Response of Portulaca olearacea and its rhizospheric microbiome to the application of slumgum from beekeeping industry transformed with Penicillium chrysogenum

Response of Portulaca olearacea and its rhizospheric microbiome to the application of slumgum from beekeeping industry transformed with Penicillium chrysogenum | 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 Response of Portulaca olearacea and its rhizospheric microbiome to the application of slumgum from beekeeping industry transformed with Penicillium chrysogenum Angel Carrascosa, Jose Antonio Pascual, Jessica Cuartero, Inmaculada García-Romera, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4480989/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract In the beekeeping industry, “slumgum” is generated as a solid organic waste during the beeswax-rendering process from old scraped honeycombs. This bio-waste could be considered as a novel organic fertilizer due to its high content in organic matter and nutrients. As a novelty in this study, we analysed the effect of application of solid and liquid slumgum transformed or not with Penicillium chrysogenum on purslane ( Portulaca oleracea ) yield and its relationship with soil rhizosphere. For this purpose, nutritional composition, enzymatic activities involved in the P, N and C cycles, fungal and bacterial community composition, diversity and potential functionality in the rhizosphere were measured. The application of solid and liquid slumgum transformed with P. chrysogenum (TS and TL, respectively) and slumgum liquid (L) significantly increased purslane shoot biomass and foliar P content, compared to the non-transformed solid slumgum (S). The different types of slumgum tested resulted in changes in the composition of both bacteria and fungi communities, resulting in distinct communities for each treatment. Moreover, changes in the functional fungal guilds were observed, with increased abundances of saprotrophs and reduced number of plant pathogens under the TS, TL, and L treatments. Solid slumgum transformed with P. chrysogenum (TS) was also the most effective in enhancing enzymatic activities related with C, N and P cycles in the rhizosphere. Conversely, the use of solid slumgum (S) led to an increase in the abundance of bacterial genes primarily associated with the denitrification process. Our preliminary results suggest that solid and liquid slumgum transformed with P. chrysogenum , as well as liquid non-transformed slumgum (TS, TL, and L, respectively), could be considered as novel organic fertilizers, amendments or additives within the circular economy context and the sustainable use of natural resources. Nevertheless, further studies are necessary to validate the positive outcomes observed, particularly under field conditions and with a variety of species. purslane circular economy wild edible species bacterial community fungal community functional genes sustainable agriculture Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Purslane ( Portulaca oleracea L.) is an important nutritional and medicinal plant, renowned for its high content of omega-3 fatty acids and other bioactive compounds (Petropoulos et al., 2016, 2015). Due to its beneficial health properties, there is a renewed interest in its integration into commercial small-scale farming systems (Carrascosa et al., 2023b; Chrysargyris et al., 2023). Moreover, purslane is recognised as a wild edible plant (WEP) which is distributed worldwide, and it grows naturally as a weed without human intervention in various agroecosystems. Purslane is also well adapted to generally adverse conditions such as drought, high temperatures, or moderate salinity levels. It has relatively low nutritional requirements, capable of achieving high yields even with low fertilisation rates (Carrascosa et al., 2023a; Jin et al., 2016). These characteristics make purslane a candidate crop to be utilised in vulnerable areas or no chemical inputs can be applied to avoid contamination of aquifers and surrounding water bodies, or in marginal areas where soils are deficient in nutrients (Cui et al., 2020; Jang and Chen, 2015). In these areas, new strategies for fertilisation should be implemented using organic amendments as substitutes of synthetic fertilizers (Alburquerque et al., 2012; Celestina et al., 2019; Reyes-Torres et al., 2018); and in particular new added-value ones could widen the options to be used under different cropping scenarios. It is well established that organic amendments (sewage sludge, pruning composts, farmyard manure, biochar, poultry manure, etc.) are a good source of essential plant nutrients and if managed properly, they can improve crop production (Ahmad et al., 2022; Luo et al., 2018). Additionally, they may enhance the physical, chemical, and biological properties of soil, presenting an eco-friendly approach to maintaining and enhancing soil fertility while contributing to natural resources preservation at the same time (Diacono and Montemurro 2010; Aytenew and Bore 2020; Moreno et al. 2022). Moreover, an increase in the content of soil organic matter contributes to carbon (C) sequestration, reducing the CO 2 atmospheric level and adverse greenhouse effects, helping to mitigate the climate change (Lorenz and Lal, 2023). In the beekeeping industry, 'slumgum' is generated as solid organic waste during the beeswax-rendering process from old scraped honeycombs, which are replaced every three or four years. This waste exhibits a dark (brown-black) colour with an appearance that falls somewhere between animal manure and vegetable compost. It is primarily composed of brood cocoons, moths, larvae of Galleria melonella L., molts, dead bees, bee droppings, pollen, propolis, and small proportions of non-extractable wax, among other elements. Usually, it is stored outdoors and currently has neither has any particular use nor it is recycled (Morales-Corts et al., 2014, 2010). The worldwide production of slumgum is estimated to 93,000 tons per year (Faostat: Agriculture Data). Until now, few studies on slumgum residue have been reported in the literature. In particular Morales-Corts et al. (2010, 2011, 2014) suggested that slumgum could be a promising candidate as an organic fertilizer due to its high content of organic matter, essential nutrients, and micronutrients. However, to the best of our knowledge the impact of slumgum on agricultural soils health is scarcely studied so far. Similar residues to slumgum such as dry olive residues (Siles et al., 2014b), are usually transformed using saprophytic fungi ( Fusarium spp, Phanerochaete spp, Pleurotus spp, Trametes versicolor and Penicillium chrysogenum, etc.) as a waste or residue pre-treatment prior of being used as organic amendments (García-Sánchez et al., 2014; Reina et al., 2013; Sampedro et al., 2007; Siles et al., 2015). The transformation process improves the quality of organic amendments, since it mobilizes nutrients, thus promoting a more efficient use of nutrients by plants. Thus, this process could be considered as an environmentally friendly and reliable bio-waste management option (Siles et al., 2015). Therefore, it seems to be an interesting approach to explore the effect of slumgum as potential new organic fertilizer, amendment or additive in agricultural soils, and its response on plants growth through soil rhizosphere. Soil microbial communities play a vital role in the functions of soil ecosystems since microbes participate in processes of organic matter decomposition, nutrient cycling, and plant productivity (Naylor et al., 2022; Saleem et al., 2019; Xia et al., 2020). Moreover, soil microorganisms rapidly respond to environmental shifts and could be considered as good indicators of soil quality. In a recently meta-analysis conducted by Shu et al. (2022), it was reported that the use of organic amendments enhances the soil microbial diversity and functionality. However, the response of microbial communities to the addition of organic residues can be affected by diverse factors: soil properties, experimental and ambient conditions, residue type, doses, crop type, etc. (Chen et al., 2023; Li et al., 2021; Ning et al., 2020; Shu et al., 2022). Thus, a detailed knowledge of microbial composition, diversity and function as well as properties which determine soil fertility and plant growth in soils amended with slumgum is essential, in order to understand the patterns and relationship between soil functioning and processes mediated by microorganisms in agroecosystems. This knowledge could have important implications for improving more sustainable agricultural management practices and conserving Mediterranean soil biodiversity in a climate changing environment. The objective of the present study is to explore the impact of short-term application of solid and liquid slumgum, both non-transformed and transformed with Penicillium chrysogenum , on the response of purslane yield and its relationship on rhizosphere soil. The nutritional composition, enzymatic activities involved in the P, N and C cycles, and fungal and bacterial community composition, diversity and functionality in the rhizosphere were deeply studied to demonstrate the beneficial effect of slumgum on purslane development and to determine the necessity or not of fungal transformation prior to its application in the soil. Materials and Methods Quisque cursus massa sed urna congue, ac convallis neque consectetur. Proin faucibus neque non metus mollis, suscipit pretium nisl blandit. In hac habitasse platea dictumst. 2.1 Experimental design and sampling The topsoil (0–20 cm) utilized in the experiment was collected from the CEBAS-CSIC Experimental Farm (Santomera, Murcia, Spain 38°06'14.0"N 1°02'00.1"W), it corresponds to a semi-arid Mediterranean climate with an annual mean temperature of 19.2 ºC, annual rainfall of 300 mm, and a potential evapotranspiration of 1000 mm y -1 . The soil is classified as Lithic xeric haploxeroll, with clay-loam texture (41% illite, 17% smectite, and 30% palygorskite). The chemical characteristics of the soil were: total nitrogen 5.7 g kg −1 , available phosphorous 6.0 mg kg −1 , available potassium 33.9 mg kg −1 , organic matter 12.6 g kg −1 , CaCO 3 3 g kg −1 , pH 7.14 and electrical conductivity 0.10 dS m -1 . The organic beekeeping residue called “slumgum” used in this experiment was collected from the “Apizafra S.L.” beekeeping company, located in Albudeite, Murcia (Spain). The slumgum residue was transformed using the saprophytic fungi: Penicillium chrysogenum (EEZ-28 ). The mycotransformation of the solid slumgum (S) was carried out by solid-state fermentation as described by Reina (Reina et al., 2013) with some modifications. Firstly, fungi were inoculated into flasks with 18 g of barley and 30 mL of sterile water for one week. After this period, 10 g of previously sterilised solid slumgum and 2.5 mL of water were added and incubated at 28 ºC for 4 weeks. The resulting biotransformed solid slumgum (TS) was then homogenised and sterilised. Furthermore, the liquid slumgum extract (L) was obtained by mixing slumgum with distilled water 1:3 (w/v ratio) and agitated for 24 hours and then filtrated. The extract was also biotransformed through the same strain of Penicillium chrysogenum (EEZ-28) , using a basal medium (MB) for 7 days at 80 rpm and at 28 °C for 14 days. After this period, slumgum liquid extract was diluted to half, (TL) and sterilised. Both transformed products, TS and TL, were stored at 4 °C, while both the raw materials before biotransformation (S and L, respectively) were autoclaved and then, stored at 4 °C. The main chemical characteristics of the studied materials are shown in Table 1. Table 1. Chemical and nutrient composition of the different types of slumgum used in this experiment. Solid Slumgum (S) Solid Slumgum Transformed (TS) Liquid extract of Slumgum (L) Liquid extract of Slumgum Transformed (TL) Moisture (%) 23.5 ± 0.4 23.5 ± 0.4 pH 4.93 ± 0.62 8.47 ± 0.52 6.25 ± 0.33 6.70 ± 0.28 Electric conductivity (EC) (dS m -1 ) 1.74 ± 0.42 2.60 ± 0.50 3.71 ± 0.11 3.10 ± 0.15 Total carbon (TC) (%) 55.60 ± 1.73 48.33 ± 1.5 1.27 ± 0.5 0.37 ± 0.6 Total organic carbon (TOC) (%) 53.49 ± 1.25 48.26 ± 1.3 Total nitrogen (TN) (%) 3.61 ± 0.08 3.63 ± 0.05 0.12 ± 0.05 0.06 ± 0.01 Relation TOC/TN 14.82 ± 0.46 13.27 ± 2.5 Total P (mg kg -1 ) 3500 ± 39 3700 ± 40 500 ± 29 346 ± 19 Total K (mg kg -1 ) 3900 ± 40 5500 ± 49 830 ± 28 745 ± 25 Total Ca (mg kg -1 ) 3600 ± 87 2250 ± 79 189 ± 19 57 ± 9 Total Mg (mg kg -1 ) 1000 ± 22 1100 ± 26 110 ± 12 67 ± 9 Total Na (mg kg -1 ) 190 ± 3 240 ± 5 35 ± 3 46 ± 4 Total Fe (mg kg -1 ) 499 ± 8 330 ± 7 10.46 ± 0.2 5.74 ± 0.2 Total Cu (mg kg - 1 ) 14.46 ± 1 11.93 ± 1 0.19 ± 0.2 15.87 ± 2 Total Mn (mg kg -1 ) 29.76 ± 1 25.72 ± 1 2.85 ± 0.1 1.66 ± 0.1 Total Al (mg kg -1 ) 257 ± 5 209 ± 4 8.09 ± 0.2 1.58 ± 0.1 Total Zn (mg kg -1 ) 255 ± 3 176 ± 2 11.63 ± 1 9.59 ± 1 Total Pb (mg kg -1 ) 3.74 ± 1 3.17 ± 1 < 0.01 < 0.01 Total Ni (mg kg -1 ) 1.86 ± 0.5 1.15 ± 0.3 0.09 ± 0.01 0.06 ± 0.01 Total Cd (mg kg -1 ) < 0.025 < 0.025 < 0.01 < 0.01 Total Co (mg kg -1 ) 0.57 ± 0.01 0.21 ± 0.01 < 0.01 < 0.01 Total Li (mg kg -1 ) 0.91 ± 0.02 1.08 ± 0.01 0.09 ± 0.01 0.12 ± 0.02 Total Se (mg kg -1 ) < 0.01 1.14 ± 0.01 < 0.01 < 0.01 In May 2022, purslane ( Portulaca oleracea L.) seeds, supplied by the Department of Agriculture Crop Production and Rural Environment (University of Thessaly, Greece) were sown under nursery conditions, in peat substrate trays for 3 weeks until reached the proper size for transplanting to the 0.3 L plastic pots. Each pot was filled with a soil:sand mixture (1:1 w:w) and one plant was transplanted into each one of them. Then plants were treated with the biotransformed and non-transformed slumgum (TS, TL, S and L, respectively) and a non-slumgum treatment was also used as a control treatment. For each treatment seven replicates were established (n=7; 35 pots in total). For soil amendment, both solid treatments (TS and S) were mixed with the soil:sand mix at a rate of 5% (17.5 g), at the beginning of the experiment. Similarly, both liquid treatments (TL and L) were added at a rate of 5% (17.5 mL) to the pots through the irrigation water one week after transplantation. The experiment was conducted for six weeks without additional fertilization under greenhouse conditions at CEBAS-CSIC Experimental Farm located in Santomera, Murcia, Spain. The day/night temperature was maintained at 30 ºC/25 ºC. Plants were regularly irrigated through sprinklers maintaining the 60% of water holding capacity. When the experiment concluded, plants were harvested, and rhizospheric soil was collected for each replicate. The soil was sieved through a 2-mm mesh and divided in a portion stored at 4 ºC for chemical and biochemical analysis and other at -20 ºC for molecular analysis. 2.2 Analyses of plant tissue Shoot fresh and dry biomass (dried at 105 ºC for 5 h) was measured. The dried shoots were then ground using a mill before nutrient analysis. Total N in the shoots was determined using a TruSpec CN Analyzer (LECO, St. Joseph, MI, USA). Analysis of shoot total P, K, Ca, Mg and Fe contents was performed using ICP-OES (ICAP 6500 DUO, Thermo Fisher Scientific, Hayward, California, USA). 