Effects of Selected Arbuscular Mycorrhizal Fungi, Rock Phosphate, and Compost on Growth and Nutrient Uptake of Citrus Seedlings

Effects of Selected Arbuscular Mycorrhizal Fungi, Rock Phosphate, and Compost on Growth and Nutrient Uptake of Citrus Seedlings | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Selected Arbuscular Mycorrhizal Fungi, Rock Phosphate, and Compost on Growth and Nutrient Uptake of Citrus Seedlings Ibrahim Ortas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8019594/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Citrus trees are among the most essential fruit plants grown in southern Turkey. Nutrient uptake in the Mediterranean region is limited by factors such as low organic matter, high soil pH, and limited availability of phosphorus and micronutrients. To evaluate, under greenhouse conditions, how rock phosphate (RP), compost, and different arbuscular mycorrhizal fungi (AMF) species influence growth, root colonization, and nutrient uptake (with emphasis on P, Zn, Fe, Mn, Cu) in Citrus (sour orange) seedlings. The experiment was conducted under greenhouse conditions with three compost levels (0, 20, and 40 g compost/kg soil), three rock phosphate levels (0, 2, and 4 g rock phosphate/kg soil), and seven mycorrhizal species (Control, G. mosseae , G. caledonium , G. etunicatum , G. clarium , indigenous mycorrhiza, and a cocktail mixture) as a completely randomized factorial design with three replications. Before the experiment began, compost and rock phosphate were mixed with the sterilised soil and incubated for 3 weeks. Plants were grown for 10 months. Root colonization, shoot and root dry weights, and tissue nutrient concentrations were determined. Results obtained after 180 days of cultivation showed that mycorrhizal-inoculated plants grew significantly more than control plants. The combination of arbuscular mycorrhizal (AM) fungi species, rock phosphate, and compost application led to a remarkable increase in dry matter production, root colonisation, and nutrient uptake. An increase in RP application resulted in higher MD across all mycorrhizal species and compost treatments. However, increased compost application reduced MD for all mycorrhizal species. Citrus seedlings were colonised by several mycorrhizal species, and the mycorrhizal dependency (MD) of these seedlings was assessed. The highest MD (91%) was observed in seedlings inoculated with G. mosseae and treated with 20 g of compost and 4 g of RP per kg of soil. Citrus seedlings inoculated with G. mosseae exhibited the highest MD (81%), while those inoculated with native mycorrhizal spores showed the lowest MD (59%). Citrus seedlings inoculated with mycorrhiza contained higher levels of phosphorus, zinc, and other micronutrients compared to non-inoculated control plants. AM fungi, particularly G. mosseae , in combination with 20 g kg-1 compost and 4 g kg − 1 RP addition, significantly improved citrus seedling growth and P and Zn uptake. Compared with the sterile control, AMF inoculation markedly increased plant height and biomass. Responses differed among fungal species; G. mosseae and the mixed “cocktail” inoculum generally produced the highest growth and root colonization. Higher RP doses tended to suppress colonisation; the effect of compost on colonisation depended on species × dose interactions. AMF treatments raised shoot P concentrations from ~ 0.04–0.07% in control/ineffective-local treatments to ~ 0.08–0.12%, and increased Zn, Fe, Mn, and Cu concentrations. RP and compost alone had limited effects, whereas their combined use with AMF supported both growth and nutrition; the full agronomic effect of RP likely requires a longer incubation period for solubilization. Arbuscular mycorrhizal fungi (AMF) Rock phosphate Compost amendment Citrus (sour orange) seedlings Phosphorus uptake Micronutrient acquisition (Zn Fe Mn Cu) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In many countries, rock phosphate (RP) is classified as a mineral resource reserve, characterised by varying concentrations of phosphorus. Many phosphorus fertilisers are obtained by treating high-P 2 O 5 -content rock phosphate (RP) with acids such as sulfuric acid (H 2 SO 4 ) and phosphoric acid (H 3 PO 4 ). It is not advantageous for developing countries to obtain fertilizer from high- and medium-value rock phosphates. There are several ways to dissolve the RP; one method is to use compost or organic manure, and another is to use pyrite. Farmers can either adopt more organic methods or resort to purchasing expensive mineral fertilisers, which can cause environmental pollution. It is possible to increase the solubility of rock phosphates by using different nutrient sources (nitrogen forms and organic fertilizers). However, these resources are often difficult to obtain, and they contain environmental pollutants. Rock phosphates are generally compared with highly soluble phosphorus fertilizers. Only 15–20% of the phosphorus in apatite, which is soluble in calcareous and pH-neutral soils, is accessible to plants. The remaining portion gradually transforms into a form that cannot be absorbed. Because the activators in rock phosphate mobilise the phosphorus, its use is believed to have little economic value. Humic and fulvic acids in organic matter (Amberger, 2006 ) and Oxalic, citric, succinic and malic acid (Gaind, 2017 ) dissolve phosphorus in rock phosphate. A similar study (Mwangi; et al., 2020 ) reported that dissolving phosphate rock with lemon juice and applying it in combination with compost at planting improves nutrient uptake, phosphorus use efficiency, and crop yields. Since abundant humic and fulvic acids are produced during the composting process, insoluble phosphates are converted to a soluble form. Phosphorus mobilization increases as a result of composting rock phosphate with wheat straw and other organic materials. The effect of organic waste and compost material on rock phosphate depends on the organic acids produced by microorganisms. Plant species differ in their impact on rock phosphate and in their capacity to utilise P from RP, due to variations in their rates of demand for calcium and phosphate ions from the soil (Mwangi; et al., 2020 ). The effect of rhizosphere and mycorrhizosphere organisms on rock phosphate solubilization is important. The development of mycorrhiza alters the biological and physical-chemical characteristics of the rhizosphere, which can dissolve minerals through root secretions. Some plants acidify their rhizosphere more than others; for example, buckwheat ( Fayopyron esculentum L.) can acidify its rhizosphere and cause the dissolution of RP, but corn cannot. Legumes better utilise P from RP than non-legumes. Some plants have more extensive root systems, which utilise more P from RP because of the greater soil volume available for P uptake. However, less is known about citrus seedling ability. Phosphorus is a nutrient that is slowly taken up by plants in the soil, and its uptake depends on the microbial population, especially the presence of the arbuscular mycorrhizal (AM) fungus and rhizosphere pH. AM infection is a common association between plant roots and microorganisms that increases plant nutrient uptake, especially P, Zn, Cu, and ammonium-N, particularly in soils with low fertilisation. Many studies show that under greenhouse and field conditions, mycorrhizal inoculation is necessary to obtain healthy, thriving mycorrhizal-dependent plants. Some plants have a mycorrhizal system that can absorb P from RP more effectively than non-mycorrhizal plants (Jaitieng; et al., 2021 ). Early establishment and growth require mycorrhizal formation in plant species that depend heavily on mycorrhizal associations. Plant species differ in their mycorrhizal dependency (Ortas, 2019a ). The dependency is closely related to P acquisition from low-P soils (Marschner 2023). The contribution of mycorrhizal fungi to plant nutrient uptake, especially phosphorus, has been extensively studied. Under nutrient-limited soil conditions, mycorrhizas can enhance plant nutrient uptake through various mechanisms, thereby alleviating plant stress. Under these conditions, plants compete for limited nutrients, and mycorrhizas may modify the root system and rhizosphere dynamics (Butler; Warren, 2025 ). Studies have shown that mycorrhizae can effectively take up P in rock phosphate. The P uptake of cowpea plants with mycorrhiza inoculation was twice as high as that of uninfected ones (Ma; et al., 2025 ). It has been reported that the application of rock phosphate and mycorrhiza is effective in improving P nutrition of citrus species in Brazilian conditions (Antunes; Cardoso, 1991 ). Other researchers have reported that applying rock phosphate in combination with mycorrhiza can lead to a significant increase in yield (Chatterjee; Margenot, 2023 ). Compost, made from various plant wastes, is rich in both macro- and micronutrients and beneficial microbes. Although rock phosphate (RP) is an alternative phosphorus source, it is relatively insoluble in soil and is slowly absorbed by plant roots. Compost and mycorrhizal fungi can effectively dissolve RP and enhance plant nutrient uptake. Srivastava; Singh; Marathe ( 2002 ) showed that the phosphorus nutrition of mycorrhizal-treated citrus trees was best improved by using rock phosphate as opposed to other sources. Miranda; Mello; Kupper ( 2018 ) reported that rootstocks are significantly different in terms of mycorrhizal dependency, and P. trifoliata was found to be the most dependent rootstock. In most of the Mediterranean, citrus is grown in subtropical climates. Sour oranges ( Citrus aurantium L. ) are the most common rootstocks presently used in Turkey. In this region, citrus plants are primarily cultivated and produced using heavy chemical fertilisers. However, in addition to their environmental impact, chemical fertilizers are expensive (especially nitrogen and phosphorus) and can degrade soil and food quality. Heavy P application causes Zn deficiency in plant tissue. Ortas ( 2019b ) showed that high P levels depressed mycorrhizal inoculation; as a result, plants were unable to acquire additional Zn and other micronutrients. Since plants are strongly mycorrhizal-dependent, producing mycorrhizal inoculated seedlings has become more important (Ortas, 2022). Citrus plants require mycorrhizal inoculation; therefore, without enough mycorrhizal colonisation, the plants cannot grow. Recent studies have shown that mycorrhiza is effective in the uptake of zinc, copper, and phosphorus. Ortas; Demirbas; Akpinar ( 2018 ) found that G clarium fungi-inoculated citrus seedlings have higher P and Zn concentrations than others. An experiment can be designed to assess the effects of several mycorrhizal species and compost doses on RP solubilization. This can help to develop a mycorrhizae species for future commercial production. The primary hypothesis is that compost and mycorrhizal fungi can dissolve rock phosphate, and that citrus plants, aided by mycorrhizae, can benefit from enhanced growth and development. This study aimed to investigate the role of mycorrhizal species and compost in mobilising rock phosphate, with a focus on their impact on citrus plant growth and nutrient uptake. Materials and Methods In this study, the importance of rock phosphate, compost, and mycorrhizal species for phosphorus uptake by citrus plants grown under greenhouse conditions was investigated. Menzilat Series soil was used as a growth medium. Sour orange ( Citrus sinensis L.) seedlings were produced from seeds in a randomized complete block design