Integrating synthetic community with chemical fungicide improves the control of Rhizoctonia bare patch disease in wheat

Integrating synthetic community with chemical fungicide improves the control of Rhizoctonia bare patch disease in wheat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrating synthetic community with chemical fungicide improves the control of Rhizoctonia bare patch disease in wheat Wajira Nandanee Gamachchige Galhena, Juntao Wang1, Bruna D Batista1, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7398546/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The soil borne pathogen Rhizoctonia solani AG8 causes Rhizoctonia bare patch disease, a major constraint on global wheat production, particularly in no- or minimal -till systems. Current control strategies such as crop rotation, chemical fungicides, and tillage provide only partial protection, while fungicides can accelerate resistance development and negatively impact crop, soil, and human health. Microbial biological control agents (BCAs) and synthetic communities (SynComs, combining two or more BCAs) represent a sustainable alternative for managing Rhizoctonia bare patch. However, inconsistent efficacy and limited compatibility with fungicides remain key challenges to adoption. Here, we demonstrated that individual BCAs and a SynCom (comprising Diaporthe sp. and Pseudomonas sp. KAR75) reduced disease incidence at the seedling stage by 51–71% compared with fungicide treatment. When combined with fungicide, disease incidence was further reduced by 65–100%. The BCA treatments also improved grain yield by 9–12% compared with fungicide treatment and by 11–15% compared to no-pathogen control, demonstrating their ability to promote wheat growth in the absence of disease. Grain yield increased most with the fungal-bacterial SynCom, particularly when applied together with fungicide. These results demonstrated that integrating BCAs with chemical fungicides has strong potential to improve control of soil-borne diseases in grain crops. Agroecology SynCom biological control Rhizoctonia bare patch integrated pest management soil-borne disease grain crops Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights BCAs and SynComs decreased Rhizoctonia bare patch incidence more effectively than chemical fungicide alone. Combining BCAs with fungicide enhanced disease suppression to a greater extent than single treatments. BCA treatment improved grain yield compared to control. The SynCom of Diaporthe sp and Pseudomonas sp. KAR75 provided the most promising result. Introduction Soil-borne diseases caused by fungal and oomycete pathogens significantly constrain farm productivity and represent a major threat to global food security. Wheat, which serves as the staple food for nearly 35% of the global population (Grote et al., 2021 ), is particularly affected. In Australia alone, fungal diseases of wheat cause an estimated annual loss of A $ 840 million (Murray and Brennan, 2010 ). Among these devastating pathogens, the soil borne fungus Rhizoctonia solani Kuhn AG8 (formerly, teleomorph: Thanatephorus cucumeris ) causes Rhizoctonia bare patch, which is prevalent in dryland cropping systems of Australia and the northwest United States. Impacts on cereals and grain crops can be severe; in Australia, yield loss can reach approximately 25% in infected wheat fields (MacNish and Neate, 1996 ). On a national scale, this represents an estimated A $ 77 million annual loss in wheat and barley (Murray & Brennan, 2010 ). Although some fungicides are used, no effective control strategy is currently available for R. solani (Barnett et al., 2019 ; Paulitz, 2006 ). The absence of resistant wheat varieties and the wide host range of the pathogen (cereal crops, broad leaf crops, grasses, and weeds) further complicate Rhizoctonia bare patch management (Hayden et al., 2018 ). In recent years, chemical fungicides including ipconazole, metalaxyl, sedaxane, difenoconazole and Penflufen have been reported to suppress Rhizoctonia bare patch (Almasudy et al., 2015 ; Hüberli et al., 2014 ). However, their efficacy remains limited and inconsistent under Australian field conditions (Barnett et al., 2019 ), and growers must weigh cost-effectiveness against seasonal conditions and soil pathogen load. Furthermore, increasing market demand for low-residue agricultural products, combined with the development of fungicide resistance, have intensified the search for sustainable alternatives (Bhatta, 2022 ; Jiménez-Reyes et al., 2019 ; Batista and Singh 2021 ). Biological control agents (BCAs) have been extensively studied for plant growth promotion and disease suppression (Li et al., 2022 ; Singh et al 2023 ; 2025 ). They can directly improve plant growth by controlling pathogens (Martins et al., 2018 ; McGahan et al., 2014 ; Trivedi et al., 2017 ) or indirectly through growth hormone production and improved soil health (Agaras et al., 2015 ; Mukherjee et al., 2021 ). BCAs may also enhance soil health and fertility by solubilizing or fixing macro and micro nutrients such as phosphate, potassium, and nitrogen, increasing their availability for plant uptake (e.g. Das et al., 2021 ; El Enshasy et al., 2020 ). Despite these benefits, widespread adoption of BCAs remain limited due to inconsistent performance under variable field conditions (Batista and Singh, 2021 ; Ons et al., 2020 ). Synthetic microbial communities (SynComs) are emerging as a potentially more versatile alternative to single-strain BCA inoculants (Liang et al., 2022 ; Minchev et al., 2021 : Delgado-Baquerizo et al., 2025 ). A SynCom, comprising different microbial groups, can provide synergistic responses to enhance crop productivity and pathogen suppression (Mishra et al., 2021 ; Mukherjee et al., 2021 ). Their diversity allows for multiple plant growth promoting mechanisms, potentially improving performance over a single-microbe inoculants (Berg et al., 2017 ; Liu et al., 2021 ). Integrating BCAs with fungicides has also been proposed as a way to achieve effective and sustainable disease control (Ons et al., 2020 ; Palmieri et al., 2022 ). In such combinations, fungicides may stabilize BCA activity, making biological products more attractive for large-scale application, and may allow reduced fungicide use in the future (Lima et al., 2008 ). On the other hand, BCAs may expand the spectrum of pathogen control and reduce the risk of fungicide resistance development (Eshel et al., 2009 ). However, the combined efficacy of BCAs with fungicides in suppressing Rhizoctonia bare patch disease in wheat remain largely unexplored. Here, we screened bacterial and fungal isolates for effectiveness against R. solani AG8 and characterized their plant growth promoting traits. Their efficacy in suppressing Rhizoctonia bare patch was assessed individually, jointly as Syncom, and in combination with a commercial fungicide. We evaluated disease incidence, plant growth parameters (including grain yield), and pathogen abundance using qPCR. Materials and methods Experiment 1: Isolation and screening of R. solani AG8-suppressive microorganisms from wheat rhizosphere and endosphere To isolate potential biocontrol agents against R. solani AG8 , field sampling was conducted in three healthy and three infected wheat fields in Young, NSW, Australia in October 2020 (Figure S1). All wheat plants were at the same phenological stage (flowering, Zadock growth stage GS60–69) (GRDC 2005) at the time of sampling. At each site, a plot was established from which samples were collected. Plants were categorized based on visible health status as healthy (plants from healthy farms), asymptomatic (plants from infected fields without visible stunting or bare patch symptoms), or symptomatic (plants from infected fields showing stunting and bare patch symptoms). Within each plot, 5 individual plants were collected from healthy fields and 10 from infected fields (5 asymptomatic and 5 symptomatic). Plants were spaced at least 25 m apart. For each plant, the entire root system was excavated within a 10 cm radius and to a depth of 20 cm. The soil monolith was removed with the plant in the centre. Bulk soil was detached from the roots by vigorous shaking, and each plant with its rhizosphere was placed in a labelled zip lock bag and stored on ice during transport. From each farm, three rhizosphere and three root samples representing each plant health status (healthy, asymptomatic, symptomatic) were used for isolations. Details of the isolation and screening of potential biocontrol agents are provided in the Supplemental materials. In total, 93 bacterial and 12 fungal isolates were screened for disease-suppressive capacity. Following dual-plate screening assays, 17 isolates (12 bacterial and five fungal) that exhibited the highest pathogen inhibition and strong siderophore production, phosphate solubilization, and zinc solubilisation (Table S1) were selected for further seedling bioassays (see supplemental materials and methods). From these assays, three BCAs were selected for subsequent testing in Experiment 2. Experiment 2: In vitro evaluation of BCAs and chemical fungicide for disease suppression Four BCA treatments were used: two single bacterial strains (A and E), one fungal strain (M), and a SynCom combining strains E (bacterial) and M (fungal), hereafter referred to as ‘SynCom’. These BCAs were evaluated for their efficacy in controlling Rhizoctonia bare patch disease in wheat under controlled environmental conditions. In addition, these BCAs were tested in combination with a commercial fungicide, Evergol® Energy (Bayer Australia/New Zealand), containing penflufen, prothioconazole, and metalaxyl (active ingredients: 38.4 g/L Penflufen, 76.8 g/L Prothioconazole, and 61.4 g/L Metalaxyl). Fungicide effect on R. solani AG8 and BCA growth in vitro The effect of the fungicide on the mycelial growth of R. solani AG8 and the fungal BCA (M) was tested following Myresiotis et al. ( 2012 ). Potato dextrose agar (PDA) plates were amended with fungicide at concentrations of 0 (control), 1, 10, and 100 ppm. Mycelial plugs (5 mm diameter) were placed at the centre of the PDA plates and incubated in the dark at 25 ºC for 7 days. For each fungus, the experiment was repeated twice using five replicates (plates) per concentration. Fungal colony diameter was measured, and percentage growth inhibition was calculated according to Myresiotis et al. ( 2012 ) using the formula: Inhibition (%) = \(\:\frac{\left(Dc-Dt\right)}{Dc}\times\:100\) Where Dc is the average colony diameter on control plates and Dt the average diameter on fungicide-amended plates. The average fungicide concentration capable of 50% inhibition of pathogen mycelium (LD 50 ) was calculated (Elslahi et al., 2014 ) using the AAT Bioquest LD50 Calculator (AAT Bioquest 2015 ). This concentration was used in the subsequent