Use of Biosolids to Enhance Tomato Growth and Tolerance to Fusarium oxysporum f. sp. radicis-lycopersici

The European Green Deal proposes the reuse of any kind of waste that can be safely repurposed; sludge may also be reused as a biosolid. The present research aims to evaluate a biosolid made from sludge, originating from a wastewater treatment plant (WWTP) for its effect on tomato growth and enhancement of tolerance against the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici (Forl). Peat and/or two soil types were amended with biosolid (0, 80 and 160 tons/ha) to form substrates on which tomato plants were grown either under controlled conditions, in a growth chamber or outdoors, in a net protected area. Plant growth and disease parameters were recorded 4–7 weeks after application of Forl inoculum in the substrates. Results showed that biosolid addition enhanced plant growth and increased tolerance against tomato foot and root rot caused by Forl. Specifically, fresh weight, root weight, stem height and leaf number of tomato plants in Forl-inoculated soils that had received biosolid, were increased when compared to Forl-inoculated control soils. Forl resulted in higher disease severity on plants grown outdoors in biosolid plus clay soil than in biosolid plus sandy soil, while the opposite occurred under controlled conditions where higher disease index was recorded on plants grown in peat plus sandy soil than in peat plus clay soil. These findings strongly suggest that this biosolid may act as an organic fertilizer and possible stimulant of tomato tolerance against Forl, and the effect is soil type dependent. Therefore, this biosolid should be considered for its possible use in agriculture according to the principles of circular economy and waste minimization. • Biosolid addition enhances tomato growth in different substrates and conditions. • Disease is less severe on plants grown on soil mixed with biosolid. • In the outdoors trial biosolid renders higher disease tolerance in sandy soil. • Fusarium disease is less aggressive on tomato plants grown on biosolid mix. • Tolerance to disease is higher in sandy soil+biosolid than in clay soil+biosolid.


Introduction
Management of environmental resources is an important issue, especially in an era of energy decarbonization and resource diminishment (Vagiona 2016;Musmarra et al. 2019). EU has launched the Green Deal initiative which leads the transition to a circular economy and to investments in environment friendly technologies. Industrial symbiosis may come true for different production sectors, where waste from one sector may become a raw material for another, as for example between side-streams from food processing and biosubstrates for cosmetic ingredients, pharmaceuticals and chemicals (Cherif et al. 2020). Sludge produced from wastewater treatment plants (WWTP) is a noxious waste, inexorably produced during their operation (Bezirgiannidis et al. 2020); its main disposal method has been landfilling due to its relatively low cost. However, this method is now abandoned in many countries because of strict environmental regulations (Collivignarelli et al. 2019;Inglezakis et al. 2014;Joo et al. 2015;Wang et al. 2008a). Furthermore, in the context of sustainable management and circular economy, this waste can become a valuable resource, recirculating raw materials (Bezirgiannidis et al. 2020;Papastergiadis et al. 2014;Rufí-Salís et al. 2020;Svarnas et al. 2020;Xue et al. 2015). As such, field application of treated sludge (biosolids) improves soil properties such as organic matter content, soil density and structure (Shan et al. 2021;Fischer et al. 2020;Zuo et al. 2019). Moreover, biosolids provide nitrogen and phosphorus to the plants, restore soil fertility, and improve water circulation (Brown et al. 2020;Giannakis et al. 2020). Nevertheless, precautions should be taken when using sludge-based biosolids as soil amendments, to avoid soil pollution with toxic metals and toxic organic compounds or soil contamination with dangerous pathogens (Clarke et al. 2016;Gonzalez-Ollauri et al. 2020). For this reason, European legislation has set limit values for metals of toxicological concern when sludge is used for fertilization (Council Directive 86/278/EEC). Due to advances in environmental protection, this Directive will have to be updated (European Commission-DG Environment 2014).