with three replications. Production of healthy citrus seedlings is a priority in countries that grow citrus. Sour orange is a key citrus rootstock widely used in Turkey for citrus cultivation. The treatments in this study comprised three levels of compost and rock phosphate: 0, 20, and 40 g compost/kg soil (4 kg soil each plot) and 0, 2, and 4 g rock phosphate/kg soil. These were tested with different mycorrhizae species in the present experiment. The compost dose used in the experiment was determined by a preliminary incubation experiment. Compost was applied in three doses: 0, 20, and 40 g compost/kg soil. Two months before planting, rock phosphate and compost were mixed with soil and incubated; during this process, chemical reactions involving rock phosphate occurred. The rock phosphate (RP) used in the experiment was prepared by incubation and applied at 0, 2, and 4 g RP/kg soil. Mycorrhiza species 1- Control, 2- G. mosseae , 3- G. caledonium , 4- G. etunicatum , 5- G. clarium , 6- Indigenous, 7- Cocktail. Rock phosphate was supplied at Mardin Mazdagı. Composting was conducted at Çukurova Research and Application Farm using mixed grasses and other straw feedstock. Soil Sterilization and Soil Analyses The soil was sieved through a 4-mm sieve for physical, chemical, and biological analyses. Micronutrient concentrations (Zn, Fe, Mn, and Cu) were analysed by atomic absorption spectrophotometry. Olsen-extractable soil phosphorus was determined using the method of Olsen; Sommers (1982). Soil properties are given in Table 1. Before the experiment was set up, half of the soil was sterilized for 2 hours at 120 °C in an autoclave. After sterilisation, the growth medium soil was left to rest for 18 days before use. Inoculum Source The experimental treatments consisted of selected species of arbuscular mycorrhizal fungi (Control, G. mosseae, G. caledonium, G. etunicatum, G. clarium , indigenous mycorrhizae, and Cocktail). The inoculum was previously multiplied in the greenhouse with Sudan grass [Sorghum bicolour (L.) Moench] as the host plant in a mixture of andesitic tuff + soil + peat (6+3+1, V:V: V) medium. Plant Growth Condition The plants were grown in a greenhouse at 25-28 °C and a relative humidity of 70-80%, with a 16 h day and 8 h dark photoperiod, under a light intensity of μmol m-2 s-1. Distilled water was added to maintain the moisture at 80% of field capacity. Plants were grown for 10 months. Root Colonization The root-clearing and staining procedure and the degree of mycorrhizal infection in the root cortex were assessed using the method described by Koske; Gemma (1989). Root colonisation was determined using a gridline-intersect method for the AM treatments (Gioannetti; Mosse, 1980). Plant analysis After harvest, the fresh and dry weights of shoots (leaf and stem) and roots were determined following drying at 65 o C. The dry material was ground in the Tema Mill. Then, 0.2 g of ground plant materials was ashed at 550 °C, followed by dissolution in 3.3 % HCl. After digestion of the plant material, the phosphorus concentration in the solution was determined colourimetrically using a flame photometer. An ICP-OES (PerkinElmer) was used to determine the concentrations of Zn, Fe, Mn, and Cu. Statistical analysis For all statistical analyses, using ANOVA, the main significant interactive effects of mycorrhizal inoculation and growing medium were separated by Duncan's multiple range test at P<0.05 using the Statistical Analysis System (SAS) (SAS, 2008). Mycorrhizal dependency (MD) was determined by expressing the difference between the dry weight of the mycorrhizal plant and the dry weight of the non-mycorrhizal plant as a percentage of the dry weight of the mycorrhizal plant (Plenchette; Fortin; Furlan, 1983). Results and Discussion At harvest, plant height, shoot and root dry weight, root colonisation, P concentration in shoot tissue, and micronutrients were determined. All inoculated plant species were colonised with arbuscular mycorrhizal fungi (Figure 1). In addition, small colonisation ranging from 1% to 2% was observed in non-inoculated plants. Indigenous mycorrhizae have a mean root colonisation of 86%. The rest of the mycorrhizae species have more than 50% of root colonisation. The lowest mycorrhizal colonisation, 3%, was observed with indigenous mycorrhiza inoculation in G. mosseae , whereas the highest colonisation, 68%, occurred with G. mosseae inoculation. Higher compost levels increased root colonisation rates. Conversely, the RP application resulted in decreased root colonisation. We noticed that root colonisation decreased at high RP. Plant height development data show that the selected mycorrhizal fungi effectively increase plant height compared to the control. The naturally occurring mycorrhizas isolated from the soil are less effective than the control. However, the effect of compost and rock phosphate application is not as effective as the mycorrhizal application (Figure 2). Sour orange seedlings inoculated with the AM species grew better than non-mycorrhizal seedlings except for indigenous inoculation (Table 2 and Figure 3). There were significant differences between the mycorrhizal species and the organic fertiliser sources. Since the soils were sterilised at the beginning of the experiment, citrus plants did not grow in sterile soil without mycorrhizal inoculation. As the citrus plants are strongly mycorrhizal-dependent, citrus seedlings are not able to obtain sufficient nutrients for growth without indigenous mycorrhizal spores. The aim of the study, based on the current results, was to test the number of spores and the effectiveness of mycorrhiza spores used in citrus seedling production. It seems that indigenous mycorrhizal spores are less efficient; however, cocktail mycorrhizal inocula are very efficient. Previously, we obtained similar results in many experiments conducted in the rhizosphere Laboratuvar (Ortas, 2019b). Mycorrhizal inoculation significantly enhanced root colonisation. Without considering the effects of RP and compost addition, the highest root colonisation occurred with G. mosseae inoculation. When selected, mycorrhiza spores are added to the seedling root area, excluding native mycorrhizae spores, and they considerably improve the dry weight of citrus seedling shoots. Compared with the control, indigenous and selected mycorrhizal inoculations significantly increased the dry weight (SDW) of citrus seedlings (Table 2). The effects of indigenous mycorrhiza are less effective than those of selected other species. Similarly, the results of Jbar; et al. (2023) demonstrated that inoculation with the fungus G. mosseae resulted in a considerable increase in plant growth and available soil phosphorus. Mycorrhizal species are different in their effects on plant growth. Five AM fungi were screened for their symbiotic response with sour orange seedlings in growth media. G. mosseae, G. clarium, and Cocktail inoculated plants grew better. Citrus seedling root development is generally similar to shoot development. G. mosseae increased SDW more than any other species. Mycorrhizal species are more beneficial than other treatments alone for the growth and nutrition of citrus plants. G. mosseae was identified as an active species . These findings are in connection with those of Wu; Zou (2009), who reported that the sole AMF inoculation significantly increased the total dry weight and leaf and root nutrient contents of the citrus seedlings compared to the non-AMF control. In the Spanish citrus agroecosystem, it has been determined that G. intraradices is the most effective mycorrhizal fungus for plant growth (Camprubi; Calvet, 1996), and G. mosseae is the least effective among several tested species (Viyanak; Bagyaraj, 1990). In sterile soil, plants such as citrus, which are highly dependent on mycorrhizal fungi, often fail to grow. The current study, like Ortas (2019b) study, found that plant growth almost stopped in a sterile soil environment without mycorrhizae spores. Among the species, differences were observed, and although G. mosseae was determined to be the most efficient mycorrhizal species, the application of indigenous mycorrhizae was found to be the least effective. Usually, indigenous mycorrhizae significantly increase citrus growth (Ortaş; Varma, 2007). Most probably, in the present experiment, indigenous mycorrhizae did not efficiently form associations with plant roots. This situation may also be due to the fact that natural mycorrhiza spores would establish relationships with other organisms, but are prevented from doing so by soil sterilisation. The adverse effects created by sterilisation should be reinvestigated from the perspective of the natural microflora-mycorrhiza relationship. Mycorrhizae inoculation alone contributed more than the RP and compost dose applications. Increasing RP doses up to 2 g kg -1 soil and 40 g kg -1 compost increased citrus seedlings' SDW for all mycorrhizal inoculations. The results of Indriani; et al. (2016) indicated that the optimal application rate of rock phosphate was 200 kg ha -1 of P 2 O 5 . Increasing RP doses up to 2 g kg -1 soil and 40 g kg -1 compost increased citrus seedlings' SDW for all mycorrhizal inoculations. The results of Indriani; et al. (2016) indicated that the optimal application rate of Rock Phosphate was 200 kg ha -1 of P 2 O 5 . Applying 20 g of compost per kg of soil increases RDW compared to no compost addition, but 40 g per kg of soil results in a greater increase. Mycorrhizae inoculation alone contributed more to seedling growth and development than either RP or compost. Compared to control and indigenous mycorrhizae, the selected mycorrhizal inoculation significantly increased nutrient concentrations, especially phosphorus (P). Citrus growth enhancement caused by AM fungi is typically attributed to the enhanced phosphorus nutrition of AM plants, which is effective only when soil P levels are low. G. etincatunium mycorrhizae inoculation of seedlings results in a mean P concentration of 0.08%. G. mosseae inoculated seedlings have a mean of 0.12% of P concentration (Figure 4). In terms of shoot P concentration, applications of rock phosphate and compost had little effect on citrus plants. P concentration varied between 0.04% and 0.07% in both the control and ineffective indigenous mycorrhiza inoculation. However, it ranged from 0.08% to 0.12% when specific mycorrhizae were inoculated. Because there was not enough time for both the incubation and planting periods, the experiment continued to track the effects of compost and mycorrhizae on the mobilisation of rock phosphate over time. Di Tomassi et al. (2021) found that struvite-P fertilization increased biomass by 22% and shoot P uptake by 32%. Since the soil was sterilised before transplanting the seedlings, the natural mycorrhizae were destroyed. Zn, Fe, Mn and Cu concentrations were significantly affected by mycorrhizae inoculation (Table 3). The concentration range of mineral nutrients are within the critical levels. Mycorrhiza inoculation shows a significant increase compared to control treatments. The concentration of Zn in the shoots increased significantly with mycorrhizae inoculation. The average Zn content for the control plant is 19.3 mg Zn kg -1 DW, while the native mycorrhizae-infected plant has 23.3 mg Zn kg -1 DW. The highest mean of Zn content in the shoots of cocktail and G. mosseae inoculated seedlings is 31, 8 and 31.2 mg Zn/kg, respectively (Table 3). G. mosseae and G. caledonium inoculation tends to increase Zn concentrations. The highest Zn concentration is observed with