Biochar reduces containerized pepper blight caused by Phytophthora capsici

Biochar reduces containerized pepper blight caused by Phytophthora capsici | 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 Article Biochar reduces containerized pepper blight caused by Phytophthora capsici Ping Yu, Kuan Qin, Kevin Crosby, Kevin Ong, Terry Gentry, Mengmeng Gu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4243906/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Phytophthora blight caused by Phytophthora capsici is a serious disease affecting a wide range of plants. Biochar as a soil amendment could partially replace peat moss and has the potential to suppress plant diseases, but its effects on controlling phytophthora blight of container-grown peppers have less been explored, especially in combination of biological control using Trichoderma . In vitro (petri dish) and in vivo (greenhouse) studies were conducted to test sugarcane bagasse biochar (SBB) and mixed hardwood biochar (HB) controlling effects on pepper phytophthora blight disease with and without Trichoderma . Sugarcane bagasse biochar and HB were blended with the commercial substrate (CS, peat-based) at 10% (SBB10, by volume), and 10%, 30%, 50%, 70% (HB10, HB30, HB50, and HB70, by volume), respectively, and CS (CS100) was used as the control. Both in vitro and in vivo studies used randomized complete block design with three treatment factors: pathogen (without or with inoculation of P. capsici ), biochar (different biochar treatments), and Trichoderma (without or with inoculation). In vitro results showed that Trichoderma inhibited P. capsici growth while biochar did not have significant beneficial effects. In vivo results showed that plants grown in HB30 and HB50 had similar or higher plant growth index and shoot dry weight than the control regardless of pathogen presence. In the presence of the pathogen, plants grown in HB30, HB50, and HB70 had significantly lower disease severity, and disease incidence ratings than the control, while Trichoderma did not show beneficial effects on controlling the disease. In conclusion, HB replacing 30% and 50% peat moss in substrate could reduce pepper blight disease caused by P. capsici without negatively affecting plant growth. Biological sciences/Biotechnology Biological sciences/Plant sciences Disease incidence Disease severity Growth index In vitro and in vivio Pathogen inhibition Trichoderma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Phytophthora capsici is a hemi-biotrophic fungal-like oomycete pathogen causing destructive disease across a diverse range of crops in cucurbitaceous , fabaceous , and solanaceous families 1 . Phytophthora blight of pepper caused by P. capsici is one of the gravest soil-borne diseases affecting pepper growth globally 2 . The symptoms of the disease appear on main stem close to the soil line as small brown (early infection) to dark purplish (late infection) water-soaked lesions 3 . Under moist conditions, the disease could affect the whole plant from roots, crown, foliage, to fruit at any growth stages 4 , 5 . Biochar is a carbon-rich pyrolysis by-product derived from a wide variety of organic materials, involves the thermochemical decomposition of biomass under specific time, temperature, and oxygen-depleted or oxygen-limited conditions 6 , 7 . Biochar has been used as a soil amendment due to its positive effects on altering the biological and physicochemical properties of soils 8 , it also has the potential use as a substrate in container nurseries to improve substrate water and nutrient holding capacity, ameliorate acidity, and provide suitable environments for plant growth 9 , 10 . Studies had shown that biochar could partially replace peat moss-based substrate for greenhouse ornamental and vegetable plants production 11 , 12 or used as an additional amendment in substrate to improve crop growth, yield, and quality 13 – 16 . As an organic amendment, biochar has shown the potential to suppress soil-borne diseases, such as Fusarium root rot (caused by Fusarium oxysporum ) of asparagus, bacterial wilt (caused by Ralstonia solanacearum ) of tomato, damping-off and root rot (caused by Rhizoctonia solani ) of cucumber and some ornamental crops 17 , 18 . Studies have also demonstrated that amended soil with biochar suppressed the pathogen abundance of Phytophthora blight of pepper: amended loam and clay loam soil with 3% (w/w) softwood-derived biochar (pH 6.5) improved root and shoot development of sweet pepper that were infested with P. capsici , the population and percentages of root infection by Phytophthora spp. were reduced 19 ; amended sandy loam soil with 1.33% (w/w) corn straw-derived biochar (pH 9.7) before transplanting reduced the disease index of Phytophthora blight of pepper and the population of P. capsici while increased the abundance of beneficial bacteria, such as Bacillus spp., Pseudomonas spp. and Streptomyces spp. 2 ; their following study using the same setup further illustrated the suppression of pepper blight could also due to the improved abundance of potential rhizosphere-associated biocontrol fungi and enhanced soil organic matter, available nutrients (P, K) 20 . Interestingly, they found biochar amendment increased the abundance of Trichoderma that led to a high control efficacy in P. capsici 20 . Trichoderma spp. has been reported as a reliable biological control agent for P. capsici : T. harzianum was proven to suppress pepper root rot caused by P. capsici through antimicrobial substances production 21 ; in a vitro test, T. harzianum inhibited P. capsici by 65.3% 22 ; similarly, T. harzianum , T . viride and T . reesei displayed over 85.5% inhibition of mycelial growth of P. capsici 23 , and T . longibranchiatum , T . asperellum showed the inhibition on P. capsici up to 22.5% 24 . The synergistic use of external Trichoderma in biochar amended soil could further strengthen the controlling effects on P. capsici . However, studies using biochar to control P. capsici were mostly conducted using sandy, loam, or clay soils with low amount of biochar amendment (less than 3% based on weight), while the higher percentage use of biochar to replace soilless substrate (e.g., peat moss) and reduce the severity of pepper blight in container production has been less reported. Our previous study already showed that partially replacing peat moss with biochar could significantly reduce the disease incidence and severity of poinsettia root rot caused by Pythium aphanidermatum 25 . Therefore, to evaluate the controlling effects of different types of biochar and Trichoderma on pepper blight in container pepper production, we conducted both in vitro petri dish tests and in vivo greenhouse trials. Inhibition of P. capsici , disease assessment, and plant growth response were evaluated. This study could provide guidance of using biochar in container soilless substrates production and demonstrate evidence of suppressing soil-borne disease from biochar. Material and methods Biochar-mixed substrate , Trichoderma , and pathogen isolation and propagation Two types of biochar, sugarcane bagasse-derived biochar (SBB; American Biocarbon LLC White Castle, LA, USA) and mixed hardwood-derived biochar (HB; Proton Power Inc. Lenoir City, TN, USA) were used in this study. The SBB was produced using proprietary methods and was provided by USDA-ARS, Sugarcane Research Unit (Houma, LA, USA), it had a pH of 5.9 and an electrical conductivity (EC) of 0.75 dS/m; the HB was a by-product from fast pyrolysis of mixed hardwood and had a pH of 10.1 and an EC of 1.06 dS/m 26,27 . These two types of biochar were separately mixed with the commercial peat moss-based substrates (Jolly Gardener C/20, Oldcastle Lawn & Garden Inc., Atlanta, GA, USA) that contain 80% Canadian Sphagnum peat and 20% perlite. Sugarcane bagasse-derived biochar was mixed with the commercial substrate at a rate of 10% by volume (SBB10), while HB was mixed with the commercial substrate at rates of 10%, 30%, 50%, and 70% by volume (HB10, HB30, HB50, HB70). Commercial substrate without mixing biochar was also included (CS). These substrates were measured for their physical properties based on Fonteno, et al. 28 used the North Carolina State University Porometer (Raleigh, NC, USA), including bulk density, air space, container capacity, and total porosity. Their chemical properties of leachate electrical conductivity (EC) and pH were measured following the pour-through method 29 with a portable EC/pH meter (Hanna Instrument, Woonsocket, RI, USA). The study utilized the Root shield Plus-WP (BioWorks, Victor, NY, USA) that consisted of two active strains of Trichoderma , T. harzianum strain T-22 and T. virens strain G-41, as the biological control agent. Pathogen Phytophthora capsici was isolated and identified from an infected pepper plant, followed by pathogen propagation, where P. capsici was isolated and maintained in the darkness on a V8 juice agar that is selective for oomycete organisms 30 . In vitro test Water extracts of commercial substrate and biochar-mixed substrates Water extracts of commercial substrate and biochar-mixed substrates were obtained following the method outlined by Gravel, et al. 31 . In summary, commercial substrate and five types of biochar-mixed substrate were separately mixed with deionized water in a 1:1 ratio by volume in 500 mL flasks and agitated for 24 hours using a shaker (Orbital; Laboratory Supply Network, Atkinson, NH, USA). The resulting mixtures were filtered through filter papers, and 25 mL of the extracts from each type were collected and sterilized (autoclaved for 1 hour) for the in vitro test. An equal amount of sterilized deionized water was used as a control. The potato dextrose agar (PDA) media was therefore produced in petri dishes (100 × 15 mm) by either incorporating the sterilized deionized water (DI water control) or commercial substrate and biochar-mixed water extracts (CS, SBB10, HB10, HB30, HB50, HB70) into a 25% PDA sterilized solution before the media solidified. Trichoderma treatment and pathogen growth Half of the PDA petri dishes produced using different water extracts were inoculated with 5 mm square plugs of actively growing P. capsici in the center of each petri dish, while the other half petri dishes were introduced with dual confrontation technique for the inoculation of Trichoderma and P. capsici 32 . Specifically, we placed a drop of Trichoderma -containing solution mixed at the recommended rate by the manufacturer (0.30 g/L) opposite to a 5 mm square plug of actively growing P. capsici within the petri dish, such that both elements positioned at equal distances from each other and to the border of the petri dish. Next, all the petri dishes were placed in a dark environment at room temperature. After four days, the radial growth of mycelium was measured, and the inhibition percentage of pathogen growth was then computed using this formula from Nawaz, et al. 23 : Inhibition = ((A1 - A2) × 100%) / A2, where A1 represents the area of pathogen growth in commercial substrate or biochar-mixed water extracts, and A2 represents the area of pathogen growth in the DI water control. In vivo greenhouse trial Plant materials, treatment setup, and growth environment Hot cherry pepper ( Capsicum annuum cv. ‘Capperino’) seeds (F1 self-selected seeds from Johnny’s Selected Seeds, Fairfield, ME, USA) were pretreated with 10% bleach for 3 minutes and then rinsed with DI water. Treated-seeds were sown in the commercial propagation media (BM2 Berger; Saint-Modeste, Quebec, Canada) and grown for 3 weeks until the true leaves came out. Uniform seedlings were then transplanted into growing pots with dimensions of 7.5 cm at the top, 6 cm at the bottom, and 8.2 cm in depth, with a total volume of 375 mL. These pots were previously filled with either commercial or biochar-mixed substrates. After transplanting, slow-released fertilizer (15N-4P-10K Osmocote Plus; Scotts-Sierra Horticultural Products Company, Marysville, OH, USA) was incorporated with the surface substrate at the rate of 450 g/m 2 (or 2 g/container). One week after transplanting, Trichoderma treatment was introduced by drenching solution to the substrate surface at the rate of 0.30 g/L. Pathogen P. capsici was inoculated with actively growing mycelium at the rate of 5 plugs with plastic inoculation loops (VWR, Radnor, PA, USA). When conducted pathogen inoculation, a 5-mm diameter agar plug was taken from the margin of an actively growing colony of the pathogen and placed on the surface of the substrate contacting plant stem. Standard propagation trays were placed underneath the pots to create a moisturized environment. All the pots were placed in a P. capcisi -permitted greenhouse at Texas A&M University, Sommerville, TX, USA. During the experiment, plants were regularly watered. Inside the greenhouse, the average temperature was 30.2°C, with a relative humidity of 77.2% and a dew point of 25.0°C. Plant growth measurements and disease assessment From 3 weeks after transplanting, weekly measurements were conducted for plant height (PH) and two-sided widths (PW1 and PW2). Plant growth index (GI) was then calculated by: GI = PH/2 + (PW1 + PW2)/4 25 . Pepper plants shoots were harvested and dried at 80°C for 3 days in the oven at the end of the experiment, shoot dry weight (SDW) was recorded thereafter. Symptoms of the disease were monitored and documented every 5 days, starting 3 days after inoculating the pathogen. Disease severity was assessed on a scale of 0–4, following the