture, Isfahan University of Technology, 8415683111 Isfahan, Iran. Key words: mycorrhizal fungi, nutrient acquisition, organic matter, symbiosis, urban trees. Abstract: Chlorosis disorder is a major problem affecting the growth and physi- ological processes of many trees including plane trees (Platanus orientalis L.). This experiment was conducted to study the relationship between leaf chloro- sis disorder and the nutritional status and some important physiological charac- teristics of plane trees. The experiment was arranged in a randomized com- plete block design with six replications and four treatments including control, manure (M), manure + fertilizer (20-5-10) (MF), and manure + fertilizer + myc- orrhizal fungi (MFA) (Glomus intraradices + G. mosseae). The results showed that although all treatments significantly improved the nutrients content, solu- ble carbohydrates content, photosynthesis rate and chlorophyll content in the leaves, they mostly reached their peak in the mycorrhizal inoculated plants. Nitrogen (N), phosphorus (P) and zinc (Zn) were increased in the AMF amended trees compared to the control plants. The photosynthesis rate was enhanced by all the mixtures at least by 60% compared to the control. The most Chlorosis (17.5%) to the leaves recorded on the control plants, while leaf damage dropped to less than 2.9% at mycorrhizal treatment leading to the improved nutritional balance in the plane trees. The results proved the effectiveness of including mycorrhizal inoculation to the common fertilization practices to pre- vent leaf chlorosis in the plane trees. 1. Introduction Plane tree (Platanus orientalis L.) is among the most common orna- mental and street trees planted in the urban landscape in Iran and some Mediterranean countries (Anselmi et al., 1994; Khorsandi et al., 2016). They are known for their longevity and wide distribution in the temperate zones. However, the chlorosis as an important physiological disorder in the plane trees has affected a majority of them in Iran in recent years (Khorsandi et al., 2016). The problem is a common physiological disorder affecting many plants around the globe. It is especially a major problem in (*) Corresponding author: h.ali@ag.iut.ac.ir Citation: AALIPOUR H., NIKBAKHT A., ETEMADI N., 2019 - Relationship between chlorosis, photosynthesis and the nutrient content of plane trees in the pre- sence of chemical and organic fertilizers. - Adv. Hort. Sci., 33(2): 171-177 Copyright: © 2019 Aalipour H., Nikbakht A., Etemadi N. This is an open access, peer reviewed article publi- shed by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 31 May 2018 Accepted for publication 10 January 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(2): 171-177 172 the calcareous soils and soils with high pH (Wallace, 1982). The chemical properties of the soil and the ade- quate supply of nutrients are major factors affecting natural plant growth and extension (Cekstere and Osvalde, 2013), therefore important factor in nutri- ent uptake is the availability of the nutrients in the soil. Most trees cultivated in the alkaline and calcare- ous soils are exposed to the incidence of chlorosis which is reported to be basically due to Fe deficiency (Mortvedt, 1986). Several factors can contribute to leaf chlorosis including nutritional disorders and a disorder in the chlorophyll biosynthesis. Indeed, the lack of some nutrient elements such as nitrogen (N), zinc (Zn) and expecially iron (Fe), lead to the chlorosis in plants (Godde and Dannehl, 1994). Moreoever, fol- lowing the lack of sufficient chlorophyll, the affected plant will not be able to operate photosynthesis process, resulting in stunted growth (Miller et al., 1984). Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs that colonize the roots of the most land plants and increase host nutrient acquisition (Desiro et al., 2014) and it is claimed that virtually all trees acquire nutrients through symbiotic mycorrhizal fungi (Brundrett, 2009). Mycorrhizal inoculation is documented as a method to the improve nutrient uptake in many plants (Lehmann et al., 2014; Varga, 2015; Young et al., 2015). AMF are effective sym- bionts for plants, and their symbiotic relationship can i n c r e a s e p l a n t g r o w t h ( V a f a d a r e t a l . , 2 0 1 4 ) . Moreover, there is a lack of information on symbiosis relationship between the plane tree and AMF fungi. In the present study, we added AMF to the com- mon fertilization program of the plane trees in the urban landscape to study the following items. Firstly study the effect of mycorrhizal association on the trees response and then, observe how nutrient con- tent and different physiological processes are associ- ated with the leaf chlorosis disorder. To the best of knowledge, this is the first report attempting to dis- cover the correlation between different plane tree physiological processes and leaf chlorosis disorder under AMF inoculation. 