2.3 Chemical, biochemical, and biological analyses of rhizospheric soil Soil pH and electrical conductivity were determined at 1:2.5 and 1:5 soil:water ratios, respectively. Available P was extracted with bicarbonate following the method of (Olsen et al., 1954) and determined using molybdate‐reactive P as described by Murphy and Riley (1962). Organic carbon (%) was measured using the dichromate oxidation method (Walkley and Black, 1934). The organic carbon values were transformed into organic matter using an empirical factor (1.724). Total nitrogen (TN) and total carbon (TC) were determined using an elemental CHNS-O analyzer (EA- 1108, Carlo Erba, Barcelona, Spain); total phosphorous (TP) and total potassium (TK) were assessed using ICP-OES (ICAP 6500 DUO, Thermo Fisher Scientific, Hayward, California, USA). Dehydrogenase activity (DHA) in the soil was determined measuring the amount of triphenyl formazan (TPF) released during incubation with 2,3,5 triphenyltetrazolium chloride (Tabatabai, 2018). The urease activity was assessed as described by (Kandeler et al., 1999), utilizing urea as a substrate (Nannipieri et al., 1980); β-glucosidase activity (β-Glu) was determined using p-nitrophenyl-β-D-glucopyranoside (Eivazi and Tabatabai, 1988), and alkaline phosphomonoesterase activities were assessed with p-nitrophenyl phosphate disodium as a substrate (Naseby and Lynch, 1997) with subsequent colorimetric determination of the p-nitrophenol released. 2.4 DNA extraction and Illumina sequencing DNA was extracted from 0.25 g of rhizospheric soil using the DNeasy PowerSoil DNA Isolation kit (Qiagen), following the manufacturer´s protocol. DNA yield and quality were assessed through electrophoresis in 0.8% ( w / v ) agarose gels stained with GelRed and visualized under UV light. Additionally, DNA quality was verified using a Qubit 3.0 fluorometer (Life Technologies, Grand Island, NY). DNA from each sample was sequenced on the Illumina MiSeq platform at the genomics service of the Institute of Parasitology and Biomedicine “López Neyra” (CSIC), Granada, Spain. Prokaryotic libraries were constructed by amplifying the hyper-variable V3–V4 regions of the 16S rRNA gene using the primer pair, 341F and 806R, according to Takahashi et al. (2014). These amplicons were tagged for attachment to peptide nucleic acid (PNA) PCR clamps to reduce amplification of plastid and mitochondrial DNA (Lundberg et al., 2013). Fungal libraries were constructed by amplifying the ITS2 region with the primer pair ITS4 (White et al., 1990) and fITS7 (Ihrmark et al., 2012). Both runs were sequenced using a paired-end 2x300bp (PE 300) strategy. 2.5 Sequencing data processing The raw sequence data from the sequencer were processed using QIIME version 2022.2 (Caporaso et al., 2010). Before the analysis, raw sequences were checked using the fastqQC program (FastQC: A Quality Control Tool for High Throughput Sequence Data). After that, sequences were uploaded to QIIME and primers were removed using cutadapt (Martin, 2011), removing sequences with less than 250 pb. To denoise the reads, the DADA2 algorithm was employed (Callahan et al., 2016) and sequences were truncated to an average length with Q score > 30. The resulting amplicon sequence variants (ASVs) were classified using SILVA v138 released in November 2020 or UNITE v9 released in October 2022 in the bacterial and fungal community, respectively. Functional analysis of the bacterial community was performed using the PICRUSt2 algorithm (Douglas et al., 2020), by predicting N, C, and P bacteria metabolism. Fungal functional guilds were determined by using the genus-guild database Fungal Traits (Põlme et al., 2020). 2.6 Statistical analyses A two-way ANOVA was performed to determine the influences of the slumgum type and transformation with P. chrysogenum , as well as their interactions, through a Tukey’s HSD test (Honestly Significant Difference, P < 0.05). Correlation analyses were performed using Pearson's method. For statistical analyses IBM-SPSS v.29 was utilized. For microbial communities' datasets, the Amplicon Sequence Variants (ASVs) per sample were rarefied to mitigate potential bias in further analyses arising from variations in sequencing effort among samples. A non-metric multidimensional scaling ordination (NMDS) based on the Bray-Curtis distance was carried out to compare the composition of the bacterial and fungal communities. This analysis utilized the 'metaMDS' function implemented in the 'vegan' package for R (Oksanen et al., 2019). The effect of the two experimental factors – slumgum type and transformation with P. chrysogenum - on the rhizosphere bacterial and fungal communities’ composition and structure were tested using a permutational multivariate analysis of variance (perMANOVA) with 999 permutations on the same Bray-Curtis matrix using the “adonis” function in “vegan” package for R (Oksanen et al., 2019). Heat maps were also constructed using the “vegan” package for R. Results 3.1 Impacts of the experimental factors on plant parameters Shoot dry biomass was significantly affected by slumgum type (solid or liquid), P. chrysogenum transformation and the interaction between both factors (Fig. 1). The dry purslane biomass under TS, L and TL treatments showed significantly higher values than control (an increase between 60-77%); however, the application of non-transformed solid treatment (S) significantly reduced dry mass compared to the control (69%). The slumgum type had a significant effect on the content of foliar P, K , Ca and Mg; whereas its fungal transformation significantly affected only the P content (Table S1). The N, P, Ca and Mg contents were also significantly affected by the interaction between both factors. All treatments significantly decreased the K and Ca content compared to the control treatment, while Mg and Fe contents were reduced in treatments S, L and TL and TS, L and TL, respectively. In contrast, P content was significantly increased by TS, L and TL treatments, while N content was the highest for the TS treatment. 3.2 Impacts of the experimental factors on rhizospheric soil parameters The electrical conductivity of the soil was only affected by slumgum type, where TS treatment recorded the significantly highest value (Table S2). On the other hand, the dehydrogenase activity was affected by the two experimental factors and their interaction, with TS treatment showing significantly higher dehydrogenase activity than the rest of the treatments and the control (Fig. 2A). The same treatment (TS) beneficially affected the activity of b-glucosidase without being significantly different from the control and L treatment (Fig. 2B). Both alkaline phosphatase and urease activities were significantly affected by the slumgum type, being the solid slumgum (S and TS treatments) more efficient to significantly increase both enzymatic activities concerning to the control and the liquid form (L and TL treatments; Fig. 2 C, D). 3.3 Impacts of the experimental factors on the fungal community composition and functionality of rhizospheric soil The sequencing of the fungal ITS region produced a total of 7,171,200 high-quality sequences, which were clustered into 2,260 fungal Amplicon Sequence Variants (ASVs) before rarefaction at 91,201 reads per sample. The ANOVA showed that all experimental factors and their interactions, except the transformation for fungal richness, had a significant effect on α-diversity; the S, TS and TL treatments significantly decreased the fungal richness and Shannon diversity index (Fig. S1A, B). The rhizosphere fungal communities differed significantly among slumgum types and the transformation process (Table S3), as well as an interaction between both factors was recorded with the perMANOVA analysis. According to the results of the perMANOVA, the NMDS ordination showed clusters of fungal communities well defined for each treatment (Fig. 3A) being the fungal communities from L and TL treatments more similar to the control treatment than S and TS treatments. The fungal ASVs were assigned to 13 phyla. The most abundant phylum was Ascomycota (82.7% of the total relative abundance), followed by Mortierellomycota (10.5%), Basidiomycota (3%), Chytridiomycota (2.3%) and Mucoromycota (1.3%) (Fig. S2A, Table S4). The remaining identified phyla accounted for less than 0.1% of the fungal sequences. Ascomycota and Basidiomycota were significantly affected by the slumgum type, decreasing their relative abundance under the S and TS treatments compared to the control soil and the rest of the treatments (L and TL). In contrast, Mortierellomycota and Mucoromycota showed opposite trends and significantly increased their relative abundance under the S and TS treatments. At the genus level, Humicola was the dominant fungal genus, with a relative abundance of 15.4%, which significantly increased after the transformation of slumgum of both types (TS and TL treatments) (Fig. S2B, Table S5). Between the genera, Mortierella and Mucor were the most significantly influenced by the slumgum type, with relative abundances that increased in solid slumgum (S and TS), while Preussia , Hormiactis and Myceliophthora also significantly decreased under the same treatments (S and TS). Only Alternaria and Ochroconis were significantly affected by the two experimental factors and their interaction, showing the highest relative abundance under the S treatment. The analysis of fungal functional guilds assigned 1,224 Amplicon Sequence Variants (ASVs), which represents 54% of the total ASVs, to the six main functional guild groups: plant pathogens, parasites, lichenized, symbiotrophs, endophytes, and saprotrophs (Fig. 4; Table S6). The plant pathogens and saprotrophs were significantly affected by the slumgum type, the transformation process and the interaction between both factors, suggesting that both fungal guilds showed opposite trends under the TS, L and TL treatments (when saprotrophs increased the plant pathogens decreased). The parasites were significantly decreased by the treatments with slumgum transformation (TS and TL), as shown also with the ANOVA. Related to the endophytes and symbiotrophs, the most influential factor was slumgum type, showing the lowest relative abundance in the treatments with solid slumgum (S and TS). 3.4 Impacts of the experimental factors on the bacterial community composition and functionality of rhizospheric soil A total of 5,452,140 reads with 5,567 different ASVs were obtained before rarefaction at 270,840 reads per sample. Bacterial richness and Shannon diversity index were significantly affected by both experimental factors and their interactions (Fig. S1C, D). The richness was significantly higher in the S and TL treatments than the rest of the treatments, while the significantly lowest values were recorded for the TS treatment. The perMANOVA analysis showed that both factors, slumgum type and transformation, as well as their interaction (S x T) significantly influenced the composition and structure of the rhizospheric bacterial communities (Table S3B). The NMDS ordination of soil bacterial communities exhibited a stress value of 0.08 and it was clustered by slumgum treatment, showing the same pattern as fungal communities (Fig. 3B). The bacterial community was composed of 17 different bacterial phyla. Actinomycetota and Pseudomonadota were the most abundant phyla (32% and 26% respectively of total relative abundance) and were affected by both experimental factors and their interaction (Fig. S2C, Table S7). The TS and S treatments were the most efficient to significantly increase Actinomycetota and Pseudomonadota, respectively. The Bacillota phylum abundance (24%) was significantly decreased by both factors, being the solid slumgum treatments (S and TS) those with the lowest values. Myxococcota phyla abundance, although accounted for only 1.43% of the bacterial sequences, was strongly affected by the two experimental factors; and it significantly increased under TS, S and L treatments. With respect to the bacterial genus, the most significant factor was the slumgum type (Table S8). The genera such as Microbacterium , Virgibacillus , Ilumatobacter and IMCC26256 decreased their relative abundance under the treatments with solid slumgum addition (S and TS). In contrast, TM7a and unknown bacterial genus significantly increased their abundances under the solid treatment (S and TS; Fig. S2D). With respect to the bacterial functionality, the relative abundance of predicted genes, involved in nitrification and nitrogen fixation process of N cycling, were not significantly affected by the slumgum addition (Table S9, Fig. S3). Instead, in the denitrification process the relative abundance of nitrate reductase genes (narG, narH), and nitric oxide reductase (norB, norC) were significantly increased by both experimental factors. The S treatment increased all four genes compared to the control, while the TS treatment only significantly increased only the values of nitrate reductase genes (narG, narH) but not the ones of nitric oxide reductase (norB, norC). The nitrite reductase gen (nirK) was also significantly increased by the S treatment. In the same way, the relative abundance of C-cycling genes participating in organic matter decomposition (cellulose (bglX) and hemicellulose (FUCA) biodegradation or C fixation (ppc)) showed significantly higher values in the S treatment compared to the others. Regarding the P-cycling pathway genes, they were not significantly affected by any of the studied treatments. 