Use of Biocontrol Agents and Plant Resistance Inducers for the control of potato late blight

Use of Biocontrol Agents and Plant Resistance Inducers for the control of potato late blight | 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 Use of Biocontrol Agents and Plant Resistance Inducers for the control of potato late blight Mati Koppel, Marian Põldmets, Britt Puidet, Isaac Kwesi Abulei, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6104485/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jul, 2025 Read the published version in Potato Research → Version 1 posted 5 You are reading this latest preprint version Abstract Late blight ( Phytophthora infestans ) is a major disease in potato cultivation, managed primarily with synthetic fungicides. This reliance makes potatoes one of the crops with the highest fungicide load. To reduce chemical dependence and promote Integrated Pest Management (IPM), alternative solutions are needed. This study explores biological control agents (BCAs) and plant resistance inducers (PRIs) as sustainable alternatives. We screened 17 BCAs and 3 PRIs in a detached leaf assay (DLA) and selected the most effective for further trials. The chosen BCAs — Polyversum ( Pythium oligandrum ), Nvs 2 ( Bacillus amyloliquefaciens ), TC 4 ( Trichoderma atroviride ), TC 6 ( T. harzianum )—and the PRI ChiProPlant (Chitosan hydrochloride) were tested in greenhouse and field trials across five European countries. In greenhouse trials, ChiProPlant and T. atroviride significantly controlled late blight. Dosage did not affect the efficacy of the BCAs and PRI, but application timing was crucial. Treatments applied 1 and 5 days before inoculation were most effective, while post-inoculation treatments were least effective. Field trials showed that weekly applications of ChiProPlant, Polyversum, and Nvs 2 significantly reduced late blight infection. However, none matched the efficacy of synthetic fungicides. This study highlights the potential of BCAs and PRIs in late blight management but underscores the need for further research to optimize their integration into IPM strategies. Biological control agent Phytophthora infestans Solanum tuberosum Trichoderma spp. Chitosan hydrochloride Bacillus spp Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Potato late blight, caused by the oomycete Phytophthora infestans (Mont.) de Bary, is a significant disease worldwide, posing a substantial threat to potato production (Haverkort et al. 2008 ). The disease can lead to complete crop failure under conducive environmental conditions (Dowley et al. 2008 ). Current management strategies for late blight are dependent on the intensive use of synthetic pesticides (Cooke et al. 2011 ), which has resulted in the emergence of fungicide-resistant strains of the pathogen (Schepers et al. 2018 , Abuley et al. 2023 ) and which poses a potential risk to the environment and human health (EFSA 2020). To promote Integrated Pest Management (IPM) strategies and reduce reliance on chemical inputs in the agricultural sector, several registered synthetic fungicides have been delisted through restrictions imposed by The Sustainable Use of Pesticides Directive (SUD) by the European Commission (European Parliament & Council, 2009). To promote ecologically sustainable disease control measures, a search for alternative approaches, such as biological control for late blight management, is of utmost importance. Microbial biocontrol agents (BCAs) and plant resistance inducers (PRIs) could be environmentally sustainable alternatives to combat agriculturally relevant plant diseases (Alexandersson et al. 2016 ). Several beneficial bacterial, oomycetes and fungal strains have demonstrated biocontrol against P. infestans in vitro and under glasshouse conditions, with a few studies also investigating these effects in the field ( in agro ) (as reviewed in Hashemi et al. 2022 ). Most of the in vitro and in planta (glasshouse) studies have focused on the biocontrol potential of antagonistic bacteria, oomycetes and fungi from the genera Bacillus , Pseudomonas, Streptomyces, Lysobacter, Pythium, Chaetomium and Trichoderma (Caulier et al. 2018 ; Yan et al. 2021 ; De Vrieze et al. 2020 ; Fu et al. 2022 ; Lazazzara et al. 2017; Kurzawińska & Mazur, 2007 ; Shanthiyaa et al. 2013 ; Mollah & Hassan, 2023 ). Moreover, biologically active compounds including chitosan have also been shown to successfully control late blight. Huang et al. ( 2021 ) reported a pronounced antimicrobial effect of chitosan against potato late blight