G. caledonium at 2 g RP kg -1, reaching an average of 33.1 mg Zn kg -1 . Generally, the RP 2 g RP kg-1 application has a higher Zn concentration than the 0 and 4 g RP kg-1 soil applications. In terms of compost application, increasing the compost and Zn doses significantly decreased the Zn concentration from 27.66 to 25.41 mg Zn kg -1 . Mousavi; Srivastava; Raiesi (2024) found that high P application reduced Zn uptake. Additionally, a relationship between Zn concentration and AM infection ratio was observed. Also, Ortas; Ortakçi; Kaya (2002) tested the effect of P and Zn levels and mycorrhizal inoculation on citrus growth; inoculated plants were significantly stimulated by mycorrhizal infection. The present results show that mycorrhiza inoculation significantly increased P and Zn concentrations. Since mycorrhizae-inoculated plants have higher P and Zn content compared to uninoculated plants, it is concluded that when root colonisation is efficient, plants obtain more nutrients with increased uptake. Results show that mycorrhizal inoculation, particularly with G. caledonium and G. mosseae , significantly increases the concentrations of Zn, Fe, Mn, and Cu. However, in terms of compost, the application of up to 20 g of compost increased mineral nutrient levels, whereas at 40 mg compost kg-1 soil, the mean decreased (Table 3). Iron mean concentration decreased with increasing doses of compost and rock phosphates. Compared to other species and the control plant, G. caledonium inoculations have a higher Fe concentration . Mn concentrations range from 22.90 to 84.87 mg Mn kg -1 . G. mosseae and G. caledonium inoculations tend to enhance Mn concentration. For instance, G. mosseae at 40 g kg -1 compost shows a high Mn concentration of 84.87 mg Mn kg -1 . The highest Mn concentration is observed with G. mosseae at 40 g Mn kg -1 compost, reaching 84.87 mg kg -1 . G. caledonium and G. etunicatum species inoculation generally affects Cu concentration. G. caledonium inoculation at 2 g kg -1 compost results in a Cu concentration of 18.95 mg Cu kg -1 . The highest concentration is observed with the Cocktail mycorrhizae treatment at 40 g kg -1 compost, with a value of 24.14 mg Cu kg -1 . This aligns with the established function of mycorrhizae in enhancing nutrient solubility and extending the hyphal network, thereby improving nutrient acquisition. The rates at which compost is applied also affect the concentrations of mineral nutrients. Higher compost rates (20 and 40 g kg -1 ) typically lead to higher nutrient levels, but the effect varies by nutrient and mycorrhizal treatment. Variability in nutrient concentrations raises the possibility that mycorrhizal species, rock phosphate, and partially composted rates affect micronutrient effectiveness. Youpensuka, Lordkaewb, and Rerkasemc (2008) reported that mycorrhizal-treated citrus trees had better plant growth and nutrient uptake, such as P, Ca, Zn, Cu, and Fe, than non-mycorrhizal trees. Similarly, according to the findings of Burni; et al. (2023), the combined application of compost and mycorrhizal inoculation can be a successful method for improving plant nutrition, as these studies partly support the present results. According to our findings, the application of mycorrhizal species increased nutrient uptake and seedling growth more efficiently than rock phosphate and/or compost. Mycorrhizal species typically respond well to low applications of compost and rock phosphate. Compost's ability to solubilize RP appears to be less successful. The results of Burni; et al. (2023) suggested that AMF inoculation may improve the solubility of phosphorus from organic manure and rock phosphate. Gaind (2017) showed that moisture content affects RP release. Before seedlings are transferred to RP and compost-treated soils, further time may be required for RP incubation. Applying compost and PR dissolved in lemon juice simultaneously at planting significantly increased plant yields, phosphorus absorption, and phosphorus use efficiency (Mwangi; et al., 2020). General Analyses Citrus plants have shown different responses to varying mycorrhizal dependency (Table 4). G. mosseae inoculation increased plant growth and had a mean of mycorrhizae dependency of 88%, while the indigenous mycorrhizae inoculated treatment had 48% of mycorrhizae dependency. Cocktail treatment shows relatively high dependency, ranging from 79% to 93%. For all mycorrhizal species, increasing RP dosages increased MD. However, increasing compost dosage did not consistently affect MD. In a similar study, Viyanak; Bagyaraj (1990) found that 18 distinct mycorrhizal fungi produced plants with stem diameters and heights that were noticeably different. G. mosseae has a mean colonisation of 91% at 0 g/kg compost, which decreases to 87 % at 40 g/kg compost. It seems that higher compost levels may negatively affect colonisation and dependency. G. etunicatum may be more suitable for soils with higher compost levels, while G. mosseae may perform better in low-compost applications. When all data were correlated, they showed strong, positive correlations, indicating concerted increases in plant height, shoot, and root biomass (Table 5). Growth performance is primarily coupled to P and Zn nutrition and to AMF colonisation. Shoot Fe, Man and Cu were not strongly related to growth parameters. Also, data were used for Principal Component Analysis, and the results were shown in Figure 5. Cumulative PCA accounts for 65% of the variation, which is mainly in the PC1 axis. PC1 loads positively on plant height, shoot/root fresh and dry weights, Shoot P, Shoot Zn, and mycorrhizal infection. Ellipses show the most apparent separation. Some AMF groups cluster on the positive PC1 side (aligned with better growth, P, and Zn), others on the negative side, with a few differing along PC2 (Fe/Mn). Thus, AMF identity is a primary driver of the overall phenotype. Results show that mycorrhizae No. 1 (control) plants do not affect plant parameters. Indigenous mycorrhiza also have less contribution. G. etinicatinium, G. clarium and Cokteyle mycorrhizae are in the core of the contribution to plant parameters. Usually, the growth parameters of G. etinicatinium, G. clarium, and cocktail-inoculated plants are highly correlated with each other. Also with P and Zn concentrations. G. mosseea have a high impact on root parameters . Plant parameters strongly influence compost and rock phosphate. Ellipses substantially overlap; compost alone does not clearly separate multivariate profiles. However, mycorrhizal species exhibit substantial variation in their effects on plant parameters. Compost contribution to plant parameters showed that 20 g per kg of soil was more effective than the control, and 40 g per kg of soil was even more effective. What effects of rock phosphate have been analyzed. It seems that 2 and 4 g RP kg -1 soil are more related to soil parameters. Mycorrhizal species vary in their effects on plant growth. G. mosseae, G. clarium, and cocktail-inoculated plants grew better . According to the findings, it is unclear how compost affects plant development, whereas mycorrhizae have a considerably greater positive impact. Mycorrhiza inoculation alone significantly affected other plant metrics, including shoot and root dry weight, root infection, and P and Zn concentrations. The order of effectiveness of species was as follows: G. mosseae > Cocktail > G. clarium > G. caledonium > G. etunicatum > Indigenous mycorrhiza > Control. It seems that indigenous mycorrhizal spores are less efficient than cultivated mycorrhizal spores in promoting plant growth. In our previous work, we found that indigenous mycorrhizae promote the growth of citrus seedlings (Ortas, Demirbas, and Akpinar 2018a). There is a need to investigate the effects of indigenous and cocktail mycorrhizae on plant growth, nutrient uptake, and other soil and plant parameters. Conclusion G. mosseae was determined to be the most efficient among the mycorrhizae species for sour orange seedlings development. For sustainable agriculture, using rock phosphate, compost, and mycorrhizae as sources of fertilizer is an encouraging practice. Generally, 20 g kg − 1 compost and 4 g kg − 1 RP increased SDW. A similar pattern was observed in root dry weight change. All levels of compost had minimal effects on nutrient uptake, but using 40g of compost increased root inoculation. The brief study period prevented a comprehensive understanding of the impact of compost application on rock phosphate mobilization. The findings highlight the role of mycorrhizal fungi in boosting root infection compared to non-inoculated control seedlings. Citrus plant strongly depends on mycorrhizal inoculation. Increasing RP increased MD; however, compost dose increases did not have a significant effect on MD. AMF maximize nutrient use efficiency. AMF selection exerts a more substantial multivariate effect than either compost or rock phosphate. Because higher RP can suppress colonization, a moderate RP rate (2 g kg⁻¹) combined with intermediate compost (20–40 g kg⁻¹) and a well-performing AMF inoculum is advisable. Increasing rock phosphate produces a moderate rightward (positive PC1) shift consistent with improved P (and associated Zn) nutrition, but the effects of compost are limited in this dataset. Plant P, Zn, Fe, Mn and Cu absorption was greatly boosted by mycorrhizal inoculation as well. AMF species substantially enhanced P and Zn concentration and also increased both shoot and root dry matter. Shoot P and Zn status, together with AMF colonisation, best explain variation in growth traits. Species choice is pivotal. Among the AMF tested, G. mosseae (and, secondarily, the mixed “cocktail”) delivered the most consistent gains in growth, root colonization, and P/micronutrient uptake; the indigenous inoculum performed poorest. Declarations Competing Interests: The authors declare that they have no competing interests. Ethics approval: Ethic approval was accepted Consent for publication: Manuscript can be published Funding: No financial support was available. Author Contribution I have prepared the manuscript “Effects of Selected Arbuscular Mycorrhizal Fungi, Rock Phosphate, and Compost on Growth and Nutrient Uptake of Citrus Seedlings”, and we would like to publish the enclosed article in Applied Fruit Science. The manuscript was not published in any journals. Acknowledgement: Thanks to Dr. Çağdaş Akpinar and Dr. Ahmet Demirbaş for their contributions. Thanks to Dr. Imran for proofreading. Availability of data and material: If it is going to be requested Code availability: We used university software References Amberger, A. (2006): Soil fertility and plant nutrition in the tropics and subtropics.(IFA, International Fertilizer Industry Association). Antunes, V.; Cardoso, E. (1991): Growth and nutrient status of citrus plants as influenced by mycorrhiza and phosphorus application. Plant and Soil 131, 1, 11–19, ://WOS:A1991EZ29300002 http://download.springer.com/static/pdf/ 317/art%253A10.1007%252FBF00010415.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2FBF00010415&token2=exp=1497614588~ acl=%2Fstatic%2Fpdf%2F317%2Fart%25253A10.1007%25252FBF00010415.pdf %3ForiginUrl%3Dhttp%253A%252F%252Flink .springer.com%252Farticle%252F10.1007%252FBF00010415* ~hmac=4df9c9bab1ed1d 766f3d2c8075 bc43a8138483a96976efed20c9467e04b7d6e9. Burni, T.; Hussain, F.; Bibi, S.; et al. (2023): The salutary impacts of AMF species, rock phosphates (RP), and organic matter (FYM) fertilizers on the development and chemical behavior of Mentha arvensis L. Acta Ecologica Sinica 43, 5, 835–841. 