2. Materials and Methods Experimental site and treatments The experiment was conducted during 2013-2014, o n t h e c a m p u s o f t h e I s f a h a n U n i v e r s i t y o f Technology in Isfahan (32°39’ N, 51°40’ E; 1600 m), Iran. The site is characterized as having an arid cli- mate with cold winters, 122.8 mm average annual rainfall and 23.4°C average annual temperature. Twenty-four uniform 15-year-old plane trees (P. ori- entalis L.) were selected. The experiment was a randomized complete block design (RCBD) with four treatments. Treatments included control, manure (M), manure + fertilizer (water soluble 20-5-10 N-P-K compound fertilizer with 12.8% sulfur, 1.3% magnesium oxide, NovaTec Solub, Compo, Germany) (MF), and manure + fertiliz- er + mycorrhizal fungi (MFA). Six replications were prepared for each treatment. The plants were inocu- lated with two AMF inoculations including Glomus intraradices and G. mosseae (both of them have been transferred to new genera, so Index Fungorum con- siders them now as Rhizophagus intraradices (N.C. Schenck & G.S. Sm.) and Funneliformis mosseae (Nicolson & Gerd.) (Schüßler and Walker, 2010). The AM fungi were provided by the Institute of Soil and Water Research, Tehran, Iran. Inoculum was com- prised of a mixture of spores (80 spores g-¹ for G. intraradices and 80 spores g-¹ for G. mosseae). The mixtures of filling materials were placed into 0.5 × 0.5 m holes, depending on the treatment in early spring. This technique provides a nutrients in a zone in and around each hole. With the first wetting, the nutrients are released from the fertilizer into the soil and the manure slowly lower the pH of the soil sur- rounding the hole. Over a period of time, a zone of soil around each hole is modified to be lower in pH and rich in micronutrients in approximately the cor- rect proportions. Two identical holes were drilled around each tree about one meter away from the tree trunk and filled up with the corresponding mix- ture. During the process, we avoided drilling into large buttress roots. In M treatment, trees received 5 kg of manure per hole mixed with the soil of the drilled hole. In MF treatment, 100 g of fertilizer per hole was added to the manure. Trees of MFA treat- ment received the AMF inoculums by adding 250 g of mycorrhizal inoculums into each hole mixed with manure and fertilizer (500 grams of inoculum per each tree in total). The control group did not receive any treatment (two identical holes were drilled). The trees were irrigated once a week. Some chemical and physical properties of the soil and cow manure are presented in Table 1. Measurements Various morphological and physiological parame- ters were measured 5 months after treatment. The Aalipour et al. - Platanus orientalis disorders and nutritional status 173 mineral contents of the plant leaves were deter- mined in the second year of the experiment. Plant samples were oven-dried at 65°C for 48 h and then were ground to determine their mineral composi- tion. The determination of the total N in the leaf samples was based on the Kjeldahl method (Baker and Thompson, 1992). The extraction of P, K, Fe, and Zn from the plant tissue material was performed by using 2 M hydrochloric acid (HCl) after dry ashing at 550°C for 5.5 h. The concentrations of Fe and Zn were determined by atomic absorption spectropho- tometer (670 Shimadzu, Kyoto, Japan) (AOAC, 2006). P concentration was determined by vanado molyb- date phosphoric acid method with a spectropho- t o m e t e r ( U V - 1 6 0 A U V - V i s i b l e R e c o r d i n g S p e c t r o p h o t o m e t e r , S h i m a d z u , T o k y o , J a p a n ) (Cottenie, 1980). LCI Portable photosynthesis and transpiration rate analyzer (Li - 6400; LICOR, Lincoln, NE, USA) was used to measure the net photosynthe- sis rate (A) between 09.30-11.30 h on 10 fully expanded current-season leaves situated at the mid- canopy height. The soluble sugars were measured according to the phenol-sulfuric acid method (Dubois et al., 1956). The extraction of the leaf chlorophyll pigments was carried out using 100% acetone according to Lichtenthaler (1987). For evaluation of the leaf chlorosis extent, 100 leaves from each tree were selected randomly and both leaf surfaces were scanned by a scanner (Canon i-SENSYS MF4010, Canon Inc., Korea). The leaf chlorosis was determined by digital image processing u s i n g M A T L A B s o f t w a r e . A r a n g e o f c o l o r w a s defined for the leaf chlorosis in the program and total leaf area was examined pixel by pixel by the software and the percentage of pixels which was defined as chlorotic areas were calculated by the software (Rathod et al., 2013). Data analysis Data were assessed for normality and log-trans- formed used to make data conform to normality when necessary prior to analysis. Non-homogeneity data were observed in leaf chlorosis, being the data transformed with the formula arcsin Ö(leaf chlorosis/100) to obtain homogeneity. The experimental data were statistically analyzed by the analysis of variance ( A N O V A ) . T h e s i g n i f i c a n c e o f t h e d i f f e r e n c e s between treatments was estimated using the least significant difference (LSD) test at P≤0.05, and graphs were drawn using Excel 2010. Statistical correlation was calculated by Pearson’s correlation coefficient (r). This test was used to measure the strength of a linear association between the leaf chlorosis and other variables including nutrients content, photo- synthesis rate and chlorophyll and soluble sugar con- tents. The value r = 1 means a perfect positive corre- lation and the value r = -1 means a perfect negative correlation. The experimental data were statistically analyzed with Statistical Analysis Systems (SAS) soft- ware, version 9.1 and Statistics, version 8.0. 