glasshouse experiments. The manufacturer’s recommended field dose (0.2 ppm) was ineffective at inhibiting pathogen growth in vitro . To test bacterial BCAs compatibility with the fungicide, 100 µL of each bacterial culture was spread on PDA plates amended with fungicide at 0 ppm (control), 1 ppm, 10 ppm and 100 ppm (duplicates per treatment). Plates were incubated in the dark at 25 ºC for 48 h, and bacterial growth was qualitatively assessed. In vitro compatibility among BCAs for SynCom design In vitro compatibility among the selected bacterial and fungal BCAs was tested using the dual culture technique described by Siddiqui & Shaukat ( 2003 ) with minor modifications. Bacterial BCAs were grown in LB broth overnight (25 ºC, 150 rpm). One hundred microliters of bacterial culture was streaked on the sides of a PDA plate, and a 5mm-diameter mycelial disc of the fungal BCA (7-day-old) was placed in the centre. Plates were incubated at 25 ºC for 3–5 days. All possible BCA combinations were tested with 3 replicate plates. The presence of a growth inhibition zone was considered evidence of incompatibility, whereas fungal overgrowth of bacterial streaks without inhibition indicated compatibility. Compatibility tests showed a negative interaction between BCA_M and BCA_A (Figure S4b), but no negative interaction between BCA_M and BCA_E (Figure S4c). Thus, BCA_M and BCA_E were combined for the “SynCom” treatment. Glasshouse experiments Two glasshouse experiments were conducted simultaneously (July - December 2021) to determine the effect of BCA treatments in controlling Rhizoctonia bare patch infection in wheat. Glasshouse experiment 1 was a short-term seedling assay (8 weeks) designed to assess seedling disease incidence (destructive assessment). Glasshouse experiment 2 ran for 20 weeks (seedling to harvest) to evaluate long-term effects of BCAs on crop yield and soil parameters. In both trials, BCAs were tested alone and in combination with the chemical fungicide. Inoculation of pots with R. solani AG8 and experimental design The pots used in this experiment were inoculated with R. solani AG8 using mycelial discs (60 discs per pot of 5 kg soil), placed at a depth of 5 cm, covered with aluminium foil, and incubated at 15 ℃ for two weeks to allow pathogen establishment. Four pre-germinated wheat seeds were then sown (3 cm deep) in each pot (180mm × 130 mm, diameter and height). Seeds were treated with 1 mL of BCA suspension (see supplemental material and methods). Both experiments followed a randomised block design with 10 treatments and 5 replicates each. For glasshouse experiment 2, an additional two treatments were established, no pathogen no fungicide; no pathogen plus fungicide to determine treatment effects relative to non-pathogen controls. Based on preliminary in vitro assays (Figure S3), chemical fungicide was applied in-furrow at 2.1 ppm (the LD 50 dose determined for pathogen control in vitro ). For application, a furrow (6 cm × 1 cm, depth and width) was made at the centre of the pot using a sterile scalpel. Fungicide was pipetted into the furrow, which was then covered with a 3 cm layer of soil before sowing 4 seeds per pot. At sowing, pots received 100 mL of 25% Hoagland’s nutrient solution, followed by 100 ml of 50% Hoagland’s nutrient solution every three weeks thereafter (Bach et al. 2016 ; Kim et al. 1997 ). Fourteen days after sowing, seedlings were thinned to one plant per pot. Plants were watered twice weekly with 100 mL of water and kept at 20º/15 ºC (day/night), 12 h light/12 h dark, for 5 months. Assessment of pathogen load in soil Bulk soil cores (top 5cm, 15mm diameter) were collected at the second, third, and fifth months after sowing. Samples were stored at -80 ºC until DNA extraction. Pathogen load was determined using quantitative polymerase chain reaction (qPCR) with primers specific for R. solani AG8 (Jaaffar et al. 2017 ). DNA was extracted from 500 mg soil per sample using the DNeasy® PowerSoil Pro Kit (Qiagen, Germany). DNA concentration was quantified using a Nanodrop spectrophotometer (DenNovix, DS- 11 FX, DeNovix Inc., USA.) and adjusted to 5 ng/ µL with ddH 2 O. qPCR was carried out using the LightCycler®480 system (Roche Holding AG, Switzerland) using R. solani AG8-specific primers (IDT, USA) for ITS1 region (AG-8_F - AGT TGG TTG TAG CTG GTC CAT TAA T ITS1; AG-8_R - AGT AGA CAG AGG GGT CCA ATA AAT GA ITS1; Budge et al., 2009 ). The 9 µL master mixture contained 1.5 µL of primer mix, 5 µL of SYBR Green PCR master mix (LightCycler 480 SYBR Green I Master), and 2.5µL of deionized water, with 1 µL of DNA template. PCR conditions were 95 ºC for 3min, followed by 40 cycles of 95 ºC for 20 s, 60 ºC for 30 s and 72 ºC for 30 s and by a melting curve program (Yin et al., 2021 ). Products were visualized on 2% agarose gels with 100 bp ladder (BIOLINE) at 75 mv for 45 min. All reactions were carried out in duplicate. DNA was excised from gel bands, purified using PCR clean-up kit (Wizard® SV Gel and PCR Clean-Up System, Promega, USA), and quantified. Standard curves were constructed from tenfold serial dilutions (10 − 1 to 10 − 7 of amplified fDNA from four random samples, relating cycle threshold (Ct) to gene copy number (Liu et al., 2019 ). Pathogen load in soil samples was calculated by interpolating Ct values against the standard curve. Assessment of disease incidence and plant growth In the short-term glasshouse experiment, plants were harvested 8 weeks after sowing. Roots were washed and the number of spear tip roots (a characteristic symptom of Rhizoctonia bare patch disease) were counted in each plant and recorded. Disease incidence was calculated as described in supplemental materials. In the long-term glasshouse experiment, plant growth attributes (plant height, number of leaves, and number of tillers) were recorded. At harvest (five months after sowing), wheat plants were uprooted, roots washed and shoot length (length from the residue seed piece to the tip of the longest leaf) measured. Shoots and roots were separated, dried at 60 ºC oven for 72 h, and weighed. Grain, spike and straw weights were recorded. For nutrient analyses of grains, dried wheat grain samples were milled to a fine powder in a Mixer mill (Retsch MM400, Germany) at 30/s for 1 min. Carbon (C) and nitrogen (N) were quantified using a LECO TruMac CN analyser (LECO cooperation, USA.) at the Hawksbury Institute for the Environment. Phosphorous (P), potassium (K), calcium (Ca), iron (Fe), zinc (Zn), and total protein contents were determined following acid digestion (nitric acid hotblock digestion) followed by Inductively Coupled Plasma Mass Spectroscopic (ICP - MS) analysis that was conducted by the Environmental Analysis Laboratory, Southern Cross University Lismore, New South Wales, Australia. Statistical analysis For both glasshouse experiments, the effects of BCA and fungicide treatments on disease incidence and plant growth were assessed using a two-way ANOVA followed by Holm-Sidak pairwise comparisons. Data that was not normally distributed and/or had unequal variance was square-root or log-transformed prior to the ANOVA. All analyses were performed in SigmaPlot 15 (Systat Software, Inc.). Results Effect of fungicide on the in vitro growth of R . solani AG8 and biocontrol agents in vitro and compatibility of bacterial and fungal biocontrol agents. The pathogen R. solani AG8 was sensitive to the fungicide, showing 32.65%, 84.92%, and 100% inhibition of mycelial growth at 1, 10, and 100 ppm, respectively (Table 1 ). In contrast, the fungal BCA_M was less impacted, with 7.42%, 39.00%, and 97.62% inhibition of mycelial growth at 1, 10 ppm and 100 ppm, respectively (Table 1 ). The growth of bacterial BCA_E was unaffected by fungicide at all concentrations tested. The calculated LD 50 value of the fungicide against R . solani AG8 was 2.1 ppm. At this concentration, inhibition of BCA_M growth was relatively low (12%), and there was no impact on bacterial BCA growth. Thus, 2.1 ppm was selected for in-furrow fungicide application in subsequent glasshouse assays. Table 1 Effect of fungicide concentration on mycelial growth of the pathogen Rhizoctonia solani AG8, bacterial biocontrol agents (BCA) A and C and the fungal BCA M. Numbers in brackets represent ± one standard error (n = 3). Fungicide concentration (ppm) R. solani AG8 BCA A BCA E BCA M 0 0 No inhibition observed No inhibition observed 0 1 32.65% (± 1.41) No inhibition observed No inhibition observed 7.42% (± 0.95) 10 84.92% (± 1.10) No inhibition observed No inhibition observed 39.00% (± 1.97) 100 100% (± 0.00) No inhibition observed No inhibition observed 97.62% (± 0.36) Effect of BCA treatment on disease incidence after eight weeks of wheat growth After eight weeks, disease incidence was significantly affected by BCA treatment (p < 0.001), fungicide treatment (p < 0.001), and by their interaction (p < 0.01; Fig. 1 a). In control plants (no BCA), fungicide treatment alone reduced disease incidence by 21.5%, although this was not significant. In the absence of fungicide, the SynCom and BCA_M treatments significantly reduced disease incidence by 71 and 51%, respectively, compared to the fungicide-free control. However, when fungicide was used in conjunction with BCA treatments, disease incidence was reduced across all BCA treatments by 65 to 100%. Notably, a 100% reduction in disease incidence was observed when the SynCom was combined with fungicide. Seedling shoot and root biomass were not significantly affected by BCA treatment alone but were significantly impacted by fungicide treatment (Fig. 1 b and 1 c). Shoot and root biomass were 80.5% and 42.6% higher, respectively, in the presence of fungicide (p < 0.001, Fig. 1 b and 1 c). There was also a significant interaction between BCA and fungicide on the number of roots exhibiting the characteristic Rhizoctonia spear-tip morphology (Fig. 1 d). Without the fungicide, the fungal BCA_M and the SynCom had significantly fewer spear tips than the control or the two bacterial BCAs (A and E). With fungicide, all BCA treatments showed significantly fewer spear tips than the control (Fig. 1 d, p < 0.01), and no spear tips were observed in the SynCom + fungicide treatment after 8 weeks. Effect of BCA treatment on plant growth The BCA treatments significantly affected several plant growth measures, but effects were dependant on fungicide treatment (Fig. 2 ). Generally, BCAs increased total grain weight compared to the pathogen control (Fig. 2 a). However, significant increases relative to the pathogen-only control occurred only when BCAs were combined with fungicide. For example, with fungicide, BCA_E, BCA_A, and the SynCom increased grain weight by 9–12% compared to the pathogen control ( P < 0.01) and by 11–15% compared to the no-pathogen control ( P < 0.01). Similar, though non-significant, trends were observed in