methodology outlined by Wang, et al. 2 . The scales were illustrated in Fig. 1 : 0 indicated healthy plants, 1 represented plants with small brown lesions on the stem or slightly wilted leaves, 2 denoted plants with moderate brown lesions on the stem and moderate wilted leaves, 3 indicated plants with large brown lesions on the stem and significantly wilted leaves, and 4 represented dead plants. Disease Severity Index (DS) was calculated using the following formula: DS = \(\sum ( \frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{d}\text{i}\text{s}\text{e}\text{a}\text{s}\text{e}\text{d} \text{p}\text{l}\text{a}\text{n}\text{t}\text{s} \text{i}\text{n} \text{t}\text{h}\text{i}\text{s} \text{i}\text{n}\text{d}\text{e}\text{x}\hspace{0.17em}\times \hspace{0.17em}\text{d}\text{i}\text{s}\text{e}\text{a}\text{s}\text{e} \text{i}\text{n}\text{d}\text{e}\text{x} \text{r}\text{a}\text{t}\text{i}\text{n}\text{g} \text{f}\text{r}\text{o}\text{m} 0 \text{t}\text{o} 4}{4\hspace{0.17em}\times \hspace{0.17em}\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{p}\text{l}\text{a}\text{n}\text{t}\text{s} \text{i}\text{n}\text{v}\text{e}\text{s}\text{t}\text{i}\text{g}\text{a}\text{t}\text{e}\text{d}} )\times 100\%\) 2 . The disease severity obtained at different times after inoculation was used to calculate areas under disease progress curves (AUDPC) following the formula: \(\text{A}\text{U}\text{D}\text{P}\text{C}={\sum }_{i=0}^{n-1}\frac{({y}_{i}+{y}_{i+1})}{2}\left({t}_{i+1}-{t}_{i}\right)\) . Where \({y}_{i}\) is the scale rating at the \(i\) th observation, \({t}_{i}\) is the day of the \(i\) th observation, and \(n\) is the total number of observations 33 . During the trials, Disease Incidence (DI) was assessed by tallying the number of diseased plants in each pot, following the specified formula: DI = \(\frac{number of diseased plants}{number of total plants}\) × 100 34 . Experimental design and data analysis The in vitro test followed a randomized complete block design (RCBD) with five blocks. Within each block, treatments were randomly assigned to petri dishes based on a 7 x 2 factorial design involving substrate treatments (water extracts of CS100, SBB10, HB10, HB30, HB50, HB70, and DI water as control) and Trichoderma application (with/without). In the greenhouse experiment, an RCBD with eight blocks was utilized. Each block consisted of a 1.5 m x 1 m area on a raised bench. Treatments were randomly allocated to pots within each block, following a 6 x 2 x 2 factorial design involving substrate treatments (CS100 as control, SBB10, HB10, HB30, HB50, and HB70 as biochar-mixed treatments), Trichoderma application (with/without), and pathogen presence (non-pathogen, pathogen-inoculated). The pathogen growth area in the in vitro test was estimated using Image J, version 1.53a 35 . Data analysis was conducted using one-way analysis of variance (ANOVA) in R program software, version 3.5.1 36 . In the greenhouse trial, treatments with non-pathogens and those inoculated with pathogens were analyzed separately. Means were separated using Dunnett’s test when treatments significantly differed from the control at p ≤ 0.05 or the least significant difference (LSD) when treatments significantly differed from each other at p ≤ 0.05. Results Substrate physical and chemical properties The majority of the mixes met the recommended range for container substrate properties, as detailed in Yeager, et al. 37 , with the exception of the bulk density in all the substrates, which were lower than the recommended value (Table 1 ). The HB50 and HB70 mixes exhibited significantly lower total porosity, container compacity, and bulk density compared to the control (CS100). All the HB mixes showed significantly higher pH levels than the control, and except for HB10 and HB30, all biochar-mixed substrates displayed significantly lower EC levels than the control. Table 1 Physical and chemical properties of commercial and biochar-mixed substrates: total porosity (TP), container compacity (CC), air space (AS), bulk density (BD), pH and electrical conductivity (EC). Substrates TP (%) CC (%) AS (%) BD (g/cm 3 ) pH EC (dS/m) CS100 74 56 18 0.10 6.8 2.06 SBB10 73 61 13 0.10 6.6 1.07*** HB10 72 54 17 0.09 7.5*** 1.96 HB30 70 52 18 0.11** 7.9*** 1.83 HB50 68* 50* 18 0.12*** 8.0*** 1.58** HB70 68* 47*** 21 0.13*** 8.4*** 1.40 *** Suitable range a 50–80 45–65 10–30 0.19–0.7 5.4–6.5 < 1.5 SBB = Sugarcane bagasse biochar, HB = Mixed hardwood biochar, CS = Peat moss based commercial substrate. Numbers after CS, SBB, and HB indicated the ratio of different components, by volume *, **, and *** indicates significant difference from the commercial substrate (CS100) according to Dunnett’s test at p ≤ 0.1, 0.05, and 0.01, respectively. a Recommended container substrate properties 37 . Pathogen growth from in vitro test Results from the in vitro test showed that without Trichoderma present, the extracts from biochar-mixed substrate showed no significant difference in inhibiting P. capsici growth compared to CS extracts (Fig. 2 , 3 A). However, HB10 extracts exhibited positive inhibition values, indicating stimulation of P. capsici growth, while all other extracts displayed negative inhibition values, indicating suppression of the pathogen. Among all the pathogen-suppressed biochar extracts, HB30 had a significantly higher inhibition percentage compared with SBB10. When Trichoderma was present, all the extracts effectively suppressed P. capsici growth, while the biochar-mixed substrate extracts showed no significant difference in inhibiting P. capsici growth compared to the CS extracts, except for HB70, which exhibited a relatively lower inhibition percentage (Fig. 2 , 3 B). Plant growth from in vivo greenhouse trial There were no significant interaction effects between substrate and Trichoderma treatments on any of the growth parameters regardless the presence or absence of the pathogen (Table 2 ). For non-pathogen treatments, pepper plant growth index from 3 weeks after transplanting and final shoot dry weight were both significantly influenced by substrate and Trichoderma treatments. However, plant growth index from 4 and 5 weeks after transplanting was only significantly influenced by substrate treatment. Under pathogen-inoculated conditions, none of the factors significantly influenced any of the growth parameters. Table 2 Pepper plant growth index (GI) from 3, 4, and 5 weeks after transplanting (3, 4, 5 WAT) and final shoot dry weight (SDW) as affected by substrate and Trichoderma treatment factors under non-pathogen and pathogen-inoculated conditions. Factors GI-3WAT GI-4WAT GI-5WAT SDW Non-Pathogen Substrate * * ** ** Trichoderma *** NS NS *** Substrate × Trichoderma NS NS NS NS Pathogen-inoculated Substrate NS NS NS NS Trichoderma NS NS NS NS Substrate × Trichoderma NS NS NS NS NS means not significant. *, **, *** indicate significance at p ≤ 0.05, 0.01, and 0.001, respectively. Biochar types and application rates did not significantly affect the shoot dry weights of healthy pepper plants (non-pathogen), whether with or without Trichoderma , except for HB70 mix, which showed significantly lower biomass compared to the control in both non- Trichoderma and Trichoderma -treated conditions (Fig. 4 ). Furthermore, Biochar did not significantly influence the growth index from 3 and 4 weeks after transplanting. However, plants grown under HB30 and HB50 substrates showed significantly higher growth index from 5 weeks after transplanting compared to the control (Fig. 5 A). The use of Trichoderma did not have a significant impact on any of the growth indexes (Fig. 5 B). While under pathogen-inoculated conditions, neither substrate nor Trichoderma treatments had significant impacts on pepper plant growth index and biomass accumulation (date not shown). Disease development from in vivo greenhouse trial Under pathogen-inoculate conditions, plants grown in all the treatments showed disease symptoms 3 days after transplanting except for HB70, which showed symptoms 7 days after transplanting (Fig. 6 A). Compared with CS100 treatment, HB50 and HB70 treatments reduced disease severity at 12 days after transplanting by 10.94% and 10.16%, respectively, and at 17 days after transplanting by 9.59% and 9.59%, respectively. The application of Trichoderma did not significantly reduce disease severity during the entire experiment (Fig. 6 B). Biochar-mixed substrates had significant impacts on disease incidence, especially HB-amended (30%-70%) mixes (Fig. 7 A). Compared with CS100 treatment, HB50, HB70, and SBB10 treatments reduced disease incidence at 7 days after transplanting by 25.0%, 25.0%, and 18.8%, respectively, and at 12 days after transplanting by 25.0%, 18.8%, and 6.3%, respectively. The application of Trichoderma did not significantly reduce disease incidence during the entire experiment (Fig. 7 B). All the biochar-mixed substrates had significantly lower AUDPC values (except for HB10) than the CS100. The HB50 and HB70 mixes reduced the AUDPC value by 9.6 and 9.4 respectively (Fig. 8 A). The application of Trichoderma did not significantly reduce AUDPC during the entire experiment (Fig. 8 B). Discussions Effects of biochar on controlling pathogen growth and disease development involve both direct and indirect mechanisms. Directly, chemical compounds naturally contained in biochar, such as benzoic acid, ethylene glycol, propylene glycol, hydroxypropionic acid, quinones, or some specific phenols and carboxylic acids that generated during the pyrolysis process, these compounds have shown the capacity to inhibit microbial growth; indirectly, synergistic effects from biochar's physical and chemical properties could lead to the beneficial outcomes: biochar absorbs and deactivates toxic metabolites or enzymes (e.g., cell wall degrading enzymes) produced by pathogens, reducing their virulence, boosting plant growth, inducing systemic plant resistance, and enhancing the abundance and activity of beneficial bacteria and fungi 2 , 18 , 20 , 38 – 40 . In vitro test isolated the physical properties of biochar by only testing the effects of chemical compounds on P. capsici development using water extracts from biochar-mixed substrates. Results showed that the extracts from high percentage biochar mixes suppressed P. capsici growth when using hardwood as the raw material. Our previous study found that used the water extracts from hardwood-derived biochar led to neutral or negative effects on suppressing pathogen Pythium aphanidermatum growth in poinsettia 25 , indicated the chemical compounds from the same type of biochar could impose different effects on different soil-borne pathogens. Different types of raw material-derived biochar could also lead to different pathogen controlling effects, as eucalyptus wood and organic waste-derived biochar extracts showed no effects on suppressing Fusarium colonization 41 . In the presence of Trichoderma , the inhibiting effects of biochar-mixed extracts on P. capsici growth were enhanced due to Trichoderma’ s biocontrol impact on suppressing the growth of P. capsici . This in vitro test showed that synergistically using the chemical compounds extracted from biochar and biocontrol components could provide positive effects on controlling pathogen P. capsici . In general, increasing biochar application rate improved the disease suppression percentage, and using hull, wood, straw and organic waste as the raw material has better controlling effects for disease compared to using bark as the raw material 42 . Results from the in vivo greenhouse study showed that compared with commercial peat-based substrate and sugarcane-derived biochar, using high-percentage hardwood-derived biochar significantly reduced disease incidence, severity, and disease intensity over time (AUDPC) of P. capsici induced Phytophthora blight. Although the highest percentage biochar-mix (HB70) significantly decreased the final plant biomass, using a moderate rate of biochar (HB50) maintained and even promoted plant growth index. Biochar application has been reported to improve crop biomass and yield by enhancing soil structure with increased nutrient availability and water holding capacity, immobilize inorganic (heavy metals) and organic contaminants, and reduce abiotic and biotic stresses 43 . Our results have further encouraged the use of biochar as a substitute for peat-based substrates 12 , offering additional benefits in suppressing soil-borne diseases due to its chemical and physical properties. Unlike the in vitro test, Trichoderma treatment only numerically decreased the disease development in the greenhouse study. And the interaction effects between biochar and Trichoderma were marginally significant, indicating that under the in vivo conditions, other factors could reduce the beneficial effects from Trichoderma . In order to better understand the complexed effects of Trichoderma and two types of biochar on plant growth and disease development, we employed a principal component analysis (PCA) to illustrate the distinct variables influenced by different treatment factors. For non-pathogen plants, 86% of the variability was explained by the first two components (Fig. 9 A). PC1 accounted for 65% variance, with HB50-TN (HB50 without Trichoderma ), HB70-TN, HB70-TY (HB70 with Trichoderma ), and CS100-TN differing from the rest of treatments. Biochar-mixed substrate at relatively lower rates (HB10-TN, HB10-TY, HB30-TN, HB30-TY, and HB50-TY) along with CS100 were associated more with the yield (SDW) and GIs (GI WK3, GI WK4, and GI WK5). PC2 