3.5 Correlations among soil properties, plant biomass, and the fungal and bacterial communities in the rhizospheric soil Correlation heat maps were performed to explore the relationships between the fungal and bacterial communities with respect to the plant biomass and the soil chemical and biological properties (Fig. S4A, B). In general, fungi and bacteria showed stronger correlations with the soil biological properties than its chemical properties. With respect to the fungal community, it was observed that Mortierellomycota and Mucoromycota phyla and their respective genera, Mortierrella and Mucor were the ones that showed the highest significant relationship with urease and phosphatase activities, as well as with EC. The Ochroconis and Alternaria genera also revealed significant positive correlations with the same enzymatic activities, but with lower significance levels. On the other hand, only Geomyces genus revealed a significant positive correlation with b-Glucosidase. The majority of fungal phyla and genera showed negative correlations with phosphatase and urease activities and EC. In the case of bacterial phyla, Pseudomonadota phylum showed a significant positive correlation with phosphatase, urease, EC and Total N, while Actinomycetota was positively correlated with b-glucosidase and dehydrogenase activities (Fig. S4C, D). In contrast, Bacillota showed a more significant negative correlation for the same soil parameters than Pseudomonadota. Also, Chloroflexota revealed significant negative correlations with all the enzymatic activities measured. At the bacterial genus level, the most significant positive correlation was found between the unknown and the TM7a genera with phosphatase, urease and EC; Rhizobium and Flavitalea showed less notable positive correlations with the same parameters; and the Myxococcus was the only genus showing significant positive relations with soil chemical properties (TC, TK, TP). The rest of the bacterial genera showed negative correlations with phosphatase and urease activities and EC. Discussion New alternative crops capable to adapt to challenging climate conditions and with low input requirements are needed in order to redesign farming systems, especially in areas with degraded soils where intensive conventional cropping is not recommended or even non-economically viable, focusing on sustainable strategies and the preservation of natural resources. Purslane has been proposed as a novel crop that could be cultivated under these conditions, being a crop of high nutritional value and easy to manage for farmers that could be very responsive to organic fertilizers (Carrascosa et al., 2023a). Our results indicate that the treatments involving transformed slumgum (ST, SL) and the non-transformed liquid form (L) significantly increased purslane dry biomass and P uptake. In studies where the addition in the soil of others organic materials such as struvite has been assayed, e.g. González-Ponce et al. (2009) and Bastida et al. (2019), it was found that greater yield and plant P uptake were attributable to the higher plant tissues Mg uptake. In contrast to these reports, in our study we observed a decrease of Mg uptake in plants grown under all the treatments with slumgum, as well as a decrease of other macro and micronutrients such as K, Ca and Fe which were also decreased. Despite the observed increase in plant P content, we did not record a corresponding increase in the available soil P. This discrepancy could be mainly attributed to two reasons. Firstly, the P taken up by the crop resulted in decreased P contents in the soil. Secondly, a more plausible explanation is that in soils with a high pH, typically around 8, P could have precipitated as Ca and Fe phosphates; hence the lower availability in the soil (Kruse et al., 2015). Consequently, the recorded decrease in K, Ca and Fe content in plants could be due to the phosphate formation during slumgum mineralization. On the other hand, it was observed that solid slumgum (S), but not the transformed form (TS), decreased the plant biomass compared to the control plants. This outcome might be attributed to the presence of residual beeswax in this raw solid residue, potentially exerting detrimental effects and reducing the availability of soil nutrients to plants. In support of this finding, Cozzolino et al. (2016) reported that a specific molecular composition of the residue could adversely affect plant growth, counterbalancing the positive effects derived from the provision of carbon (C) and nutrients. Numerous investigations have demonstrated the positive impact of using organic residues or materials in agricultural ecosystems on soil fertility and environmental quality (Aytenew and Bore, 2020). Soil enzyme activities are considered sensitive indicators of soil fertility, as they catalyse key biochemical reactions involved in soil nutrient cycling and organic matter degradation (Nannipieri et al., 2012). In our study, we observed significant effects of the slumgum type on N cycling enzymes, specifically urease (Kandeler et al., 1999), and P cycling enzymes, namely phosphomonoesterase (Naseby and Lynch, 1997). However, we did not observe any significant impact on C cycling enzymes, such as β-glucosidase (Eivazi and Tabatabai, 1988). In general, both transformed and non-transformed solid slumgum (TS and S), as well as transformed and non-transformed liquid slumgum (TL and L), significantly increased phosphomonoesterase levels compared to the control treatment. However, the liquid treatments exhibited lower enzymatic activity than the solid treatments. Regarding urease, only the solid slumgum treatments (TS and S) showed significantly higher values than the control, whereas the liquid slumgum treatments (TL and L) did not show significant differences. Enhanced enzyme activities involved in nutrient cycling due to the application of organic residues have been previously reported in different soils (Dămătîrcă et al., 2023; Das et al., 2017; López-Piñeiro et al., 2011), especially the application of dried olive residues transformed with saprophytic fungi similarly to the treatments we used in the present work (Siles et al., 2014b) The observed increase in enzymatic activities could suggest higher turnover rates of soil P and N, with the effect being more pronounced in soils amended with solid slumgum compared to those amended with liquid slumgum (Das et al., 2017). In line with this statement, our findings revealed that only the transformed solid slumgum (ST) significantly increased dehydrogenase activity in the soil. Dehydrogenase is an enzyme that measures soil microbial activity through oxidoreductases (Nannipieri et al., 2012). Previous studies have attributed the increase in this activity to the proliferation of soil microorganisms due to higher availability of organic substrates, consequently resulting in increased levels of microbial biomass (Macci et al., 2012). In accordance with our findings, Siles et al. (2014b) also observed an increase in this activity in soils amended with residues of dried olive residues bio-transformed with saprotrophic fungi. However, we did not observe an effect of solid slumgum in its raw state (S) on this activity. It is suggested that the saprotrophic fungus P. chrysogenum may have a potential strategy in slumgum transformation, thereby producing readily available organic materials for soil microorganisms. This process involves the biodegradation of recalcitrant organic compounds and the mineralization of nutrients through a diverse set of extracellular enzymes, potentially influencing soil microbial activities (García-Sánchez et al., 2019). To delve into the effect of slumgum on plant yield through microorganisms, we studied the soil microbiome (Luo et al., 2022; Siles et al., 2014b). It is important to emphasize that the observed changes in the purslane rhizosphere microbial community are the direct result of slumgum's impact on the resident soil microbiome. Notably, these changes do not arise from slumgum itself, as the inhabiting microbes within it were inactivated by sterilization before soil application, aimed at eliminating P. chrysogenum as the final step. Therefore, slumgum's influence on the natural soil microbiome most likely is related to its physical and chemical characteristics, which may include the presence of compounds like cellulose, hemicellulose, among others (van der Wal et al., 2013), given its origin as residual material left after beeswax removal from honeycomb. In relation to the fungal community, our findings revealed a significant change in the fungal community composition upon the addition of slumgum to the soil, resulting in decreased fungal diversity and richness across all slumgum treatments, except for the non-transformed liquid slumgum (L). Other studies where residues transformed with saprobic fungi were applied in Mediterranean soils have reported a decrease in fungal functional diversity and changes in the structure of fungal communities (Siles et al., 2014b), whereas others have found no changes in fungal diversity and richness (Siles et al., 2014a). Moreover, Luo et al. (2022) conducted a study in soils amended with sterile composts and they did not observe changes in microbial communities. They suggested that the reduction in fungal diversity might be attributed to the adaptation and selective proliferation of specific fungal groups toward the new organic source at the expense of the added nutrients. This phenomenon might be applicable to our study, as we observed, consistently with Siles et al. (2014b), a negative correlation between fungal richness and soil total N (R pearson -0.534, P<0.05). Furthermore, our study revealed a shift in the functional fungal guild due to the effect of slumgum type and transformation with P. chrysogenum , with an increase in saprotrophs and a decrease in plant pathogens and parasites fungi being recorded. This finding is aligned with studies such as the ones of Xiong et al. (2017) and Siegel-Hertz et al. (2018), who attributed the increase in saprotrophic fungi to the reduction of plant pathogens, due to competitive interactions for space and resources between pathogenic and saprotrophic fungi (Kepler et al., 2017; Shu et al., 2022). Numerous studies over the years have documented how the use of organic residues, such as compost, manure, bone meal, poultry litter slurry, straw, wood residues, among others, can exert suppressive effects on certain phytopathogenic microorganisms by modifying indigenous microbial communities and stimulating microorganisms antagonistic to soil-borne pathogens(Chen et al., 2023; Reardon and Wuest, 2016; Shu et al., 2022; Tao et al., 2020). In contrast, in our study the incorporation of slumgum in its raw state (S) resulted in a diminished response to saprotrophic fungi. This decrease might be attributed to the presence of higher concentration of inhibitory secondary metabolites, such as hydrophobic compounds present in the beeswax remained within the slumgum. Consequently, the chemical composition and quality of organic residues, alongside the saprotrophic capabilities of microorganisms inhabiting the rhizosphere, emerge as pivotal factors influencing the microbial communities of residue decomposers (Bonanomi et al., 2018; Clocchiatti et al., 2020; Liu et al., 2021). On the other hand, the composition of root exudates is an important factor in selecting a subset of soil microorganisms (Afzal et al., 2019; Haichar et al., 2014). Literature reports have shown an increase of saprotrophic fungi involved in consuming rhizodeposits in soil amended with sawdust (Clocchiatti et al., 2021). These fungi are known to assimilate soluble sugars, utilizing labile monomeric exudate compounds (de Vries and Caruso, 2016). In our study, the observed increase in plant yield might have led to a higher quantity of root exudates. Consequently, the complex soluble compounds within these exudates could have significantly influenced interactions among rhizosphere microorganisms (Zhalnina et al., 2018). Moreover, our study revealed a positive correlation between the relative abundance of saprotrophic fungi and plant dry biomass (R pearson = 0.73; P < 0.001), while indicating a negative correlation between plant dry biomass and the relative abundance of plant-pathogenic fungi (R pearson = -0.74; P < 0.001) and parasites (R pearson = -0.65; P = 0.003). Interestingly, our study revealed an increase in the Mortierellomycota and Mucoromycota phyla under the treatment involving solid slumgum. Clocchiatti et al. (2020) attributed the rise in Mortierellomycota to the introduction of nitrogen-rich organic materials derived from soil amended with cover crop fragments, paper pulp, and wood sawdust. In our investigation, these phyla exhibited a strong correlation with increased enzymatic activities, specifically urease (R pearson = 0.82; P < 0.001 for Mortierellomycota; R pearson = 0.93; P < 0.001 for Mucoromycota) and phosphatase (R pearson = 0.82; P < 0.001 for Mortierellomycota; R pearson = 0.88; P < 0.001 for Mucoromycota). A detailed analysis of the effect of slumgum on the fungal genus level revealed that the relative abundance of Alternaria spp. and Ochroconis spp. increased with the addition of non-transformed solid slumgum (S) to the soil. However, these abundances decreased notably following the transformation of slumgum and the application of liquid slumgum (TS, TL, and L). Therefore, applying this residue in its raw state might prove inconvenient, as it could potentially negatively impact purslane development and biomass, as indicated in our study by the observed negative correlation between plant biomass and these fungal genera (R pearson = -0.632; p = 0.004 for Alternaria and R pearson = -0.561; p = 0.012 for Ochroconis ). The bacterial community displayed a pattern similar to that of the fungal community, undergoing changes in response to all slumgum addition treatments, with the most significant differences observed in treatments involving the addition of solid slumgum (S and ST) compared to the control. In contrast to findings from other studies (Shu et al., 2022; Zhaoxiang et al., 2020), we did not observe a higher sensitivity of the soil bacterial community compared to the fungal community in response to organic amendment addition. However, it's essential to note that these studies might not be directly comparable to ours. The previous studies were conducted with organic residues-associated active microbiota, potentially introducing a competition effect or alterations in habitat for the active microbiota of the residue (Luo et al., 2022). In our study, the predominant bacterial phyla identified were Actinomycetota, Pseudomonadota, and Bacillota, showing significant variations based on the type of slumgum applied (solid or liquid) and the transformation with P. chrysogenum . Actinomycetota and Pseudomonadota, known as fast-growing microbes, have previously been linked to cellulose degradation (Kramer et al., 2016). We found that these two bacterial phyla exhibited a positive correlation with increased microbial activity in the rhizosphere. Particularly noteworthy was the significant increase in Actinomycetota under the TS treatment, which in conjunction with the substantial rise in the Mortierellomycota fungal phylum, suggests that Actinomycetota may contribute not only to slumgum decomposition but also to the consumption of breakdown products resulting from fungal slumgum decomposition (de Menezes et al., 2017), given their known engagement in fungal-bacterial interactions (Sathya et al., 2017). Pseudomonadota have also been linked with the decomposition of organic residues, suggesting their role in providing an additional nitrogen source to fungal saprotrophs in exchange for breakdown products from residues (Johnston et al., 2016). Consequently, the significant increase in Pseudomonadota and N content observed in the rhizosphere only under the non-transformed slumgum (S) treatment could elucidate their function as N-suppliers and decomposers within the microenvironment generated under this specific treatment. Notably, only the TS treatment significantly increased the N content in purslane, coinciding with the highest relative abundance of the Rhizobium genus. It is plausible that these plant growth-promoting rhizobacteria played a significant role in facilitating plant N uptake and promoting plant growth through direct and/or indirect