Crop production is strongly affected by several persisting diseases and phytopathogenic fungi may cause devastating losses for millions of crops worldwide (Yoon et al. 2013). Tomato fusarium foot and root rot (TFFR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici (Forl), is a serious disease that infects tomato crop grown both under greenhouse and field conditions (Lagopodi et al. 2002). This and other soil-borne fungal diseases are difficult to control with synthetic fungicides, and for this reason alternative to chemical control methods have been used (Kamou et al. 2020;Saligkarias et al. 2002;Romero-Arenas et al. 2017). Among these methods, organic soil amendments such as green waste compost mixtures were found effective against diverse plant diseases and especially against soil-borne pathogens (Tubeileh and Stephenson 2020;Milinković et al. 2019). Other compost mixtures made from sewage sludge were also able to suppress plant diseases caused by F. oxysporum f. sp. lycopersici race 1, F. oxysporum f sp. basilici, Pythium ultimum and F. oxysporum f. sp. radicis-cucumerinum (Cotxarrera et al. 2002;Ferrara et al. 1996;Markakis et al. 2016). Therefore, the use of sludge-based biosolids may be an appealing option against soil-borne plant diseases. For this reason, confirmation of these biosolids' performance against important plant pathogens, in various commercially important crops, is necessary in order to promote their use in agriculture.
The use of biosolids agrees with the principles of circular economy and of sustainable development because waste is transformed into a product with economic and environmental benefits (Pradel et al. 2016). In soils that exhibit low organic matter as do most of the soils in Greece (Kouloubis and Tsantilas 2008), biosolids could well be used to enhance soil fertility, improve plant growth, and suppress plant diseases (Bonanomi et al. 2020a;Khaleel et al. 1981;Pascual et al. 2009;Stewart-Wade 2020a, b). Furthermore, this is an option for disease suppression devoid of the polluting effects of chemical fungicides (Yoon et al. 2013). As such, the present work was conducted to investigate the possible use of a biosolid made from sludge, as a medium that improves soil properties, enhances tomato growth, and increases tomato tolerance to Forl, suggesting its potential use as a soil amendment in agricultural land. This research may highlight novel advantages of biosolids made from WWTP sludge and point towards their successful repurpose. Such a reuse is indeed a sustainable, environmentally-friendly solution that may promote crop yield through fertilization and disease suppression.

Culture of Fusarium oxysporum F. sp. radicis-lycopersici and Preparation of Inocula
A virulent strain of Forl, deposited in the Centraalbureau voor Schimmelcultures, The Netherlands (CBS 101587) was used for artificial inoculations of tomato plants. The fungus, which was kept on potato dextrose agar (PDA; Lab M, Lancashire, UK), on Petri dishes at 4 o C, was inoculated on surface-sterilized tomato fruit and re-isolated on PDA from time to time in order to maintain its pathogenicity. For the production of inoculum used in artificial inoculations, the fungus was grown in liquid cultures, as described in Kamou et al. (2015) and Lagopodi et al. (2002). Conidia were separated from mycelium by filtering through Miracloth (Calbiochem, USA), washed with sterile distilled water by centrifugation at 5,000 rpm for 10 min (RC5C; Sorvall Instruments, Waltham, USA), and re-suspended in sterile distilled water. Their concentration was adjusted to 10 6 spores/mL. Conidia inoculum was used in experiments performed under controlled conditions to inoculate tender plantlets grown indoors. Furthermore, hyphal fragments were obtained by macerating the mycelium in a blender after washing with sterile distilled water. Another mixed suspension of colony-forming units (cfu), consisting of fine hyphal fragments and conidia 1:2 (v:v) was prepared and adjusted to 10 6 cfu/mL. The mixture of hyphal fragments and conidia was used in experiments carried out in the net protected field to ensure inoculum strength under variable outdoor conditions. Treatments with and without Forl were designated as F1 and F2, respectively.

Pot Experiment under Controlled Conditions
Six different substrates prepared from biosolid, peat and two soil types (Table 1) were used for tomato growth. The substrate treatments consisted of: two levels of biosolid (-, +), two levels of inoculum (-, +), and 3 peat treatments (peat, peat plus clay soil, peat plus sandy soil). Some of the biosolid properties, which were kindly provided by the WWTP Company, EYATH S.A., are shown in Table 2. Clay soil showed the following characteristics: 40 % silt, 30 % clay, 30 % sand, 2.5 % organic matter, pH 7.9, while sandy soil had the following texture: 71 % sand, 20 % silt, 9 % clay, 1.15 % organic matter, pH 8.1. Biosolid, peat and soil mixture of each substrate was inoculated with Forl conidia at a final concentration of 10 5 spores/g of the substrate, by mixing the conidia inoculum prepared as described above, with the substrates, in a 1/10 volume ratio. Non-inoculated substrates were mixed with water.