in in vitro assays and Cooke and Little (2001) demonstrated that foliar sprays of phosphonic acid reduced late blight infection of potato tubers in glasshouse and field experiments. However, a universal challenge in selecting and employing BCAs for disease control is their susceptibility to various environmental conditions. Microbial agents demonstrating effectiveness in vitro often fail to control plant pathogens under field conditions (Dorn et al. 2011). Furthermore, determining the optimal dosage and timing of application can contribute significantly to the ability of biocontrol agents to suppress target pathogens (Raziq and Fox, 2004 ; Kim et al. 2008 ). Reliable data demonstrating efficacy of BCAs under field conditions are necessary for the successful integration of biological control into potato IPM strategies. To the best of our knowledge, no previous large-scale field studies evaluating biocontrol agents against P. infestans across multiple regions of Europe have been conducted to date. In the present study, biological agents (BCAs) and plant resistance inducers (PRIs) were tested for their ability to control late blight in laboratory, glasshouse and field trials on potato cultivars with varying levels of host resistance. The study was aimed at selection of effective BCAs and PRIs in controlling of potato late blight and determination of optimal dosages and timing of applications. The following hypothesis were studied: (1) timing of application is critical for the efficacy of BCAs, and that BCAs are more effective when applied before the onset/arrival of pathogen, (2) BCAs are cultivar dependent, with some BCAs showing more effect on certain cultivars than others. 2. Materials and methods 2.1 Types of experiments Three types of experiment, including a detached leaf assay (DLA) in petri dishes, whole plant assay in the glasshouse (WPA), and field trials ( in agro ) were carried out to study the effect of BCAs and PRIs on late blight potatoes. 2.2. Products Seventeen biological control agents (BCAs) and three plant resistance inducers (PRIs) were tested for their ability to control late blight, using DLA (Table 1 ). The five best-performing BCAs/PRIs from the DLA (TC 4, TC 6, Nvs 2, Polyversum, ChiProPlant) were then selected for testing using whole plant assay (WPA) and in agro trials. All the treatments and dosages used are shown in Table 1 . Table 1 List of biological control agents and plant-resistant inducers tested in detached leaf assay Product Active ingredient Type 1 Dosage Water volume (l/ha) Nvs 1 Bacillus velezensis BCA 5kg/ha 500 Nvs 2 B. amyloliquefaciens BCA 5kg/ha 500 Nvs 3 B. velezensis B. amyloliquefaciens BCA 5kg/ha 500 Nvs 4 B. velezensis (liquid formulation) BCA 10 l/ha 500 Nvs 5 B. amyloliquefaciens (liquid formulation) BCA 10 l/ha 500 Nvs 6 B. velezensis and B. amyloliquefaciens (liquid formulation) BCA 10 l/ha 500 Polyversum 1x10 6 oospore/g Pythium oligandrum M1 BCA 200 g/ha 300 Seranade ASO 1x10 11 cfu/l Bacillus amyloliquefaciens QST 713 BCA 4 l/ha 300 TC 1* T. harzianum BCA 10 4 cfu/ml 300 TC 2* T. capillare BP-B2-1 BCA 10 4 cfu/ml 300 TC 3* T. virens Gv-29 BCA 10 4 cfu/ml 300 TC 4* T. atroviride JWB BCA 10 4 cfu/ml 300 TC 5* T. atroviride BCA 10 4 cfu/ml 300 TC 6* T. harzianum BCA 10 4 cfu/ml 300 TC 7* T. tomentosum BCA 10 4 cfu/ml 300 TC 8* T. reesei TMW4.2103 BCA 10 4 cfu/ml 300 TC 9* T. afroharzianum T-22 BCA 10 4 cfu/ml 300 Resistim Potassium phosphite: 6.8% phosphonate, 10.9% potassium PRI 4 l/ha 300 Fytosol 12.5 g/L COS-OGA PRI 4 l/ha 300 ChiProPlant Chitosan (80%) hydrochloride (20%) PRI 300 g/ha 300 Ranman Top 160 g/l cyazofamid Fungicide 80 g/ha 300 * The pure organisms, not formulated product were used; 1The type of product were (1) biological control agent (BCA), Plant resistant inducer (PRI), and synthetic or conventional fungicide (Fungicide) Bacterial products Nvs 1–6 were kindly provided by Novonesis A/S (Denmark). Spore suspensions of the Trichoderma species, obtained from Laura Grenville-Briggs Didymus, Swedish Agricultural University, were cultured in Petri dishes containing Potato Dextrose Agar (PDA). The plates were covered, sealed and incubated at 12h light (UV-A)/12 hr darkness at 20–22°C. Once the cultures fully covered the plates and showed the characteristic green color of Trichoderma spp., spore suspensions were prepared by flooding with distilled water and scraping the spores off into a beaker after filtering through a sterilized gauze. The solution was adjusted to 1x10 5 spores/ml with a haemocytometer. 