effects (Adesemoye et al., 2010). The non-transformed solid slumgum (S) significantly influenced bacterial gene expression, particularly the genes related to N recycling in the denitrification process, e.g. nitrate reductase (narG, narH), nitrite reductase (nirk), and nitric oxide reductase (norB, norC). However, there was no significant effect on the nosZ gene, which plays a vital role in reducing nitrous oxide (N 2 O) to nitrogen gas (N 2 ) in the last step of denitrification and acts as unique known sink for N 2 O in soil (Krause et al., 2017). Therefore, the application of non-transformed slumgum (S) to soil might contribute to increased greenhouse gas emissions, specifically through N 2 O, due to the enhanced expression of functional genes involved in nitrite and nitric oxide reduction. However, this increased expression does not affect the final step, thereby leading to the accumulation of N 2 O. According to Yoon et al. (2015), denitrification typically prevails in soils characterized by low C/N ratios, which is aligned with our observations in the non-transformed slumgum (S) treatment. Moreover, non-transformed solid slumgum (S) increased the expression of ppc genes associated with C fixation, as well as genes related to the degradation of cellulose (beta-glucosidase, bglx) and hemicellulose (FUCA). This increased gene expression could be attributed to the greater complexity of non-transformed slumgum in comparison to other treatments. The non-transformed slumgum lacks P. chrysogenum transformation, thus containing these complex polysaccharides, which potentially foster a higher abundance of bacteria capable of metabolizing them (Jiménez et al., 2014). Conclusions The use of bio-waste residuals as soil amendments is a novel agronomic practice that can significantly contribute to the improvement of soil fertility and quality improvement while at the same time facilitates the concepts of circular economy and conservation of natural resources. To the best of our knowledge, this is the first study reporting the influence of slumgum, a residue coming from the beekeeping industry, on the purslane biomass and its rhizosphere microbial community. The application of solid and liquid slumgum transformed with P. chrysogenum (TS and TL, respectively) and liquid slumgum (L) significantly increased the purslane shoot biomass and the foliar P content. The different slumgum amendments assayed also changed and clearly differentiated the purslane bacterial and fungal community composition, despite the very short period experimental period (6 weeks). A change in the functional fungal guilds increasing the abundances of saprotrophs and decreasing plant pathogens under the treatments TS, TL and L was recorded. The solid slumgum transformed with P. chrysogenum (TS) was the most efficient to significantly increase the soil enzymatic activities related to C, N and P cycles. Instead, the slumgum in its raw state (S) increased the abundance of the majority of bacterial genes related to the denitrification process, which could contribute to the negative effect of N 2 O gas emissions. Therefore, solid and liquid slumgum transformed with P. chrysogenum and the liquid non-transformed slumgum (TS, TL and L, respectively) can be considered as effective sustainable organic fertilizers, additive or soil amendments that can improve crop yield and beneficially affect soil microbial communities. However, further studies are needed to extrapolate the beneficial results obtained to field conditions, while more crops should be tested to validate the recorded effects. Declarations Data availability The raw sequencing data is available from the the NCBI Sequence Read Archive repository (www.ncbi.nlm.nih.gov/sra) under the BioProject PRJNA1051377. Author contribution Angel Carrascosa: investigation, empirical analysis and data curation. Jose Antonio Pascual: writing—review and editing. Jessica Cuartero: sequencing analysis and review. Inmaculada García-Romera and Gloria Andrea Silva-Castro: methodology and experimental design. Ana De Santiago: soil samples analysis. 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Zhaoxiang, W., Huihu, L., Qiaoli, L., Changyan, Y., and Faxin, Y.: Application of bio-organic fertilizer, not biochar, in degraded red soil improves soil nutrients and plant growth, Rhizosphere, 16, 100264, https://doi.org/10.1016/j.rhisph.2020.100264, 2020. Additional Declarations The authors declare no competing interests. Supplementary Files Suppplementalmaterial.docx Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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TS= solid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e ; L= liquid slumgum; TL= liquid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e ) (n = 5). Significance of effects of slumgum type, transformation with \u003cem\u003eP.\u003c/em\u003e \u003cem\u003echrysogenum\u003c/em\u003e and their interaction on the measured variable is also shown (P- values). For each treatment, bars followed by the same letter are not significantly different according to Tukey's HSD-test (p \u0026lt; 0.05). Bars represent standard error.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4480989/v2/156d87c0eebfba43765e95b5.png"},{"id":64048520,"identity":"51e87c8d-e5d0-4c74-92f3-9b8140dbc99a","added_by":"auto","created_at":"2024-09-05 15:11:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36717,"visible":true,"origin":"","legend":"\u003cp\u003eBiological properties of the rhizosperic soil of purslane plants grown under different slumgum treatments (S= solid slumgum; TS= solid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e ; L= liquid slumgum; TL= liquid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e ). Mean ± standard error, n = 5. Significance of effects of slumgum type, transformation with \u003cem\u003ePenicillium\u003c/em\u003e \u003cem\u003echrysogenum\u003c/em\u003e and their interaction on the measured variables is also shown (P- values). For each treatment, bars followed by the same letter are not significantly different according to Tukey's HSD-test (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4480989/v2/c9f5bce58bf03003ab85b6ec.png"},{"id":64048185,"identity":"183091c4-037d-41fa-aec3-90b3213785df","added_by":"auto","created_at":"2024-09-05 15:03:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55849,"visible":true,"origin":"","legend":"\u003cp\u003eNon-metric multidimensional scaling (NMDS) analysis on a Bray-Curtis dissimilarity matrix based on the ASVs dataset retrieved from the fungal (A) and bacterial (B) communities of the rhizospheric soil of purslane grown under different slumgum treatments (S= solid slumgum; TS= solid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e ; L= liquid slumgum; TL= liquid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e ). Ellipsoids represent 95% confidence for each treatment mean.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4480989/v2/74d61c4df8d80a0c5bf37f6a.png"},{"id":64048183,"identity":"8e4aa5fe-92c2-4487-a069-aa8253abf905","added_by":"auto","created_at":"2024-09-05 15:03:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56088,"visible":true,"origin":"","legend":"\u003cp\u003eMean relative abundance of fungal OTUs belonging to six major functional guilds in rhizospheric soil under different slumgum treatments (S= solid slumgum; TS= solid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e ; L= liquid slumgum; TL= liquid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e ). N=4 soil samples per treatment. For each functional guilds, significant differences were assessed by the Tukey´s post-hoc test calculated at \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05 and indicated by an asterisk, *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4480989/v2/43990d403a1db68570459436.png"},{"id":64048837,"identity":"54403ac0-b135-460a-83f5-35b7d593ccd1","added_by":"auto","created_at":"2024-09-05 15:19:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":937191,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4480989/v2/ffeb2404-9690-4f1e-92d7-9d12a1fe8ae9.pdf"},{"id":64048187,"identity":"060f0871-7d55-4071-8cc0-d716e6c4447b","added_by":"auto","created_at":"2024-09-05 15:03:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1621537,"visible":true,"origin":"","legend":"","description":"","filename":"Suppplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4480989/v2/8bd81e7dfbe756bbaa1ad77a.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eResponse of Portulaca olearacea and its rhizospheric microbiome to the application of slumgum from beekeeping industry transformed with Penicillium chrysogenum\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePurslane (\u003cem\u003ePortulaca oleracea\u003c/em\u003e L.) is an important nutritional and medicinal plant, renowned for its high content of omega-3 fatty acids and other bioactive compounds\u0026nbsp;(Petropoulos et al., 2016, 2015). Due to its beneficial health properties, there is a renewed interest in its integration into commercial small-scale farming systems\u0026nbsp;(Carrascosa et al., 2023b; Chrysargyris et al., 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, purslane is recognised as a wild edible plant (WEP) which is distributed worldwide, and it grows naturally as a weed without human intervention in various agroecosystems. Purslane is also well adapted to generally adverse conditions such as drought, high temperatures, or moderate salinity levels. It has relatively low nutritional requirements, capable of achieving high yields even with low fertilisation rates\u0026nbsp;(Carrascosa et al., 2023a; Jin et al., 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese characteristics make purslane a candidate crop to be utilised in vulnerable areas or no chemical inputs can be applied to avoid contamination of aquifers and surrounding water bodies, or in marginal areas where soils are deficient in nutrients\u0026nbsp;(Cui et al., 2020; Jang and Chen, 2015). In these areas, new strategies for fertilisation should be implemented using organic amendments as substitutes of synthetic fertilizers\u0026nbsp;(Alburquerque et al., 2012; Celestina et al., 2019; Reyes-Torres et al., 2018); and in particular new added-value ones could widen the options to be used under different cropping scenarios.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is well established that organic amendments (sewage sludge, pruning composts, farmyard manure, biochar, poultry manure, etc.) are a good source of essential plant nutrients and if managed properly, they can improve crop production\u0026nbsp;(Ahmad et al., 2022; Luo et al., 2018). Additionally, they may enhance the physical, chemical, and biological properties of soil, presenting an eco-friendly approach to maintaining and enhancing soil fertility while contributing to natural resources preservation at the same time\u0026nbsp;(Diacono and Montemurro 2010; Aytenew and Bore 2020; Moreno et al. 2022). Moreover, an increase in the content of soil organic matter contributes to carbon (C) sequestration, reducing the CO\u003csub\u003e2\u003c/sub\u003e atmospheric level and adverse greenhouse effects, helping to mitigate the climate change\u0026nbsp;(Lorenz and Lal, 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the beekeeping industry, \u0026apos;slumgum\u0026apos; is generated as solid organic waste during the beeswax-rendering process from old scraped honeycombs, which are replaced every three or four years. This waste exhibits a dark (brown-black) colour with an appearance that falls somewhere between animal manure and vegetable compost. It is primarily composed of brood cocoons, moths, larvae of \u003cem\u003eGalleria melonella\u003c/em\u003e L., molts, dead bees, bee droppings, pollen, propolis, and small proportions of non-extractable wax, among other elements. Usually, it is stored outdoors and currently has neither has any particular use nor it is recycled\u0026nbsp;(Morales-Corts et al., 2014, 2010). The worldwide production of slumgum is estimated to 93,000 tons per year\u0026nbsp;(Faostat: Agriculture Data). Until now, few studies on slumgum residue have been reported in the literature. In particular Morales-Corts et al. \u0026nbsp;(2010, 2011, 2014)\u0026nbsp;suggested that slumgum could be a promising candidate as an organic fertilizer due to its high content of organic matter, essential nutrients, and micronutrients. However, to the best of our knowledge the impact of slumgum on agricultural soils health is scarcely studied so far.\u003c/p\u003e\n\u003cp\u003eSimilar residues to slumgum such as dry olive residues\u0026nbsp;(Siles et al., 2014b), are usually transformed using saprophytic fungi (\u003cem\u003eFusarium\u0026nbsp;\u003c/em\u003espp, \u003cem\u003ePhanerochaete\u0026nbsp;\u003c/em\u003espp, \u003cem\u003ePleurotus\u0026nbsp;\u003c/em\u003espp, \u003cem\u003eTrametes versicolor\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003ePenicillium chrysogenum,\u0026nbsp;\u003c/em\u003eetc.) as a waste or residue pre-treatment prior of being used as organic amendments\u0026nbsp;(Garc\u0026iacute;a-S\u0026aacute;nchez et al., 2014; Reina et al., 2013; Sampedro et al., 2007; Siles et al., 2015). The transformation process improves the quality of organic amendments, since it mobilizes nutrients, thus promoting a more efficient use of nutrients by plants. Thus, this process could be considered as an environmentally friendly and reliable bio-waste management option\u0026nbsp;(Siles et al., 2015). Therefore, it seems to be an interesting approach to explore the effect of slumgum as potential new organic fertilizer, amendment or additive in agricultural soils, and its response on plants growth through soil rhizosphere.\u003c/p\u003e\n\u003cp\u003eSoil microbial communities play a vital role in the functions of soil ecosystems since microbes participate in processes of organic matter decomposition, nutrient cycling, and plant productivity\u0026nbsp;(Naylor et al., 2022; Saleem et al., 2019; Xia et al., 2020). Moreover, soil microorganisms rapidly respond to environmental shifts and could be considered as good indicators of soil quality.\u0026nbsp;In a recently meta-analysis conducted by Shu et al.\u0026nbsp;(2022), it was reported that the use of organic amendments enhances the soil microbial diversity and functionality. However, the response of microbial communities to the addition of organic residues can be affected by diverse factors: soil properties, experimental and ambient conditions, residue type, doses, crop type, etc.