fungicide-free soils. Total spike weight also generally responded positively to BCA treatment, and this effect was not dependant on fungicide. For example, spike weight in the SynCom, BCA_E, and BCA_A treatments was 4–9% greater than in both the pathogen-only and pathogen-free controls, irrespective of fungicide application (Fig. 2 b, P < 0.05). In the absence of fungicide, the presence of pathogen (no BCA) reduced biomass by 7% compared to the non-pathogen control (Fig. 2 c, P < 0.01). However, BCA treatments did not significantly affect plant biomass compared to the pathogen control (Fig. 2 c), and this trend was also reflected in straw weight (Fig. 2 d). Plant height generally increased (4–18%) under BCA treatments, both with and without fungicide (Fig. 2 e). The strongest effect was observed with BCA_M, which increased plant height by 12% relative to the pathogen control and by 10% relative to the pathogen-free control in the absence of fungicide (Fig. 2 e, P < 0.001). The strong positive effect of BCA_M on plant height was also evident in the presence of fungicide (Fig. 2 e, P < 0.001). By contrast, BCA treatments generally reduced root biomass (Fig. 2 f), with effects typically more pronounced in the presence of fungicide. Effect of BCA treatment on nutrient content in wheat grains Grain crude protein content was unaffected by either BCA or fungicide treatment (Fig. 3 a). However, BCA treatment did impact grain C-content (Fig. 3 b). For example, in fungicide-free treatments, SynCom and BCA_A increased grain C content by 4–6% compared with the pathogen control (Fig. 3 b, P < 0.05). However, these increases were not observed when BCAs were combined with fungicide. Effects of BCAs on other micronutrients varied (Fig. 3 , Table S2). For example, BCA_M decreased grain Mg by 8% compared to the pathogen-free control (Fig. 3 c P < 0.05). For Mn, all BCA treatments significantly reduced content by 9–15% (Fig. 3 d), with the greatest decline under BCA_M. Conversely, grain Cu content was 15% higher in the BCA_A treatment compared to the pathogen-free control (Fig. 3 e). Although effects on other macro- and micronutrients were not statistically significant, there was a general trend towards higher N, P, K, Ca, Mg, Fe, Zn, Cu, and crude protein contents in SynCom and BCA_A treatments compared to the controls. Fungicide treatment generally had little impact on grain nutrient content, with the notable exception of a 4% reduction grain P (Fig. 3 f, P < 0.01). Effect of BCA treatment on pathogen load over five months Pathogen load in the bulk soil, assessed by qPCR targeting R. solani AG8 ITS1 biomarker genes, decreased over the five-month study (Fig. 4 ). Fungicide treatment had no significant effect at any time point, whereas BCA treatments significantly reduced pathogen load at T2 and T3. At T2, BCA_M and BCA_E reduced pathogen load by 75 and 61%, respectively, compared to the control treatments (Fig. 4 a). By T3, BCA effects were stronger: pathogen load was reduced by 95% with BCA_E, 89% with SynCom, 78% with BCA_M, and 60% with BCA_A compared to the control (Fig. 4 b). After 5 months (T5), pathogen loads did not differ significantly between BCA treatments (Fig. 4 c). Discussion Here we demonstrated individual and SynCom BCAs reduced the incidence of Rhizoctonia bare patch disease in wheat. Further, when selected BCAs were used in conjunction with the chemical fungicide (containing the active ingredients penflufen, prothioconazole, and metalaxyl), reduction in disease incidence, and gains in grain yield were greater than when either the BCA of the fungicide were used alone. Compatibility of such beneficial microbes with chemical products, including fungicides, commonly used by farmers is important for their successful adoption as an alternative or complementary method for disease management (Ferrarezi et al., 2022 ). While a previous study conducted in wheat demonstrated compatibility of a SynCom treatment containing Bacillus subtilis and Trichoderma harzianum with a fungicide (a.i. metalaxyl M + fludioxonil), in controlling root rot caused by R. solani and Fusarium graminearum , the SynCOM + fungicide combination did not produced significantly higher disease control over the fungicide alone treatment (Sameer, 2019 ). In contrast, our results support the fact that the compatible BCAs could be combined with a chemical fungicide for an improved level of protection against R. solani AG8. The best performing BCAs of the present study (both invitro and in plant assays) included the bacterial Pseudomonas spp. and the fungal genera Diaporthe spp. Successful application of Pseudomonas spp. as BCAs against numerous fungal pathogens across a variety of crops has been widely demonstrated (e.g. Islam et al., 2014 ; Lally et al., 2017 ; Siddiqui and Shaukat, 2003 ; Weller, 2007 ). Pseudomonas is one of the most extensively studied genus of BCA with many commercialized biocontrol products in the market (Bonaterra et al., 2022 ). They possess many beneficial traits such as strong antimicrobial activities against a variety of plant pathogens, high ecological fitness, and production of a wide variety of bioactive compounds including antibiotics, enzymes, and cyclic peptides (Bonaterra et al., 2022 ; Haas and Keel, 2003 ). On the other hand, fungal Diaporthe spp. are more commonly associated with plants as pathogens of different crops (Markell et al., 2015 ; Mena et al., 2020 ). Nevertheless, they have demonstrated biocontrol activity against many important plant pathogens including Verticillium dahliae, Botrytis cinerea, F. avenaceum, F. sprotrichioides, Alternaria alternata , and Trichothecium roseum (Abramczyk et al., 2022 ; Xu et al., 2021 ). Further, many studies reported that endophytic Diaporthe sp. fungi are rich in anti-fungal compounds which make them successful BCA candidates against fungal plant pathogens (Gao et al., 2020 ) and our results demonstrate that combining bacterial and fungal BCA can lead to further improved benefits of either BCA alone. The SynCom ( Diaporthe sp. + Pseudomonas sp. KAR75) used in the present study performed better than the respective single strain BCAs and outperformed single strain BCAs in increasing grain yield, plant height, and tiller production when combined with the fungicide. Although not all reported SynComs are more effective than single strains in reducing disease incidence of R solani , AG8 in wheat (Yin et al., 2022 ), there is a growing body of literature reporting the benefits of SynComs and microbial consortia in improving disease suppression, plant growth and yield in several crops including wheat, canola and maize over single strain BCAs (Ferrarezi et al., 2022 ; Kumar et al., 2014 ; Lally et al., 2017 ). They include microbes from different bacterial species of Pseudomonas (Lally et al., 2017 ), Bacillus thuringiensis RZ2MS9 and Azospirillum brasilense Ab-V5 (Ferrarezi et al., 2022 ) and Bacillus megaterium, Arthrobacter chlorophenolicus , and Enterobacter sp. (Kumar et al., 2014 ). Although none of the studies have applied the same combination of fungal Diaporthe sp + bacterial Pseudomonas sp. KAR75 used in the present study, they all suggest that microbial consortia and SynCom could provide higher biological control and growth improvement over the single strain BCAs. Our work provide further evidence on the compatibility of BCAs with chemical fungicide treatment. In addition, as consortia and SynCom consist of different microbes with a variety of biocontrol mechanisms, they can also provide an increased level of protection against plant pathogens (Amirthalingam et al., 2020 ; Sarma et al., 2015 ; Thakkar and Saraf, 2015 ). Validating the best performing candidates of the present study, under field conditions and against a wider range of pathogens and plant hosts is required to facilitate product development and successful adoption in integrated pest management. Several microbial genera have been identified as having biocontrol activity against R. solani AG8 and increasing crop yield including Bacillus, Streptomyces (Broadbent et al., 1971), Trichoderma (Ishtiaq Ch et al., 2019 ; Worasatit et al., 1994 ), Pantoea , Exiguobacterium , and Microbacteria (Barnett et al., 2006 ) and Pseudomonas spp. (Mavrodi et al., 2012 ; Yang et al., 2014 ). Most studies that explored fungal BCAs against R. solani AG8, have focused on different species of Trichoderma as BCA. However, in the present study, the fungal Daiporthe sp. demonstrated biocontrol potential against R. solani AG8 and potential to promote wheat growth. Although there are many reports around potential fungal and bacterial BCAs against R. solani AG8, no commercial biocontrol products have been successfully developed yet. Our work demonstrates, diversifying screening studies to assess a wider range of fungal and bacterial genera could increase the chances of successfully prospecting effective biocontrol candidates. Moreover, it might be worthwhile to understand the mechanisms of biocontrol of these isolates in the future to confirm its validity as a good candidate BCA against R. solani AG8. Many studies report that the plant growth promoting bacteria can significantly increase plant macronutrients (i.e., nitrogen, phosphorus, and potassium; e.g. Wang et al., 2020 ) and micronutrients content (i.e., iron, copper, manganese, and zinc; Kumar et al., 2014 ) of wheat under glasshouse and field conditions. The results showed higher (but statistically insignificant) N, P, K, Ca, Mg, Fe, Zn, Cu, and crude protein contents compared to the controls, particularly when the SynCom and BCA-A were applied. study provided evidence of increased grain Cu content under BCA_A ( Pseudomonas sp. 21-C3-ER). Several Pseudomonas spp . have been reported to facilitate phytoextraction of Cu from soils (e.g. Plociniczak et al., 2013 ), however less is known about their ability to facilitate uptake when Cu availability is scarce. Considering recent evidence of decreasing grain Cu content because of wheat breeding programmes in recent decades (Hao et al., 2022 ), identifying BCAs that not only decrease disease incidence but also increase nutritional quality of grain is an important step to improved food security and nutrition. Interestingly, the fungal BCA (M) decreased grain Mg and Mn content. Both Mg and Mn play an important role in the interaction between plants and fungi and some fungi are known to actively bio-protect wheat against Mn toxicity (Brito et al., 2014 ). Understanding how potential BCA affect plant micro-nutrients is an important aspect in the successful development of integrated management systems and further investigations on grain, leaf, and soil nutrients with differing nutritional status may provide more insights into the potential of the BCAs to improve plant nutritional quality. Conclusion and Future directions The focus of this