Each substrate (Μ1-Μ3Β) was used to fill 100 mL pots where one tomato seedling, of cv. 'ACE 55', at the two-leaf stage was transplanted. All plants were placed in the same growth chamber, with 16 h photoperiod, at 20-25 o C and 60 % RH, and they were scored for disease severity four weeks after transplanting (WAT), using the 1-5 disease index scale applied by Kamou et al. (2015) slightly modified, as follows: (1) apparently healthy plant, no visible symptoms; (2) symptomless aboveground part, root and crown slightly symptomatic, with one or two light brown lesions; (3) symptomless aboveground part, severe symptoms on the crown and root, with extended dark brown lesions; (4) advanced foot and root rot and wilting of the aboveground part of the plant; and (5) dead or almost dead plant. Isolations from diseased and apparently healthy plants were done on PDA in order to confirm the presence of the pathogen according to Koch' postulates. In addition, fresh weight, root weight, stem height and number of tomato plant leaves were recorded. A 2 × 2 × 3 [2 (-, +) biosolid by 2 (-, +) inoculum states by 3 peat treatments (peat, peat plus clay soil, peat plus sandy soil)] factorial experiment was used. This experiment was carried out three times using a Completely Randomized Design (CRD) with 15, 15, 10 replications for each treatment in the first, second and third repetition, respectively. Treatments with and without biosolid were designated as B1 and B2, respectively. Treatments with substrates peat, peat plus clay soil and peat plus sandy soil were designated as P, PC and PS, respectively.

Pot Experiment under Natural Conditions
Another experiment was performed under natural conditions meaning open field climatic conditions, excluding peat from the substrates, in a net protected area. The biosolid was mixed with sandy or clay soil, at rates of 0 %, 2 % or 4 % w/w, corresponding to 0, 80 or 160 tons/ha, respectively. After thorough mixing in a big container, each substrate was used to fill 3 L pots where two uniform tomato seedlings at the two-leaf stage of cv. 'ACE 55' were transplanted. Forl was applied during transplanting, as soil drenching, using 30 mL of inoculum, at a concentration of 10 6 cfu/mL, prepared as described above, and poured in the vicinity of each plant's roots. Placing the inoculum on the roots directly in such a way, was chosen to promote inoculation of vigorous roots of plants growing outside, in contrast to mixing the inoculum with the substrate to inoculate tender plantlets growing inside the growth camber. Tomato seedlings transplanted in each substrate without fungal inoculum were used as controls. A 2 × 3 × 2 factorial experiment, in a split-plot arrangement was used, where the two soil types were the main plots, the three biosolid rates were the sub-plots, and the two (-, +) inoculation states were the sub-sub plots. A Randomized Complete Block Design (RCBD) was used with four replicate pots and two plants per pot, for each combined treatment. After transplanting, all pots were placed outdoors in a net protected area at the farm of the School of Agriculture. Half of the plants (i.e., one plant from each pot) were scored for disease severity, at 5 WAT, using the disease index scale described above. Isolations from diseased and apparently healthy plants were also done as described before in order to confirm the presence of the pathogen according to Koch' postulates. Growth parameters (response variables), namely fresh weight, root weight, stem height, number of leaves and number of flowers were also recorded. Disease severity scoring and growth parameters were also determined in the other half of the plants, at 7 WAT. The experiment was repeated simultaneously at two different sites of the farm. Treatments with biosolid rates 0 %, 2 and 4 % w/w were designated as B0, B2 and B4, respectively.

Statistical Analyses
Tomato disease index, fresh weight, root weight, stem height and number leaves data obtained from the three experiments conducted under controlled conditions were used in the statistical analysis. Therefore, a combined analysis of variance (ANOVA) over the three experiments was performed using a 2 × 3 × 3 (x 10-15) [2 (-, +) biosolid by 2 (-, +) inoculum by 3 peat mixtures (peat, peat and clay soil, peat, and sandy soil) with 9 replications per combined treatment] factorial experiment. This experimental setup considers the variability of the data between and within the three experiments and consequently increases the precision and reliability of the results. Differences among treatment means were compared at the 5 % level of significance (P ≤ 0.05) using the Least Significant Difference (LSD) criterion. Tomato disease index, fresh weight, root weight, stem height and number leaves data obtained from the two experiments conducted outdoors were used in the statistical analysis. More specifically, a combined analysis of variance (ANOVA) over the two experiments was performed using a 2 × 3 × 2 (x 4) (2 soil types by 3 biosolid concentrations by 2 (-, +) inoculum states with 4 replications per combined treatment, 2 × 4 = 8 in total) factorial experiment based on the RCBD in a split plot arrangement. Differences among treatment means were compared at the 5 % level of significance using the Least Significant Difference (LSD) criterion. All statistical analyses were performed using SPSS v.25 (IBM, USA).