\u0026nbsp;(Chen et al., 2023; Li et al., 2021; Ning et al., 2020; Shu et al., 2022). Thus, a detailed knowledge of microbial composition, diversity and function as well as properties which determine soil fertility and plant growth in soils amended with slumgum is essential, in order to understand the patterns and relationship between soil functioning and processes mediated by microorganisms in agroecosystems. This knowledge could have important implications for improving more sustainable agricultural management practices and conserving Mediterranean soil biodiversity in a climate changing environment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe objective of the present study is to explore the impact of short-term application of solid and liquid slumgum, both non-transformed and transformed with \u003cem\u003ePenicillium\u003c/em\u003e \u003cem\u003echrysogenum\u003c/em\u003e, on the response of purslane yield and its relationship on rhizosphere soil. The nutritional composition, enzymatic activities involved in the P, N and C cycles, and fungal and bacterial community composition, diversity and functionality in the rhizosphere were deeply studied to demonstrate the beneficial effect of slumgum on purslane development and to determine the necessity or not of fungal transformation prior to its application in the soil.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eQuisque cursus massa sed urna congue, ac convallis neque consectetur. Proin faucibus neque non metus mollis, suscipit pretium nisl blandit. In hac habitasse platea dictumst.\u003c/p\u003e\n\u003ch2\u003e2.1 Experimental design and sampling\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe topsoil (0\u0026ndash;20 cm) utilized in the experiment was collected from the CEBAS-CSIC Experimental Farm (Santomera, Murcia, Spain 38\u0026deg;06\u0026apos;14.0\u0026quot;N 1\u0026deg;02\u0026apos;00.1\u0026quot;W), it corresponds to a semi-arid Mediterranean climate with an annual mean temperature of 19.2 \u0026ordm;C, annual rainfall of 300 mm, and a potential evapotranspiration of 1000 mm y\u003csup\u003e-1\u003c/sup\u003e. The soil is classified as Lithic xeric haploxeroll, with clay-loam texture (41% illite, 17% smectite, and 30% palygorskite). The chemical characteristics of the soil were: total nitrogen 5.7 g kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e, available phosphorous 6.0 mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e, available potassium 33.9 mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e, organic matter 12.6 g kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e, CaCO\u003csub\u003e3\u003c/sub\u003e 3 g kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e, pH 7.14 and electrical conductivity 0.10 dS m\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe organic beekeeping residue called \u0026ldquo;slumgum\u0026rdquo; used in this experiment was collected from the \u0026ldquo;Apizafra S.L.\u0026rdquo; beekeeping company, located in Albudeite, Murcia (Spain). The slumgum residue was transformed using the saprophytic fungi: \u003cem\u003ePenicillium chrysogenum\u003c/em\u003e (EEZ-28\u003cem\u003e).\u003c/em\u003e The mycotransformation of the solid slumgum (S) was carried out by solid-state fermentation as described by Reina (Reina et al., 2013) with some modifications. Firstly, fungi were inoculated into flasks with 18 g of barley and 30 mL of sterile water for one week. After this period, 10 g of previously sterilised solid slumgum and 2.5 mL of water were added and incubated at 28 \u0026ordm;C for 4 weeks. The resulting biotransformed solid slumgum (TS) was then homogenised and sterilised. Furthermore, the liquid slumgum extract (L) was obtained by mixing slumgum with distilled water 1:3 (w/v ratio) and agitated for 24 hours and then filtrated. The extract was also biotransformed through the same strain of \u003cem\u003ePenicillium chrysogenum\u003c/em\u003e (EEZ-28)\u003cem\u003e,\u0026nbsp;\u003c/em\u003eusing a basal medium (MB) for 7 days at 80 rpm and at 28 \u0026deg;C for 14 days. After this period, slumgum liquid extract was diluted to half, (TL) and sterilised. Both transformed products, TS and TL, were stored at 4 \u0026deg;C, while both the raw materials before biotransformation (S and L, respectively) were autoclaved and then, stored at 4 \u0026deg;C. The main chemical characteristics of the studied materials are shown in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin:0in;text-align:justify;line-height:150%;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\n \u003ctable style=\"border: none;width:518.25pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width:518.25pt;border:none;border-bottom:double windowtext 1.5pt;padding:0in 3.5pt 0in 3.5pt;height:.15in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;text-align:justify;font-size:12px;font-family:\"Times New Roman\",serif;font-weight:bold;'\u003eTable 1. Chemical and nutrient composition of the different types of slumgum used in this experiment.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.85pt;border:double windowtext 1.5pt;border-top:none;padding:0in 3.5pt 0in 3.5pt;height:.15in;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width:74.8pt;border-top:none;border-left:none;border-bottom:double windowtext 1.5pt;border-right:double windowtext 1.5pt;padding:0in 3.5pt 0in 3.5pt;height:.15in;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eSolid Slumgum\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003e(S)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:95.3pt;border-top:none;border-left:none;border-bottom:double windowtext 1.5pt;border-right:double windowtext 1.5pt;padding:0in 3.5pt 0in 3.5pt;height:.15in;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eSolid Slumgum Transformed\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003e(TS)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border-top:none;border-left:none;border-bottom:double windowtext 1.5pt;border-right:double windowtext 1.5pt;padding:0in 3.5pt 0in 3.5pt;height:.15in;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eLiquid extract of Slumgum\u003c/span\u003e\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cem\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003e(L)\u003c/span\u003e\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:114.25pt;border-top:none;border-left:none;border-bottom:double windowtext 1.5pt;border-right:double windowtext 1.5pt;padding:0in 3.5pt 0in 3.5pt;height:.15in;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eLiquid extract of Slumgum Transformed\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003e(TL)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 12.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eMoisture (%)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 12.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e23.5 \u0026plusmn; 0.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 12.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e23.5 \u0026plusmn; 0.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 12.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 12.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003epH \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e4.93 \u0026plusmn; 0.62\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e8.47 \u0026plusmn; 0.52\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e6.25 \u0026plusmn; 0.33\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e6.70 \u0026plusmn; 0.28\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 11.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eElectric conductivity (EC) (dS m\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 11.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1.74 \u0026plusmn; 0.42\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 11.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e2.60 \u0026plusmn; 0.50\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 11.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3.71 \u0026plusmn; 0.11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 11.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3.10 \u0026plusmn; 0.15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal carbon (TC) (%)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e55.60 \u0026plusmn; 1.73\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e48.33 \u0026plusmn; 1.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1.27 \u0026plusmn; 0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.37 \u0026plusmn; 0.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal organic carbon (TOC) (%) \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e53.49 \u0026plusmn; 1.25\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e48.26 \u0026plusmn; 1.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal nitrogen (TN) (%)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3.61 \u0026plusmn; 0.08\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3.63 \u0026plusmn; 0.05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.12 \u0026plusmn; 0.05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.06 \u0026plusmn; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eRelation TOC/TN\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e14.82 \u0026plusmn; 0.46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e13.27 \u0026plusmn; 2.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal P (mg kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3500 \u0026plusmn; 39\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3700 \u0026plusmn; 40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e500 \u0026plusmn; 29\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e346 \u0026plusmn; 19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal K (mg kg\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3900 \u0026plusmn; 40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e5500 \u0026plusmn; 49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e830 \u0026plusmn; 28\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e745 \u0026plusmn; 25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Ca (mg kg\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3600 \u0026plusmn; 87\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e2250 \u0026plusmn; 79\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e189 \u0026plusmn; 19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e57 \u0026plusmn; 9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Mg (mg kg\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1000 \u0026plusmn; 22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1100 \u0026plusmn; 26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e110 \u0026plusmn; 12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e67 \u0026plusmn; 9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Na (mg kg\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e190 \u0026plusmn; 3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e240 \u0026plusmn; 5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e35 \u0026plusmn; 3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e46 \u0026plusmn; 4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Fe (mg kg\u003csup\u003e-1\u003c/sup\u003e)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e499 \u0026plusmn; 8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e330 \u0026plusmn; 7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e10.46 \u0026plusmn; 0.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e5.74 \u0026plusmn; 0.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Cu (mg kg\u003cstrong\u003e\u003csup\u003e-\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e1\u003c/sup\u003e)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e14.46 \u0026plusmn; 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e11.93 \u0026plusmn; 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.19 \u0026plusmn; 0.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e15.87 \u0026plusmn; 2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Mn (mg kg\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e29.76 \u0026plusmn; 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e25.72 \u0026plusmn; 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e2.85 \u0026plusmn; 0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1.66 \u0026plusmn; 0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Al (mg kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e257 \u0026plusmn; 5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e209 \u0026plusmn; 4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e8.09 \u0026plusmn; 0.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1.58 \u0026plusmn; 0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Zn (mg kg\u003csup\u003e-1\u003c/sup\u003e)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e255 \u0026plusmn; 3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e176 \u0026plusmn; 2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e11.63 \u0026plusmn; 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e9.59 \u0026plusmn; 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Pb (mg kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3.74 \u0026plusmn; 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e3.17 \u0026plusmn; 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Ni (mg kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1.86 \u0026plusmn; 0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1.15 \u0026plusmn; 0.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.09 \u0026plusmn; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.06 \u0026plusmn; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Cd (mg kg\u003csup\u003e-1\u003c/sup\u003e)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.025\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.025\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Co (mg kg\u003csup\u003e-1\u003c/sup\u003e)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;0.57 \u0026plusmn; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.21 \u0026plusmn; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-top: none;border-left: 1.5pt double windowtext;border-bottom: none;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Li (mg kg\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.91 \u0026plusmn; 0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1.08 \u0026plusmn; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.09 \u0026plusmn; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e0.12 \u0026plusmn; 0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 148.85pt;border-right: 1.5pt double windowtext;border-bottom: 1.5pt double windowtext;border-left: 1.5pt double windowtext;border-image: initial;border-top: none;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003eTotal Se (mg kg\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-left: none;border-bottom: 1.5pt double windowtext;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95.3pt;border-top: none;border-left: none;border-bottom: 1.5pt double windowtext;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e1.14 \u0026plusmn; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1.5pt double windowtext;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114.25pt;border-top: none;border-left: none;border-bottom: 1.5pt double windowtext;border-right: 1.5pt double windowtext;padding: 0in 3.5pt;height: 0.15in;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style=\"color:black;\"\u003e\u0026lt; 0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eIn May 2022, purslane (\u003cem\u003ePortulaca oleracea\u0026nbsp;\u003c/em\u003eL.) seeds, supplied by the Department of Agriculture Crop Production and Rural Environment (University of Thessaly, Greece) were sown under nursery conditions, in peat substrate trays for 3 weeks until reached the proper size for transplanting to the 0.3 L plastic pots. Each pot was filled with a soil:sand mixture (1:1 w:w) and one plant was transplanted into each one of them. Then plants were treated with the biotransformed and non-transformed slumgum (TS, TL, S and L, respectively) and a non-slumgum treatment was also used as a control treatment. For each treatment seven replicates were established (n=7; 35 pots in total). For soil amendment, both solid treatments (TS and S) were\u0026nbsp;mixed with the soil:sand mix at a rate of 5% (17.5 g), at the beginning of the experiment. Similarly, both liquid treatments (TL and L) were added at a rate of 5% (17.5 mL) to the pots through the irrigation water one week after transplantation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted for six weeks without additional fertilization under greenhouse conditions at CEBAS-CSIC Experimental Farm located in Santomera, Murcia, Spain. The day/night temperature was maintained at 30 \u0026ordm;C/25 \u0026ordm;C. Plants were regularly irrigated through sprinklers maintaining the 60% of water holding capacity. When the experiment concluded, plants were harvested, and rhizospheric soil was collected for each replicate. The soil was sieved through a 2-mm mesh and divided in a portion stored at 4 \u0026ordm;C for chemical and biochemical analysis and other at -20 \u0026ordm;C for molecular analysis.