work was to investigate the efficacy of BCAs and their combined application with a chemical fungicide commonly adopted by farmers in Australia to control R. solani AG8. Findings of the current work provide strong evidence that compatible BCAs could be used in combination with chemical fungicides and improved grain yield could be achieved. The SynCom of Diaporthe sp. + Pseudomonas sp. KAR75 showed promising results for Rhizoctonia bare patch disease control, wheat grain yield and tiller production improvement when combined with the fungicide. However, it must be noted that in the present study the LD 50 dose of the fungicide to control R. solani AG8 mycelial growth in vitro (2.1 ppm) was applied into pots which was higher than the recommended dose (0.2 ppm) by the manufacturer. Thus, further work is required to determine efficacy of biocontrol across reduced doses of fungicide. For developing successful and commercially viable BCAs, testing under field conditions is critical to ensure BCAs retain the protective characteristics demonstrated under laboratory screening and glasshouse experiments (Ferrarezi et al., 2022 ). Developing high throughput in planta assays at an early stage of screening to maximise discovery of effective strains to control disease under field level conditions (Barnett et al., 2017 ). Further, tracing the activity of bioinoculants in soil would help in understanding the fate and persistence of these inoculants in field conditions. The work presented here sets the foundations for future field scale assessments for controlling Rhizoctonia bare patch of wheat. Declarations AUTHOR CONTRIBUTIONS All authors contributed to the conceptualization and design; WNGG conducted the work with assistance from JW and BDB. WNGG, and CAM analysed the data and wrote the original draft, with all authors providing input to the final manuscript. ACKNOWLEDGEMENTS This work was funded by the AHEAD project Sri Lanka awarded to WNGG. We are grateful to Peter Hambling (Kalyx Ltd, Australia) for assistance in field access and sampling. Microbial control works in B.K.S lab is funded by the Australian Research Council (DP230101448), the CRC-Future Food System, the Department of Agriculture, Fisheries and Forestry (DAFF) Soil Science Challenge grants (ID: 4-H4SSYXD; 4-H4T24R2), CONFLICT OF INTEST The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ETHICS STATEMENT Ethics statement not applicable to this study. References AAT Bioquest (2015) LD50 Calculator. Abramczyk B, Marzec-Grządziel A, Grządziel J, Król E, Gałązka A, Oleszek W (2022) Biocontrol potential and catabolic profile of endophytic Diaportheeres strain 1420S from Prunus domestica L. in Poland—A preliminary study. 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Macdonald","email":"data:image/png;base64,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","orcid":"","institution":"Western Sydney University","correspondingAuthor":true,"prefix":"","firstName":"Catriona","middleName":"A.","lastName":"Macdonald","suffix":""}],"badges":[],"createdAt":"2025-08-18 10:21:07","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7398546/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7398546/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89368651,"identity":"1bad7844-7742-4477-a0d8-81b81a157822","added_by":"auto","created_at":"2025-08-19 09:41:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88592,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCA treatment application on (a) disease incidence (%), (b) shoot biomass, (c) root biomass and (c) number of spear roots of wheat seedlings grown for eight weeks under glasshouse conditions. Light blue bars represent BCA treatments without the fungicide application, while the dark bars are BCA treatments combined with the chemical fungicide. Error bars represent ± one standard error (n=5). Different letters within a fungicide treatment indicate statistically significant differences among means (Holm-Sidak, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7398546/v1/e671f26a6c4cf79b6cdc9667.jpg"},{"id":89367796,"identity":"ace0ac5b-5c93-4a8d-b092-6e5afb028a9d","added_by":"auto","created_at":"2025-08-19 09:33:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122375,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCA treatments on mean (± one standard error) (a) grain weight, (b) spike weight, (c) total plant biomass, (d) straw weight (e) plant height and (f) root weight in the absence (light bars) and presence (dark bars) of fungicide after 5 months growth. Hatched bars represent no pathogen controls. Within a fungicide treatment, bars sharing the same letter are not significantly different (n = 5). Capital letters indicate overall BCA treatment effect in the absence of a BCA x fungicide interaction.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7398546/v1/b21f110b15c0d6632f879a99.jpg"},{"id":89368653,"identity":"9e8aeee3-b900-4317-9aa4-a02ba5e0a5dc","added_by":"auto","created_at":"2025-08-19 09:41:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114175,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCA treatments on mean (± one standard error) wheat grain (a), protein (b) % C, (c) Mg, (d) Mn, (e) Cu and (f) P content the absence (light bars) and presence (dark bars) of fungicide after 5 months growth. Hatched bars represent no pathogen controls. Within a fungicide treatment, bars sharing the same letter are not significantly different (n = 5). Capital letters indicate overall BCA treatment effect in the absence of a BCA x fungicide interaction.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7398546/v1/6ad12652672ced422d99ac38.jpg"},{"id":89368656,"identity":"a542afc2-f2d7-4137-93d8-b5fd3e3229fe","added_by":"auto","created_at":"2025-08-19 09:41:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":78082,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCA treatments on R. solani AG8 load in bulk soil (copies of ITS1 gene per gram of soil) over the three time points assessed (2, 3, and 5 months after sowing = T2, T3, and T5, respectively) with (light blue) and without (dark blue) fungicide treatment. Error bars represent ± 95% CI (n=5). Within a time period, bars with different letters are significantly different (P \u0026lt; 0.05). Red dashed line and dotted lines represents the mean pathogen load and upper and lower 95% CI at T0.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7398546/v1/6ab18b74918e5dadd0b2a3ea.jpg"},{"id":89370482,"identity":"b4b8ad6f-398d-4df8-a40d-6ea3ca33efbd","added_by":"auto","created_at":"2025-08-19 10:05:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1409547,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7398546/v1/6ddecdbe-9a86-4cb8-bca1-8cc8aa315a4d.pdf"},{"id":89369022,"identity":"ab4255cd-c6da-4b5a-b81f-c9c0d1bf9271","added_by":"auto","created_at":"2025-08-19 09:49:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30330,"visible":true,"origin":"","legend":"\u003cp\u003esupplementary materials\u003c/p\u003e","description":"","filename":"SupplementalMaterialandMethodsFINAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-7398546/v1/b75174a90b6f3564fad6bef4.docx"},{"id":89369026,"identity":"855861ff-7bb9-4fa7-ba4e-7694a80590c2","added_by":"auto","created_at":"2025-08-19 09:49:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3168154,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTablesandFiguresFINAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-7398546/v1/fa280522ffbdc900f0bc185f.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eIntegrating synthetic community with chemical fungicide improves the control of Rhizoctonia bare patch disease in wheat\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eBCAs and SynComs decreased Rhizoctonia bare patch incidence more effectively than chemical fungicide alone.\u003c/li\u003e\n \u003cli\u003eCombining BCAs with fungicide enhanced disease suppression to a greater extent than single treatments.\u003c/li\u003e\n \u003cli\u003eBCA treatment improved grain yield compared to control.\u003c/li\u003e\n \u003cli\u003eThe SynCom of\u0026nbsp;\u003cem\u003eDiaporthe sp\u003c/em\u003e and\u0026nbsp;\u003cem\u003ePseudomonas\u0026nbsp;\u003c/em\u003esp. KAR75 provided the most promising result.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eSoil-borne diseases caused by fungal and oomycete pathogens significantly constrain farm productivity and represent a major threat to global food security. Wheat, which serves as the staple food for nearly 35% of the global population (Grote et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), is particularly affected. In Australia alone, fungal diseases of wheat cause an estimated annual loss of A\u003cspan\u003e$\u003c/span\u003e 840\u0026nbsp;million (Murray and Brennan, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Among these devastating pathogens, the soil borne fungus \u003cem\u003eRhizoctonia solani\u003c/em\u003e Kuhn AG8 (formerly, teleomorph: \u003cem\u003eThanatephorus cucumeris\u003c/em\u003e) causes Rhizoctonia bare patch, which is prevalent in dryland cropping systems of Australia and the northwest United States. Impacts on cereals and grain crops can be severe; in Australia, yield loss can reach approximately 25% in infected wheat fields (MacNish and Neate, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). On a national scale, this represents an estimated A\u003cspan\u003e$\u003c/span\u003e 77\u0026nbsp;million annual loss in wheat and barley (Murray \u0026amp; Brennan, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Although some fungicides are used, no effective control strategy is currently available for \u003cem\u003eR. solani\u003c/em\u003e (Barnett et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Paulitz, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The absence of resistant wheat varieties and the wide host range of the pathogen (cereal crops, broad leaf crops, grasses, and weeds) further complicate Rhizoctonia bare patch management (Hayden et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, chemical fungicides including ipconazole, metalaxyl, sedaxane, difenoconazole and Penflufen have been reported to suppress Rhizoctonia bare patch (Almasudy et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; H\u0026uuml;berli et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, their efficacy remains limited and inconsistent under Australian field conditions (Barnett et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and growers must weigh cost-effectiveness against seasonal conditions and soil pathogen load. Furthermore, increasing market demand for low-residue agricultural products, combined with the development of fungicide resistance, have intensified the search for sustainable alternatives (Bhatta, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jim\u0026eacute;nez-Reyes