ANOVA method was accomplished for both cases within the methodological frame of mixed linear models (Steel et al. 1997). In all implementations of the ANOVA method the normality and the homoscedasticity of the corresponding mixed linear models' residuals were tested and verified. No serious violations of the two previously mentioned assumptions were detected. Consequently, data transformations were not needed.

Pot Experiment under Controlled Conditions
For the data obtained from tomato plants grown under controlled conditions, in a growth chamber, ANOVA results showed that the biosolid (-, +), inoculum (-, +) and peat mixtures along with the interactions "biosolid by inoculum" and "inoculum by peat mixture" had a statistically significant effect (P ≤ 0.05) on the parameters disease index, fresh weight, root weight, stem height and number of leaves. Inoculum presence (averaged over peat treatments) showed a lower disease index in the presence of biosolid. Consequently, it decreased less the parameters fresh weight, root weight, stem height, and leaf number when compared with no biosolid treatment (Fig. 1).
Inoculum presence (averaged over biosolid presence/absence) caused lower disease index in peat plus clay soil substrate as compared with peat or peat plus sandy soil substrates, and thus resulted in more fresh weight, stem height, and number of leaves in peat plus clay soil substrate than in peat or peat plus sandy soil (Fig. 2). However, root weight of plants grown in peat plus clay soil substrate was lower than in peat substrate.

Pot Experiment under Natural Conditions
For the data obtained from tomato plants grown under natural conditions at 5 WAT, ANOVA results showed that biosolid concentrations, inoculum (-, +) and soil types along with the interactions "biosolid by inoculum" and "inoculum by soil type" had a statistically significant effect (P ≤ 0.05) on disease index, fresh weight, root weight, stem height, number of leaves and number of flowers. Inoculum presence (averaged over clay and sandy soil) at 4 % of biosolid caused lower disease index on tomato plants than in 2 % biosolid (Fig. 3), but all growth parameters were reduced less at both 2 and 4 % of biosolid as compared with no biosolid treatment. Moreover, regardless of the presence or the absence of the inoculum, all growth parameters of the plants were similar or higher in the presence of 2 and 4 % of biosolid, as compared to the plants grown in the absence of biosolid (Figs. 3 and 4).
Inoculum presence (averaged over biosolid concentrations) caused a lower disease index on tomato plants grown in the sandy soil as compared to the clay soil at 5 WAT (Fig. 5). In addition, it caused a lower reduction on all parameters of tomato plants grown in the sandy soil than in the clay soil, whereas, in the absence of inoculum, all growth parameters of tomato plants were higher in the sandy soil than in the clay soil (Fig. 4).
For the data obtained from tomato plants grown under natural conditions at 7 WAT, ANOVA results showed that biosolid levels, inoculum levels and soil type along with the interactions "biosolid by inoculum" and "inoculum by soil type" had a statistically significant effect (P ≤ 0.05) on disease index, fresh weight, root weight, stem height, number of leaves and number of flowers. In particular, disease index (averaged over the two soil types) at 7 WAT was slightly higher on tomato grown in the presence of both 2 and 4 % biosolid (Fig. 6), as compared to 5 WAT (Fig. 3). However, regardless of the higher disease index at 7 WAT, all growth parameters were similar or higher in the presence of 2 and 4 % of biosolid, as compared to no biosolid and to the non-inoculated controls.
Although Forl at 7 WAT caused similar disease index (averaged over biosolid levels) in both soils, all growth parameters of plants grown in the sandy soil were higher than in the clay soil, and the same growth response was observed in the absence of inoculum (Fig. 7).