\u003c/p\u003e\n\u003ch2\u003e2.2 Analyses of plant tissue\u003c/h2\u003e\n\u003cp\u003eShoot fresh and dry biomass (dried at 105 \u0026ordm;C for 5 h) was measured. The dried shoots were then ground using a mill before nutrient analysis. Total N in the shoots was determined using a TruSpec CN Analyzer (LECO, St. Joseph, MI, USA). Analysis of shoot total P, K, Ca, Mg and Fe contents was performed using ICP-OES (ICAP 6500 DUO, Thermo Fisher Scientific, Hayward, California, USA).\u003c/p\u003e\n\u003ch2\u003e2.3\u0026nbsp;Chemical, biochemical, and biological analyses of rhizospheric soil\u003c/h2\u003e\n\u003cp\u003eSoil pH and electrical conductivity\u0026nbsp;were determined at 1:2.5 and 1:5 soil:water ratios, respectively. Available P was extracted with bicarbonate\u0026nbsp;following the method of\u0026nbsp;(Olsen et al., 1954) and determined using molybdate‐reactive P as described by Murphy and Riley (1962).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Organic carbon (%) was measured using the dichromate oxidation method (Walkley and Black, 1934). The organic carbon values were transformed into organic matter using an empirical factor (1.724). Total nitrogen (TN) and total carbon (TC) were determined using an elemental CHNS-O analyzer (EA- 1108, Carlo Erba, Barcelona, Spain); total phosphorous (TP) and total potassium (TK) were assessed using ICP-OES (ICAP 6500 DUO, Thermo Fisher Scientific, Hayward, California, USA). Dehydrogenase activity (DHA) in the soil was determined measuring the amount of triphenyl formazan (TPF) released during incubation with 2,3,5 triphenyltetrazolium chloride (Tabatabai, 2018). The urease activity was assessed as described by (Kandeler et al., 1999), utilizing urea as a substrate (Nannipieri et al., 1980); \u0026beta;-glucosidase activity (\u0026beta;-Glu) was determined using p-nitrophenyl-\u0026beta;-D-glucopyranoside (Eivazi and Tabatabai, 1988), and alkaline phosphomonoesterase activities were assessed with p-nitrophenyl phosphate disodium as a substrate (Naseby and Lynch, 1997) with subsequent colorimetric determination of the p-nitrophenol released.\u003c/p\u003e\n\u003ch2\u003e2.4 DNA extraction and Illumina sequencing\u003c/h2\u003e\n\u003cp\u003eDNA was extracted from 0.25 g of rhizospheric soil using the DNeasy PowerSoil DNA Isolation kit (Qiagen), following the manufacturer\u0026acute;s protocol. DNA yield and quality were assessed through electrophoresis in 0.8% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) agarose gels stained with GelRed and visualized under UV light. Additionally, DNA quality was verified using a Qubit 3.0 fluorometer (Life Technologies, Grand Island, NY).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDNA from each sample was sequenced on the Illumina MiSeq platform at the genomics service of the Institute of Parasitology and Biomedicine \u0026ldquo;L\u0026oacute;pez Neyra\u0026rdquo; (CSIC), Granada, Spain. Prokaryotic libraries were constructed by amplifying the hyper-variable V3\u0026ndash;V4 regions of the 16S rRNA gene using the primer pair, 341F and 806R, according to Takahashi et al. (2014). These amplicons were tagged for attachment to peptide nucleic acid\u0026nbsp;(PNA) PCR clamps to reduce amplification of plastid and mitochondrial DNA (Lundberg et al., 2013). \u0026nbsp;Fungal libraries were constructed by amplifying the ITS2 region with the primer pair ITS4 (White et al., 1990) and fITS7 (Ihrmark et al., 2012). Both runs were sequenced using a paired-end 2x300bp (PE 300) strategy.\u003c/p\u003e\n\u003ch2\u003e2.5 Sequencing data processing\u003c/h2\u003e\n\u003cp\u003eThe raw sequence data from the sequencer were processed using QIIME version 2022.2 (Caporaso et al., 2010). Before the analysis, raw sequences were checked using the fastqQC program (FastQC: \u0026nbsp;A Quality Control Tool for High Throughput Sequence Data). After that, sequences were uploaded to QIIME and primers were removed using cutadapt (Martin, 2011), removing sequences with less than 250 pb. To denoise the reads, the DADA2 algorithm was employed (Callahan et al., 2016) and sequences were truncated to an average length with Q score \u0026gt; 30. The resulting amplicon sequence variants (ASVs) were classified using SILVA v138 released in November 2020 or UNITE v9 released in October 2022 in the bacterial and fungal community, respectively.\u0026nbsp;Functional analysis of the bacterial community was performed using the PICRUSt2 algorithm (Douglas et al., 2020), by predicting N, C, and P bacteria metabolism.\u0026nbsp;Fungal functional guilds were determined by using the genus-guild database Fungal Traits (P\u0026otilde;lme et al., 2020).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.6 Statistical analyses\u003c/h2\u003e\n\u003cp\u003eA two-way ANOVA was performed to determine the influences of the slumgum type and transformation with\u0026nbsp;\u003cem\u003eP. chrysogenum\u003c/em\u003e,\u0026nbsp;as well as their interactions, through a Tukey\u0026rsquo;s HSD test (Honestly Significant Difference, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u0026nbsp;Correlation analyses were performed using Pearson\u0026apos;s method. For statistical analyses IBM-SPSS v.29 was utilized.\u0026nbsp;For microbial communities\u0026apos; datasets, the Amplicon Sequence Variants (ASVs) per sample were rarefied to mitigate potential bias in further analyses arising from variations in sequencing effort among samples. A non-metric multidimensional scaling ordination (NMDS) based on the Bray-Curtis distance was carried out to compare the composition of the bacterial and fungal communities. This analysis utilized the \u0026apos;metaMDS\u0026apos; function implemented in the \u0026apos;vegan\u0026apos; package for R (Oksanen et al., 2019). The effect of the two experimental factors \u0026ndash; slumgum type and transformation with\u0026nbsp;\u003cem\u003eP. chrysogenum\u003c/em\u003e- on the rhizosphere bacterial and fungal communities\u0026rsquo; composition and structure were tested using a permutational multivariate analysis of variance (perMANOVA) with 999 permutations on the same Bray-Curtis matrix using the \u0026ldquo;adonis\u0026rdquo; function in \u0026ldquo;vegan\u0026rdquo; package for R (Oksanen et al., 2019). Heat maps were also constructed using the \u0026ldquo;vegan\u0026rdquo; package for R.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e3.1\u0026nbsp;Impacts of the experimental factors on plant parameters\u003c/h2\u003e\n\u003cp\u003eShoot dry biomass was significantly affected by slumgum type (solid or liquid), \u003cem\u003eP. chrysogenum\u003c/em\u003e transformation and the interaction between both factors (Fig. 1). The dry purslane biomass under TS, L and TL treatments showed significantly higher values than control (an increase between 60-77%); however, the application of non-transformed solid treatment (S) significantly reduced dry mass compared to the control (69%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe slumgum type had a significant effect on the content of foliar P, K , Ca and Mg; whereas its fungal transformation significantly affected only the P content (Table S1). The N, P, Ca and Mg contents were also significantly affected by the interaction between both factors. All treatments significantly decreased the K and Ca content compared to the control treatment, while Mg and Fe contents were reduced in treatments S, L and TL and TS, L and TL, respectively. In contrast, P content was significantly increased by TS, L and TL treatments, while N content was the highest for the TS treatment.\u003c/p\u003e\n\u003ch2\u003e3.2\u0026nbsp;Impacts of the experimental factors on rhizospheric soil parameters\u003c/h2\u003e\n\u003cp\u003eThe electrical conductivity of the soil was only affected by slumgum type, where TS treatment recorded the significantly highest value (Table S2). On the other hand, the dehydrogenase activity was affected by the two experimental factors and their interaction, with TS treatment showing significantly higher dehydrogenase activity than the rest of the treatments and the control (Fig. 2A). The same treatment (TS) beneficially affected the activity of b-glucosidase without being significantly different from the control and L treatment (Fig. 2B). Both alkaline phosphatase and urease activities were significantly affected by the slumgum type, being the solid slumgum (S and TS treatments) more efficient to significantly increase both enzymatic activities concerning to the control and the liquid form (L and TL treatments; Fig. 2 C, D).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.3 Impacts of the experimental factors on the fungal community composition and functionality of rhizospheric soil\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe sequencing of the fungal ITS region produced a total of 7,171,200 high-quality sequences, which were clustered into 2,260 fungal Amplicon Sequence Variants (ASVs) before rarefaction at 91,201 reads per sample. The ANOVA showed that all experimental factors and their interactions, except the transformation for fungal richness, had a significant effect on \u0026alpha;-diversity; the S, TS and TL treatments significantly decreased the fungal richness and Shannon diversity index (Fig. S1A, B). The rhizosphere fungal communities differed significantly among slumgum types and the transformation process (Table S3), as well as an interaction between both factors was recorded with the perMANOVA analysis. According to the results of the perMANOVA, the NMDS ordination showed clusters of fungal communities well defined for each treatment (Fig. 3A) being the fungal communities from L and TL treatments more similar to the control treatment than S and TS treatments.\u003c/p\u003e\n\u003cp\u003eThe fungal ASVs were assigned to 13 phyla. The most abundant phylum was Ascomycota (82.7% of the total relative abundance), followed by Mortierellomycota (10.5%), Basidiomycota (3%), Chytridiomycota (2.3%) and Mucoromycota (1.3%) (Fig. S2A, Table S4). The remaining identified phyla accounted for less than 0.1% of the fungal sequences. Ascomycota and Basidiomycota were significantly affected by the slumgum type, decreasing their relative abundance under the S and TS treatments compared to the control soil and the rest of the treatments (L and TL). In contrast, Mortierellomycota and Mucoromycota showed opposite trends and significantly increased their relative abundance under the S and TS treatments.\u003c/p\u003e\n\u003cp\u003eAt the genus level, \u003cem\u003eHumicola\u003c/em\u003e was the dominant fungal genus, with a relative abundance of 15.4%, which significantly increased after the transformation of slumgum of both types (TS and TL treatments) (Fig. S2B, Table S5). Between the genera, \u003cem\u003eMortierella\u003c/em\u003e and \u003cem\u003eMucor\u003c/em\u003e were the most significantly influenced by the slumgum type, with relative abundances that increased in solid slumgum (S and TS), while \u003cem\u003ePreussia\u003c/em\u003e, \u003cem\u003eHormiactis\u003c/em\u003e and \u003cem\u003eMyceliophthora\u003c/em\u003e also significantly decreased under the same treatments (S and TS). Only \u003cem\u003eAlternaria\u003c/em\u003e and \u003cem\u003eOchroconis\u003c/em\u003e were significantly affected by the two experimental factors and their interaction, showing the highest relative abundance under the S treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe analysis of fungal functional guilds assigned 1,224 Amplicon Sequence Variants (ASVs), which represents 54% of the total ASVs, to the six main functional guild groups: plant pathogens, parasites, lichenized, symbiotrophs, endophytes, and saprotrophs (Fig. 4; Table S6). The plant pathogens and saprotrophs were significantly affected by the slumgum type, the transformation process and the interaction between both factors, suggesting that both fungal guilds showed opposite trends under the TS, L and TL treatments (when saprotrophs increased the plant pathogens decreased). The parasites were significantly decreased by the treatments with slumgum transformation (TS and TL), as shown also with the ANOVA. Related to the endophytes and symbiotrophs, the most influential factor was slumgum type, showing the lowest relative abundance in the treatments with solid slumgum (S and TS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Impacts of the experimental factors on the bacterial community composition and functionality of rhizospheric soil\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 5,452,140 reads with 5,567 different ASVs were obtained before rarefaction at 270,840 reads per sample. Bacterial richness and Shannon diversity index were significantly affected by both experimental factors and their interactions (Fig. S1C, D). The richness was significantly higher in the S and TL treatments than the rest of the treatments, while the significantly lowest values were recorded for the TS treatment. The perMANOVA analysis showed that both factors, slumgum type and transformation, as well as their interaction (S x T) significantly influenced the composition and structure of the rhizospheric bacterial communities (Table S3B). The NMDS ordination of soil bacterial communities exhibited a stress value of 0.08 and it was clustered by slumgum treatment, showing the