et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Batista and Singh \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBiological control agents (BCAs) have been extensively studied for plant growth promotion and disease suppression (Li et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Singh et al \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). They can directly improve plant growth by controlling pathogens (Martins et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; McGahan et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Trivedi et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) or indirectly through growth hormone production and improved soil health (Agaras et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mukherjee et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). BCAs may also enhance soil health and fertility by solubilizing or fixing macro and micro nutrients such as phosphate, potassium, and nitrogen, increasing their availability for plant uptake (e.g. Das et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; El Enshasy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite these benefits, widespread adoption of BCAs remain limited due to inconsistent performance under variable field conditions (Batista and Singh, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ons et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Synthetic microbial communities (SynComs) are emerging as a potentially more versatile alternative to single-strain BCA inoculants (Liang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Minchev et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e: Delgado-Baquerizo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). A SynCom, comprising different microbial groups, can provide synergistic responses to enhance crop productivity and pathogen suppression (Mishra et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mukherjee et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Their diversity allows for multiple plant growth promoting mechanisms, potentially improving performance over a single-microbe inoculants (Berg et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Integrating BCAs with fungicides has also been proposed as a way to achieve effective and sustainable disease control (Ons et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Palmieri et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In such combinations, fungicides may stabilize BCA activity, making biological products more attractive for large-scale application, and may allow reduced fungicide use in the future (Lima et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). On the other hand, BCAs may expand the spectrum of pathogen control and reduce the risk of fungicide resistance development (Eshel et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, the combined efficacy of BCAs with fungicides in suppressing Rhizoctonia bare patch disease in wheat remain largely unexplored.\u003c/p\u003e\u003cp\u003eHere, we screened bacterial and fungal isolates for effectiveness against \u003cem\u003eR. solani\u003c/em\u003e AG8 and characterized their plant growth promoting traits. Their efficacy in suppressing Rhizoctonia bare patch was assessed individually, jointly as Syncom, and in combination with a commercial fungicide. We evaluated disease incidence, plant growth parameters (including grain yield), and pathogen abundance using qPCR.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eExperiment 1: Isolation and screening of\u003c/b\u003e \u003cb\u003eR. solani\u003c/b\u003e \u003cb\u003eAG8-suppressive microorganisms from wheat rhizosphere and endosphere\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo isolate potential biocontrol agents against \u003cem\u003eR. solani AG8\u003c/em\u003e, field sampling was conducted in three healthy and three infected wheat fields in Young, NSW, Australia in October 2020 (Figure S1). All wheat plants were at the same phenological stage (flowering, Zadock growth stage GS60\u0026ndash;69) (GRDC 2005) at the time of sampling. At each site, a plot was established from which samples were collected. Plants were categorized based on visible health status as healthy (plants from healthy farms), asymptomatic (plants from infected fields without visible stunting or bare patch symptoms), or symptomatic (plants from infected fields showing stunting and bare patch symptoms). Within each plot, 5 individual plants were collected from healthy fields and 10 from infected fields (5 asymptomatic and 5 symptomatic). Plants were spaced at least 25 m apart. For each plant, the entire root system was excavated within a 10 cm radius and to a depth of 20 cm. The soil monolith was removed with the plant in the centre. Bulk soil was detached from the roots by vigorous shaking, and each plant with its rhizosphere was placed in a labelled zip lock bag and stored on ice during transport.\u003c/p\u003e\u003cp\u003eFrom each farm, three rhizosphere and three root samples representing each plant health status (healthy, asymptomatic, symptomatic) were used for isolations. Details of the isolation and screening of potential biocontrol agents are provided in the Supplemental materials. In total, 93 bacterial and 12 fungal isolates were screened for disease-suppressive capacity. Following dual-plate screening assays, 17 isolates (12 bacterial and five fungal) that exhibited the highest pathogen inhibition and strong siderophore production, phosphate solubilization, and zinc solubilisation (Table S1) were selected for further seedling bioassays (see supplemental materials and methods). From these assays, three BCAs were selected for subsequent testing in Experiment 2.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperiment 2: In vitro evaluation of BCAs and chemical fungicide for disease suppression\u003c/h2\u003e\u003cp\u003eFour BCA treatments were used: two single bacterial strains (A and E), one fungal strain (M), and a SynCom combining strains E (bacterial) and M (fungal), hereafter referred to as \u0026lsquo;SynCom\u0026rsquo;. These BCAs were evaluated for their efficacy in controlling Rhizoctonia bare patch disease in wheat under controlled environmental conditions. In addition, these BCAs were tested in combination with a commercial fungicide, Evergol\u0026reg; Energy (Bayer Australia/New Zealand), containing penflufen, prothioconazole, and metalaxyl (active ingredients: 38.4 g/L Penflufen, 76.8 g/L Prothioconazole, and 61.4 g/L Metalaxyl).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFungicide effect on\u003c/b\u003e \u003cb\u003eR. solani\u003c/b\u003e \u003cb\u003eAG8 and BCA growth\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe effect of the fungicide on the mycelial growth of \u003cem\u003eR. solani\u003c/em\u003e AG8 and the fungal BCA (M) was tested following Myresiotis et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Potato dextrose agar (PDA) plates were amended with fungicide at concentrations of 0 (control), 1, 10, and 100 ppm. Mycelial plugs (5 mm diameter) were placed at the centre of the PDA plates and incubated in the dark at 25 \u0026ordm;C for 7 days. For each fungus, the experiment was repeated twice using five replicates (plates) per concentration. Fungal colony diameter was measured, and percentage growth inhibition was calculated according to Myresiotis et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) using the formula:\u003c/p\u003e\u003cp\u003eInhibition (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\left(Dc-Dt\\right)}{Dc}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWhere Dc is the average colony diameter on control plates and Dt the average diameter on fungicide-amended plates.\u003c/p\u003e\u003cp\u003eThe average fungicide concentration capable of 50% inhibition of pathogen mycelium (LD\u003csub\u003e50\u003c/sub\u003e) was calculated (Elslahi et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) using the AAT Bioquest LD50 Calculator (AAT Bioquest \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This concentration was used in the subsequent glasshouse experiments. The manufacturer\u0026rsquo;s recommended field dose (0.2 ppm) was ineffective at inhibiting pathogen growth \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo test bacterial BCAs compatibility with the fungicide, 100 \u0026micro;L of each bacterial culture was spread on PDA plates amended with fungicide at 0 ppm (control), 1 ppm, 10 ppm and 100 ppm (duplicates per treatment). Plates were incubated in the dark at 25 \u0026ordm;C for 48 h, and bacterial growth was qualitatively assessed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecompatibility among BCAs for SynCom design\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e compatibility among the selected bacterial and fungal BCAs was tested using the dual culture technique described by Siddiqui \u0026amp; Shaukat (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) with minor modifications. Bacterial BCAs were grown in LB broth overnight (25 \u0026ordm;C, 150 rpm). One hundred microliters of bacterial culture was streaked on the sides of a PDA plate, and a 5mm-diameter mycelial disc of the fungal BCA (7-day-old) was placed in the centre. Plates were incubated at 25 \u0026ordm;C for 3\u0026ndash;5 days. All possible BCA combinations were tested with 3 replicate plates. The presence of a growth inhibition zone was considered evidence of incompatibility, whereas fungal overgrowth of bacterial streaks without inhibition indicated compatibility. Compatibility tests showed a negative interaction between BCA_M and BCA_A (Figure S4b), but no negative interaction between BCA_M and BCA_E (Figure S4c). Thus, BCA_M and BCA_E were combined for the \u0026ldquo;SynCom\u0026rdquo; treatment.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGlasshouse experiments\u003c/h3\u003e\n\u003cp\u003eTwo glasshouse experiments were conducted simultaneously (July - December 2021) to determine the effect of BCA treatments in controlling Rhizoctonia bare patch infection in wheat. Glasshouse experiment 1 was a short-term seedling assay (8 weeks) designed to assess seedling disease incidence (destructive assessment). Glasshouse experiment 2 ran for 20 weeks (seedling to harvest) to evaluate long-term effects of BCAs on crop yield and soil parameters. In both trials, BCAs were tested alone and in combination with the chemical fungicide.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInoculation of pots with\u003c/b\u003e \u003cb\u003eR. solani\u003c/b\u003e \u003cb\u003eAG8 and experimental design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe pots used in this experiment were inoculated with \u003cem\u003eR. solani\u003c/em\u003e AG8 using mycelial discs (60 discs per pot of 5 kg soil), placed at a depth of 5 cm, covered with aluminium foil, and incubated at 15 ℃ for two weeks to allow pathogen establishment. Four pre-germinated wheat seeds were then sown (3 cm deep) in each pot (180mm \u0026times; 130 mm, diameter and height). Seeds were treated with 1 mL of BCA suspension (see supplemental material and methods).