10.1016/j.chnaes.2022.12.003 . Butler, O. M.; Warren, C. R. (2025): Microbial phosphorus in loamy, basalt-derived forest soil is altered by plant nutritional status: a root-splitting study. Plant and Soil, 1–15. 10.1007/s11104-025-07224-w . Camprubi, A.; Calvet, C. (1996): Isolation and screening of mycorrhizal fungi from citrus nurseries and orchards and inoculation studies. Hortscience 31, 3, 366–369, ://WOS:A1996UQ16300018. Chatterjee, N.; Margenot, A. J. (2023): Crop growth is increased by arbuscular mycorrhizae for both phosphate rock and soluble phosphorus fertilizers, but fertilizer solubility primarily determines crop growth. Biology and Fertility of Soils 59, 7, 843–862. 10.1007/s00374-023-01751-3 . Gaind, S. (2017): Exploitation of Orange Peel for Fungal Solubilization of Rock Phosphate by Solid State Fermentation. Waste Biomass Valor 8, 4, 1351–1360. 10.1007/s12649-016-9682-2 . Gioannetti, M.; Mosse, B. (1980): An evaluation of techniques for measuring vesicular-arbuscular mycorrhiza in roots. New Phytologist 84, 489–500. 10.1111/j.1469-8137.1980.tb04556.x . Indriani, N. P.; Yuwariah, Y.; Rochana, A.; et al. (2016): The role of Vesicular Arbuscular Mycorrhiza (VAM) and rock phosphate application on production and nutritional value of centro legumes ( Centrosema pubescens). Legume Research 39, 6, 987–990. 10.18805/lr.v0iOF.6645 . Jaitieng, S.; Sinma, K.; Rungcharoenthong, P.; et al. (2021): Arbuscular mycorrhiza fungi applications and rock phosphate fertilizers enhance available phosphorus in soil and promote plant immunity in robusta coffee. Soil Science and Plant Nutrition 67, 1, 97–101. 10.1080/00380768.2020.1848343 . Jbar, A. K.; Noon, G. B.; Al-Taweel, L. S. J.; et al. (2023): Effect of bio fertilization and phosphate rock under sterilization conditions on the activity of phosphatase enzyme, phosphorus availability and growth of wheat plant triticum aestivum L.. International Journal of Agricultural and Statistical Sciences 19, 1493–1499. 10.59467/ijass.2023.19.1493 . Koske, R.; Gemma, J. (1989): A modified procedure for staining roots to detect VA mycorrhizas. Mycological Research 92, 4, 486–488. Ma, H.; Zhang, H.; Gao, Q.; et al. (2025): The Relative Contribution of Root Morphology and Arbuscular Mycorrhizal Fungal Colonization on Phosphorus Uptake in Rice/Soybean Intercropping Under Dry Cultivation. Plants (Basel) 14, 1, 106. 10.3390/plants14010106 . Miranda, P. B.; Mello, A. H.; Kupper, K. C. (2018): Mycorrhizal dependence of citrus rootstock. Revista Brasileira de Fruticultura 40, 3, e-762. Mousavi, S. M.; Srivastava, A. K.; Raiesi, T. (2024): Citrus nutrition in Iran: lessons from calcareous soils. Journal of Plant Nutrition 47, 19, 3367–3392. 10.1080/01904167.2024.2379571 . Mwangi, E.; Ngamau, C.; Wesonga, J.; et al. (2020): Managing Phosphate Rock to Improve Nutrient Uptake, Phosphorus Use Efficiency, and Carrot Yields. J. Soil Sci. Plant Nutr. 20, 3, 1350–1365. 10.1007/s42729-020-00217-x . Olsen, S. R.; Sommers, L. E. (1982): Methods of soil analysis. Part 2, 1982, 403–430. Ortas, I. (2019a): Do horticultural tree plant species depend on mycorrhizal inoculation under marginal soil conditions? In 30th International Horticultural Congress (IHC) / International Symposium on Water and Nutrient Relations and Management of Horticultural Crops. (Istanbul, TURKEY), pp 85–92. Ortas, I. (2019b): Effect of mycorrhizal inoculation on citrus seedling growth and nutrient uptake. In 30th International Horticultural Congress (IHC) / International Symposium on Water and Nutrient Relations and Management of Horticultural Crops. (Istanbul, TURKEY), pp 77–83. Ortas, I. (2022): What is Ecological/Organic Farming? Soil Science and microbiological Point of view. In Introduction and Application of Organic Fertilizers as Protectors of Our Environment, Ozturk, M.; Akram, N. A.; Unal, B. T.; et al., eds. (Newcastle, UK, Cambridge Scholars Publishing), p 90. Ortas, I.; Demirbas, A.; Akpinar, C. (2018): Time period and nutrient contents alter the mycorrhizal responsiveness of citrus seedlings. European Journal of Horticultural Science 83, 2, 72–80. 10.17660/eJHS.2018/83.2.2 . Ortas, I.; Ortakçi, D.; Kaya, Z. (2002): Various mycorrhizal fungi propagated on different hosts have different effects on citrus growth and nutrient uptake. Communications in Soil Science and Plant Analysis 33, 1–2, 259–272. Doi 10.1081/Css-120002392 . Ortaş, I.; Varma, A. (2007): Field trials of bioinoculants. In Advanced Techniques in Soil Microbiology. (Springer), pp 397–413. Plenchette, C.; Fortin, J. A.; Furlan, V. (1983): Growth-responses of several plant-species to mycorrhizae in a soil of moderate P-fertılıty.1. mycorrhizal dependency under field conditions. Plant and Soil 70, 2, 199–209, ://WOS:A1983QM70100005 http://download.springer.com/static/pdf/ 117/art%253A10.1007%252FBF02374780.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2FBF02374780&token2=exp=1497614703~ acl=%2Fstatic%2Fpdf%2F117%2Fart%25253A10.1007%25252FBF02374780.pd f%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007 %252FBF02374780*~hmac=326241feee2c99e4d7f4cbb2e70079e51 fce4f8bd6bb11b296cdb7b62a656776. SAS (2008): Statistical Analysis Systems user's guide (SAS/STAT® 9.2 User’s Guide).(North Carolina, USA: SAS Institute Inc.). Srivastava, A. K.; Singh, S.; Marathe, R. A. (2002): Organic citrus: Soil fertility and plant nutrition. Journal of Sustainable Agriculture 19, 3, 5–29. 10.1300/J064v19n03_03 . Viyanak, K.; Bagyaraj, D. J. (1990): Selection of efficient VA mycorrhizal fungi for trifoliate orange. Biological Agriculture & Horticulture 6, 4, 305–311, ://WOS:A1990CZ24000004. Wu, Q. S.; Zou, Y. N. (2009): The effect of Dual Application of Arbuscular Mycorrhizal Fungi and Polyamines upon Growth and Nutrient Uptake on Trifoliate Orange (Poncirus trifoliata) Seedlings. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 37, 2, 95–98, ://WOS:000272606600015. Tables Table 1 to 5 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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many countries, rock phosphate (RP) is classified as a mineral resource reserve, characterised by varying concentrations of phosphorus. Many phosphorus fertilisers are obtained by treating high-P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e-content rock phosphate (RP) with acids such as sulfuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) and phosphoric acid (H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e). It is not advantageous for developing countries to obtain fertilizer from high- and medium-value rock phosphates. There are several ways to dissolve the RP; one method is to use compost or organic manure, and another is to use pyrite. Farmers can either adopt more organic methods or resort to purchasing expensive mineral fertilisers, which can cause environmental pollution. It is possible to increase the solubility of rock phosphates by using different nutrient sources (nitrogen forms and organic fertilizers). However, these resources are often difficult to obtain, and they contain environmental pollutants. Rock phosphates are generally compared with highly soluble phosphorus fertilizers. Only 15\u0026ndash;20% of the phosphorus in apatite, which is soluble in calcareous and pH-neutral soils, is accessible to plants. The remaining portion gradually transforms into a form that cannot be absorbed. Because the activators in rock phosphate mobilise the phosphorus, its use is believed to have little economic value. Humic and fulvic acids in organic matter (Amberger, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and Oxalic, citric, succinic and malic acid (Gaind, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) dissolve phosphorus in rock phosphate. A similar study (Mwangi; et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that dissolving phosphate rock with lemon juice and applying it in combination with compost at planting improves nutrient uptake, phosphorus use efficiency, and crop yields.\u003c/p\u003e\u003cp\u003eSince abundant humic and fulvic acids are produced during the composting process, insoluble phosphates are converted to a soluble form. Phosphorus mobilization increases as a result of composting rock phosphate with wheat straw and other organic materials. The effect of organic waste and compost material on rock phosphate depends on the organic acids produced by microorganisms. Plant species differ in their impact on rock phosphate and in their capacity to utilise P from RP, due to variations in their rates of demand for calcium and phosphate ions from the soil (Mwangi; et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The effect of rhizosphere and mycorrhizosphere organisms on rock phosphate solubilization is important. The development of mycorrhiza alters the biological and physical-chemical characteristics of the rhizosphere, which can dissolve minerals through root secretions. Some plants acidify their rhizosphere more than others; for example, buckwheat (\u003cem\u003eFayopyron esculentum\u003c/em\u003e L.) can acidify its rhizosphere and cause the dissolution of RP, but corn cannot. Legumes better utilise P from RP than non-legumes. Some plants have more extensive root systems, which utilise more P from RP because of the greater soil volume available for P uptake. However, less is known about citrus seedling ability. Phosphorus is a nutrient that is slowly taken up by plants in the soil, and its uptake depends on the microbial population, especially the presence of the arbuscular mycorrhizal (AM) fungus and rhizosphere pH.\u003c/p\u003e\u003cp\u003eAM infection is a common association between plant roots and microorganisms that increases plant nutrient uptake, especially P, Zn, Cu, and ammonium-N, particularly in soils with low fertilisation. Many studies show that under greenhouse and field conditions, mycorrhizal inoculation is necessary to obtain healthy, thriving mycorrhizal-dependent plants. Some plants have a mycorrhizal system that can absorb P from RP more effectively than non-mycorrhizal plants (Jaitieng; et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Early establishment and growth require mycorrhizal formation in plant species that depend heavily on mycorrhizal associations. Plant species differ in their mycorrhizal dependency (Ortas, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). The dependency is closely related to P acquisition from low-P soils (Marschner 2023). The contribution of mycorrhizal fungi to plant nutrient uptake, especially phosphorus, has been extensively studied. Under nutrient-limited soil conditions, mycorrhizas can enhance plant nutrient uptake through various mechanisms, thereby alleviating plant stress. Under these conditions, plants compete for limited nutrients, and mycorrhizas may modify the root system and rhizosphere dynamics (Butler; Warren, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStudies have shown that mycorrhizae can effectively take up P in rock phosphate. The P uptake of cowpea plants with mycorrhiza inoculation was twice as high as that of uninfected ones (Ma; et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It has been reported that the application of rock phosphate and mycorrhiza is effective in improving P nutrition of citrus species in Brazilian conditions (Antunes; Cardoso, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Other researchers have reported that applying rock phosphate in combination with mycorrhiza can lead to a significant increase in yield (Chatterjee; Margenot, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Compost, made from various plant wastes, is rich in both macro- and micronutrients and beneficial microbes. Although rock phosphate (RP) is an alternative phosphorus source, it is relatively insoluble in soil and is slowly absorbed by plant roots. Compost and mycorrhizal fungi can effectively dissolve RP and enhance plant nutrient uptake. Srivastava; Singh; Marathe (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) showed that the phosphorus nutrition of mycorrhizal-treated citrus trees was best improved by using rock phosphate as opposed to other sources. Miranda; Mello; Kupper (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that rootstocks are significantly different in terms of mycorrhizal dependency, and P. trifoliata was found to be the most dependent rootstock.