accounted for 21% variance, distinguishing the CS100 and SBB10 from HB mixes. Commercial substrate and SBB-amended mixes tended to be affiliated with plant biomass and GI WK3 and GI WK4, however, HB30, HB50 mixes appeared to be related to GI WK5. For pathogen-inoculated plants, 87% of the variability was explained by the first two components (Fig. 9 B). PC1 accounted for 63% variance, differing biochar-mixed substrate with Trichoderma (except for HB30-TN) and CS100-TY from those without Trichoderma treatments. The treatment HB30-TN and CS100 were associated more with the yield (SDW) and plant late growth (GI WK4 and GI WK5). PC2 accounted for 24% variance, distinguishing mixes without Trichoderma from those with Trichoderma . Commercial substrate and biochar-amended mixes with Trichoderma tended to be affiliated with GI WK3 while HB30-TN appeared to be related to GI WK4. For pathogen-inoculated plants, 94% of the variability was explained by the first two components (Fig. 10 ). PC1 accounted for 77% variance, differing CS100, HB10, HB70-TN, and SBB10-TY mixes from the rest biochar-amended treatments. The treatments CS100, HB10, HB70-TN, and SBB10-TY were positively associated with all the disease parameters while the rest of the treatments were negatively associated with them. PC2 accounted for 17% variance, distinguishing CS100, SBB10-TY, HB30, HB50-TY and HB70-TY mixes from the rest of the treatments. CS100-TY, SBB10-TY, HB30-TY, and HB30-TN mixes tended to be affiliated with DI1, DS1, DS2 and AUDPC while HB10-TN, HB10-TY, and HB70-TN, appeared to be related to DI2, DI3, DI4, DS3, and DS4. This indicated that Trichoderma -treated plant (TY) tended to have an early disease development, while during the plant late growth period, Trichoderma positively suppressed disease development and intensity when biochar was present. Although studies testing the effects of combined use of biochar with other materials on pathogen suppression have been less reported, promising research had shown that the combined use of compost and biochar could suppress Rhizoctonia solani induced disease 44 , and the combined use of biochar and arbuscular mycorrhizal fungi could control Fusarium oxysporum by altering root exudates 43 . Future studies could focus on exploring more reliable bioactive materials that could be combined used with biochar to promote the synergistic effects. Conclusions The mixed hardwood biochar blended with commercial peat moss-based substrate at 50% and 70% (by volume) could significantly reduce Phytophthora pepper blight disease severity, incidence, and intensity over time. The chemical compounds contained in biochar could contribute to its disease inhibition capability for P. capsici to a certain extent. In the absence and presence of the pathogen, mixed hardwood biochar could replace commercial peat moss-based substrate up to 50% (by volume) for container pepper plant production without negative impact on its growth. Combined use of moderate rate of biochar amendment (up to 50% by volume) and Trichoderma could improve plant growth while reducing pathogen P. capsici growth. Declarations Data Availability Statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments We thank Dr. Thomas Isakeit from the Department of Plant pathogen and Microbiology, Texas AM University for assisting in the isolation and identification of P. capsici . We would also like to thank Dr. Lan Zhou for her statistical consultant. Author Contributions Statement This work was a product of the combined effort of all the authors. All authors conceptualized and designed the study. Ping Yu performed the experiments, collected and analysed the data, and wrote the manuscript with assistance from all other authors, mainly Mengmeng Gu. Kuan Qin, Kevin Crosby and Kevin Ong provided technical advice and assistance when the study was conducted and revised and improved the manuscript. Terry Gentry revised the manuscript. All authors have read and agreed to the published version of the manuscript. Additional Information Funding This research received no external funding. Competing Interests Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Kousik, C. S., Parada, C. & Quesada-Ocampo, L. First Report of Phytophthora Fruit Rot on Bitter Gourd (Mormodica charantia) and Sponge Gourd (Luffa cylindrica) Caused by Phytophthora capsici. Plant Health Progress 16, 93–94, doi: 10.1094/php-br-15-0005 (2015). Wang, G. et al. Suppression of Phytophthora blight of pepper by biochar amendment is associated with improved soil bacterial properties. Biol. Fertil. Soils 55, 813–824, doi: 10.1007/s00374-019-01391-6 (2019). Erwin, D. C. & Ribeiro, O. K. Phytophthora diseases worldwide . (American Phytopathological Society (APS Press), 1996). Lamour, K. H., Stam, R., Jupe, J. & Huitema, E. The oomycete broad-host-range pathogen Phytophthora capsici. Molecular Plant Pathology 13, 329–337, doi: https://doi.org/10.1111/j.1364-3703.2011.00754.x (2012). Parada-Rojas, C. H. & Quesada-Ocampo, L. M. Characterizing Sources of Resistance to Phytophthora Blight of Pepper Caused by Phytophthora capsici in North Carolina. Plant Health Progress 20, 112–119, doi: 10.1094/php-09-18-0054-rs (2019). Demirbas, A. & Arin, G. An Overview of Biomass Pyrolysis. Energy Sources 24, 471–482, doi: 10.1080/00908310252889979 (2002). Singh, B., Singh, B. P. & Cowie, A. L. Characterisation and evaluation of biochars for their application as a soil amendment. Soil Research 48, 516–525 (2010). Brassard, P. et al. in Char and carbon materials derived from biomass 109–146 (Elsevier, 2019). Dumroese, R. K., Heiskanen, J., Englund, K. & Tervahauta, A. Pelleted biochar: Chemical and physical properties show potential use as a substrate in container nurseries. Biomass and Bioenergy 35, 2018–2027, doi: https://doi.org/10.1016/j.biombioe.2011.01.053 (2011). Vaughn, S. F., Kenar, J. A., Thompson, A. R. & Peterson, S. C. Comparison of biochars derived from wood pellets and pelletized wheat straw as replacements for peat in potting substrates. Ind. Crop. Prod. 51, 437–443, doi: https://doi.org/10.1016/j.indcrop.2013.10.010 (2013). Yu, P. et al. The Effects of Mixed Hardwood Biochar, Mycorrhizae, and Fertigation on Container Tomato and Pepper Plant Growth. Sustainability 12, 7072 (2020). Huang, L., Yu, P. & Gu, M. Evaluation of Biochar and Compost Mixes as Substitutes to a Commercial Propagation Mix. Appl. Sci. 9, 4394 (2019). Méndez, A., Cárdenas-Aguiar, E., Paz-Ferreiro, J., Plaza, C. & Gascó, G. The effect of sewage sludge biochar on peat-based growing media. Biol. Agric. Hortic. 33, 40–51, doi: 10.1080/01448765.2016.1185645 (2017). Nieto, A. et al. The effect of pruning waste and biochar addition on brown peat based growing media properties. Sci Hortic. 199, 142–148, doi: https://doi.org/10.1016/j.scienta.2015.12.012 (2016). Tian, Y. et al. Biochar made from green waste as peat substitute in growth media for Calathea rotundifola cv. Fasciata. Sci Hortic. 143, 15–18, doi: https://doi.org/10.1016/j.scienta.2012.05.018 (2012). Zhang, L., Sun, X.-y., Tian, Y. & Gong, X.-q. Biochar and humic acid amendments improve the quality of composted green waste as a growth medium for the ornamental plant Calathea insignis. Sci Hortic. 176, 70–78, doi: https://doi.org/10.1016/j.scienta.2014.06.021 (2014). Frenkel, O. et al. The effect of biochar on plant diseases: what should we learn while designing biochar substrates? Journal of Environmental Engineering and Landscape Management 25, 105–113 (2017). Graber, E. R., Frenkel, O., Jaiswal, A. K. & Elad, Y. How may biochar influence severity of diseases caused by soilborne pathogens? Carbon Management 5, 169–183, doi: 10.1080/17583004.2014.913360 (2014). Shoaf, N., Hoagland, L. & Egel, D. S. Suppression of Phytophthora Blight in Sweet Pepper Depends on Biochar Amendment and Soil Type. Hortscience. 51, 518–524, doi: 10.21273/hortsci.51.5.518 (2016). Wang, G. et al. Biochar-Mediated Control of Phytophthora Blight of Pepper Is Closely Related to the Improvement of the Rhizosphere Fungal Community. Front. Microbiol. 11, doi: 10.3389/fmicb.2020.01427 (2020). Ezziyyani, M., Requena, M. E., Egea-Gilabert, C. & Candela, M. E. Biological Control of Phytophthora Root Rot of Pepper Using Trichoderma harzianum and Streptomyces rochei in Combination. Journal of Phytopathology 155, 342–349, doi: https://doi.org/10.1111/j.1439-0434.2007.01237.x (2007). Mousumi Das, M., Haridas, M. & Sabu, A. Biological control of black pepper and ginger pathogens, Fusarium oxysporum, Rhizoctonia solani and Phytophthora capsici, using Trichoderma spp. Biocatal Agric Biotechnol. 17, 177–183, doi: https://doi.org/10.1016/j.bcab.2018.11.021 (2019). Nawaz, K. et al. Diversity of Trichoderma species in chili rhizosphere that promote vigor and antagonism against virulent Phytophthora capsici. Sci Hortic. 239, 242–252, doi: https://doi.org/10.1016/j.scienta.2018.05.048 (2018). De la Cruz-Quiroz, R., Roussos, S., Rodríguez-Herrera, R., Hernandez-Castillo, D. & Aguilar, C. N. Growth inhibition of Colletotrichum gloeosporioides and Phytophthora capsici by native Mexican Trichoderma strains. Karbala International Journal of Modern Science 4, 237–243, doi: https://doi.org/10.1016/j.kijoms.2018.03.002 (2018). Yu, P., Ong, K., Ueckert, J. & Gu, M. Biochar and Trichoderma Reduce Containerized Poinsettia Root Rot Caused by Pythium aphanidermatum. Hortscience. 58, 846–854, doi: 10.21273/hortsci17203-23 (2023). Yu, P., Li, Q., Huang, L., Niu, G. & Gu, M. Mixed Hardwood and Sugarcane Bagasse Biochar as Potting Mix Components for Container Tomato and Basil Seedling Production. Appl. Sci. 9, 4713 (2019). Webber III, C. L. et al. Sugarcane and pine biochar as amendments for greenhouse growing media for the production of bean ( Phaseolus vulgaris L.) seedlings. Journal of Agricultural Science 10, 58–68, doi: https://doi.org/10.5539/jas.v10n4p58 (2018). Fonteno, W., Hardin, C. & Brewster, J. Procedures for determining physical properties of horticultural substrates using the NCSU Porometer. Horticultural Substrates Laboratory, North Carolina State University (1995). LeBude, A. & Bilderback, T. The pour-through extraction method: A nutrient management tool for nursery crops. NC State Univ. Coop. Ext (2009). Parada-Rojas, C. H. et al. A Diagnostic Guide for Phytophthora capsici Infecting Vegetable Crops. Plant Health Progress 22, 404–414, doi: 10.1094/php-02-21-0027-fi (2021). Gravel, V., Dorais, M. & Ménard, C. Organic potted plants amended with biochar: its effect on growth and Pythium colonization. Can. J. Plant Sci. 93, 1217–1227, doi: 10.4141/cjps2013-315 (2013). Sinclair, J. B. & Dhingra, O. D. Basic plant pathology methods . (CRC press, 2017). Madden, L. V., Hughes, G. & van den Bosch, F. The Study of Plant Disease Epidemics . (2017). Bellini, A. et al. A Compost Treatment Acts as a Suppressive Agent in Phytophthora capsici – Cucurbita pepo Pathosystem by Modifying the Rhizosphere Microbiota. Front.Plant Sci. 11, doi: 10.3389/fpls.2020.00885 (2020). Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682, doi: 10.1038/nmeth.2019 (2012). R-4.3.1 for Windows (R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org . [accessed 10 Sep 2023], 2023). Yeager, T. et al. Best management practices: Guide for producing container-grown plants. Southern Nursery Association. Atlanta, GA 7 (1997). Bonanomi, G., Ippolito, F. & Scala, F. A “black” future for plant pathology? Biochar as a new soil amendment for controlling plant diseases. Journal of Plant Pathology 97, 223–234, doi: 10.4454/jpp.v97i2.3381 (2015). Klinke, H. B., Thomsen, A. B. & Ahring, B. K. Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol. 66, 10–26, doi: 10.1007/s00253-004-1642-2 (2004). Jaiswal, A. K., Frenkel, O., Tsechansky, L., Elad, Y. & Graber, E. R. Immobilization and deactivation of pathogenic enzymes and toxic metabolites by biochar: A possible mechanism involved in soilborne disease suppression. Soil Biol. Biochem. 121, 59–66, doi: https://doi.org/10.1016/j.soilbio.2018.03.001 (2018). Jaiswal, A. K. et al. Linking the Belowground Microbial Composition, Diversity and Activity to Soilborne Disease Suppression and Growth Promotion of Tomato Amended with Biochar. Sci Rep 7, 44382, doi: 10.1038/srep44382 (2017). Iacomino, G., Idbella, M., Laudonia, S., Vinale, F. & Bonanomi, G. The Suppressive Effects of Biochar on Above- and Belowground Plant Pathogens and Pests: A Review. Plants (Basel) 11, doi: 10.3390/plants11223144 (2022). Poveda, J., Martínez-Gómez, Á., Fenoll, C. & Escobar, C. The Use of Biochar for Plant Pathogen Control. Phytopathology® 111, 1490–1499, doi: 10.1094/phyto-06-20-0248-rvw (2021). Debode, J. et al. Has compost with biochar added during the process added value over biochar or compost to increase disease suppression? Appl. Soil Ecol. 153, 103571, doi: https://doi.org/10.1016/j.apsoil.2020.103571 (2020). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 29 Jul, 2024 Reviewers agreed at journal 15 May, 2024 Reviews received at journal 02 May, 2024 Reviewers agreed at journal 25 Apr, 2024 Reviewers invited by journal 25 Apr, 2024 Editor assigned by journal 25 Apr, 2024 Editor invited by journal 22 Apr, 2024 Submission checks completed at journal 22 Apr, 2024 First submitted to journal 09 Apr, 2024 You are reading this latest preprint version 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4243906","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":294049015,"identity":"db6e6688-91c4-4460-86fb-b65a60965c19","order_by":0,"name":"Ping Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYDACZjBpA+HwkKAljRQtEHCYBC3m7LyHX/O2nU+c736A8cHbNiK0WDbzpVnztt1O3HgmgdlwLjFaDA7zmBmDtTQksEnzkqDlXOLG/gfsv4nVYvyYt+1A4nyJBDZmorRYNvOYMc45l2y8QeJhs+Scc0RoMec/Y/zhTZmd7Pz+5INABjEOY2BgkwJFh8EBxgYi1EO0MH/8AWTIE6lhFIyCUTAKRiAAABfaNVfEhobVAAAAAElFTkSuQmCC","orcid":"","institution":"University of Georgia","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Yu","suffix":""},{"id":294049016,"identity":"ec226241-ce86-4654-8f47-4a9c8d2e182d","order_by":1,"name":"Kuan Qin","email":"","orcid":"","institution":"University of Georgia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kuan","middleName":"","lastName":"Qin","suffix":""},{"id":294049026,"identity":"c8f75f43-af1c-4f78-ac59-2d28d1d31e33","order_by":2,"name":"Kevin Crosby","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Crosby","suffix":""},{"id":294049027,"identity":"86b5b05b-8d25-4604-86ea-5c593eee482b","order_by":3,"name":"Kevin Ong","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Ong","suffix":""},{"id":294049028,"identity":"e978e806-362f-4216-b2d9-c9e32d2f07af","order_by":4,"name":"Terry Gentry","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Terry","middleName":"","lastName":"Gentry","suffix":""},{"id":294049029,"identity":"3e4de5af-e515-467a-8bc8-0ccc6b410649","order_by":5,"name":"Mengmeng Gu","email":"","orcid":"","institution":"Colorado State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengmeng","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2024-04-09 20:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4243906/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4243906/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-76712-3","type":"published","date":"2024-12-28T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55285871,"identity":"25ab13ae-68cd-4f6b-8464-e758f94cc8be","added_by":"auto","created_at":"2024-04-25 08:02:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2781145,"visible":true,"origin":"","legend":"\u003cp\u003eVisual scales used for the pepper blight caused by \u003cem\u003ePhytophthora capcisi\u003c/em\u003e disease severity rating used in this study. 0 indicated healthy plants, 1 represented plants with small brown lesions on the stem or slightly wilted leaves, 2 denoted plants with moderate brown lesions on the stem and moderate wilted leaves, 3 indicated plants with large brown lesions on the stem and significantly wilted leaves, and 4 represented dead plants.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/fc05c4e45e058ae0cf34e999.png"},{"id":55285870,"identity":"e1d4a271-cca5-4ccc-9634-fc8a7497b496","added_by":"auto","created_at":"2024-04-25 08:02:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39749,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition percentage of \u003cem\u003ePhytophthora capsici\u003c/em\u003efrom in vitro test (n = 5) without (A) and with the presence of \u003cem\u003eTrichoderma \u003c/em\u003e(B), values with the same letters are not significantly different based on LSD's multiple comparison test at p ≤ 0.05. CS100 represents water extract from peat moss-based commercial substrate; SBB10, HB10, HB30, HB50, and HB70 represent water extracts from 10% sugarcane bagasse biochar, 10%, 30%, 50%, and 70% mixed hardwood biochar-mixed substrates (by volume), respectively.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/350906341e8f410b32a04f2d.png"},{"id":55286476,"identity":"464a1cb0-688b-4eee-926d-b2f718bdb932","added_by":"auto","created_at":"2024-04-25 08:10:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1095751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhytophthora capsici\u003c/em\u003e grown on potato dextrose agar (PDA) media without (A) and with the presence of \u003cem\u003eTrichoderma \u003c/em\u003e(B) after four days in the dark environment and room temperature. CS100 represents water extract from peat moss-based commercial substrate; SBB10, HB10, HB30, HB50, and HB70 represent water extracts from 10% sugarcane bagasse biochar, 10%, 30%, 50%, and 70% mixed hardwood biochar-mixed substrates (by volume), respectively. DI water indicates deionized water as the control.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/90b422433b6dfd71fd1b4cff.png"},{"id":55286477,"identity":"8783ce10-fa01-4394-8c5f-a81f8e23b7d2","added_by":"auto","created_at":"2024-04-25 08:10:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":35105,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of substrate on pepper plant shoot dry weight under non-pathogen conditions without (A) or with (B) \u003cem\u003eTrichoderma \u003c/em\u003eapplication. CS100 represents water extract from peat moss-based commercial substrate; SBB10, HB10, HB30, HB50, and HB70 represent water extracts from 10% sugarcane bagasse biochar, 10%, 30%, 50%, and 70% mixed hardwood biochar-mixed substrates (by volume), respectively. *, ** indicate significantly different from the control (CS100) according to the Dunnett test at \u003cem\u003ep\u003c/em\u003e ≤ 0.05, 0.01.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/d28875e8170f6594a627d899.png"},{"id":55285879,"identity":"c339d965-e03a-4b7e-9bbc-1b64eaf4f5dc","added_by":"auto","created_at":"2024-04-25 08:02:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43626,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of substrate on pepper plant growth index at week 3, 4 and 5 after transplanting (WK3, WK4, and WK5) under non-pathogen conditions without (A) or with (B) \u003cem\u003eTrichoderma \u003c/em\u003eapplication. CS100 represents water extract from peat moss-based commercial substrate; SBB10, HB10, HB30, HB50, and HB70 represent water extracts from 10% sugarcane bagasse biochar, 10%, 30%, 50%, and 70% mixed hardwood biochar-mixed substrates (by volume), respectively. * indicates significantly different from the control (CS100) according to the Dunnett test at \u003cem\u003ep\u003c/em\u003e≤ 0.05.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/2abd3e1a6b2da29fc6c925e5.png"},{"id":55285877,"identity":"248d42be-ee32-43ef-b439-c1c4a786091a","added_by":"auto","created_at":"2024-04-25 08:02:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":80704,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of substrate (A) and \u003cem\u003eTrichoderma \u003c/em\u003e(B) treatments on disease severity under pathogen-inoculate conditions. CS100 represents water extract from peat moss-based commercial substrate; SBB10, HB10, HB30, HB50, and HB70 represent water extracts from 10% sugarcane bagasse biochar, 10%, 30%, 50%, and 70% mixed hardwood biochar-mixed substrates (by volume), respectively. The same letter indicates not significantly different from each other on the same day according to LSD multiple comparison test at \u003cem\u003ep \u003c/em\u003e≤ 0.05.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/34be39ca56ee619828384e68.png"},{"id":55285872,"identity":"5b4c3a40-a1bc-4b43-a169-45f130d4c403","added_by":"auto","created_at":"2024-04-25 08:02:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":81670,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of substrate (A) and \u003cem\u003eTrichoderma \u003c/em\u003e(B) treatments on disease incidence under pathogen-inoculate conditions. CS100 represents water extract from peat moss-based commercial substrate; SBB10, HB10, HB30, HB50, and HB70 represent water extracts from 10% sugarcane bagasse biochar, 10%, 30%, 50%, and 70% mixed hardwood biochar-mixed substrates (by volume), respectively. The same letter indicates not significantly different from each other on the same day according to LSD multiple comparison test at \u003cem\u003ep \u003c/em\u003e≤ 0.05.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/f1325005e2f5f0a74abb73ee.png"},{"id":55285878,"identity":"4f8f9f76-7fe1-47ba-af51-458ee5411d4a","added_by":"auto","created_at":"2024-04-25 08:02:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":30512,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of substrate (A) and \u003cem\u003eTrichoderma \u003c/em\u003e(B) treatments on the area under disease progress curve (AUDPC) under pathogen-inoculate conditions. CS100 represents water extract from peat moss-based commercial substrate; SBB10, HB10, HB30, HB50, and HB70 represent water extracts from 10% sugarcane bagasse biochar, 10%, 30%, 50%, and 70% mixed hardwood biochar-mixed substrates (by volume), respectively. The same letter indicates not significantly different from each other on the same day according to LSD multiple comparison test at \u003cem\u003ep \u003c/em\u003e≤ 0.05.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/b1b72b69308c26cdebe535fa.png"},{"id":55286475,"identity":"86860c56-3516-4f8c-9af5-e42b362164fd","added_by":"auto","created_at":"2024-04-25 08:10:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":57000,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) depicting the relationships between selected variables and treatment factors in non-pathogen (A) and pathogen-inoculated (B) plants. Selected variables are displayed by arrows and include plant growth parameters—growth index after 3, 4, and 5 weeks of transplanting (GI WK3, GI WK4, and GI WK5) and shoot dry weight (SDW). Treatment factors are displayed by filled grey circles: CS100 represents peat-based commercial substrate, SBB10, HB10, HB30, HB50, and HB70 represent biochar-mixed substrates at different rates (by volume) with (TY) or without \u003cem\u003eTrichoderma \u003c/em\u003e(TN).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/02c97645111b5c44c848fdc9.png"},{"id":55285874,"identity":"7ff717dd-e56d-4836-a67d-0dc70987a515","added_by":"auto","created_at":"2024-04-25 08:02:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":35883,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) depicting the relationships between selected variables and treatment factors in pathogen-inoculated plants. Selected variables are displayed by arrows and include disease parameters—disease severity after 3, 7, 12, and 17 days of transplanting (DS1, DS2, DS3, and DS4), disease incidence after 3, 7, 12, and 17 days of transplanting (DI1, DI2, DI3, and DI4), and area under disease progress curve (AUDPC). Treatment factors are displayed by filled grey circles: CS100 represents peat-based commercial substrate, SBB10, HB10, HB30, HB50, and HB70 represent biochar-mixed substrates at different rates (by volume) with (TY) or without \u003cem\u003eTrichoderma \u003c/em\u003e(TN).\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/679b477752297383989ce2cc.png"},{"id":72640673,"identity":"a41022fb-ebd4-4082-961f-b47bdcf1f31b","added_by":"auto","created_at":"2024-12-30 16:08:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7673066,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4243906/v1/86e26979-a2fd-421a-831d-c04d14ac436b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biochar reduces containerized pepper blight caused by Phytophthora capsici","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePhytophthora capsici\u003c/em\u003e is a hemi-biotrophic fungal-like oomycete pathogen causing destructive disease across a diverse range of crops in \u003cem\u003ecucurbitaceous\u003c/em\u003e, \u003cem\u003efabaceous\u003c/em\u003e, and \u003cem\u003esolanaceous\u003c/em\u003e families\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePhytophthora\u003c/em\u003e blight of pepper caused by \u003cem\u003eP. capsici\u003c/em\u003e is one of the gravest soil-borne diseases affecting pepper growth globally\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The symptoms of the disease appear on main stem close to the soil line as small brown (early infection) to dark purplish (late infection) water-soaked lesions\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Under moist conditions, the disease could affect the whole plant from roots, crown, foliage, to fruit at any growth stages\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBiochar is a carbon-rich pyrolysis by-product derived from a wide variety of organic materials, involves the thermochemical decomposition of biomass under specific time, temperature, and oxygen-depleted or oxygen-limited conditions\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Biochar has been used as a soil amendment due to its positive effects on altering the biological and physicochemical properties of soils\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, it also has the potential use as a substrate in container nurseries to improve substrate water and nutrient holding capacity, ameliorate acidity, and provide suitable environments for plant growth\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Studies had shown that biochar could partially replace peat moss-based substrate for greenhouse ornamental and vegetable plants production\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e or used as an additional amendment in substrate to improve crop growth, yield, and quality\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs an organic amendment, biochar has shown the potential to suppress soil-borne diseases, such as \u003cem\u003eFusarium\u003c/em\u003e root rot (caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e) of asparagus, bacterial wilt (caused by \u003cem\u003eRalstonia solanacearum\u003c/em\u003e) of tomato, damping-off and root rot (caused by \u003cem\u003eRhizoctonia solani\u003c/em\u003e) of cucumber and some ornamental crops\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Studies have also demonstrated that amended soil with biochar suppressed the pathogen abundance of \u003cem\u003ePhytophthora\u003c/em\u003e blight of pepper: amended loam and clay loam soil with 3% (w/w) softwood-derived biochar (pH 6.5) improved root and shoot development of sweet pepper that were infested with \u003cem\u003eP. capsici\u003c/em\u003e, the population and percentages of root infection by \u003cem\u003ePhytophthora\u003c/em\u003e spp. were reduced\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e; amended sandy loam soil with 1.33% (w/w) corn straw-derived biochar (pH 9.7) before transplanting reduced the disease index of \u003cem\u003ePhytophthora\u003c/em\u003e blight of pepper and the population of \u003cem\u003eP. capsici\u003c/em\u003e while increased the abundance of beneficial bacteria, such as \u003cem\u003eBacillus\u003c/em\u003e spp., \u003cem\u003ePseudomonas\u003c/em\u003e spp. and \u003cem\u003eStreptomyces\u003c/em\u003e spp.