3. Results AMF inoculation increased all nutrients content including P, N, Fe and Zn in the leaves of the treated trees (Table 2). N, P and Zn reached their peak value only when the fertilizer mix amended by mycorrhizal inoculums. P and Zn increased by 424% and 425% Factors Texture pH EC (dS m-1) Organic matter (%) N (%) P-available (mg kg-1) K-exchangeable (mg kg-1) Fe (mg kg-1) Zn (mg kg-1) Soil Clay 7.9 1.53 1.15 0.15 140 235 1400 21 Manure - 8.02 15.23 20.4 3.07 791 2030 12300 194 Table 1 - Some chemical and physical properties of the soil and cow manure used in research Treatment Nutrient N (g kg-1) P (g kg-1) Fe (mg kg-1) Zn (mg kg-1) Control 17.47 d 1.85 c 54.93 b 4.82 d Manure 19.14 c 5.25 b 148.99 a 15.09 c Manure + Fertilizer 19.65 b 7.31 b 146.71 a 17.32 b Manure + Fertilizer + AMF 20.39 a 9.71 a 170.14 a 25.31 a Table 2 - Influence of arbuscular mycorrhizal (AM) fungi and other treatments on nutrient uptake of plane tree (Platanus orientalis L.) Means in the same column followed by the same letters are not statistically different at P≤0.05 by the Least Significant Difference test (LSD). Adv. Hort. Sci., 2019 33(2): 171-177 174 respectively, compared to the control plants. All treatments (regardless of the composition of the mix- ture) successfully enhanced Fe and N contents in the leaves compared to the control plants (Table 2). All treatments significantly improved the soluble carbohydrates content in the leaves; however, it reached the peak in the mycorrhizal inoculated plants. The soluble carbohydrates content increased by 35.44% and 13.82% compared to the control of non-fertilized treatment and non-inoculated plants, respectively (Fig. 1). All treatments increased the photosynthesis rate at least by 60% compared to the control, although no significant difference was observed between the treatments (Fig. 2). The same trend was observed in the case of chlorophyll content (Fig. 3), where it increased at least by 32% compared to the control. Leaf chlorosis was influenced dramatically by the treatments (Fig. 4). The most leaf chlorosis was recorded in control non-fertilized treatments. Leaf chlorosis reduced to less than 2.9% on inoculated trees by AMF, while 17.5% of leaves tissue were affected by chlorosis in the control plants. The AMF inoculated plants showed an increase of 13.44% com- pared to the plants that received manure + fertilizer. A strong relationship was found between leaf chlorosis and Fe, N and Zn contents (Fig. 5). A significant and linear relationship was also found between chlorosis and the chlorophyll content and net photosynthesis in the plane tree leaves (Fig. 6). Leaf chlorosis in the plane trees resulted in a dra- matic and linear decline in the soluble carbohydrate in the leaves (Fig. 6). 4. Discussion and Conclusions Two explanations can be presented for increasing Fe content under any treatments (except for the con- Fig. 1 - Influence of arbuscular mycorrhizal (AM) fungi and other treatments on soluble carbohydrate content in plane tree (Platanus orientalis L.). Means are separated by LSD test at P≤0.05. Fig. 2 - Influence of arbuscular mycorrhizal (AM) fungi and other treatments on photosynthesis rates in plane tree (Platanus orientalis L.). Means are separated by LSD test at P≤0.05. Fig. 3 - Influence of arbuscular mycorrhizal (AM) fungi and other treatments on chlorophyll content in plane tree (Platanus orientalis L.). Means are separated by LSD test at P≤0.05. Fig. 4 - Influence of arbuscular mycorrhizal (AM) fungi and other treatments on chlorosis damage in plane tree (Platanus orientalis L.). Means are separated by LSD test at P≤0.05. Aalipour et al. - Platanus orientalis disorders and nutritional status 175 trol) in this experiment (Fig. 1). Firstly, as the soil of the site was a calcareous and compacted soil, any treatment increasing the air flow in the soil could improve Fe content in the plant (Lucena, 2003). Secondly, using the manure itself can increase Fe content in the plants (Mortvedt, 1986). It is docu- mented that the manure provides micronutrients including Fe and improves the structure of the soil (Lucena, 2003). Our results at least partly confirm the findings that combining Fe fertilizers with the organic matter is more favorable in terms of Fe uptake than Fe sources applied alone (Mortvedt, 