same pattern as fungal communities (Fig. 3B). The bacterial community was composed of 17 different bacterial phyla. Actinomycetota and Pseudomonadota were the most abundant phyla (32% and 26% respectively of total relative abundance) and were affected by both experimental factors and their interaction (Fig. S2C, Table S7). The TS and S treatments were the most efficient to significantly increase Actinomycetota and Pseudomonadota, respectively. The Bacillota phylum abundance (24%) was significantly decreased by both factors, being the solid slumgum treatments (S and TS) those with the lowest values. Myxococcota phyla abundance, although accounted for only 1.43% of the bacterial sequences, was strongly affected by the two experimental factors; and it significantly increased under TS, S and L treatments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith respect to the bacterial genus, the most significant factor was the slumgum type (Table S8). The genera such as \u003cem\u003eMicrobacterium\u003c/em\u003e, \u003cem\u003eVirgibacillus\u003c/em\u003e, \u003cem\u003eIlumatobacter\u003c/em\u003e and IMCC26256 decreased their relative abundance under the treatments with solid slumgum addition (S and TS). In contrast, TM7a and unknown bacterial genus significantly increased their abundances under the solid treatment (S and TS; Fig. S2D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith respect to the bacterial functionality, the relative abundance of predicted genes, involved in nitrification and nitrogen fixation process of N cycling, were not significantly affected by the slumgum addition (Table S9, Fig. S3). Instead, in the denitrification process the relative abundance of nitrate reductase genes (narG, narH), and nitric oxide reductase (norB, norC) were significantly increased by both experimental factors. The S treatment increased all four genes compared to the control, while the TS treatment only significantly increased only the values of nitrate reductase genes (narG, narH) but not the ones of nitric oxide reductase (norB, norC). The nitrite reductase gen (nirK) was also significantly increased by the S treatment. In the same way, the relative abundance of C-cycling genes participating in organic matter decomposition (cellulose (bglX) and hemicellulose (FUCA) biodegradation or C fixation\u0026nbsp;(ppc)) showed significantly higher values in the S treatment compared to the others. Regarding the P-cycling pathway genes, they were not significantly affected by any of the studied treatments.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.5 Correlations among soil properties, plant biomass, and the fungal and bacterial communities in the rhizospheric soil\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eCorrelation heat maps were performed to explore the relationships between the fungal and bacterial communities with respect to the plant biomass and the soil chemical and biological properties (Fig. S4A, B). In general, fungi and bacteria showed stronger correlations with the soil biological properties than its chemical properties. With respect to the fungal community, it was observed that Mortierellomycota and Mucoromycota phyla and their respective genera, \u003cem\u003eMortierrella\u003c/em\u003e and \u003cem\u003eMucor\u003c/em\u003e were the ones that showed the highest significant relationship with urease and phosphatase activities, as well as with EC. The \u003cem\u003eOchroconis\u003c/em\u003e and \u003cem\u003eAlternaria\u003c/em\u003e genera also revealed significant positive correlations with the same enzymatic activities, but with lower significance levels. On the other hand, only \u003cem\u003eGeomyces\u003c/em\u003e genus revealed a significant positive correlation with\u0026nbsp;b-Glucosidase. The majority of fungal phyla and genera showed negative correlations with phosphatase and urease activities and EC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the case of bacterial phyla, Pseudomonadota phylum showed a significant positive correlation with phosphatase, urease, EC and Total N, while Actinomycetota was positively correlated with\u0026nbsp;b-glucosidase and dehydrogenase activities (Fig. S4C, D). In contrast, Bacillota showed a more significant negative correlation for the same soil parameters than Pseudomonadota. Also, Chloroflexota revealed significant negative correlations with all the enzymatic activities measured. At the bacterial genus level, the most significant positive correlation was found between the unknown and the TM7a genera with phosphatase, urease and EC; \u003cem\u003eRhizobium\u003c/em\u003e and \u003cem\u003eFlavitalea\u003c/em\u003e showed less notable positive correlations with the same parameters; and the \u003cem\u003eMyxococcus\u003c/em\u003e was the only genus showing significant positive relations with soil chemical properties (TC, TK, TP). The rest of the bacterial genera showed negative correlations with phosphatase and urease activities and EC.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNew alternative crops capable to adapt to challenging climate conditions and with low input requirements are needed in order to redesign farming systems, especially in areas with degraded soils where intensive conventional cropping is not recommended or even non-economically viable, focusing on sustainable strategies and the preservation of natural resources. Purslane has been proposed as a novel crop that could be cultivated under these conditions, being a crop of high nutritional value and easy to manage for farmers that could be very responsive to organic fertilizers (Carrascosa et al., 2023a). Our results indicate that the treatments involving transformed slumgum (ST, SL) and the non-transformed liquid form (L) significantly increased purslane dry biomass and P uptake. In studies where the addition in the soil of others organic materials such as struvite has been assayed, e.g. Gonz\u0026aacute;lez-Ponce et al. (2009) and Bastida et al. (2019), it was found that greater yield and plant P uptake were attributable to the higher plant tissues Mg uptake. In contrast to these reports, in our study we observed a decrease of Mg uptake in plants grown under all the treatments with slumgum, as well as a decrease of other macro and micronutrients such as K, Ca and Fe which were also decreased. Despite the observed increase in plant P content, we did not record a corresponding increase in the available soil P. This discrepancy could be mainly attributed to two reasons. Firstly, the P taken up by the crop resulted in decreased P contents in the soil. Secondly, a more plausible explanation is that in soils with a high pH, typically around 8, P could have precipitated as Ca and Fe phosphates; hence the lower availability in the soil (Kruse et al., 2015). Consequently, the recorded decrease in K, Ca and Fe content in plants could be due to the phosphate formation during slumgum mineralization. On the other hand, it was observed that solid slumgum (S), but not the transformed form (TS), decreased the plant biomass compared to the control plants. This outcome might be attributed to the presence of residual beeswax in this raw solid residue, potentially exerting detrimental effects and reducing the availability of soil nutrients to plants. In support of this finding, Cozzolino et al. (2016) reported that a specific molecular composition of the residue could adversely affect plant growth, counterbalancing the positive effects derived from the provision of carbon (C) and nutrients. Numerous investigations have demonstrated the positive impact of using organic residues or materials in agricultural ecosystems on soil fertility and environmental quality (Aytenew and Bore, 2020). Soil enzyme activities are considered sensitive indicators of soil fertility, as they catalyse key biochemical reactions involved in soil nutrient cycling and organic matter degradation (Nannipieri et al., 2012). In our study, we observed significant effects of the slumgum type on N cycling enzymes, specifically urease (Kandeler et al., 1999), and P cycling enzymes, namely phosphomonoesterase (Naseby and Lynch, 1997). However, we did not observe any significant impact on C cycling enzymes, such as \u0026beta;-glucosidase (Eivazi and Tabatabai, 1988).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn general, both transformed and non-transformed solid slumgum (TS and S), as well as transformed and non-transformed liquid slumgum (TL and L), significantly increased phosphomonoesterase levels compared to the control treatment. However, the liquid treatments exhibited lower enzymatic activity than the solid treatments. Regarding urease, only the solid slumgum treatments (TS and S) showed significantly higher values than the control, whereas the liquid slumgum treatments (TL and L) did not show significant differences.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEnhanced enzyme activities involved in nutrient cycling due to the application of organic residues have been previously reported in different soils (Dămăt\u0026icirc;rcă et al., 2023; Das et al., 2017; L\u0026oacute;pez-Pi\u0026ntilde;eiro et al., 2011), especially the application of dried olive residues transformed with saprophytic fungi similarly to the treatments we used in the present work (Siles et al., 2014b)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe observed increase in enzymatic activities could suggest higher turnover rates of soil P and N, with the effect being more pronounced in soils amended with solid slumgum compared to those amended with liquid slumgum (Das et al., 2017). In line with this statement, our findings revealed that only the transformed solid slumgum (ST) significantly increased dehydrogenase activity in the soil. Dehydrogenase is an enzyme that measures soil microbial activity through oxidoreductases (Nannipieri et al., 2012). Previous studies have attributed the increase in this activity to the proliferation of soil microorganisms due to higher availability of organic substrates, consequently resulting in increased levels of microbial biomass (Macci et al., 2012). In accordance with our findings, Siles et al. (2014b) also observed an increase in this activity in soils amended with residues of dried olive residues bio-transformed with saprotrophic fungi. However, we did not observe an effect of solid slumgum in its raw state (S) on this activity. It is suggested that the saprotrophic fungus \u003cem\u003eP. chrysogenum\u003c/em\u003e may have a potential strategy in slumgum transformation, thereby producing readily available organic materials for soil microorganisms. This process involves the biodegradation of recalcitrant organic compounds and the mineralization of nutrients through a diverse set of extracellular enzymes, potentially influencing soil microbial activities (Garc\u0026iacute;a-S\u0026aacute;nchez et al., 2019). To delve into the effect of slumgum on plant yield through microorganisms, we studied the soil microbiome (Luo et al., 2022; Siles et al., 2014b). It is important to emphasize that the observed changes in the purslane rhizosphere microbial community are the direct result of slumgum\u0026apos;s impact on the resident soil microbiome. Notably, these changes do not arise from slumgum itself, as the inhabiting microbes within it were inactivated by sterilization before soil application, aimed at eliminating \u003cem\u003eP. chrysogenum\u003c/em\u003e as the final step. Therefore, slumgum\u0026apos;s influence on the natural soil microbiome most likely is related to its physical and chemical characteristics, which may include the presence of compounds like cellulose, hemicellulose, among others (van der Wal et al., 2013), given its origin as residual material left after beeswax removal from honeycomb.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn relation to the fungal community, our findings revealed a significant change in the fungal community composition upon the addition of slumgum to the soil, resulting in decreased fungal diversity and richness across all slumgum treatments, except for the non-transformed liquid slumgum (L). Other studies where residues transformed with saprobic fungi were applied in Mediterranean soils have reported a decrease in fungal functional diversity and changes in the structure of fungal communities (Siles et al., 2014b), whereas others have found no changes in fungal diversity and richness (Siles et al., 2014a). Moreover, Luo et al. (2022) conducted a study in soils amended with sterile composts and they did not observe changes in microbial communities. They suggested that the reduction in fungal diversity might be attributed to the adaptation and selective proliferation of specific fungal groups toward the new organic source at the expense of the added nutrients. This phenomenon might be applicable to our study, as we observed, consistently with Siles et al. (2014b), a negative correlation between fungal richness and soil total N (R\u003csub\u003epearson\u003c/sub\u003e -0.534, P\u0026lt;0.05). Furthermore, our study revealed a shift in the functional fungal guild due to the effect of slumgum type and transformation with \u003cem\u003eP. chrysogenum\u003c/em\u003e, with an increase in saprotrophs and a decrease in plant pathogens and parasites fungi being recorded. This finding is aligned with studies such as the ones of Xiong et al. (2017) and Siegel-Hertz et al. (2018), who attributed the increase in saprotrophic fungi to the reduction of plant pathogens, due to competitive interactions for space and resources between pathogenic and saprotrophic fungi (Kepler et al., 2017; Shu et al., 2022). Numerous studies over the years have documented how the use of organic residues, such as compost, manure, bone meal, poultry litter slurry, straw, wood residues, among others, can exert suppressive effects on certain phytopathogenic microorganisms by modifying indigenous microbial communities and stimulating microorganisms antagonistic to soil-borne pathogens(Chen et al., 2023; Reardon and Wuest, 2016; Shu et al., 2022; Tao et al., 2020). In contrast, in our study the incorporation of slumgum in its raw state (S) resulted in a diminished response to saprotrophic fungi. This decrease might be attributed to the presence of higher concentration of inhibitory secondary metabolites, such as hydrophobic compounds present in the beeswax remained within the slumgum. Consequently, the chemical composition and quality of organic residues, alongside the saprotrophic capabilities of microorganisms inhabiting the rhizosphere, emerge as pivotal factors influencing the microbial communities of residue decomposers (Bonanomi et al., 2018; Clocchiatti et al., 2020; Liu et al., 2021). On the other hand, the composition of root exudates is an important factor in selecting a subset of soil microorganisms (Afzal et al., 2019; Haichar et al., 2014). Literature reports have shown an increase of saprotrophic fungi involved in consuming rhizodeposits in soil amended with sawdust (Clocchiatti et al., 2021). These fungi are known