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e; their following study using the same setup further illustrated the suppression of pepper blight could also due to the improved abundance of potential rhizosphere-associated biocontrol fungi and enhanced soil organic matter, available nutrients (P, K)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Interestingly, they found biochar amendment increased the abundance of \u003cem\u003eTrichoderma\u003c/em\u003e that led to a high control efficacy in \u003cem\u003eP. capsici\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eTrichoderma\u003c/em\u003e spp. has been reported as a reliable biological control agent for \u003cem\u003eP. capsici\u003c/em\u003e: \u003cem\u003eT. harzianum\u003c/em\u003e was proven to suppress pepper root rot caused by \u003cem\u003eP. capsici\u003c/em\u003e through antimicrobial substances production\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e; in a vitro test, \u003cem\u003eT. harzianum\u003c/em\u003e inhibited \u003cem\u003eP. capsici\u003c/em\u003e by 65.3%\u003csup\u003e22\u003c/sup\u003e; similarly, \u003cem\u003eT. harzianum\u003c/em\u003e, \u003cem\u003eT\u003c/em\u003e. \u003cem\u003eviride\u003c/em\u003e and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ereesei\u003c/em\u003e displayed over 85.5% inhibition of mycelial growth of \u003cem\u003eP. capsici\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003elongibranchiatum\u003c/em\u003e, \u003cem\u003eT\u003c/em\u003e. \u003cem\u003easperellum\u003c/em\u003e showed the inhibition on \u003cem\u003eP. capsici\u003c/em\u003e up to 22.5%\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe synergistic use of external \u003cem\u003eTrichoderma\u003c/em\u003e in biochar amended soil could further strengthen the controlling effects on \u003cem\u003eP. capsici\u003c/em\u003e. However, studies using biochar to control \u003cem\u003eP. capsici\u003c/em\u003e were mostly conducted using sandy, loam, or clay soils with low amount of biochar amendment (less than 3% based on weight), while the higher percentage use of biochar to replace soilless substrate (e.g., peat moss) and reduce the severity of pepper blight in container production has been less reported. Our previous study already showed that partially replacing peat moss with biochar could significantly reduce the disease incidence and severity of poinsettia root rot caused by \u003cem\u003ePythium aphanidermatum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, to evaluate the controlling effects of different types of biochar and \u003cem\u003eTrichoderma\u003c/em\u003e on pepper blight in container pepper production, we conducted both in vitro petri dish tests and in vivo greenhouse trials. Inhibition of \u003cem\u003eP. capsici\u003c/em\u003e, disease assessment, and plant growth response were evaluated. This study could provide guidance of using biochar in container soilless substrates production and demonstrate evidence of suppressing soil-borne disease from biochar.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e \u003cb\u003eBiochar-mixed substrate\u003c/b\u003e, \u003cb\u003eTrichoderma\u003c/b\u003e, \u003cb\u003eand pathogen isolation and propagation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwo types of biochar, sugarcane bagasse-derived biochar (SBB; American Biocarbon LLC White Castle, LA, USA) and mixed hardwood-derived biochar (HB; Proton Power Inc. Lenoir City, TN, USA) were used in this study. The SBB was produced using proprietary methods and was provided by USDA-ARS, Sugarcane Research Unit (Houma, LA, USA), it had a pH of 5.9 and an electrical conductivity (EC) of 0.75 dS/m; the HB was a by-product from fast pyrolysis of mixed hardwood and had a pH of 10.1 and an EC of 1.06 dS/m\u003csup\u003e26,27\u003c/sup\u003e. These two types of biochar were separately mixed with the commercial peat moss-based substrates (Jolly Gardener C/20, Oldcastle Lawn \u0026amp; Garden Inc., Atlanta, GA, USA) that contain 80% Canadian Sphagnum peat and 20% perlite. Sugarcane bagasse-derived biochar was mixed with the commercial substrate at a rate of 10% by volume (SBB10), while HB was mixed with the commercial substrate at rates of 10%, 30%, 50%, and 70% by volume (HB10, HB30, HB50, HB70). Commercial substrate without mixing biochar was also included (CS). These substrates were measured for their physical properties based on Fonteno, et al. \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e used the North Carolina State University Porometer (Raleigh, NC, USA), including bulk density, air space, container capacity, and total porosity. Their chemical properties of leachate electrical conductivity (EC) and pH were measured following the pour-through method\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e with a portable EC/pH meter (Hanna Instrument, Woonsocket, RI, USA).\u003c/p\u003e \u003cp\u003eThe study utilized the Root shield Plus-WP (BioWorks, Victor, NY, USA) that consisted of two active strains of \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eT. harzianum\u003c/em\u003e strain T-22 and \u003cem\u003eT. virens\u003c/em\u003e strain G-41, as the biological control agent. Pathogen \u003cem\u003ePhytophthora capsici\u003c/em\u003e was isolated and identified from an infected pepper plant, followed by pathogen propagation, where \u003cem\u003eP. capsici\u003c/em\u003e was isolated and maintained in the darkness on a V8 juice agar that is selective for oomycete organisms\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro test\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eWater extracts of commercial substrate and biochar-mixed substrates\u003c/h2\u003e \u003cp\u003eWater extracts of commercial substrate and biochar-mixed substrates were obtained following the method outlined by Gravel, et al. \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In summary, commercial substrate and five types of biochar-mixed substrate were separately mixed with deionized water in a 1:1 ratio by volume in 500 mL flasks and agitated for 24 hours using a shaker (Orbital; Laboratory Supply Network, Atkinson, NH, USA). The resulting mixtures were filtered through filter papers, and 25 mL of the extracts from each type were collected and sterilized (autoclaved for 1 hour) for the in vitro test. An equal amount of sterilized deionized water was used as a control. The potato dextrose agar (PDA) media was therefore produced in petri dishes (100 \u0026times; 15 mm) by either incorporating the sterilized deionized water (DI water control) or commercial substrate and biochar-mixed water extracts (CS, SBB10, HB10, HB30, HB50, HB70) into a 25% PDA sterilized solution before the media solidified.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTrichoderma treatment and pathogen growth\u003c/h2\u003e \u003cp\u003eHalf of the PDA petri dishes produced using different water extracts were inoculated with 5 mm square plugs of actively growing \u003cem\u003eP. capsici\u003c/em\u003e in the center of each petri dish, while the other half petri dishes were introduced with dual confrontation technique for the inoculation of \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eP. capsici\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Specifically, we placed a drop of \u003cem\u003eTrichoderma\u003c/em\u003e-containing solution mixed at the recommended rate by the manufacturer (0.30 g/L) opposite to a 5 mm square plug of actively growing \u003cem\u003eP. capsici\u003c/em\u003e within the petri dish, such that both elements positioned at equal distances from each other and to the border of the petri dish. Next, all the petri dishes were placed in a dark environment at room temperature. After four days, the radial growth of mycelium was measured, and the inhibition percentage of pathogen growth was then computed using this formula from Nawaz, et al. \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e: Inhibition = ((A1 - A2) \u0026times; 100%) / A2, where A1 represents the area of pathogen growth in commercial substrate or biochar-mixed water extracts, and A2 represents the area of pathogen growth in the DI water control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo greenhouse trial\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003ePlant materials, treatment setup, and growth environment\u003c/h2\u003e \u003cp\u003eHot cherry pepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e cv. \u0026lsquo;Capperino\u0026rsquo;) seeds (F1 self-selected seeds from Johnny\u0026rsquo;s Selected Seeds, Fairfield, ME, USA) were pretreated with 10% bleach for 3 minutes and then rinsed with DI water. Treated-seeds were sown in the commercial propagation media (BM2 Berger; Saint-Modeste, Quebec, Canada) and grown for 3 weeks until the true leaves came out. Uniform seedlings were then transplanted into growing pots with dimensions of 7.5 cm at the top, 6 cm at the bottom, and 8.2 cm in depth, with a total volume of 375 mL. These pots were previously filled with either commercial or biochar-mixed substrates. After transplanting, slow-released fertilizer (15N-4P-10K Osmocote Plus; Scotts-Sierra Horticultural Products Company, Marysville, OH, USA) was incorporated with the surface substrate at the rate of 450 g/m\u003csup\u003e2\u003c/sup\u003e (or 2 g/container). One week after transplanting, \u003cem\u003eTrichoderma\u003c/em\u003e treatment was introduced by drenching solution to the substrate surface at the rate of 0.30 g/L. Pathogen \u003cem\u003eP. capsici\u003c/em\u003e was inoculated with actively growing mycelium at the rate of 5 plugs with plastic inoculation loops (VWR, Radnor, PA, USA). When conducted pathogen inoculation, a 5-mm diameter agar plug was taken from the margin of an actively growing colony of the pathogen and placed on the surface of the substrate contacting plant stem. Standard propagation trays were placed underneath the pots to create a moisturized environment. All the pots were placed in a \u003cem\u003eP. capcisi\u003c/em\u003e-permitted greenhouse at Texas A\u0026amp;M University, Sommerville, TX, USA. During the experiment, plants were regularly watered. Inside the greenhouse, the average temperature was 30.2\u0026deg;C, with a relative humidity of 77.2% and a dew point of 25.0\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003ePlant growth measurements and disease assessment\u003c/h2\u003e \u003cp\u003eFrom 3 weeks after transplanting, weekly measurements were conducted for plant height (PH) and two-sided widths (PW1 and PW2). Plant growth index (GI) was then calculated by: GI\u0026thinsp;=\u0026thinsp;PH/2 + (PW1\u0026thinsp;+\u0026thinsp;PW2)/4\u003csup\u003e25\u003c/sup\u003e. Pepper plants shoots were harvested and dried at 80\u0026deg;C for 3 days in the oven at the end of the experiment, shoot dry weight (SDW) was recorded thereafter.\u003c/p\u003e \u003cp\u003eSymptoms of the disease were monitored and documented every 5 days, starting 3 days after inoculating the pathogen. Disease severity was assessed on a scale of 0\u0026ndash;4, following the methodology outlined by Wang, et al. \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The scales were illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: 0 indicated healthy plants, 1 represented plants with small brown lesions on the stem or slightly wilted leaves, 2 denoted plants with moderate brown lesions on the stem and moderate wilted leaves, 3 indicated plants with large brown lesions on the stem and significantly wilted leaves, and 4 represented dead plants. Disease Severity Index (DS) was calculated using the following formula: DS = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\sum ( \\frac{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{d}\\text{i}\\text{s}\\text{e}\\text{a}\\text{s}\\text{e}\\text{d} \\text{p}\\text{l}\\text{a}\\text{n}\\text{t}\\text{s} \\text{i}\\text{n} \\text{t}\\text{h}\\text{i}\\text{s} \\text{i}\\text{n}\\text{d}\\text{e}\\text{x}\\hspace{0.17em}\\times \\hspace{0.17em}\\text{d}\\text{i}\\text{s}\\text{e}\\text{a}\\text{s}\\text{e} \\text{i}\\text{n}\\text{d}\\text{e}\\text{x} \\text{r}\\text{a}\\text{t}\\text{i}\\text{n}\\text{g} \\text{f}\\text{r}\\text{o}\\text{m} 0 \\text{t}\\text{o} 4}{4\\hspace{0.17em}\\times \\hspace{0.17em}\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{p}\\text{l}\\text{a}\\text{n}\\text{t}\\text{s} \\text{i}\\text{n}\\text{v}\\text{e}\\text{s}\\text{t}\\text{i}\\text{g}\\text{a}\\text{t}\\text{e}\\text{d}} )\\times 100\\%\\)\u003c/span\u003e\u003c/span\u003e\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe disease severity obtained at different times after inoculation was used to calculate areas under disease progress curves (AUDPC) following the formula: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{A}\\text{U}\\text{D}\\text{P}\\text{C}={\\sum }_{i=0}^{n-1}\\frac{({y}_{i}+{y}_{i+1})}{2}\\left({t}_{i+1}-{t}_{i}\\right)\\)\u003c/span\u003e\u003c/span\u003e. Where \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({y}_{i}\\)\u003c/span\u003e\u003c/span\u003e is the scale rating at the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(i\\)\u003c/span\u003e\u003c/span\u003eth observation, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({t}_{i}\\)\u003c/span\u003e\u003c/span\u003eis the day of the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(i\\)\u003c/span\u003e\u003c/span\u003eth observation, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(n\\)\u003c/span\u003e\u003c/span\u003e is the total number of observations\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. During the trials, Disease Incidence (DI) was assessed by tallying the number of diseased plants in each pot, following the specified formula: DI = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{number of diseased plants}{number of total plants}\\)\u003c/span\u003e\u003c/span\u003e\u0026times; 100\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eExperimental design and data analysis\u003c/h2\u003e \u003cp\u003eThe in vitro test followed a randomized complete block design (RCBD) with five blocks. Within each block, treatments were randomly assigned to petri dishes based on a 7 x 2 factorial design involving substrate treatments (water extracts of CS100, SBB10, HB10, HB30, HB50, HB70, and DI water as control) and \u003cem\u003eTrichoderma\u003c/em\u003e application (with/without).