1986). It has been reported that the application of FeSO4 (4-8 kg tree-¹) mixed with manure, cotton seed cake, or other organic substances in 8 to 10 holes in the soil around the crowns of apple trees (Malus sylvestris Mill.) r e s u l t e d i n m a r k e d c o r r e c t i o n o f F e c h l o r o s i s (Zheng-Qing and Cang-Zhen, 1982). It is also well established that as a result of the decomposition of organic matters in the soil, compounds such as humic acid (HA) and fulvic acid (FA) are produced in the soil (Nardi et al., 2002). These acids are well known as naturally-occurring chelating agents (Mortvedt, 1986; Nardi et al., 2002). There are many reports showing enhanced micronutrients uptake by the plants receiv- ing HA, FA (Nikbakht et al., 2008; 2014) or organic matter (Atiyeh et al., 2002). It is shown that inocula- tion turfgrass (Lolium prenne L.) with AMF receiving HA not only improved plant growth but also showed more elevated nutrients content in the leaves than in non-inoculated (control) plants or plants receiving only HA (Nikbakht et al., 2014). Researchers believed that the role of AMF in NO-3 t r a n s p o r t t o t h e r o o t s u r f a c e i s s i g n i fi c a n t (Subramanian and Charest, 1999; Javaid, 2009). They especially insist that the role of AMF is of value and importance in nitrate uptake in Mediterranean and (semi-) desert ecosystems which are characterized by calcareous soils. N, P and Zn uptake reached their peak value when the fertilizer mixture amended by AMF (Table 2). These results confirm the well-documented effect of AMF inoculation on nutrients uptake (Brundrett, 2009; Varga, 2015; Young et al., 2015). A strong rela- tionship between leaf chlorosis and Fe, N and Zn con- tents implies that the chlorosis is not only because of Fe deficiency in the plant, but also other nutrients including N and Zn play they own role (Fig. 5). It indi- cates that leaf chlorosis in the plane trees was not simply due to Fe deficiency. It is well documented that Fe is an essential element for many vital process- es in a plant including photosynthesis, respiration, N Fig. 5 - Relationship between chlorosis with Fe (A), Zn (B) and N concentration (C) in plane tree (Platanus orientalis L.). Fig. 6 - Relationship between chlorosis with Photosynthesis rate (A), Chlorophyll Content (B) and Soluble Carbohydrate (C) in plane tree (Platanus orientalis L.). 176 Adv. Hort. Sci., 2019 33(2): 171-177 fixation, chlorophyll and hormone synthesis; Fe is also a constituent of heme proteins (cytochromes, catalase, and peroxidase) (Briat and Lobreaux, 1997). As a result, affected plants by Fe deficiency suffer severe metabolic and structural disorders (Javaid, 2009). There are also some reports indicating that the major cause of Fe deficiency is the very low solu- bility of Fe oxides in the soil (Mortvedt, 1986). It shows the importance of the fact that the role and priority of each element in the plane tree chlorosis remain to be investigated further. Fe deficiency depresses the synthesis of chloro- phyll, which results in the decrease of photosynthetic products, which in turn affect plant growth (Wang et al., 2008). As a result of carbohydrates synthesis reduction in chlorotic leaves, which slows the move- ment of K+ from the leaf to the phloem vessels, a decline in the production of biomass is reported (Maldonado-Torres et al., 2006). These explain why we found a relationship between chlorosis and chlorophyll content, net photosynthesis and soluble c a r b o h y d r a t e c o n t e n t o f t h e l e a v e s ( F i g . 6 ) . Moreover, increased photosynthetic capacity by AMF is in agreement with the results of the previous study by Birhane et al. (2012). It seems this process has improved nutrition, leading to higher photosynthetic rates (Vafadar et al., 2014). To the best of our knowl- edge, no similar information has yet been provided for interaction effect of fertilizers and AMF inocula- tion on plane trees and its relationship with the leaf chlorosis disorder. This study demonstrated that AMF inoculation added to the common fertilizer program served suc- cessfully as a biological and environmental-friendly method to overcome chlorosis disorder of the plane trees. In addition, the findings of this study suggest that in calcareous soils drill hole nutrition should be considered as a standard method to prevent nutri- tional disorders in the urban landscape. The results also revealed that Fe is not the only nutrient partici- pating in the leaf chlorosis of plane trees. It suggests further investigations to study the weight and impor- tance of each nutritional element in chlorosis disor- der of the plane trees. In this study we mainly focused on the effect of improved media around the plane trees, rather than specific effect of AMF. Indeed, this study was one of the primary trail in a series of experiments we want done later. In the later, we specifically will studied the AMF effect on the plane trees. 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