\u003c/p\u003e\u003cp\u003eIn most of the Mediterranean, citrus is grown in subtropical climates. Sour oranges (\u003cem\u003eCitrus aurantium\u003c/em\u003e L.\u003cem\u003e)\u003c/em\u003e are the most common rootstocks presently used in Turkey. In this region, citrus plants are primarily cultivated and produced using heavy chemical fertilisers. However, in addition to their environmental impact, chemical fertilizers are expensive (especially nitrogen and phosphorus) and can degrade soil and food quality. Heavy P application causes Zn deficiency in plant tissue. Ortas (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e) showed that high P levels depressed mycorrhizal inoculation; as a result, plants were unable to acquire additional Zn and other micronutrients. Since plants are strongly mycorrhizal-dependent, producing mycorrhizal inoculated seedlings has become more important (Ortas, 2022). Citrus plants require mycorrhizal inoculation; therefore, without enough mycorrhizal colonisation, the plants cannot grow. Recent studies have shown that mycorrhiza is effective in the uptake of zinc, copper, and phosphorus. Ortas; Demirbas; Akpinar (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that \u003cem\u003eG clarium\u003c/em\u003e fungi-inoculated citrus seedlings have higher P and Zn concentrations than others.\u003c/p\u003e\u003cp\u003eAn experiment can be designed to assess the effects of several mycorrhizal species and compost doses on RP solubilization. This can help to develop a mycorrhizae species for future commercial production. The primary hypothesis is that compost and mycorrhizal fungi can dissolve rock phosphate, and that citrus plants, aided by mycorrhizae, can benefit from enhanced growth and development. This study aimed to investigate the role of mycorrhizal species and compost in mobilising rock phosphate, with a focus on their impact on citrus plant growth and nutrient uptake.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eIn this study, the importance of rock phosphate, compost, and mycorrhizal species for phosphorus uptake by citrus plants grown under greenhouse conditions was investigated. Menzilat Series soil was used as a growth medium. Sour orange (\u003cem\u003eCitrus sinensis\u003c/em\u003e L.) seedlings were produced from seeds in a randomized complete block design with three replications. Production of healthy citrus seedlings is a priority in countries that grow citrus. Sour orange is a key citrus rootstock widely used in Turkey for citrus cultivation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe treatments in this study comprised three levels of compost and rock phosphate: 0, 20, and 40 g compost/kg soil (4 kg soil each plot) and 0, 2, and 4 g rock phosphate/kg soil. These were tested with different mycorrhizae species in the present experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe compost dose used in the experiment was determined by a preliminary incubation experiment. Compost was applied in three doses: 0, 20, and 40 g compost/kg soil. Two months before planting, rock phosphate and compost were mixed with soil and incubated; during this process, chemical reactions involving rock phosphate occurred. The rock phosphate (RP) used in the experiment was prepared by incubation and applied at 0, 2, and 4 g RP/kg soil. Mycorrhiza species 1- Control, 2- \u003cem\u003eG. mosseae\u003c/em\u003e, 3- \u003cem\u003eG. caledonium\u003c/em\u003e, 4- \u003cem\u003eG. etunicatum\u003c/em\u003e, 5- \u003cem\u003eG. clarium\u003c/em\u003e, 6- Indigenous, 7- Cocktail. Rock phosphate was supplied at Mardin Mazdagı. Composting was conducted at \u0026Ccedil;ukurova Research and Application Farm using mixed grasses and other straw feedstock.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil Sterilization and Soil Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe soil was sieved through a 4-mm sieve for physical, chemical, and biological analyses. Micronutrient concentrations (Zn, Fe, Mn, and Cu) were analysed by atomic absorption spectrophotometry. Olsen-extractable soil phosphorus was determined using the method of Olsen; Sommers (1982).\u0026nbsp;Soil properties are given in Table 1.\u003c/p\u003e\n\u003cp\u003eBefore the experiment was set up, half of the soil was sterilized for 2 hours at 120 \u0026deg;C in an autoclave. After sterilisation, the growth medium soil was left to rest for 18 days before use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInoculum Source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental treatments consisted of selected species of arbuscular mycorrhizal fungi (Control, \u003cem\u003eG. mosseae, G. caledonium, G. etunicatum, G. clarium\u003c/em\u003e, indigenous mycorrhizae, and Cocktail). The inoculum was previously multiplied in the greenhouse with Sudan grass [Sorghum bicolour (L.) Moench] as the host plant in a mixture of andesitic tuff + soil + peat (6+3+1, V:V: V) medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant Growth Condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plants were grown in a greenhouse at 25-28 \u0026deg;C and a relative humidity of 70-80%, with a 16 h day and 8 h dark photoperiod, under a light intensity of \u0026mu;mol m-2 s-1. Distilled water was added to maintain the moisture at 80% of field capacity. Plants were grown for 10 months.\u003c/p\u003e\n\u003cp\u003eRoot Colonization\u003c/p\u003e\n\u003cp\u003eThe root-clearing and staining procedure and the degree of mycorrhizal infection in the root cortex were assessed using the method described by Koske; Gemma (1989). Root colonisation was determined using a gridline-intersect method for the AM treatments (Gioannetti; Mosse, 1980).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlant analysis\u003c/p\u003e\n\u003cp\u003eAfter harvest, the fresh and dry weights of shoots (leaf and stem) and roots were determined following drying at 65 \u003csup\u003eo\u003c/sup\u003eC. The dry material was ground in the Tema Mill.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThen, 0.2 g of ground plant materials was ashed at 550 \u0026deg;C, followed by dissolution in 3.3 % HCl. After digestion of the plant material, the phosphorus concentration in the solution was determined colourimetrically using a flame photometer. An ICP-OES (PerkinElmer) was used to determine the concentrations of Zn, Fe, Mn, and Cu.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor all statistical analyses, using ANOVA, the main significant interactive effects of mycorrhizal inoculation and growing medium were separated by Duncan\u0026apos;s multiple range test at P\u0026lt;0.05 using the Statistical Analysis System (SAS) (SAS, 2008).\u003c/p\u003e\n\u003cp\u003eMycorrhizal dependency (MD) was determined by expressing the difference between the dry weight of the mycorrhizal plant and the dry weight of the non-mycorrhizal plant as a percentage of the dry weight of the mycorrhizal plant (Plenchette; Fortin; Furlan, 1983).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eAt harvest, plant height, shoot and root dry weight, root colonisation, P concentration in shoot tissue, and micronutrients were determined. All inoculated plant species were colonised with arbuscular mycorrhizal fungi (Figure 1). In addition, small colonisation ranging from 1% to 2% was observed in non-inoculated plants. Indigenous mycorrhizae have a mean root colonisation of 86%. The rest of the mycorrhizae species have more than 50% of root colonisation. The lowest mycorrhizal colonisation, 3%, was observed with indigenous mycorrhiza inoculation in \u003cem\u003eG. mosseae\u003c/em\u003e, whereas the highest colonisation, 68%, occurred with \u003cem\u003eG. mosseae\u003c/em\u003e inoculation. Higher compost levels increased root colonisation rates. Conversely, the RP application resulted in decreased root colonisation. We noticed that root colonisation decreased at high RP.\u003c/p\u003e\n\u003cp\u003ePlant height development data show that the selected mycorrhizal fungi effectively increase plant height compared to the control. The naturally occurring mycorrhizas isolated from the soil are less effective than the control. However, the effect of compost and rock phosphate application is not as effective as the mycorrhizal application (Figure 2).\u003c/p\u003e\n\u003cp\u003eSour orange seedlings inoculated with the AM species grew better than non-mycorrhizal seedlings except for indigenous inoculation (Table 2 and Figure 3). There were significant differences between the mycorrhizal species and the organic fertiliser sources. Since the soils were sterilised at the beginning of the experiment, citrus plants did not grow in sterile soil without mycorrhizal inoculation. As the citrus plants are strongly mycorrhizal-dependent, citrus seedlings are not able to obtain sufficient nutrients for growth without indigenous mycorrhizal spores. The aim of the study, based on the current results, was to test the number of spores and the effectiveness of mycorrhiza spores used in citrus seedling production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt seems that indigenous mycorrhizal spores are less efficient; however, cocktail mycorrhizal inocula are very efficient. Previously, we obtained similar results in many experiments conducted in the rhizosphere Laboratuvar (Ortas, 2019b). Mycorrhizal inoculation significantly enhanced root colonisation. Without considering the effects of RP and compost addition, the highest root colonisation occurred with \u003cem\u003eG. mosseae\u003c/em\u003e inoculation. When selected, mycorrhiza spores are added to the seedling root area, excluding native mycorrhizae spores, and they considerably improve the dry weight of citrus seedling shoots. Compared with the control, indigenous and selected mycorrhizal inoculations significantly increased the dry weight (SDW) of citrus seedlings (Table 2). The effects of indigenous mycorrhiza are less effective than those of selected other species. Similarly, the results of Jbar; et al. (2023) demonstrated that inoculation with the fungus\u0026nbsp;\u003cem\u003eG. mosseae\u003c/em\u003e resulted in a considerable increase in plant growth and available soil phosphorus.\u0026nbsp;Mycorrhizal species are different in their effects on plant growth. Five AM fungi were screened for their symbiotic response with sour orange seedlings in growth media. \u003cem\u003eG. mosseae, G. clarium,\u0026nbsp;\u003c/em\u003eand Cocktail inoculated plants grew better. Citrus seedling root development is generally similar to shoot development. \u003cem\u003eG. mosseae\u0026nbsp;\u003c/em\u003eincreased SDW\u003cem\u003e\u0026nbsp;\u003c/em\u003emore than any other species. Mycorrhizal species are more beneficial than other treatments alone for the growth and nutrition of citrus plants. \u003cem\u003eG. mosseae\u0026nbsp;\u003c/em\u003ewas identified as an active species\u003cem\u003e.