Discussion
In the present research, tomato plants grown on substrates inoculated with Forl showed reduction in fresh weight, root weight, stem height, and leaf number due to the TFFR disease (Agrios 2005). However, lower disease index on tomato plants grown outdoors, for 5 WAT, was noted in the presence of biosolid, as compared to no biosolid; this could be attributed to increased crop tolerance due to the indirect beneficial effect of biosolid on biotic and abiotic Fig. 1 Disease index, fresh weight, root weight, stem height, and number of leaves of tomato plants as affected by Forl inoculum (F1 = presence, F2 = absence) and biosolid levels (B1 = with, B2 = without). Treatment means and standard deviation are averaged over the three peat substrates factors (Brown et al. 2020). Hence, fresh weight, root weight, stem height, and leaf number were reduced less in inoculated plants grown on biosolid substrates, as compared to inoculated plants without biosolid. Most growth parameters of tomato plants grown outdoors in inoculated soil mixtures, in the presence of biosolids were similar to the respective parameters in non-inoculated mixtures, at 7 WAT, suggesting enhanced recovery and increased tolerance of the plants to fungus due to biosolid amendment.
Addition of biosolid from WWTP sludge to clay or sandy soil increased tomato growth as compared to unamended soils. This means that the biosolid acts as an organic fertilizer, and it may restore or even slightly increases organic soil matter (Brown et al. 2020;Cid et al. 2020;Wijesekara et al. 2017;Pascual et al. 2008). Organic amendments (various biosolids) also promote soil microbiota diversity (Abawi and Widmer 2000;Al-Kindi and Abed 2016;Curci et al. 2020;De Corato et al. 2019) and many times they suppress the activity of the pathogenic microbiota (Hoitink and Boehm 1999;Lutz et al. 2020). The suppression may be due to provision of favorable conditions and ingredients for the development of beneficial microbiota by the biosolid or to the direct enrichment of the soil with biocontrol-based microbiota found in the biosolid (De Corato 2020). Moreover, improved plant nutrition due to biosolid amendment enhances crop tolerance to disease (Noble and Coventry 2005). Various organic amendments such as compost from agriculture industry residues have caused suppression of Fig. 2 Disease index, fresh weight, root weight, stem height, and number of leaves of tomato plants as affected by Forl inoculum (F1 = presence, F2 = absence) and peat substrate P = peat, PC = peat plus clay soil, PS = peat plus sandy soil). Treatment means and standard deviation are averaged over the biosolid levels Fig. 3 Disease index, fresh weight, root weight, stem height, number of leaves and number of flowers of tomato plants as affected by Forl inoculum (F1 = presence, F2 = absence) and biosolid levels (B0 = 0 %, B2 = 2 %, B4 = 4 % w/w) at 5 weeks after transplanting. Treatment means and standard deviation are averaged over the two soil types Fig. 4 Tomato plants 5 WAT grown in the absence of the inoculum, on substrates with clay soil (left) and sandy soil (right) and with different concentrations of biosolid (4 %, 2 and 0 % w/w from the left to the right correspondingly). The respective tomato plants grown in the presence of the inoculum are not shown because they present similar results Fusarium wilt in tomato or carnation (Borrero et al. 2013), and compost from date palm wastes, suppressed Fusarium wilt of the date palm (Mohamed et al. 2020). Finally, other biodegradable wastes such as coconut fibers were proven to be the ideal substrate for inoculation with T. asperellum B1092, which was effective against Forl in cherry tomato (Hasan et al. 2020). Treated sewage sludge has been examined to a much lesser extent, regarding its ability to suppress diseases. Nevertheless, Cotxarrera et al. (2002) verified that compost, prepared from vegetable and animal wastes, sewage sludge and yard waste, suppressed tomato wilt caused by F. oxysporum f. sp. lycopersici race 1, at the early stages of plant growth, and Pinto et al. (2013) found that composted sewage sludge, incorporated into pine bark substrate, significantly reduced chrysanthemum wilting caused by F. oxysporum f. sp. crysanthemi. In the study by Markakis et al. (2016), sewage sludge compost was found superior in lowering disease presence to other tested compost amendments only in the cucumber -Fusarium (Forc-Afu-68 A) pathosystem. Finally, in the study of Ghini et al. (2007), sludge from two WWTP in Brazil failed to suppress Forl. The effects of sludge on the pathogens may be due to the inexorable increase in bacterial community diversity that it causes in soil, the increased soil enzymatic activities and the augmented soil microbial biomass, in relation to un-amended soil (Curci et al. 2020). The relative differences in disease suppression can be attributed to contents and quality of the organic matter in the substrates as well as to other characteristics (Ghini et al. 2007), such as sludge amendments (e.g., digestion type, subjection to composting). The biosolid used in the present research derives from dewatered, anaerobically digested sludge, devoid of Salmonella sp. presence (in house data), capable of successful suppression of Forl.