\u003c/p\u003e\u003cp\u003eBoth experiments followed a randomised block design with 10 treatments and 5 replicates each. For glasshouse experiment 2, an additional two treatments were established, no pathogen no fungicide; no pathogen plus fungicide to determine treatment effects relative to non-pathogen controls. Based on preliminary \u003cem\u003ein vitro\u003c/em\u003e assays (Figure S3), chemical fungicide was applied in-furrow at 2.1 ppm (the LD \u003csub\u003e50\u003c/sub\u003e dose determined for pathogen control \u003cem\u003ein vitro\u003c/em\u003e). For application, a furrow (6 cm \u0026times; 1 cm, depth and width) was made at the centre of the pot using a sterile scalpel. Fungicide was pipetted into the furrow, which was then covered with a 3 cm layer of soil before sowing 4 seeds per pot.\u003c/p\u003e\u003cp\u003eAt sowing, pots received 100 mL of 25% Hoagland\u0026rsquo;s nutrient solution, followed by 100 ml of 50% Hoagland\u0026rsquo;s nutrient solution every three weeks thereafter (Bach et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Fourteen days after sowing, seedlings were thinned to one plant per pot. Plants were watered twice weekly with 100 mL of water and kept at 20\u0026ordm;/15 \u0026ordm;C (day/night), 12 h light/12 h dark, for 5 months.\u003c/p\u003e\n\u003ch3\u003eAssessment of pathogen load in soil\u003c/h3\u003e\n\u003cp\u003eBulk soil cores (top 5cm, 15mm diameter) were collected at the second, third, and fifth months after sowing. Samples were stored at -80 \u0026ordm;C until DNA extraction. Pathogen load was determined using quantitative polymerase chain reaction (qPCR) with primers specific for \u003cem\u003eR. solani\u003c/em\u003e AG8 (Jaaffar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). DNA was extracted from 500 mg soil per sample using the DNeasy\u0026reg; PowerSoil Pro Kit (Qiagen, Germany). DNA concentration was quantified using a Nanodrop spectrophotometer (DenNovix, DS- 11 FX, DeNovix Inc., USA.) and adjusted to 5 ng/ \u0026micro;L with ddH\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\u003cp\u003eqPCR was carried out using the LightCycler\u0026reg;480 system (Roche Holding AG, Switzerland) using \u003cem\u003eR. solani\u003c/em\u003e AG8-specific primers (IDT, USA) for ITS1 region (AG-8_F - AGT TGG TTG TAG CTG GTC CAT TAA T ITS1; AG-8_R - AGT AGA CAG AGG GGT CCA ATA AAT GA ITS1; Budge et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The 9 \u0026micro;L master mixture contained 1.5 \u0026micro;L of primer mix, 5 \u0026micro;L of SYBR Green PCR master mix (LightCycler 480 SYBR Green I Master), and 2.5\u0026micro;L of deionized water, with 1 \u0026micro;L of DNA template. PCR conditions were 95 \u0026ordm;C for 3min, followed by 40 cycles of 95 \u0026ordm;C for 20 s, 60 \u0026ordm;C for 30 s and 72 \u0026ordm;C for 30 s and by a melting curve program (Yin et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Products were visualized on 2% agarose gels with 100 bp ladder (BIOLINE) at 75 mv for 45 min. All reactions were carried out in duplicate. DNA was excised from gel bands, purified using PCR clean-up kit (Wizard\u0026reg; SV Gel and PCR Clean-Up System, Promega, USA), and quantified. Standard curves were constructed from tenfold serial dilutions (10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003eof amplified fDNA from four random samples, relating cycle threshold (Ct) to gene copy number (Liu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Pathogen load in soil samples was calculated by interpolating Ct values against the standard curve.\u003c/p\u003e\n\u003ch3\u003eAssessment of disease incidence and plant growth\u003c/h3\u003e\n\u003cp\u003eIn the short-term glasshouse experiment, plants were harvested 8 weeks after sowing. Roots were washed and the number of spear tip roots (a characteristic symptom of Rhizoctonia bare patch disease) were counted in each plant and recorded. Disease incidence was calculated as described in supplemental materials.\u003c/p\u003e\u003cp\u003eIn the long-term glasshouse experiment, plant growth attributes (plant height, number of leaves, and number of tillers) were recorded. At harvest (five months after sowing), wheat plants were uprooted, roots washed and shoot length (length from the residue seed piece to the tip of the longest leaf) measured. Shoots and roots were separated, dried at 60 \u0026ordm;C oven for 72 h, and weighed. Grain, spike and straw weights were recorded.\u003c/p\u003e\u003cp\u003eFor nutrient analyses of grains, dried wheat grain samples were milled to a fine powder in a Mixer mill (Retsch MM400, Germany) at 30/s for 1 min. Carbon (C) and nitrogen (N) were quantified using a LECO TruMac CN analyser (LECO cooperation, USA.) at the Hawksbury Institute for the Environment. Phosphorous (P), potassium (K), calcium (Ca), iron (Fe), zinc (Zn), and total protein contents were determined following acid digestion (nitric acid hotblock digestion) followed by Inductively Coupled Plasma Mass Spectroscopic (ICP - MS) analysis that was conducted by the Environmental Analysis Laboratory, Southern Cross University Lismore, New South Wales, Australia.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor both glasshouse experiments, the effects of BCA and fungicide treatments on disease incidence and plant growth were assessed using a two-way ANOVA followed by Holm-Sidak pairwise comparisons. Data that was not normally distributed and/or had unequal variance was square-root or log-transformed prior to the ANOVA. All analyses were performed in SigmaPlot 15 (Systat Software, Inc.).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eEffect of fungicide on the\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003egrowth of\u003c/b\u003e \u003cb\u003eR\u003c/b\u003e. \u003cb\u003esolani\u003c/b\u003e \u003cb\u003eAG8 and biocontrol agents\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand compatibility of bacterial and fungal biocontrol agents.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe pathogen \u003cem\u003eR. solani\u003c/em\u003e AG8 was sensitive to the fungicide, showing 32.65%, 84.92%, and 100% inhibition of mycelial growth at 1, 10, and 100 ppm, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the fungal BCA_M was less impacted, with 7.42%, 39.00%, and 97.62% inhibition of mycelial growth at 1, 10 ppm and 100 ppm, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The growth of bacterial BCA_E was unaffected by fungicide at all concentrations tested. The calculated LD\u003csub\u003e50\u003c/sub\u003e value of the fungicide against \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolani\u003c/em\u003e AG8 was 2.1 ppm. At this concentration, inhibition of BCA_M growth was relatively low (12%), and there was no impact on bacterial BCA growth. Thus, 2.1 ppm was selected for in-furrow fungicide application in subsequent glasshouse assays.\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\u003eEffect of fungicide concentration on mycelial growth of the pathogen \u003cem\u003eRhizoctonia solani\u003c/em\u003e AG8, bacterial biocontrol agents (BCA) A and C and the fungal BCA M. Numbers in brackets represent\u0026thinsp;\u0026plusmn;\u0026thinsp;one standard error (n\u0026thinsp;=\u0026thinsp;3).\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\u003eFungicide concentration (ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eR. solani\u003c/em\u003e AG8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBCA A\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBCA E\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBCA M\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo inhibition observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo inhibition observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32.65% (\u0026plusmn;\u0026thinsp;1.41)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo inhibition observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo inhibition observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.42% (\u0026plusmn;\u0026thinsp;0.95)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e84.92% (\u0026plusmn;\u0026thinsp;1.10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo inhibition observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo inhibition observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e39.00% (\u0026plusmn;\u0026thinsp;1.97)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100% (\u0026plusmn;\u0026thinsp;0.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo inhibition observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo inhibition observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e97.62% (\u0026plusmn;\u0026thinsp;0.36)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eEffect of BCA treatment on disease incidence after eight weeks of wheat growth\u003c/h3\u003e\n\u003cp\u003eAfter eight weeks, disease incidence was significantly affected by BCA treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), fungicide treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and by their interaction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In control plants (no BCA), fungicide treatment alone reduced disease incidence by 21.5%, although this was not significant.\u003c/p\u003e\u003cp\u003eIn the absence of fungicide, the SynCom and BCA_M treatments significantly reduced disease incidence by 71 and 51%, respectively, compared to the fungicide-free control. However, when fungicide was used in conjunction with BCA treatments, disease incidence was reduced across all BCA treatments by 65 to 100%. Notably, a 100% reduction in disease incidence was observed when the SynCom was combined with fungicide.\u003c/p\u003e\u003cp\u003eSeedling shoot and root biomass were not significantly affected by BCA treatment alone but were significantly impacted by fungicide treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Shoot and root biomass were 80.5% and 42.6% higher, respectively, in the presence of fungicide (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eThere was also a significant interaction between BCA and fungicide on the number of roots exhibiting the characteristic Rhizoctonia spear-tip morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Without the fungicide, the fungal BCA_M and the SynCom had significantly fewer spear tips than the control or the two bacterial BCAs (A and E). With fungicide, all BCA treatments showed significantly fewer spear tips than the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and no spear tips were observed in the SynCom\u0026thinsp;+\u0026thinsp;fungicide treatment after 8 weeks.