\u003c/em\u003e These findings are in connection with those of Wu; Zou (2009), who reported that the sole AMF inoculation significantly increased the total dry weight and leaf and root nutrient contents of the citrus seedlings compared to the non-AMF control.\u0026nbsp;In the Spanish citrus agroecosystem, it has been determined that \u003cem\u003eG. intraradices\u003c/em\u003e is the most effective mycorrhizal fungus for plant growth (Camprubi; Calvet, 1996), and \u003cem\u003eG. mosseae\u003c/em\u003e is the least effective among several tested species\u0026nbsp;(Viyanak; Bagyaraj, 1990).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn sterile soil, plants such as citrus, which are highly dependent on mycorrhizal fungi, often fail to grow. The current study, like Ortas (2019b)\u0026nbsp; study, found that plant growth almost stopped in a sterile soil environment without mycorrhizae spores.\u003c/p\u003e\n\u003cp\u003eAmong the species, differences were observed, and although \u003cem\u003eG. mosseae\u003c/em\u003e was determined to be the most efficient mycorrhizal species, the application of indigenous mycorrhizae was found to be the least effective. Usually, indigenous mycorrhizae significantly increase citrus growth (Ortaş; Varma, 2007). Most probably, in the present experiment, indigenous mycorrhizae did not efficiently form associations with plant roots. This situation may also be due to the fact that natural mycorrhiza spores would establish relationships with other organisms, but are prevented from doing so by soil sterilisation. The adverse effects created by sterilisation should be reinvestigated from the perspective of the natural microflora-mycorrhiza relationship.\u003c/p\u003e\n\u003cp\u003eMycorrhizae inoculation alone contributed more than the RP and compost dose applications. Increasing RP doses up to 2 g kg\u003csup\u003e-1\u003c/sup\u003e soil and 40 g kg\u003csup\u003e-1\u003c/sup\u003e compost increased citrus seedlings\u0026apos; SDW for all mycorrhizal inoculations. The results of Indriani; et al. (2016) indicated that the optimal application rate of rock phosphate was 200 kg ha\u003csup\u003e-1\u003c/sup\u003e of P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e. Increasing RP doses up to 2 g kg\u003csup\u003e-1\u003c/sup\u003e soil and 40 g kg\u003csup\u003e-1\u003c/sup\u003e compost increased citrus seedlings\u0026apos; SDW for all mycorrhizal inoculations. The results of Indriani; et al. (2016) indicated that the optimal application rate of Rock Phosphate was 200 kg ha\u003csup\u003e-1\u003c/sup\u003e of P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e. Applying 20 g of compost per kg of soil increases RDW compared to no compost addition, but 40 g per kg of soil results in a greater increase. Mycorrhizae inoculation alone contributed more to seedling growth and development than either RP or compost. Compared to control and indigenous mycorrhizae, the selected mycorrhizal inoculation significantly increased nutrient concentrations, especially phosphorus (P). Citrus growth enhancement caused by AM fungi is typically attributed to the enhanced phosphorus nutrition of AM plants, which is effective only when soil P levels are low. \u003cem\u003eG. etincatunium\u0026nbsp;\u003c/em\u003emycorrhizae inoculation of seedlings results in a mean P concentration of 0.08%. \u003cem\u003eG. mosseae\u003c/em\u003e inoculated seedlings have a mean of 0.12% of P concentration (Figure 4).\u003cem\u003e\u0026nbsp;\u003c/em\u003eIn terms of shoot P concentration, applications of rock phosphate and compost had little effect on citrus plants. P concentration varied between 0.04% and 0.07% in both the control and ineffective indigenous mycorrhiza inoculation. However, it ranged from 0.08% to 0.12% when specific mycorrhizae were inoculated. Because there was not enough time for both the incubation and planting periods, the experiment continued to track the effects of compost and mycorrhizae on the mobilisation of rock phosphate over time.\u003cem\u003e\u0026nbsp;\u003c/em\u003eDi Tomassi et al. (2021) found that struvite-P fertilization increased biomass by 22% and shoot P uptake by 32%. Since the soil was sterilised before transplanting the seedlings, the natural mycorrhizae were destroyed.\u003c/p\u003e\n\u003cp\u003eZn, Fe, Mn and Cu concentrations were significantly affected by mycorrhizae inoculation (Table 3). The concentration range of mineral nutrients are within the critical levels. Mycorrhiza inoculation shows a significant increase compared to control treatments. The concentration of Zn in the shoots increased significantly with mycorrhizae inoculation. The average Zn content for the control plant is 19.3 mg Zn kg\u003csup\u003e-1\u003c/sup\u003e DW, while the native mycorrhizae-infected plant has 23.3 mg Zn kg\u003csup\u003e-1\u003c/sup\u003e DW. The highest mean of Zn content in the shoots of cocktail and \u003cem\u003eG. mosseae\u0026nbsp;\u003c/em\u003einoculated seedlings is 31, 8 and 31.2 mg Zn/kg, respectively (Table 3). \u003cem\u003eG. mosseae\u003c/em\u003e and \u003cem\u003eG. caledonium\u0026nbsp;\u003c/em\u003einoculation tends to increase Zn concentrations. The highest Zn concentration is observed with \u003cem\u003eG. caledonium\u003c/em\u003e at 2 g RP kg\u003csup\u003e-1,\u003c/sup\u003e reaching an average of 33.1 mg Zn kg\u003csup\u003e-1\u003c/sup\u003e. Generally, the RP 2 g RP kg-1 application has a higher Zn concentration than the 0 and 4 g RP kg-1 soil applications. In terms of compost application, increasing the compost and Zn doses significantly decreased the Zn concentration from 27.66 to 25.41 mg Zn kg\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMousavi; Srivastava; Raiesi (2024) found that high P application reduced Zn uptake. Additionally, a relationship between Zn concentration and AM infection ratio was observed. Also, Ortas; Ortak\u0026ccedil;i; Kaya (2002) tested the effect of P and Zn levels and mycorrhizal inoculation on citrus growth; \u0026nbsp; inoculated plants were significantly stimulated by mycorrhizal infection. The present results show that mycorrhiza inoculation significantly increased P and Zn concentrations. Since mycorrhizae-inoculated plants have higher P and Zn content compared to uninoculated plants, it is concluded that when root colonisation is efficient, plants obtain more nutrients with increased uptake.\u003c/p\u003e\n\u003cp\u003eResults show that mycorrhizal inoculation, particularly with \u003cem\u003eG. caledonium\u003c/em\u003e and \u003cem\u003eG. mosseae\u003c/em\u003e, significantly increases the concentrations of Zn, Fe, Mn, and Cu. However, in terms of compost, the application of up to 20 g of compost increased mineral nutrient levels, whereas at 40 mg compost kg-1 soil, the mean decreased (Table 3). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIron mean concentration decreased with increasing doses of compost and rock phosphates. Compared to other species and the control plant, \u003cem\u003eG. caledonium\u0026nbsp;\u003c/em\u003einoculations have a higher Fe concentration\u003cem\u003e.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMn concentrations range from 22.90 to 84.87 mg Mn\u0026nbsp;kg\u003csup\u003e-1\u003c/sup\u003e. \u003cem\u003eG. mosseae\u003c/em\u003e and \u003cem\u003eG. caledonium\u003c/em\u003e inoculations tend to enhance Mn concentration. For instance, \u003cem\u003eG. mosseae\u003c/em\u003e at 40 g\u0026nbsp;kg\u003csup\u003e-1\u003c/sup\u003ecompost shows a high Mn concentration of 84.87 mg Mn\u0026nbsp;kg\u003csup\u003e-1\u003c/sup\u003e. The highest Mn concentration is observed with \u003cem\u003eG. mosseae\u003c/em\u003e at 40 g Mn\u0026nbsp;kg\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ecompost, reaching 84.87 mg\u0026nbsp;kg\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eG. caledonium\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;G. etunicatum\u0026nbsp;\u003c/em\u003especies inoculation generally affects Cu concentration. \u003cem\u003eG. caledonium\u003c/em\u003e inoculation at 2 g kg\u003csup\u003e-1\u003c/sup\u003e compost results in a Cu concentration of 18.95 mg Cu kg\u003csup\u003e-1\u003c/sup\u003e. The highest concentration is observed with the Cocktail mycorrhizae treatment at 40 g kg\u003csup\u003e-1\u003c/sup\u003e compost, with a value of 24.14 mg Cu kg\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis aligns with the established function of mycorrhizae in enhancing nutrient solubility and extending the hyphal network, thereby improving nutrient acquisition. The rates at which compost is applied also affect the concentrations of mineral nutrients. Higher compost rates (20 and 40 g kg\u003csup\u003e-1\u003c/sup\u003e) typically lead to higher nutrient levels, but the effect varies by nutrient and mycorrhizal treatment. Variability in nutrient concentrations raises the possibility that mycorrhizal species, rock phosphate, and partially composted rates affect micronutrient effectiveness.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYoupensuka, Lordkaewb, and Rerkasemc (2008) reported that mycorrhizal-treated citrus trees had better plant growth and nutrient uptake, such as P, Ca, Zn, Cu, and Fe, than non-mycorrhizal trees. Similarly, according to the findings of Burni; et al. (2023), the combined application of compost and mycorrhizal inoculation can be a successful method for improving plant nutrition, as these studies partly support the present results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;According to our findings, the application of mycorrhizal species increased nutrient uptake and seedling growth more efficiently than rock phosphate and/or compost. Mycorrhizal species typically respond well to low applications of compost and rock phosphate. Compost\u0026apos;s ability to solubilize RP appears to be less successful. The results of Burni; et al. (2023) suggested that AMF inoculation may improve the solubility of phosphorus from organic manure and rock phosphate. Gaind (2017) showed that moisture content affects RP release. Before seedlings are transferred to RP and compost-treated soils, further time may be required for RP incubation. Applying compost and PR dissolved in lemon juice simultaneously at planting significantly increased plant yields, phosphorus absorption, and phosphorus use efficiency (Mwangi; et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCitrus plants have shown different responses to varying mycorrhizal dependency (Table 4).\u0026nbsp;\u003cem\u003eG. mosseae\u0026nbsp;\u003c/em\u003einoculation increased plant growth and had a mean of mycorrhizae dependency of 88%, while the indigenous mycorrhizae inoculated treatment had 48% of mycorrhizae dependency. Cocktail treatment shows relatively high dependency, ranging from 79% to 93%. For all mycorrhizal species, increasing RP dosages increased MD. However, increasing compost dosage did not consistently affect MD. In a similar study, Viyanak; Bagyaraj (1990) found that 18 distinct mycorrhizal fungi produced plants with stem diameters and heights that were noticeably different. \u003cem\u003eG. mosseae\u003c/em\u003e has a mean colonisation of 91% at 0 g/kg compost, which decreases to 87 % at 40 g/kg compost. It seems that higher compost levels may negatively affect colonisation and dependency. \u003cem\u003eG. etunicatum\u0026nbsp;\u003c/em\u003emay be more suitable for soils with higher compost levels, while \u003cem\u003eG. mosseae\u003c/em\u003e may perform better in low-compost applications.