The increased tomato growth in the mixtures with biosolid as compared to those without biosolid, regardless of the presence or the absence of Forl inoculum, strongly supports the evidence of the beneficial effects of this biosolid. In addition, the increased crop tolerance to Forl along with increasing biosolid amendment could be attributed to dose-dependent beneficial effects, such as improvement of organic matter, density and structure of soil, nitrogen and phosphorus supply to the plants, restoration of soil fertility and improvement of water circulation (Bonanomi et al. 2020b;Fischer et al. 2020;Zaman et al. 2019). Wang et al. (2021) found that single and joint application of biochar and vermicompost increased cucumber yield and quality, while Wang et al. (2008b) reported that Zoysia japonica biomass was increased by 64-316 % in biosolid treated soil as compared to control soil. In addition, Singh and Agrawal (2010) found that stem height, leaf area, root length, number of nodules Fig. 6 Disease index, fresh weight, root weight, stem height, number of leaves and number of flowers of tomato plants as affected by Forl inoculum (F1 = presence, F2 = absence) and biosolid levels (B0 = 0 %, B2 = 2 %, B4 = 4 % w/w) at 7 weeks after transplanting. Treatment means and standard deviation are averaged over the two soil types and total biomass of bean plants increased in soil amended with sludge-based biosolid at rates 60-90 tn/ha as compared to unamended soil.
The higher disease index on tomato plants grown in the growth chamber than outdoors could be attributed to conditions that favored the disease. Hibar et al. (2006) reported that the 25 o C temperature prevailing in the growth chamber (controlled conditions) is the optimum temperature for the mycelial growth of Forl. Higher growth parameters of tomato plants grown under controlled conditions were recorded in comparison to peat plus sandy soil. The opposite results were noted for outdoors plants; lower growth parameters of tomato plants grown in clay soil than those grown in sandy soil could be attributed to the different soil substrates (Stirzaker et al. 1996), environmental conditions prevailing and size of pots used. Agrios (2005) also reported that soil-borne diseases, such as Forl, are favored in heavy clay than in sandy soils.
The results of this study showed clearly that the amendment of clay and sandy soils with a biosolid made from municipal wastewater sludge enhanced tomato growth and tolerance to the fungus Forl. Previous studies showed that this biosolid was of negligible ecotoxicity and it had not enriched clay or sandy soil with metals of toxicological concern (Ni, Pb, Zn, Cu) with the exception of Ni in clay soil and Cd in sandy soil to a small extent (Giannakis et al. 2020(Giannakis et al. , 2021. The same soil samples (clay or sandy soil) were also used in the current research. Fig. 7 Disease index, fresh weight, root weight, stem height, number of leaves and number of flowers of tomato plants as affected by Forl inoculum (F1 = presence, F2 = absence) and soil type (C = clay, S = sandy) at 7 weeks after transplanting. Treatment means and standard deviation are averaged over the three biosolid levels (B0 = 0 %, B2 = 2 %, B4 = 4 % w/w) Therefore, the biosolid used here is not expected to enrich the receiving soil with toxic metals, at least in short-term usage. These findings encourage the use of this biosolid as a natural and environmentally acceptable amendment since it improved soil fertility that enhanced tomato growth and crop tolerance to the pathogenic fungus. The use of 80 and 160 tons biosolid/ha in the present study is comparable to that reported by Sharma et al. (2017), and as such this is a realistic field scenario.

Conclusions
This study has shown that the addition of a biosolid made from municipal wastewater sludge in an outdoor experiment increased tomato growth as compared to unamended soils. This suggests that this biosolid acts as an organic amendment with fertilizing properties. In addition, there was enhanced tomato tolerance to Forl when the soil was amended with biosolids, which may act as a possible stimulant of tomato tolerance against fungus disease. Moreover, the higher disease index on tomato plants grown outdoors in biosolid plus clay soil substrate than in biosolid plus sandy soil substrate suggests that biosolid activity is affected by soil type. Based on these findings, it could be concluded that the examined biosolid should be considered for its possible use in agriculture according to the principles of circular economy and waste minimization. This biosolid also exerts minimal ecotoxicological impacts, as shown in a previous study. Future research directions should concentrate on elucidating the mechanism of crop tolerance to Forl, because of biosolid addition. This may include investigation of plant defence mechanisms such as induction of defence-related genes in tomato or soil microbiota alterations due to biosolid addition.