\u003c/p\u003e\n\u003ch3\u003eEffect of BCA treatment on plant growth\u003c/h3\u003e\n\u003cp\u003eThe BCA treatments significantly affected several plant growth measures, but effects were dependant on fungicide treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Generally, BCAs increased total grain weight compared to the pathogen control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). However, significant increases relative to the pathogen-only control occurred only when BCAs were combined with fungicide. For example, with fungicide, BCA_E, BCA_A, and the SynCom increased grain weight by 9–12% compared to the pathogen control (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) and by 11–15% compared to the no-pathogen control (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Similar, though non-significant, trends were observed in fungicide-free soils.\u003c/p\u003e\u003cp\u003eTotal spike weight also generally responded positively to BCA treatment, and this effect was not dependant on fungicide. For example, spike weight in the SynCom, BCA_E, and BCA_A treatments was 4–9% greater than in both the pathogen-only and pathogen-free controls, irrespective of fungicide application (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, P \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003eIn the absence of fungicide, the presence of pathogen (no BCA) reduced biomass by 7% compared to the non-pathogen control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, P \u003cem\u003e\u0026lt;\u003c/em\u003e 0.01). However, BCA treatments did not significantly affect plant biomass compared to the pathogen control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), and this trend was also reflected in straw weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Plant height generally increased (4–18%) under BCA treatments, both with and without fungicide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The strongest effect was observed with BCA_M, which increased plant height by 12% relative to the pathogen control and by 10% relative to the pathogen-free control in the absence of fungicide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, P \u0026lt; 0.001). The strong positive effect of BCA_M on plant height was also evident in the presence of fungicide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, P \u0026lt; 0.001). By contrast, BCA treatments generally reduced root biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), with effects typically more pronounced in the presence of fungicide.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEffect of BCA treatment on nutrient content in wheat grains\u003c/h2\u003e\u003cp\u003eGrain crude protein content was unaffected by either BCA or fungicide treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, BCA treatment did impact grain C-content (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). For example, in fungicide-free treatments, SynCom and BCA_A increased grain C content by 4–6% compared with the pathogen control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, P \u003cem\u003e\u0026lt;\u003c/em\u003e 0.05). However, these increases were not observed when BCAs were combined with fungicide.\u003c/p\u003e\u003cp\u003eEffects of BCAs on other micronutrients varied (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table S2). For example, BCA_M decreased grain Mg by 8% compared to the pathogen-free control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ec \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). For Mn, all BCA treatments significantly reduced content by 9–15% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), with the greatest decline under BCA_M. Conversely, grain Cu content was 15% higher in the BCA_A treatment compared to the pathogen-free control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003eAlthough effects on other macro- and micronutrients were not statistically significant, there was a general trend towards higher N, P, K, Ca, Mg, Fe, Zn, Cu, and crude protein contents in SynCom and BCA_A treatments compared to the controls. Fungicide treatment generally had little impact on grain nutrient content, with the notable exception of a 4% reduction grain P (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, P \u0026lt; 0.01).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEffect of BCA treatment on pathogen load over five months\u003c/h2\u003e\u003cp\u003ePathogen load in the bulk soil, assessed by qPCR targeting \u003cem\u003eR. solani\u003c/em\u003e AG8 ITS1 biomarker genes, decreased over the five-month study (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Fungicide treatment had no significant effect at any time point, whereas BCA treatments significantly reduced pathogen load at T2 and T3.\u003c/p\u003e\u003cp\u003eAt T2, BCA_M and BCA_E reduced pathogen load by 75 and 61%, respectively, compared to the control treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). By T3, BCA effects were stronger: pathogen load was reduced by 95% with BCA_E, 89% with SynCom, 78% with BCA_M, and 60% with BCA_A compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). After 5 months (T5), pathogen loads did not differ significantly between BCA treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we demonstrated individual and SynCom BCAs reduced the incidence of Rhizoctonia bare patch disease in wheat. Further, when selected BCAs were used in conjunction with the chemical fungicide (containing the active ingredients penflufen, prothioconazole, and metalaxyl), reduction in disease incidence, and gains in grain yield were greater than when either the BCA of the fungicide were used alone. Compatibility of such beneficial microbes with chemical products, including fungicides, commonly used by farmers is important for their successful adoption as an alternative or complementary method for disease management (Ferrarezi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While a previous study conducted in wheat demonstrated compatibility of a SynCom treatment containing \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e with a fungicide (a.i. metalaxyl M + fludioxonil), in controlling root rot caused by \u003cem\u003eR. solani\u003c/em\u003e and \u003cem\u003eFusarium graminearum\u003c/em\u003e, the SynCOM + fungicide combination did not produced significantly higher disease control over the fungicide alone treatment (Sameer, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, our results support the fact that the compatible BCAs could be combined with a chemical fungicide for an improved level of protection against \u003cem\u003eR. solani\u003c/em\u003e AG8.\u003c/p\u003e\u003cp\u003eThe best performing BCAs of the present study (both invitro and in plant assays) included the bacterial \u003cem\u003ePseudomonas\u003c/em\u003e spp. and the fungal genera \u003cem\u003eDiaporthe\u003c/em\u003e spp. Successful application of \u003cem\u003ePseudomonas\u003c/em\u003e spp. as BCAs against numerous fungal pathogens across a variety of crops has been widely demonstrated (e.g. Islam et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lally et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Siddiqui and Shaukat, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Weller, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). \u003cem\u003ePseudomonas\u003c/em\u003e is one of the most extensively studied genus of BCA with many commercialized biocontrol products in the market (Bonaterra et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). They possess many beneficial traits such as strong antimicrobial activities against a variety of plant pathogens, high ecological fitness, and production of a wide variety of bioactive compounds including antibiotics, enzymes, and cyclic peptides (Bonaterra et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Haas and Keel, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). On the other hand, fungal \u003cem\u003eDiaporthe\u003c/em\u003e spp. are more commonly associated with plants as pathogens of different crops (Markell et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mena et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nevertheless, they have demonstrated biocontrol activity against many important plant pathogens including \u003cem\u003eVerticillium dahliae, Botrytis cinerea, F. avenaceum, F. sprotrichioides, Alternaria alternata\u003c/em\u003e, and \u003cem\u003eTrichothecium roseum\u003c/em\u003e (Abramczyk et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Further, many studies reported that endophytic \u003cem\u003eDiaporthe\u003c/em\u003e sp. fungi are rich in anti-fungal compounds which make them successful BCA candidates against fungal plant pathogens (Gao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and our results demonstrate that combining bacterial and fungal BCA can lead to further improved benefits of either BCA alone.\u003c/p\u003e\u003cp\u003eThe SynCom (\u003cem\u003eDiaporthe\u003c/em\u003e sp. + \u003cem\u003ePseudomonas\u003c/em\u003e sp. KAR75) used in the present study performed better than the respective single strain BCAs and outperformed single strain BCAs in increasing grain yield, plant height, and tiller production when combined with the fungicide. Although not all reported SynComs are more effective than single strains in reducing disease incidence of \u003cem\u003eR solani\u003c/em\u003e, AG8 in wheat (Yin et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), there is a growing body of literature reporting the benefits of SynComs and microbial consortia in improving disease suppression, plant growth and yield in several crops including wheat, canola and maize over single strain BCAs (Ferrarezi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lally et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). They include microbes from different bacterial species of \u003cem\u003ePseudomonas\u003c/em\u003e (Lally et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003eBacillus thuringiensis\u003c/em\u003e RZ2MS9 and \u003cem\u003eAzospirillum brasilense\u003c/em\u003e Ab-V5 (Ferrarezi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eBacillus megaterium, Arthrobacter chlorophenolicus\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e sp. (Kumar et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although none of the studies have applied the same combination of fungal Diaporthe sp + bacterial \u003cem\u003ePseudomonas\u003c/em\u003e sp. KAR75 used in the present study, they all suggest that microbial consortia and SynCom could provide higher biological control and growth improvement over the single strain BCAs. Our work provide further evidence on the compatibility of BCAs with chemical fungicide treatment. In addition, as consortia and SynCom consist of different microbes with a variety of biocontrol mechanisms, they can also provide an increased level of protection against plant pathogens (Amirthalingam et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sarma et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Thakkar and Saraf, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Validating the best performing candidates of the present study, under field conditions and against a wider range of pathogens and plant hosts is required to facilitate product development and successful adoption in integrated pest management.