to assimilate soluble sugars, utilizing labile monomeric exudate compounds (de Vries and Caruso, 2016). In our study, the observed increase in plant yield might have led to a higher quantity of root exudates. Consequently, the complex soluble compounds within these exudates could have significantly influenced interactions among rhizosphere microorganisms (Zhalnina et al., 2018). Moreover, our study revealed a positive correlation between the relative abundance of saprotrophic fungi and plant dry biomass (R\u003csub\u003epearson\u0026nbsp;\u003c/sub\u003e= 0.73; P \u0026lt; 0.001), while indicating a negative correlation between plant dry biomass and the relative abundance of plant-pathogenic fungi (R\u003csub\u003epearson\u003c/sub\u003e = -0.74; P \u0026lt; 0.001) and parasites (R\u003csub\u003epearson\u003c/sub\u003e = -0.65; P = 0.003). Interestingly, our study revealed an increase in the Mortierellomycota and Mucoromycota phyla under the treatment involving solid slumgum. Clocchiatti et al. (2020) attributed the rise in Mortierellomycota to the introduction of nitrogen-rich organic materials derived from soil amended with cover crop fragments, paper pulp, and wood sawdust. In our investigation, these phyla exhibited a strong correlation with increased enzymatic activities, specifically urease (R\u003csub\u003epearson\u0026nbsp;\u003c/sub\u003e= 0.82; P \u0026lt; 0.001 for Mortierellomycota; R\u003csub\u003epearson\u0026nbsp;\u003c/sub\u003e= 0.93; P \u0026lt; 0.001 for Mucoromycota) and phosphatase (R\u003csub\u003epearson\u0026nbsp;\u003c/sub\u003e= 0.82; P \u0026lt; 0.001 for Mortierellomycota; R\u003csub\u003epearson\u0026nbsp;\u003c/sub\u003e= 0.88; P \u0026lt; 0.001 for Mucoromycota). A detailed analysis of the effect of slumgum on the fungal genus level revealed that the relative abundance of \u003cem\u003eAlternaria\u0026nbsp;\u003c/em\u003espp. and \u003cem\u003eOchroconis\u003c/em\u003e spp. increased with the addition of non-transformed solid slumgum (S) to the soil. However, these abundances decreased notably following the transformation of slumgum and the application of liquid slumgum (TS, TL, and L). Therefore, applying this residue in its raw state might prove inconvenient, as it could potentially negatively impact purslane development and biomass, as indicated in our study by the observed negative correlation between plant biomass and these fungal genera (R\u003csub\u003epearson\u003c/sub\u003e = -0.632; p = 0.004 for \u003cem\u003eAlternaria\u003c/em\u003e and R\u003csub\u003epearson\u003c/sub\u003e = -0.561; p = 0.012 for \u003cem\u003eOchroconis\u003c/em\u003e). The bacterial community displayed a pattern similar to that of the fungal community, undergoing changes in response to all slumgum addition treatments, with the most significant differences observed in treatments involving the addition of solid slumgum (S and ST) compared to the control. In contrast to findings from other studies (Shu et al., 2022; Zhaoxiang et al., 2020), we did not observe a higher sensitivity of the soil bacterial community compared to the fungal community in response to organic amendment addition. However, it\u0026apos;s essential to note that these studies might not be directly comparable to ours. The previous studies were conducted with organic residues-associated active microbiota, potentially introducing a competition effect or alterations in habitat for the active microbiota of the residue (Luo et al., 2022). In our study, the predominant bacterial phyla identified were Actinomycetota, Pseudomonadota, and Bacillota, showing significant variations based on the type of slumgum applied (solid or liquid) and the transformation with \u003cem\u003eP. chrysogenum\u003c/em\u003e. Actinomycetota and Pseudomonadota, known as fast-growing microbes, have previously been linked to cellulose degradation (Kramer et al., 2016). We found that these two bacterial phyla exhibited a positive correlation with increased microbial activity in the rhizosphere. Particularly noteworthy was the significant increase in Actinomycetota under the TS treatment, which in conjunction with the substantial rise in the Mortierellomycota fungal phylum, suggests that Actinomycetota may contribute not only to slumgum decomposition but also to the consumption of breakdown products resulting from fungal slumgum decomposition (de Menezes et al., 2017), given their known engagement in fungal-bacterial interactions (Sathya et al., 2017). Pseudomonadota have also been linked with the decomposition of organic residues, suggesting their role in providing an additional nitrogen source to fungal saprotrophs in exchange for breakdown products from residues (Johnston et al., 2016). Consequently, the significant increase in Pseudomonadota and N content observed in the rhizosphere only under the non-transformed slumgum (S) treatment could elucidate their function as N-suppliers and decomposers within the microenvironment generated under this specific treatment. Notably, only the TS treatment significantly increased the N content in purslane, coinciding with the highest relative abundance of the \u003cem\u003eRhizobium\u003c/em\u003e genus. It is plausible that these plant growth-promoting rhizobacteria played a significant role in facilitating plant N uptake and promoting plant growth through direct and/or indirect effects (Adesemoye et al., 2010). The non-transformed solid slumgum (S) significantly influenced bacterial gene expression, particularly the genes related to N\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erecycling in the denitrification process, e.g. nitrate reductase (narG, narH), nitrite reductase (nirk), and nitric oxide reductase (norB, norC). However, there was no significant effect on the nosZ gene, which plays a vital role in reducing nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) to nitrogen gas (N\u003csub\u003e2\u003c/sub\u003e) in the last step of denitrification and acts as unique known sink for N\u003csub\u003e2\u003c/sub\u003eO in soil (Krause et al., 2017). Therefore, the application of non-transformed slumgum (S) to soil might contribute to increased greenhouse gas emissions, specifically through N\u003csub\u003e2\u003c/sub\u003eO, due to the enhanced expression of functional genes involved in nitrite and nitric oxide reduction. However, this increased expression does not affect the final step, thereby leading to the accumulation of N\u003csub\u003e2\u003c/sub\u003eO. According to Yoon et al. (2015), denitrification typically prevails in soils characterized by low C/N ratios, which is aligned with our observations in the non-transformed slumgum (S) treatment. Moreover, non-transformed solid slumgum (S) increased the expression of ppc genes associated with C fixation, as well as genes related to the degradation of cellulose (beta-glucosidase, bglx) and hemicellulose (FUCA). This increased gene expression could be attributed to the greater complexity of non-transformed slumgum in comparison to other treatments. The non-transformed slumgum lacks \u003cem\u003eP. chrysogenum\u003c/em\u003e transformation, thus containing these complex polysaccharides, which potentially foster a higher abundance of bacteria capable of metabolizing them (Jim\u0026eacute;nez et al., 2014).\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe use of bio-waste residuals as soil amendments is a novel agronomic practice that can significantly contribute to the improvement of soil fertility and quality improvement while at the same time facilitates the concepts of circular economy and conservation of natural resources. To the best of our knowledge, this is the first study reporting the influence of slumgum, a residue coming from the beekeeping industry, on the purslane biomass and its rhizosphere microbial community. The application of solid and liquid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e (TS and TL, respectively) and liquid slumgum (L) significantly increased the purslane shoot biomass and the foliar P content. The different slumgum amendments assayed also changed and clearly differentiated the purslane bacterial and fungal community composition, despite the very short period experimental period (6 weeks). A change in the functional fungal guilds increasing the abundances of saprotrophs and decreasing plant pathogens under the treatments TS, TL and L was recorded. The solid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e (TS) was the most efficient to significantly increase the soil enzymatic activities related to C, N and P cycles. Instead, the slumgum in its raw state (S) increased the abundance of the majority of bacterial genes related to the denitrification process, which could contribute to the negative effect of N\u003csub\u003e2\u003c/sub\u003eO gas emissions. Therefore, solid and liquid slumgum transformed with \u003cem\u003eP. chrysogenum\u0026nbsp;\u003c/em\u003eand the liquid non-transformed slumgum (TS, TL and L, respectively) can be considered as effective sustainable organic fertilizers, additive or soil amendments that can improve crop yield and beneficially affect soil microbial communities. However, further studies are needed to extrapolate the beneficial results obtained to field conditions, while more crops should be tested to validate the recorded effects.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe raw sequencing data is available from the the NCBI Sequence Read Archive repository (www.ncbi.nlm.nih.gov/sra) under the BioProject \u0026nbsp; PRJNA1051377.\u003c/p\u003e\n\u003cp\u003eAuthor contribution\u003c/p\u003e\n\u003cp\u003eAngel Carrascosa: investigation, empirical analysis and data curation.\u0026nbsp;Jose Antonio Pascual: writing\u0026mdash;review and editing. Jessica Cuartero:\u0026nbsp;sequencing analysis and review. Inmaculada Garc\u0026iacute;a-Romera and Gloria Andrea Silva-Castro: methodology and experimental design.\u0026nbsp;Ana De Santiago: soil samples analysis. Margarita Ros: review and editing. Spyridon Alexandros Petropoulos:\u0026nbsp;funding acquisition, review and validation.\u0026nbsp;Maria del Mar Alguacil:\u0026nbsp;project administration, writing, editing and funding acquisition. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe contact author has declared that none of the authors has any competing interests.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Spanish Ministerio de Ciencia e Innovaci\u0026oacute;n (project PCI2020-112091) and the PRIMA foundation under the project VALUEFARM (PRIMA/0009/2019). \u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAdesemoye, A. O., Torbert, H. A., and Kloepper, J. 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E.: Denitrification versus respiratory ammonification: environmental controls of two competing dissimilatory NO3\u0026minus;/NO2\u0026minus; reduction pathways in Shewanella loihica strain PV-4, ISME J, 9, 1093\u0026ndash;1104, https://doi.org/10.1038/ismej.2014.201, 2015.\u003c/p\u003e\n\u003cp\u003eZhalnina, K., Louie, K. B., Hao, Z., Mansoori, N., da Rocha, U. N., Shi, S., Cho, H., Karaoz, U., Loqu\u0026eacute;, D., Bowen, B. P., Firestone, M. K., Northen, T. R., and Brodie, E. L.: Dynamic root exudate chemistry and microbial substrate preferences drive patterns in rhizosphere microbial community assembly, Nat Microbiol, 3, 470\u0026ndash;480, https://doi.org/10.1038/s41564-018-0129-3, 2018.\u003c/p\u003e\n\u003cp\u003eZhaoxiang, W., Huihu, L., Qiaoli, L., Changyan, Y., and Faxin, Y.: Application of bio-organic fertilizer, not biochar, in degraded red soil improves soil nutrients and plant growth, Rhizosphere, 16, 100264, https://doi.org/10.1016/j.rhisph.2020.100264, 2020.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"purslane, circular economy, wild edible species, bacterial community, fungal community, functional genes, sustainable agriculture","lastPublishedDoi":"10.21203/rs.3.rs-4480989/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4480989/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the beekeeping industry, \u0026ldquo;slumgum\u0026rdquo; is generated as a solid organic waste during the beeswax-rendering process from old scraped honeycombs. This bio-waste could be considered as a novel organic fertilizer due to its high content in organic matter and nutrients. As a novelty in this study, we analysed the effect of application of solid and liquid slumgum transformed or not with \u003cem\u003ePenicillium chrysogenum\u003c/em\u003e on purslane (\u003cem\u003ePortulaca oleracea\u003c/em\u003e) yield and its relationship with soil rhizosphere. For this purpose, nutritional composition, enzymatic activities involved in the P, N and C cycles, fungal and bacterial community composition, diversity and potential functionality in the rhizosphere were measured. The application of solid and liquid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e (TS and TL, respectively) and slumgum liquid (L) significantly increased purslane shoot biomass and foliar P content, compared to the non-transformed solid slumgum (S). The different types of slumgum tested resulted in changes in the composition of both bacteria and fungi communities, resulting in distinct communities for each treatment. Moreover, changes in the functional fungal guilds were observed, with increased abundances of saprotrophs and reduced number of plant pathogens under the TS, TL, and L treatments. Solid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e (TS) was also the most effective in enhancing enzymatic activities related with C, N and P cycles in the rhizosphere. Conversely, the use of solid slumgum (S) led to an increase in the abundance of bacterial genes primarily associated with the denitrification process. Our preliminary results suggest that solid and liquid slumgum transformed with \u003cem\u003eP. chrysogenum\u003c/em\u003e, as well as liquid non-transformed slumgum (TS, TL, and L, respectively), could be considered as novel organic fertilizers, amendments or additives within the circular economy context and the sustainable use of natural resources. Nevertheless, further studies are necessary to validate the positive outcomes observed, particularly under field conditions and with a variety of species.\u003c/p\u003e","manuscriptTitle":"Response of Portulaca olearacea and its rhizospheric microbiome to the application of slumgum from beekeeping industry transformed with Penicillium chrysogenum","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-09-05 15:03:42","doi":"10.21203/rs.3.rs-4480989/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2024-07-02 12:16:15","doi":"10.21203/rs.3.rs-4480989/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11ed65b1-02de-4285-87ec-45aabcb7b71f","owner":[],"postedDate":"September 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T20:02:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-05 15:03:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-4480989","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4480989","identity":"rs-4480989","version":["v2"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}