\u003c/p\u003e \u003cp\u003eIn the greenhouse experiment, an RCBD with eight blocks was utilized. Each block consisted of a 1.5 m x 1 m area on a raised bench. Treatments were randomly allocated to pots within each block, following a 6 x 2 x 2 factorial design involving substrate treatments (CS100 as control, SBB10, HB10, HB30, HB50, and HB70 as biochar-mixed treatments), \u003cem\u003eTrichoderma\u003c/em\u003e application (with/without), and pathogen presence (non-pathogen, pathogen-inoculated).\u003c/p\u003e \u003cp\u003eThe pathogen growth area in the in vitro test was estimated using Image J, version 1.53a\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Data analysis was conducted using one-way analysis of variance (ANOVA) in R program software, version 3.5.1\u003csup\u003e36\u003c/sup\u003e. In the greenhouse trial, treatments with non-pathogens and those inoculated with pathogens were analyzed separately. Means were separated using Dunnett\u0026rsquo;s test when treatments significantly differed from the control at p\u0026thinsp;\u0026le;\u0026thinsp;0.05 or the least significant difference (LSD) when treatments significantly differed from each other at p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSubstrate physical and chemical properties\u003c/h2\u003e \u003cp\u003eThe majority of the mixes met the recommended range for container substrate properties, as detailed in Yeager, et al. \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, with the exception of the bulk density in all the substrates, which were lower than the recommended value (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The HB50 and HB70 mixes exhibited significantly lower total porosity, container compacity, and bulk density compared to the control (CS100). All the HB mixes showed significantly higher pH levels than the control, and except for HB10 and HB30, all biochar-mixed substrates displayed significantly lower EC levels than the control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysical and chemical properties of commercial and biochar-mixed substrates: total porosity (TP), container compacity (CC), air space (AS), bulk density (BD), pH and electrical conductivity (EC).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTP (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAS (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBD (g/cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEC (dS/m)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCS100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.07***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.5***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHB30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.11**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.9***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHB50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.0***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.58**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHB70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.13***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.4***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.40 ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuitable range\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u0026ndash;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45\u0026ndash;65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.19\u0026ndash;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.4\u0026ndash;6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSBB\u0026thinsp;=\u0026thinsp;Sugarcane bagasse biochar, HB\u0026thinsp;=\u0026thinsp;Mixed hardwood biochar, CS\u0026thinsp;=\u0026thinsp;Peat moss based commercial substrate. Numbers after CS, SBB, and HB indicated the ratio of different components, by volume *, **, and *** indicates significant difference from the commercial substrate (CS100) according to Dunnett\u0026rsquo;s test at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.1, 0.05, and 0.01, respectively. \u003csup\u003ea\u003c/sup\u003eRecommended container substrate properties\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePathogen growth from in vitro test\u003c/h2\u003e \u003cp\u003eResults from the in vitro test showed that without \u003cem\u003eTrichoderma\u003c/em\u003e present, the extracts from biochar-mixed substrate showed no significant difference in inhibiting \u003cem\u003eP. capsici\u003c/em\u003e growth compared to CS extracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, HB10 extracts exhibited positive inhibition values, indicating stimulation of \u003cem\u003eP. capsici\u003c/em\u003e growth, while all other extracts displayed negative inhibition values, indicating suppression of the pathogen. Among all the pathogen-suppressed biochar extracts, HB30 had a significantly higher inhibition percentage compared with SBB10. When \u003cem\u003eTrichoderma\u003c/em\u003e was present, all the extracts effectively suppressed \u003cem\u003eP. capsici\u003c/em\u003e growth, while the biochar-mixed substrate extracts showed no significant difference in inhibiting \u003cem\u003eP. capsici\u003c/em\u003e growth compared to the CS extracts, except for HB70, which exhibited a relatively lower inhibition percentage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth from in vivo greenhouse trial\u003c/h2\u003e \u003cp\u003eThere were no significant interaction effects between substrate and \u003cem\u003eTrichoderma\u003c/em\u003e treatments on any of the growth parameters regardless the presence or absence of the pathogen (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For non-pathogen treatments, pepper plant growth index from 3 weeks after transplanting and final shoot dry weight were both significantly influenced by substrate and \u003cem\u003eTrichoderma\u003c/em\u003e treatments. However, plant growth index from 4 and 5 weeks after transplanting was only significantly influenced by substrate treatment. Under pathogen-inoculated conditions, none of the factors significantly influenced any of the growth parameters.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePepper plant growth index (GI) from 3, 4, and 5 weeks after transplanting (3, 4, 5 WAT) and final shoot dry weight (SDW) as affected by substrate and \u003cem\u003eTrichoderma\u003c/em\u003e treatment factors under non-pathogen and pathogen-inoculated conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGI-3WAT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGI-4WAT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGI-5WAT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSDW\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNon-Pathogen\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate \u003cem\u003e\u0026times; Trichoderma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePathogen-inoculated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate \u003cem\u003e\u0026times; Trichoderma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNS means not significant. *, **, *** indicate significance at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, 0.01, and 0.001, respectively.\u003c/p\u003e \u003cp\u003eBiochar types and application rates did not significantly affect the shoot dry weights of healthy pepper plants (non-pathogen), whether with or without \u003cem\u003eTrichoderma\u003c/em\u003e, except for HB70 mix, which showed significantly lower biomass compared to the control in both non-\u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eTrichoderma\u003c/em\u003e-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, Biochar did not significantly influence the growth index from 3 and 4 weeks after transplanting. However, plants grown under HB30 and HB50 substrates showed significantly higher growth index from 5 weeks after transplanting compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The use of \u003cem\u003eTrichoderma\u003c/em\u003e did not have a significant impact on any of the growth indexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). While under pathogen-inoculated conditions, neither substrate nor \u003cem\u003eTrichoderma\u003c/em\u003e treatments had significant impacts on pepper plant growth index and biomass accumulation (date not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDisease development from in vivo greenhouse trial\u003c/h2\u003e \u003cp\u003eUnder pathogen-inoculate conditions, plants grown in all the treatments showed disease symptoms 3 days after transplanting except for HB70, which showed symptoms 7 days after transplanting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Compared with CS100 treatment, HB50 and HB70 treatments reduced disease severity at 12 days after transplanting by 10.94% and 10.16%, respectively, and at 17 days after transplanting by 9.59% and 9.59%, respectively. The application of \u003cem\u003eTrichoderma\u003c/em\u003e did not significantly reduce disease severity during the entire experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBiochar-mixed substrates had significant impacts on disease incidence, especially HB-amended (30%-70%) mixes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Compared with CS100 treatment, HB50, HB70, and SBB10 treatments reduced disease incidence at 7 days after transplanting by 25.0%, 25.0%, and 18.8%, respectively, and at 12 days after transplanting by 25.0%, 18.8%, and 6.3%, respectively. The application of \u003cem\u003eTrichoderma\u003c/em\u003e did not significantly reduce disease incidence during the entire experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll the biochar-mixed substrates had significantly lower AUDPC values (except for HB10) than the CS100. The HB50 and HB70 mixes reduced the AUDPC value by 9.6 and 9.4 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The application of \u003cem\u003eTrichoderma\u003c/em\u003e did not significantly reduce AUDPC during the entire experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cp\u003eEffects of biochar on controlling pathogen growth and disease development involve both direct and indirect mechanisms. Directly, chemical compounds naturally contained in biochar, such as benzoic acid, ethylene glycol, propylene glycol, hydroxypropionic acid, quinones, or some specific phenols and carboxylic acids that generated during the pyrolysis process, these compounds have shown the capacity to inhibit microbial growth; indirectly, synergistic effects from biochar's physical and chemical properties could lead to the beneficial outcomes: biochar absorbs and deactivates toxic metabolites or enzymes (e.g., cell wall degrading enzymes) produced by pathogens, reducing their virulence, boosting plant growth, inducing systemic plant resistance, and enhancing the abundance and activity of beneficial bacteria and fungi\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn vitro test isolated the physical properties of biochar by only testing the effects of chemical compounds on \u003cem\u003eP. capsici\u003c/em\u003e development using water extracts from biochar-mixed substrates. Results showed that the extracts from high percentage biochar mixes suppressed \u003cem\u003eP. capsici\u003c/em\u003e growth when using hardwood as the raw material. Our previous study found that used the water extracts from hardwood-derived biochar led to neutral or negative effects on suppressing pathogen \u003cem\u003ePythium aphanidermatum\u003c/em\u003e growth in poinsettia\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, indicated the chemical compounds from the same type of biochar could impose different effects on different soil-borne pathogens. Different types of raw material-derived biochar could also lead to different pathogen controlling effects, as eucalyptus wood and organic waste-derived biochar extracts showed no effects on suppressing \u003cem\u003eFusarium\u003c/em\u003e colonization\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In the presence of \u003cem\u003eTrichoderma\u003c/em\u003e, the inhibiting effects of biochar-mixed extracts on \u003cem\u003eP. capsici\u003c/em\u003e growth were enhanced due to \u003cem\u003eTrichoderma\u0026rsquo;\u003c/em\u003es biocontrol impact on suppressing the growth of \u003cem\u003eP. capsici\u003c/em\u003e. This in vitro test showed that synergistically using the chemical compounds extracted from biochar and biocontrol components could provide positive effects on controlling pathogen \u003cem\u003eP. capsici\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn general, increasing biochar application rate improved the disease suppression percentage, and using hull, wood, straw and organic waste as the raw material has better controlling effects for disease compared to using bark as the raw material\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Results from the in vivo greenhouse study showed that compared with commercial peat-based substrate and sugarcane-derived biochar, using high-percentage hardwood-derived biochar significantly reduced disease incidence, severity, and disease intensity over time (AUDPC) of \u003cem\u003eP. capsici\u003c/em\u003e induced Phytophthora blight. Although the highest percentage biochar-mix (HB70) significantly decreased the final plant biomass, using a moderate rate of biochar (HB50) maintained and even promoted plant growth index. Biochar application has been reported to improve crop biomass and yield by enhancing soil structure with increased nutrient availability and water holding capacity, immobilize inorganic (heavy metals) and organic contaminants, and reduce abiotic and biotic stresses\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Our results have further encouraged the use of biochar as a substitute for peat-based substrates\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, offering additional benefits in suppressing soil-borne diseases due to its chemical and physical properties.\u003c/p\u003e \u003cp\u003eUnlike the in vitro test, \u003cem\u003eTrichoderma\u003c/em\u003e treatment only numerically decreased the disease development in the greenhouse study. And the interaction effects between biochar and \u003cem\u003eTrichoderma\u003c/em\u003e were marginally significant, indicating that under the in vivo conditions, other factors could reduce the beneficial effects from \u003cem\u003eTrichoderma\u003c/em\u003e. In order to better understand the complexed effects of \u003cem\u003eTrichoderma\u003c/em\u003e and two types of biochar on plant growth and disease development, we employed a principal component analysis (PCA) to illustrate the distinct variables influenced by different treatment factors. For non-pathogen plants, 86% of the variability was explained by the first two components (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). PC1 accounted for 65% variance, with HB50-TN (HB50 without \u003cem\u003eTrichoderma\u003c/em\u003e), HB70-TN, HB70-TY (HB70 with \u003cem\u003eTrichoderma\u003c/em\u003e), and CS100-TN differing from the rest of treatments. Biochar-mixed substrate at relatively lower rates (HB10-TN, HB10-TY, HB30-TN, HB30-TY, and HB50-TY) along with CS100 were associated more with the yield (SDW) and GIs (GI WK3, GI WK4, and GI WK5). PC2 accounted for 21% variance, distinguishing the CS100 and SBB10 from HB mixes. Commercial substrate and SBB-amended mixes tended to be affiliated with plant biomass and GI WK3 and GI WK4, however, HB30, HB50 mixes appeared to be related to GI WK5. For pathogen-inoculated plants, 87% of the variability was explained by the first two components (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). PC1 accounted for 63% variance, differing biochar-mixed substrate with \u003cem\u003eTrichoderma\u003c/em\u003e (except for HB30-TN) and CS100-TY from those without \u003cem\u003eTrichoderma\u003c/em\u003e treatments. The treatment HB30-TN and CS100 were associated more with the yield (SDW) and plant late growth (GI WK4 and GI WK5). PC2 accounted for 24% variance, distinguishing mixes without \u003cem\u003eTrichoderma\u003c/em\u003e from those with \u003cem\u003eTrichoderma\u003c/em\u003e. Commercial substrate and biochar-amended mixes with \u003cem\u003eTrichoderma\u003c/em\u003e tended to be affiliated with GI WK3 while HB30-TN appeared to be related to GI WK4.\u003c/p\u003e \u003cp\u003eFor pathogen-inoculated plants, 94% of the variability was explained by the first two components (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). PC1 accounted for 77% variance, differing CS100, HB10, HB70-TN, and SBB10-TY mixes from the rest biochar-amended treatments. The treatments CS100, HB10, HB70-TN, and SBB10-TY were positively associated with all the disease parameters while the rest of the treatments were negatively associated with them. PC2 accounted for 17% variance, distinguishing CS100, SBB10-TY, HB30, HB50-TY and HB70-TY mixes from the rest of the treatments. CS100-TY, SBB10-TY, HB30-TY, and HB30-TN mixes tended to be affiliated with DI1, DS1, DS2 and AUDPC while HB10-TN, HB10-TY, and HB70-TN, appeared to be related to DI2, DI3, DI4, DS3, and DS4. This indicated that \u003cem\u003eTrichoderma\u003c/em\u003e-treated plant (TY) tended to have an early disease development, while during the plant late growth period, \u003cem\u003eTrichoderma\u003c/em\u003e positively suppressed disease development and intensity when biochar was present. Although studies testing the effects of combined use of biochar with other materials on pathogen suppression have been less reported, promising research had shown that the combined use of compost and biochar could suppress \u003cem\u003eRhizoctonia solani\u003c/em\u003e induced disease\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and the combined use of biochar and arbuscular mycorrhizal fungi could control \u003cem\u003eFusarium oxysporum\u003c/em\u003e by altering root exudates\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Future studies could focus on exploring more reliable bioactive materials that could be combined used with biochar to promote the synergistic effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe mixed hardwood biochar blended with commercial peat moss-based substrate at 50% and 70% (by volume) could significantly reduce Phytophthora pepper blight disease severity, incidence, and intensity over time. The chemical compounds contained in biochar could contribute to its disease inhibition capability for \u003cem\u003eP. capsici\u003c/em\u003e to a certain extent. In the absence and presence of the pathogen, mixed hardwood biochar could replace commercial peat moss-based substrate up to 50% (by volume) for container pepper plant production without negative impact on its growth. Combined use of moderate rate of biochar amendment (up to 50% by volume) and \u003cem\u003eTrichoderma\u003c/em\u003e could improve plant growth while reducing pathogen \u003cem\u003eP. capsici\u003c/em\u003e growth.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Thomas Isakeit from the Department of Plant pathogen and Microbiology, Texas AM University for assisting in the isolation and identification of \u003cem\u003eP. capsici\u003c/em\u003e. We would also like to thank Dr. Lan Zhou for her statistical consultant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was a product of the combined effort of all the authors. All authors conceptualized and designed the study. Ping Yu performed the experiments, collected and analysed the data, and wrote the manuscript with assistance from all other authors, mainly Mengmeng Gu. Kuan Qin, Kevin Crosby and Kevin Ong provided technical advice and assistance when the study was conducted and revised and improved the manuscript. Terry Gentry revised the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKousik, C. S., Parada, C. \u0026amp; Quesada-Ocampo, L. First Report of Phytophthora Fruit Rot on Bitter Gourd (Mormodica charantia) and Sponge Gourd (Luffa cylindrica) Caused by Phytophthora capsici. Plant Health Progress 16, 93\u0026ndash;94, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1094/php-br-15-0005\u003c/span\u003e\u003cspan address=\"10.1094/php-br-15-0005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, G. \u003cem\u003eet al.\u003c/em\u003e Suppression of Phytophthora blight of pepper by biochar amendment is associated with improved soil bacterial properties. Biol. Fertil. Soils 55, 813\u0026ndash;824, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00374-019-01391-6\u003c/span\u003e\u003cspan address=\"10.1007/s00374-019-01391-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErwin, D. C. \u0026amp; Ribeiro, O. K. \u003cem\u003ePhytophthora diseases worldwide\u003c/em\u003e. (American Phytopathological Society (APS Press), 1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamour, K. H., Stam, R., Jupe, J. \u0026amp; Huitema, E. The oomycete broad-host-range pathogen Phytophthora capsici. Molecular Plant Pathology 13, 329\u0026ndash;337, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1364-3703.2011.00754.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1364-3703.2011.00754.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParada-Rojas, C. H. \u0026amp; Quesada-Ocampo, L. M. Characterizing Sources of Resistance to Phytophthora Blight of Pepper Caused by Phytophthora capsici in North Carolina. Plant Health Progress 20, 112\u0026ndash;119, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1094/php-09-18-0054-rs\u003c/span\u003e\u003cspan address=\"10.1094/php-09-18-0054-rs\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDemirbas, A. \u0026amp; Arin, G. An Overview of Biomass Pyrolysis. Energy Sources 24, 471\u0026ndash;482, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/00908310252889979\u003c/span\u003e\u003cspan address=\"10.1080/00908310252889979\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh, B., Singh, B. P. \u0026amp; Cowie, A. L. Characterisation and evaluation of biochars for their application as a soil amendment. Soil Research 48, 516\u0026ndash;525 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrassard, P. \u003cem\u003eet al.\u003c/em\u003e in \u003cem\u003eChar and carbon materials derived from biomass\u003c/em\u003e 109\u0026ndash;146 (Elsevier, 2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDumroese, R. K., Heiskanen, J., Englund, K. \u0026amp; Tervahauta, A. Pelleted biochar: Chemical and physical properties show potential use as a substrate in container nurseries. Biomass and Bioenergy 35, 2018\u0026ndash;2027, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biombioe.2011.01.053\u003c/span\u003e\u003cspan address=\"10.1016/j.biombioe.2011.01.053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaughn, S. F., Kenar, J. A., Thompson, A. R. \u0026amp; Peterson, S. C. Comparison of biochars derived from wood pellets and pelletized wheat straw as replacements for peat in potting substrates. Ind. Crop. Prod. 51, 437\u0026ndash;443, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.indcrop.2013.10.010\u003c/span\u003e\u003cspan address=\"10.1016/j.indcrop.2013.10.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, P. \u003cem\u003eet al.\u003c/em\u003e The Effects of Mixed Hardwood Biochar, Mycorrhizae, and Fertigation on Container Tomato and Pepper Plant Growth. Sustainability 12, 7072 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, L., Yu, P. \u0026amp; Gu, M. Evaluation of Biochar and Compost Mixes as Substitutes to a Commercial Propagation Mix. Appl. Sci. 9, 4394 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026eacute;ndez, A., C\u0026aacute;rdenas-Aguiar, E., Paz-Ferreiro, J., Plaza, C. \u0026amp; Gasc\u0026oacute;, G. The effect of sewage sludge biochar on peat-based growing media. Biol. Agric. Hortic. 33, 40\u0026ndash;51, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/01448765.2016.1185645\u003c/span\u003e\u003cspan address=\"10.1080/01448765.2016.1185645\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNieto, A. \u003cem\u003eet al.\u003c/em\u003e The effect of pruning waste and biochar addition on brown peat based growing media properties. Sci Hortic. 199, 142\u0026ndash;148, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2015.12.012\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2015.12.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian, Y. \u003cem\u003eet al.\u003c/em\u003e Biochar made from green waste as peat substitute in growth media for Calathea rotundifola cv. Fasciata. Sci Hortic. 143, 15\u0026ndash;18, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2012.05.018\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2012.05.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, L., Sun, X.-y., Tian, Y. \u0026amp; Gong, X.-q. Biochar and humic acid amendments improve the quality of composted green waste as a growth medium for the ornamental plant Calathea insignis. Sci Hortic. 176, 70\u0026ndash;78, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2014.06.021\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2014.06.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrenkel, O. \u003cem\u003eet al.