\u003c/p\u003e\n\u003cp\u003eWhen all data were correlated, they showed strong, positive correlations, indicating concerted increases in plant height, shoot, and root biomass (Table 5). Growth performance is primarily coupled to P and Zn nutrition and to AMF colonisation. Shoot Fe, Man and Cu were not strongly related to growth parameters.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlso, data were used for Principal Component Analysis, and the results were shown in Figure 5. Cumulative PCA accounts for 65% of the variation, which is mainly in the PC1 axis. PC1 loads positively on plant height, shoot/root fresh and dry weights, Shoot P, Shoot Zn, and mycorrhizal infection. Ellipses show the most apparent separation. Some AMF groups cluster on the positive PC1 side (aligned with better growth, P, and Zn), others on the negative side, with a few differing along PC2 (Fe/Mn). Thus, AMF identity is a primary driver of the overall phenotype. Results show that mycorrhizae No. 1 (control) plants do not affect plant parameters. Indigenous mycorrhiza also have less contribution. \u003cem\u003eG. etinicatinium, G. clarium\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Cokteyle\u003c/em\u003e mycorrhizae are in the core of the contribution to plant parameters. Usually, the growth parameters of\u003cem\u003e\u0026nbsp;G. etinicatinium, G. clarium, and cocktail-inoculated plants\u003c/em\u003e are highly correlated with each other. Also with P and Zn concentrations. \u003cem\u003eG. mosseea\u0026nbsp;\u003c/em\u003ehave a high impact on root parameters\u003cem\u003e.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlant parameters strongly influence compost and rock phosphate. Ellipses substantially overlap; compost alone does not clearly separate multivariate profiles. However, mycorrhizal species exhibit substantial variation in their effects on plant parameters. Compost contribution to plant parameters showed that 20 g per kg of soil was more effective than the control, and 40 g per kg of soil was even more effective. What effects of rock phosphate have been analyzed. It seems that 2 and 4 g RP kg\u003csup\u003e-1\u003c/sup\u003e soil are more related to soil parameters. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMycorrhizal species vary in their effects on plant growth. \u003cem\u003eG. mosseae, G. clarium,\u0026nbsp;\u003c/em\u003eand cocktail-inoculated plants grew better\u003cem\u003e.\u003c/em\u003e According to the findings, it is unclear how compost affects plant development, whereas mycorrhizae have a considerably greater positive impact. Mycorrhiza inoculation alone significantly affected other plant metrics, including shoot and root dry weight, root infection, and P and Zn concentrations. The order of effectiveness of species was as follows: \u003cem\u003eG. mosseae\u003c/em\u003e \u0026gt; Cocktail \u0026gt; \u003cem\u003eG. clarium \u0026gt; G. caledonium \u0026gt; G. etunicatum\u003c/em\u003e \u0026gt; Indigenous mycorrhiza \u0026gt; Control. It seems that indigenous mycorrhizal spores are less efficient than cultivated mycorrhizal spores in promoting plant growth. In our previous work, we found that indigenous mycorrhizae promote the growth of citrus seedlings (Ortas, Demirbas, and Akpinar 2018a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere is a need to investigate the effects of indigenous and cocktail mycorrhizae on plant growth, nutrient uptake, and other soil and plant parameters.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eG. mosseae\u003c/em\u003e was determined to be the most efficient among the mycorrhizae species for sour orange seedlings development. For sustainable agriculture, using rock phosphate, compost, and mycorrhizae as sources of fertilizer is an encouraging practice. Generally, 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e compost and 4 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e RP increased SDW. A similar pattern was observed in root dry weight change. All levels of compost had minimal effects on nutrient uptake, but using 40g of compost increased root inoculation. The brief study period prevented a comprehensive understanding of the impact of compost application on rock phosphate mobilization. The findings highlight the role of mycorrhizal fungi in boosting root infection compared to non-inoculated control seedlings. Citrus plant strongly depends on mycorrhizal inoculation. Increasing RP increased MD; however, compost dose increases did not have a significant effect on MD.\u003c/p\u003e\u003cp\u003eAMF maximize nutrient use efficiency. AMF selection exerts a more substantial multivariate effect than either compost or rock phosphate. Because higher RP can suppress colonization, a moderate RP rate (2 g kg⁻\u0026sup1;) combined with intermediate compost (20\u0026ndash;40 g kg⁻\u0026sup1;) and a well-performing AMF inoculum is advisable. Increasing rock phosphate produces a moderate rightward (positive PC1) shift consistent with improved P (and associated Zn) nutrition, but the effects of compost are limited in this dataset. Plant P, Zn, Fe, Mn and Cu absorption was greatly boosted by mycorrhizal inoculation as well. AMF species substantially enhanced P and Zn concentration and also increased both shoot and root dry matter. Shoot P and Zn status, together with AMF colonisation, best explain variation in growth traits. Species choice is pivotal. Among the AMF tested, \u003cem\u003eG. mosseae\u003c/em\u003e (and, secondarily, the mixed \u0026ldquo;cocktail\u0026rdquo;) delivered the most consistent gains in growth, root colonization, and P/micronutrient uptake; the indigenous inoculum performed poorest.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003ch2\u003eEthics approval:\u003c/h2\u003e\u003cp\u003eEthic approval was accepted\u003c/p\u003e\u003ch2\u003eConsent for publication:\u003c/h2\u003e\u003cp\u003eManuscript can be published\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eNo financial support was available.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eI have prepared the manuscript \u0026ldquo;Effects of Selected Arbuscular Mycorrhizal Fungi, Rock Phosphate, and Compost on Growth and Nutrient Uptake of Citrus Seedlings\u0026rdquo;, and we would like to publish the enclosed article in Applied Fruit Science. The manuscript was not published in any journals.\u003c/p\u003e\u003ch2\u003eAcknowledgement:\u003c/h2\u003e\u003cp\u003eThanks to Dr. \u0026Ccedil;ağdaş Akpinar and Dr. Ahmet Demirbaş for their contributions. Thanks to Dr. Imran for proofreading.\u003c/p\u003e\u003ch2\u003eAvailability of data and material:\u003c/h2\u003e\u003cp\u003eIf it is going to be requested\u003c/p\u003e\u003ch2\u003eCode availability:\u003c/h2\u003e\u003cp\u003eWe used university software\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmberger, A. (2006): Soil fertility and plant nutrition in the tropics and subtropics.(IFA, International Fertilizer Industry Association).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAntunes, V.; Cardoso, E. (1991): Growth and nutrient status of citrus plants as influenced by mycorrhiza and phosphorus application. Plant and Soil 131, 1, 11\u0026ndash;19, \u0026lt;Go to ISI\u0026gt;://WOS:A1991EZ29300002\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://download.springer.com/static/pdf/\u003c/span\u003e\u003cspan address=\"http://download.springer.com/static/pdf/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e317/art%253A10.1007%252FBF00010415.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2FBF00010415\u0026amp;token2=exp=1497614588~ acl=%2Fstatic%2Fpdf%2F317%2Fart%25253A10.1007%25252FBF00010415.pdf %3ForiginUrl%3Dhttp%253A%252F%252Flink\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.springer.com%252Farticle%252F10.1007%252FBF00010415* ~hmac=4df9c9bab1ed1d\u003c/span\u003e\u003cspan address=\"http://.springer.com%252Farticle%252F10.1007%252FBF00010415* ~hmac=4df9c9bab1ed1d\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e766f3d2c8075 bc43a8138483a96976efed20c9467e04b7d6e9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBurni, T.; Hussain, F.; Bibi, S.; et al. (2023): The salutary impacts of AMF species, rock phosphates (RP), and organic matter (FYM) fertilizers on the development and chemical behavior of Mentha arvensis L. Acta Ecologica Sinica 43, 5, 835\u0026ndash;841. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chnaes.2022.12.003\u003c/span\u003e\u003cspan address=\"10.1016/j.chnaes.2022.12.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eButler, O. M.; Warren, C. R. (2025): Microbial phosphorus in loamy, basalt-derived forest soil is altered by plant nutritional status: a root-splitting study. Plant and Soil, 1\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11104-025-07224-w\u003c/span\u003e\u003cspan address=\"10.1007/s11104-025-07224-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCamprubi, A.; Calvet, C. (1996): Isolation and screening of mycorrhizal fungi from citrus nurseries and orchards and inoculation studies. Hortscience 31, 3, 366\u0026ndash;369, \u0026lt;Go to ISI\u0026gt;://WOS:A1996UQ16300018.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChatterjee, N.; Margenot, A. J. (2023): Crop growth is increased by arbuscular mycorrhizae for both phosphate rock and soluble phosphorus fertilizers, but fertilizer solubility primarily determines crop growth. Biology and Fertility of Soils 59, 7, 843\u0026ndash;862. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00374-023-01751-3\u003c/span\u003e\u003cspan address=\"10.1007/s00374-023-01751-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGaind, S. (2017): Exploitation of Orange Peel for Fungal Solubilization of Rock Phosphate by Solid State Fermentation. Waste Biomass Valor 8, 4, 1351\u0026ndash;1360. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12649-016-9682-2\u003c/span\u003e\u003cspan address=\"10.1007/s12649-016-9682-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGioannetti, M.; Mosse, B. (1980): An evaluation of techniques for measuring vesicular-arbuscular mycorrhiza in roots. New Phytologist 84, 489\u0026ndash;500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1469-8137.1980.tb04556.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1469-8137.1980.tb04556.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIndriani, N. P.; Yuwariah, Y.; Rochana, A.; et al. (2016): The role of Vesicular Arbuscular Mycorrhiza (VAM) and rock phosphate application on production and nutritional value of centro legumes (\u0026lt;\u0026thinsp;i\u0026thinsp;\u0026gt;\u0026thinsp;Centrosema pubescens). Legume Research 39, 6, 987\u0026ndash;990. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18805/lr.v0iOF.6645\u003c/span\u003e\u003cspan address=\"10.18805/lr.v0iOF.6645\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJaitieng, S.; Sinma, K.; Rungcharoenthong, P.; et al. (2021): Arbuscular mycorrhiza fungi applications and rock phosphate fertilizers enhance available phosphorus in soil and promote plant immunity in robusta coffee. Soil Science and Plant Nutrition 67, 1, 97\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/00380768.2020.1848343\u003c/span\u003e\u003cspan address=\"10.1080/00380768.2020.1848343\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJbar, A. K.; Noon, G. B.; Al-Taweel, L. S. J.; et al. (2023): Effect of bio fertilization and phosphate rock under sterilization conditions on the activity of phosphatase enzyme, phosphorus availability and growth of wheat plant triticum aestivum L.. International Journal of Agricultural and Statistical Sciences 19, 1493\u0026ndash;1499. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.59467/ijass.2023.19.1493\u003c/span\u003e\u003cspan address=\"10.59467/ijass.2023.19.1493\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoske, R.; Gemma, J. (1989): A modified procedure for staining roots to detect VA mycorrhizas. Mycological Research 92, 4, 486\u0026ndash;488.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa, H.; Zhang, H.; Gao, Q.; et al. (2025): The Relative Contribution of Root Morphology and Arbuscular Mycorrhizal Fungal Colonization on Phosphorus Uptake in Rice/Soybean Intercropping Under Dry Cultivation. Plants (Basel) 14, 1, 106. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/plants14010106\u003c/span\u003e\u003cspan address=\"10.3390/plants14010106\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiranda, P. B.; Mello, A. H.; Kupper, K. C. (2018): Mycorrhizal dependence of citrus rootstock. Revista Brasileira de Fruticultura 40, 3, e-762.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMousavi, S. M.; Srivastava, A. K.; Raiesi, T. (2024): Citrus nutrition in Iran: lessons from calcareous soils. Journal of Plant Nutrition 47, 19, 3367\u0026ndash;3392. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/01904167.2024.2379571\u003c/span\u003e\u003cspan address=\"10.1080/01904167.2024.2379571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMwangi, E.; Ngamau, C.; Wesonga, J.; et al. (2020): Managing Phosphate Rock to Improve Nutrient Uptake, Phosphorus Use Efficiency, and Carrot Yields. J. Soil Sci. Plant Nutr. 20, 3, 1350\u0026ndash;1365. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s42729-020-00217-x\u003c/span\u003e\u003cspan address=\"10.1007/s42729-020-00217-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOlsen, S. R.; Sommers, L. E. (1982): Methods of soil analysis. Part 2, 1982, 403\u0026ndash;430.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOrtas, I. (2019a): Do horticultural tree plant species depend on mycorrhizal inoculation under marginal soil conditions? In 30th International Horticultural Congress (IHC) / International Symposium on Water and Nutrient Relations and Management of Horticultural Crops. (Istanbul, TURKEY), pp 85\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOrtas, I. (2019b): Effect of mycorrhizal inoculation on citrus seedling growth and nutrient uptake. In 30th International Horticultural Congress (IHC) / International Symposium on Water and Nutrient Relations and Management of Horticultural Crops. (Istanbul, TURKEY), pp 77\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOrtas, I. (2022): What is Ecological/Organic Farming? Soil Science and microbiological Point of view. In Introduction and Application of Organic Fertilizers as Protectors of Our Environment, Ozturk, M.; Akram, N. A.; Unal, B. T.; et al., eds. (Newcastle, UK, Cambridge Scholars Publishing), p 90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOrtas, I.; Demirbas, A.; Akpinar, C. (2018): Time period and nutrient contents alter the mycorrhizal responsiveness of citrus seedlings. European Journal of Horticultural Science 83, 2, 72\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17660/eJHS.2018/83.2.2\u003c/span\u003e\u003cspan address=\"10.17660/eJHS.2018/83.2.2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOrtas, I.; Ortak\u0026ccedil;i, D.; Kaya, Z. (2002): Various mycorrhizal fungi propagated on different hosts have different effects on citrus growth and nutrient uptake. Communications in Soil Science and Plant Analysis 33, 1\u0026ndash;2, 259\u0026ndash;272. Doi \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1081/Css-120002392\u003c/span\u003e\u003cspan address=\"10.1081/Css-120002392\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOrtaş, I.; Varma, A. (2007): Field trials of bioinoculants. In Advanced Techniques in Soil Microbiology. (Springer), pp 397\u0026ndash;413.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePlenchette, C.; Fortin, J. A.; Furlan, V. (1983): Growth-responses of several plant-species to mycorrhizae in a soil of moderate P-fertılıty.1. mycorrhizal dependency under field conditions. Plant and Soil 70, 2, 199\u0026ndash;209, \u0026lt;Go to ISI\u0026gt;://WOS:A1983QM70100005\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://download.springer.com/static/pdf/\u003c/span\u003e\u003cspan address=\"http://download.springer.com/static/pdf/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e117/art%253A10.1007%252FBF02374780.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2FBF02374780\u0026amp;token2=exp=1497614703~ acl=%2Fstatic%2Fpdf%2F117%2Fart%25253A10.1007%25252FBF02374780.pd f%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007 %252FBF02374780*~hmac=326241feee2c99e4d7f4cbb2e70079e51 fce4f8bd6bb11b296cdb7b62a656776.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSAS (2008): Statistical Analysis Systems user's guide (SAS/STAT\u0026reg; 9.2 User\u0026rsquo;s Guide).(North Carolina, USA: SAS Institute Inc.).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSrivastava, A. K.; Singh, S.; Marathe, R. A. (2002): Organic citrus: Soil fertility and plant nutrition. Journal of Sustainable Agriculture 19, 3, 5\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1300/J064v19n03_03\u003c/span\u003e\u003cspan address=\"10.1300/J064v19n03_03\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eViyanak, K.; Bagyaraj, D. J. (1990): Selection of efficient VA mycorrhizal fungi for trifoliate orange. Biological Agriculture \u0026amp; Horticulture 6, 4, 305\u0026ndash;311, \u0026lt;Go to ISI\u0026gt;://WOS:A1990CZ24000004.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, Q. S.; Zou, Y. N. (2009): The effect of Dual Application of Arbuscular Mycorrhizal Fungi and Polyamines upon Growth and Nutrient Uptake on Trifoliate Orange (Poncirus trifoliata) Seedlings. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 37, 2, 95\u0026ndash;98, \u0026lt;Go to ISI\u0026gt;://WOS:000272606600015.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"applied-fruit-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Applied Fruit Science","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Arbuscular mycorrhizal fungi (AMF), Rock phosphate, Compost amendment, Citrus (sour orange) seedlings, Phosphorus uptake, Micronutrient acquisition (Zn, Fe, Mn, Cu)","lastPublishedDoi":"10.21203/rs.3.rs-8019594/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8019594/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCitrus trees are among the most essential fruit plants grown in southern Turkey. Nutrient uptake in the Mediterranean region is limited by factors such as low organic matter, high soil pH, and limited availability of phosphorus and micronutrients. To evaluate, under greenhouse conditions, how rock phosphate (RP), compost, and different arbuscular mycorrhizal fungi (AMF) species influence growth, root colonization, and nutrient uptake (with emphasis on P, Zn, Fe, Mn, Cu) in Citrus (sour orange) seedlings.\u003c/p\u003e\u003cp\u003eThe experiment was conducted under greenhouse conditions with three compost levels (0, 20, and 40 g compost/kg soil), three rock phosphate levels (0, 2, and 4 g rock phosphate/kg soil), and seven mycorrhizal species (Control, \u003cem\u003eG. mosseae\u003c/em\u003e, \u003cem\u003eG. caledonium\u003c/em\u003e, \u003cem\u003eG. etunicatum\u003c/em\u003e, \u003cem\u003eG. clarium\u003c/em\u003e, indigenous mycorrhiza, and a cocktail mixture) as a completely randomized factorial design with three replications. Before the experiment began, compost and rock phosphate were mixed with the sterilised soil and incubated for 3 weeks. Plants were grown for 10 months. Root colonization, shoot and root dry weights, and tissue nutrient concentrations were determined.\u003c/p\u003e\u003cp\u003eResults obtained after 180 days of cultivation showed that mycorrhizal-inoculated plants grew significantly more than control plants. The combination of arbuscular mycorrhizal (AM) fungi species, rock phosphate, and compost application led to a remarkable increase in dry matter production, root colonisation, and nutrient uptake. An increase in RP application resulted in higher MD across all mycorrhizal species and compost treatments. However, increased compost application reduced MD for all mycorrhizal species. Citrus seedlings were colonised by several mycorrhizal species, and the mycorrhizal dependency (MD) of these seedlings was assessed. The highest MD (91%) was observed in seedlings inoculated with \u003cem\u003eG. mosseae\u003c/em\u003e and treated with 20 g of compost and 4 g of RP per kg of soil. Citrus seedlings inoculated with \u003cem\u003eG. mosseae\u003c/em\u003e exhibited the highest MD (81%), while those inoculated with native mycorrhizal spores showed the lowest MD (59%). Citrus seedlings inoculated with mycorrhiza contained higher levels of phosphorus, zinc, and other micronutrients compared to non-inoculated control plants.\u003c/p\u003e\u003cp\u003eAM fungi, particularly \u003cem\u003eG. mosseae\u003c/em\u003e, in combination with 20 g kg-1 compost and 4 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e RP addition, significantly improved citrus seedling growth and P and Zn uptake.\u003c/p\u003e\u003cp\u003eCompared with the sterile control, AMF inoculation markedly increased plant height and biomass. Responses differed among fungal species; \u003cem\u003eG. mosseae\u003c/em\u003e and the mixed \u0026ldquo;cocktail\u0026rdquo; inoculum generally produced the highest growth and root colonization. Higher RP doses tended to suppress colonisation; the effect of compost on colonisation depended on species \u0026times; dose interactions. AMF treatments raised shoot P concentrations from ~\u0026thinsp;0.04\u0026ndash;0.07% in control/ineffective-local treatments to ~\u0026thinsp;0.08\u0026ndash;0.12%, and increased Zn, Fe, Mn, and Cu concentrations. RP and compost alone had limited effects, whereas their combined use with AMF supported both growth and nutrition; the full agronomic effect of RP likely requires a longer incubation period for solubilization.\u003c/p\u003e","manuscriptTitle":"Effects of Selected Arbuscular Mycorrhizal Fungi, Rock Phosphate, and Compost on Growth and Nutrient Uptake of Citrus Seedlings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 18:38:50","doi":"10.21203/rs.3.rs-8019594/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-16T10:46:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-13T07:43:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-27T16:17:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151240174828624009337068359549257489023","date":"2025-11-17T12:47:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49270214787681704094984384098844394607","date":"2025-11-17T10:46:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-07T09:26:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-03T19:33:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-03T19:32:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Fruit Science","date":"2025-11-03T13:12:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"applied-fruit-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Applied Fruit Science","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a2a3d30e-21fb-4447-b160-a6da69a8cdca","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T10:38:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 18:38:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8019594","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8019594","identity":"rs-8019594","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}