\u003c/p\u003e\u003cp\u003eSeveral microbial genera have been identified as having biocontrol activity against \u003cem\u003eR. solani\u003c/em\u003e AG8 and increasing crop yield including \u003cem\u003eBacillus, Streptomyces\u003c/em\u003e (Broadbent et al., 1971), \u003cem\u003eTrichoderma\u003c/em\u003e (Ishtiaq Ch et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Worasatit et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), \u003cem\u003ePantoea\u003c/em\u003e, \u003cem\u003eExiguobacterium\u003c/em\u003e, and \u003cem\u003eMicrobacteria\u003c/em\u003e (Barnett et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and \u003cem\u003ePseudomonas\u003c/em\u003e spp. (Mavrodi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Most studies that explored fungal BCAs against \u003cem\u003eR. solani\u003c/em\u003e AG8, have focused on different species of \u003cem\u003eTrichoderma\u003c/em\u003e as BCA. However, in the present study, the fungal \u003cem\u003eDaiporthe\u003c/em\u003e sp. demonstrated biocontrol potential against \u003cem\u003eR. solani\u003c/em\u003e AG8 and potential to promote wheat growth. Although there are many reports around potential fungal and bacterial BCAs against \u003cem\u003eR. solani\u003c/em\u003e AG8, no commercial biocontrol products have been successfully developed yet. Our work demonstrates, diversifying screening studies to assess a wider range of fungal and bacterial genera could increase the chances of successfully prospecting effective biocontrol candidates. Moreover, it might be worthwhile to understand the mechanisms of biocontrol of these isolates in the future to confirm its validity as a good candidate BCA against \u003cem\u003eR. solani\u003c/em\u003e AG8.\u003c/p\u003e\u003cp\u003eMany studies report that the plant growth promoting bacteria can significantly increase plant macronutrients (i.e., nitrogen, phosphorus, and potassium; e.g. Wang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and micronutrients content (i.e., iron, copper, manganese, and zinc; Kumar et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) of wheat under glasshouse and field conditions. The results showed higher (but statistically insignificant) N, P, K, Ca, Mg, Fe, Zn, Cu, and crude protein contents compared to the controls, particularly when the SynCom and BCA-A were applied. study provided evidence of increased grain Cu content under BCA_A (\u003cem\u003ePseudomonas sp.\u003c/em\u003e 21-C3-ER). Several \u003cem\u003ePseudomonas spp\u003c/em\u003e. have been reported to facilitate phytoextraction of Cu from soils (e.g. Plociniczak et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), however less is known about their ability to facilitate uptake when Cu availability is scarce. Considering recent evidence of decreasing grain Cu content because of wheat breeding programmes in recent decades (Hao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), identifying BCAs that not only decrease disease incidence but also increase nutritional quality of grain is an important step to improved food security and nutrition. Interestingly, the fungal BCA (M) decreased grain Mg and Mn content. Both Mg and Mn play an important role in the interaction between plants and fungi and some fungi are known to actively bio-protect wheat against Mn toxicity (Brito et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Understanding how potential BCA affect plant micro-nutrients is an important aspect in the successful development of integrated management systems and further investigations on grain, leaf, and soil nutrients with differing nutritional status may provide more insights into the potential of the BCAs to improve plant nutritional quality.\u003c/p\u003e"},{"header":"Conclusion and Future directions","content":"\u003cp\u003eThe focus of this work was to investigate the efficacy of BCAs and their combined application with a chemical fungicide commonly adopted by farmers in Australia to control \u003cem\u003eR. solani\u003c/em\u003e AG8. Findings of the current work provide strong evidence that compatible BCAs could be used in combination with chemical fungicides and improved grain yield could be achieved. The SynCom of \u003cem\u003eDiaporthe\u003c/em\u003e sp. + \u003cem\u003ePseudomonas\u003c/em\u003e sp. KAR75 showed promising results for Rhizoctonia bare patch disease control, wheat grain yield and tiller production improvement when combined with the fungicide. However, it must be noted that in the present study the LD\u003csub\u003e50\u003c/sub\u003e dose of the fungicide to control \u003cem\u003eR. solani\u003c/em\u003e AG8 mycelial growth \u003cem\u003ein vitro\u003c/em\u003e (2.1 ppm) was applied into pots which was higher than the recommended dose (0.2 ppm) by the manufacturer. Thus, further work is required to determine efficacy of biocontrol across reduced doses of fungicide. For developing successful and commercially viable BCAs, testing under field conditions is critical to ensure BCAs retain the protective characteristics demonstrated under laboratory screening and glasshouse experiments (Ferrarezi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Developing high throughput \u003cem\u003ein planta\u003c/em\u003e assays at an early stage of screening to maximise discovery of effective strains to control disease under field level conditions (Barnett et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Further, tracing the activity of bioinoculants in soil would help in understanding the fate and persistence of these inoculants in field conditions. The work presented here sets the foundations for future field scale assessments for controlling Rhizoctonia bare patch of wheat.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conceptualization and design; WNGG conducted the work with assistance from JW and BDB. \u0026nbsp; WNGG, and CAM analysed the data and wrote the original draft, with all authors providing input to the final manuscript. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the AHEAD project Sri Lanka awarded to WNGG. \u0026nbsp;We are grateful to Peter Hambling (Kalyx Ltd, Australia) for assistance in field access and sampling. Microbial control works in B.K.S lab is funded by the Australian Research Council (DP230101448), the CRC-Future Food System, the Department of Agriculture, Fisheries and Forestry (DAFF) Soil Science Challenge grants (ID: 4-H4SSYXD; 4-H4T24R2),\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics statement not applicable to this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAAT Bioquest (2015) LD50 Calculator. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003c/span\u003e\u003cspan address=\"http://www.aatbio.com/tools/ld50-calculator\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbramczyk B, Marzec-Grządziel A, Grządziel J, Kr\u0026oacute;l E, Gałązka A, Oleszek W (2022) Biocontrol potential and catabolic profile of endophytic \u003cem\u003eDiaportheeres\u003c/em\u003e strain 1420S from \u003cem\u003ePrunus domestica\u003c/em\u003e L. in Poland\u0026mdash;A preliminary study. 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Front Microbiol 13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2022.908981\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2022.908981\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"19966eb1-cb95-4c87-9461-ef77eb32845a","identifier":"10.13039/501100000923","name":"Australian Research Council","awardNumber":"DP230101448","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Western Sydney University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SynCom, biological control, Rhizoctonia bare patch, integrated pest management, soil-borne disease, grain crops","lastPublishedDoi":"10.21203/rs.3.rs-7398546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7398546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe soil borne pathogen \u003cem\u003eRhizoctonia solani\u003c/em\u003e AG8 causes Rhizoctonia bare patch disease, a major constraint on global wheat production, particularly in no- or minimal -till systems. Current control strategies such as crop rotation, chemical fungicides, and tillage provide only partial protection, while fungicides can accelerate resistance development and negatively impact crop, soil, and human health. Microbial biological control agents (BCAs) and synthetic communities (SynComs, combining two or more BCAs) represent a sustainable alternative for managing Rhizoctonia bare patch. However, inconsistent efficacy and limited compatibility with fungicides remain key challenges to adoption. Here, we demonstrated that individual BCAs and a SynCom (comprising \u003cem\u003eDiaporthe sp.\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e sp. KAR75) reduced disease incidence at the seedling stage by 51\u0026ndash;71% compared with fungicide treatment. When combined with fungicide, disease incidence was further reduced by 65\u0026ndash;100%. The BCA treatments also improved grain yield by 9\u0026ndash;12% compared with fungicide treatment and by 11\u0026ndash;15% compared to no-pathogen control, demonstrating their ability to promote wheat growth in the absence of disease. Grain yield increased most with the fungal-bacterial SynCom, particularly when applied together with fungicide. These results demonstrated that integrating BCAs with chemical fungicides has strong potential to improve control of soil-borne diseases in grain crops.\u003c/p\u003e","manuscriptTitle":"Integrating synthetic community with chemical fungicide improves the control of Rhizoctonia bare patch disease in wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 09:32:58","doi":"10.21203/rs.3.rs-7398546/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9e4eb966-38b3-40a6-9fd1-b6ecbdab077a","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53305746,"name":"Agroecology"}],"tags":[],"updatedAt":"2025-08-19T09:32:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 09:32:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7398546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7398546","identity":"rs-7398546","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}