\u003c/em\u003e The effect of biochar on plant diseases: what should we learn while designing biochar substrates? Journal of Environmental Engineering and Landscape Management 25, 105\u0026ndash;113 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraber, E. R., Frenkel, O., Jaiswal, A. K. \u0026amp; Elad, Y. How may biochar influence severity of diseases caused by soilborne pathogens? Carbon Management 5, 169\u0026ndash;183, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/17583004.2014.913360\u003c/span\u003e\u003cspan address=\"10.1080/17583004.2014.913360\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShoaf, N., Hoagland, L. \u0026amp; Egel, D. S. Suppression of Phytophthora Blight in Sweet Pepper Depends on Biochar Amendment and Soil Type. Hortscience. 51, 518\u0026ndash;524, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21273/hortsci.51.5.518\u003c/span\u003e\u003cspan address=\"10.21273/hortsci.51.5.518\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, G. \u003cem\u003eet al.\u003c/em\u003e Biochar-Mediated Control of Phytophthora Blight of Pepper Is Closely Related to the Improvement of the Rhizosphere Fungal Community. Front. Microbiol. 11, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2020.01427\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2020.01427\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEzziyyani, M., Requena, M. E., Egea-Gilabert, C. \u0026amp; Candela, M. E. Biological Control of Phytophthora Root Rot of Pepper Using Trichoderma harzianum and Streptomyces rochei in Combination. Journal of Phytopathology 155, 342\u0026ndash;349, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1439-0434.2007.01237.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1439-0434.2007.01237.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMousumi Das, M., Haridas, M. \u0026amp; Sabu, A. Biological control of black pepper and ginger pathogens, Fusarium oxysporum, Rhizoctonia solani and Phytophthora capsici, using Trichoderma spp. Biocatal Agric Biotechnol. 17, 177\u0026ndash;183, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcab.2018.11.021\u003c/span\u003e\u003cspan address=\"10.1016/j.bcab.2018.11.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNawaz, K. \u003cem\u003eet al.\u003c/em\u003e Diversity of Trichoderma species in chili rhizosphere that promote vigor and antagonism against virulent Phytophthora capsici. Sci Hortic. 239, 242\u0026ndash;252, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2018.05.048\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2018.05.048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe la Cruz-Quiroz, R., Roussos, S., Rodr\u0026iacute;guez-Herrera, R., Hernandez-Castillo, D. \u0026amp; Aguilar, C. N. Growth inhibition of Colletotrichum gloeosporioides and Phytophthora capsici by native Mexican Trichoderma strains. Karbala International Journal of Modern Science 4, 237\u0026ndash;243, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.kijoms.2018.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.kijoms.2018.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, P., Ong, K., Ueckert, J. \u0026amp; Gu, M. Biochar and Trichoderma Reduce Containerized Poinsettia Root Rot Caused by Pythium aphanidermatum. Hortscience. 58, 846\u0026ndash;854, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21273/hortsci17203-23\u003c/span\u003e\u003cspan address=\"10.21273/hortsci17203-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, P., Li, Q., Huang, L., Niu, G. \u0026amp; Gu, M. Mixed Hardwood and Sugarcane Bagasse Biochar as Potting Mix Components for Container Tomato and Basil Seedling Production. Appl. Sci. 9, 4713 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWebber III, C. L. \u003cem\u003eet al.\u003c/em\u003e Sugarcane and pine biochar as amendments for greenhouse growing media for the production of bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.) seedlings. Journal of Agricultural Science 10, 58\u0026ndash;68, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5539/jas.v10n4p58\u003c/span\u003e\u003cspan address=\"10.5539/jas.v10n4p58\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonteno, W., Hardin, C. \u0026amp; Brewster, J. Procedures for determining physical properties of horticultural substrates using the NCSU Porometer. \u003cem\u003eHorticultural Substrates Laboratory, North Carolina State University\u003c/em\u003e (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeBude, A. \u0026amp; Bilderback, T. The pour-through extraction method: A nutrient management tool for nursery crops. NC State Univ. Coop. Ext (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParada-Rojas, C. H. \u003cem\u003eet al.\u003c/em\u003e A Diagnostic Guide for Phytophthora capsici Infecting Vegetable Crops. Plant Health Progress 22, 404\u0026ndash;414, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1094/php-02-21-0027-fi\u003c/span\u003e\u003cspan address=\"10.1094/php-02-21-0027-fi\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGravel, V., Dorais, M. \u0026amp; M\u0026eacute;nard, C. Organic potted plants amended with biochar: its effect on growth and Pythium colonization. Can. J. Plant Sci. 93, 1217\u0026ndash;1227, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4141/cjps2013-315\u003c/span\u003e\u003cspan address=\"10.4141/cjps2013-315\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinclair, J. B. \u0026amp; Dhingra, O. D. \u003cem\u003eBasic plant pathology methods\u003c/em\u003e. (CRC press, 2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadden, L. V., Hughes, G. \u0026amp; van den Bosch, F. \u003cem\u003eThe Study of Plant Disease Epidemics\u003c/em\u003e. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBellini, A. \u003cem\u003eet al.\u003c/em\u003e A Compost Treatment Acts as a Suppressive Agent in Phytophthora capsici \u0026ndash; Cucurbita pepo Pathosystem by Modifying the Rhizosphere Microbiota. Front.Plant Sci. 11, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fpls.2020.00885\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2020.00885\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindelin, J. \u003cem\u003eet al.\u003c/em\u003e Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676\u0026ndash;682, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nmeth.2019\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.2019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR-4.3.1 for Windows (R Foundation for Statistical Computing, Vienna, Austria. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org\u003c/span\u003e\u003cspan address=\"https://www.R-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. [accessed 10 Sep 2023], 2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeager, T. \u003cem\u003eet al.\u003c/em\u003e Best management practices: Guide for producing container-grown plants. \u003cem\u003eSouthern Nursery Association. Atlanta, GA\u003c/em\u003e 7 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonanomi, G., Ippolito, F. \u0026amp; Scala, F. A \u0026ldquo;black\u0026rdquo; future for plant pathology? Biochar as a new soil amendment for controlling plant diseases. Journal of Plant Pathology 97, 223\u0026ndash;234, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4454/jpp.v97i2.3381\u003c/span\u003e\u003cspan address=\"10.4454/jpp.v97i2.3381\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlinke, H. B., Thomsen, A. B. \u0026amp; Ahring, B. K. Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol. 66, 10\u0026ndash;26, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00253-004-1642-2\u003c/span\u003e\u003cspan address=\"10.1007/s00253-004-1642-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaiswal, A. K., Frenkel, O., Tsechansky, L., Elad, Y. \u0026amp; Graber, E. R. Immobilization and deactivation of pathogenic enzymes and toxic metabolites by biochar: A possible mechanism involved in soilborne disease suppression. Soil Biol. Biochem. 121, 59\u0026ndash;66, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.soilbio.2018.03.001\u003c/span\u003e\u003cspan address=\"10.1016/j.soilbio.2018.03.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaiswal, A. K. \u003cem\u003eet al.\u003c/em\u003e Linking the Belowground Microbial Composition, Diversity and Activity to Soilborne Disease Suppression and Growth Promotion of Tomato Amended with Biochar. Sci Rep 7, 44382, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep44382\u003c/span\u003e\u003cspan address=\"10.1038/srep44382\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIacomino, G., Idbella, M., Laudonia, S., Vinale, F. \u0026amp; Bonanomi, G. The Suppressive Effects of Biochar on Above- and Belowground Plant Pathogens and Pests: A Review. Plants (Basel) 11, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/plants11223144\u003c/span\u003e\u003cspan address=\"10.3390/plants11223144\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoveda, J., Mart\u0026iacute;nez-G\u0026oacute;mez, \u0026Aacute;., Fenoll, C. \u0026amp; Escobar, C. The Use of Biochar for Plant Pathogen Control. \u003cem\u003ePhytopathology\u0026reg;\u003c/em\u003e 111, 1490\u0026ndash;1499, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1094/phyto-06-20-0248-rvw\u003c/span\u003e\u003cspan address=\"10.1094/phyto-06-20-0248-rvw\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDebode, J. \u003cem\u003eet al.\u003c/em\u003e Has compost with biochar added during the process added value over biochar or compost to increase disease suppression? Appl. Soil Ecol. 153, 103571, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.apsoil.2020.103571\u003c/span\u003e\u003cspan address=\"10.1016/j.apsoil.2020.103571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Disease incidence, Disease severity, Growth index, In vitro and in vivio, Pathogen inhibition, Trichoderma","lastPublishedDoi":"10.21203/rs.3.rs-4243906/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4243906/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003ePhytophthora\u003c/em\u003e blight caused by \u003cem\u003ePhytophthora capsici\u003c/em\u003e is a serious disease affecting a wide range of plants. Biochar as a soil amendment could partially replace peat moss and has the potential to suppress plant diseases, but its effects on controlling \u003cem\u003ephytophthora\u003c/em\u003e blight of container-grown peppers have less been explored, especially in combination of biological control using \u003cem\u003eTrichoderma\u003c/em\u003e. In vitro (petri dish) and in vivo (greenhouse) studies were conducted to test sugarcane bagasse biochar (SBB) and mixed hardwood biochar (HB) controlling effects on pepper \u003cem\u003ephytophthora\u003c/em\u003e blight disease with and without \u003cem\u003eTrichoderma\u003c/em\u003e. Sugarcane bagasse biochar and HB were blended with the commercial substrate (CS, peat-based) at 10% (SBB10, by volume), and 10%, 30%, 50%, 70% (HB10, HB30, HB50, and HB70, by volume), respectively, and CS (CS100) was used as the control. Both in vitro and in vivo studies used randomized complete block design with three treatment factors: pathogen (without or with inoculation of \u003cem\u003eP. capsici\u003c/em\u003e), biochar (different biochar treatments), and \u003cem\u003eTrichoderma\u003c/em\u003e (without or with inoculation). In vitro results showed that \u003cem\u003eTrichoderma\u003c/em\u003e inhibited \u003cem\u003eP. capsici\u003c/em\u003e growth while biochar did not have significant beneficial effects. In vivo results showed that plants grown in HB30 and HB50 had similar or higher plant growth index and shoot dry weight than the control regardless of pathogen presence. In the presence of the pathogen, plants grown in HB30, HB50, and HB70 had significantly lower disease severity, and disease incidence ratings than the control, while \u003cem\u003eTrichoderma\u003c/em\u003e did not show beneficial effects on controlling the disease. In conclusion, HB replacing 30% and 50% peat moss in substrate could reduce pepper blight disease caused by \u003cem\u003eP. capsici\u003c/em\u003e without negatively affecting plant growth.\u003c/p\u003e","manuscriptTitle":"Biochar reduces containerized pepper blight caused by Phytophthora capsici","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 08:01:58","doi":"10.21203/rs.3.rs-4243906/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-29T07:05:38+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"122403446448168413162968128907265322419","date":"2024-05-15T17:05:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-02T16:02:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68a59923-043e-44f5-87a5-5c41d03d2c9f_SNPRID","date":"2024-04-26T00:52:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-26T00:48:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-26T00:41:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-22T11:14:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-22T10:48:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-09T20:08:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e42a8a9-0edb-44d8-913e-e585154c51ed","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30992617,"name":"Biological sciences/Biotechnology"},{"id":30992618,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2024-12-30T16:03:02+00:00","versionOfRecord":{"articleIdentity":"rs-4243906","link":"https://doi.org/10.1038/s41598-024-76712-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-12-28 15:57:48","publishedOnDateReadable":"December 28th, 2024"},"versionCreatedAt":"2024-04-25 08:01:58","video":"","vorDoi":"10.1038/s41598-024-76712-3","vorDoiUrl":"https://doi.org/10.1038/s41598-024-76712-3","workflowStages":[]},"version":"v1","identity":"rs-4243906","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4243906","identity":"rs-4243906","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}