2.2 Inoculum preparation and inoculation Inoculum for the DLA was prepared in three steps. Phytophthora infestans isolates, including EU_43_A1, EU_41_A2 and one unique multilocus genotype (MLG) were grown on rye agar B media and incubated under fluorescent light at 17 ± 2°C for 14 days, as described in Abuley et al. ( 2023 ). Detached leaves of the late blight susceptible cv. Bintje were inoculated with a sporangial suspension of the pathogen inoculum and incubated at 17 ± 2°C for 10 days. Sporangia that had formed on the leaves 10 days after inoculation were washed off with sterilized distilled water into a beaker and adjusted to a final concentration of 1x10 4 sporangia ml − 1 . The suspension was incubated at 4 ° C for 4 h before the inoculation to promote zoospore release. Six droplets (10µl) of the sporangial suspension were then placed on the abaxial leaf surface in a humid chamber, covered with lids and placed in an incubation room at 17°C under 12 h/day of fluorescent light. For the WPA and in agro experiments, P. infestans isolates were grown on rye agar B at 18°C for 14 days. In the Estonian assays, two isolates with unique multilocus genotypes were used. In the Danish assay, three genotypes, including EU_43_A2, EU_41_A1 and one unique multilocus genotype were used. In Scotland, EU36 genotype was used. The sporangial suspension was prepared by flooding the plates with sterilized distilled water and washing the sporangia off into the beaker via a gauze. The suspension was adjusted to 1x10 5 sporangia/ml with a haemocytometer and incubated at 4 ° C for 4 h to promote zoospore release. Each plant was inoculated by 5 mL of suspension. 2.3 Detached leaf assay Two late blight susceptible (‘Folva’ and ‘Bintje’), one moderately susceptible (‘Kuras’), and two resistant (‘Sarpo Mira’ and ‘Nofy’) potato cultivars were used in the experiment. Seed tubers of each of the cultivars were planted in 5L plastic pots containing a peat-sandy/clay soil mixture in a ratio of 2:1 v/v. Nutrients were supplied in the following amounts per 100 L soil-peat mixture: 90 g KSO 4 , 60 g (NH 4 ) 2 HPO 4 , 33 g CaNO 3 , 7 g MgSO 4 , 7 g CuSO 4 , 133 g CaMg (CO 3 ) 2 . The greenhouse was maintained at 20°C during the day and 18°C at night with a 16h photoperiod. Plants were drip-irrigated at a rate of 1mm/5min. For DLAs, leaves were harvested from 6-week-old potato plants and moistened cotton wool was wrapped around leaf petioles to keep the leaflets alive during the experiment. Leaves were placed in a Petri dish lined with moistened filter paper. The experiment was a 5x19x2 factorial completely randomized design with 5 levels of cultivars (Kuras’, ‘Bintje’, ‘Folva’, ‘Sarpo Mira’, and ‘Nofy’), 19 levels of products and 2 product timings (7 days before the inoculation with P. infestans or 1 day before the inoculation). The BCAs and PRIs were applied according to the dosages shown in Table 1 . In addition to the BCA products, a treatment with Ranman Top (160 g/l cyazofamid) was included as a fungicide reference and an untreated control. Applications were made until the run-off. Inoculation was done by dropping 6 10µl droplets of sporangial suspension on a leaflet. Four replications for each variant were included for each cultivar. Leaves were monitored visually for late blight symptoms from 24 hours to 7 days after pathogen inoculation. Disease severity was evaluated daily as the percentage of leaf area covered with blight symptoms. The disease severity data taken over time was used to calculate the area under the disease progress curve (AUDPC) using the mid-point method (Shaner & Finney, 1977). The relative area under the disease progress curve (rAUDPC) was calculated by dividing the AUDPC by the maximum potential AUDPC as described by Fry et al . 1978. The average rAUDPC value of untreated control was then used to calculate efficacy (i.e., the disease control offered by a product relative to an untreated control), using the method of Abuley and Nielsen (2017). The performance of products in controlling late blight was ranked based on their efficacy to reduce the severity of disease. Products with the same efficacy were given the same value. This calculation was done separately for each of the three cultivars and two application timings; thus, six values were obtained for a given product. The mean rank of the product across both application timing and all three cultivars was then calculated and assigned as the composite rank, giving a measure of average performance. 2.4 Whole plant assays Three sub-experiments were carried out in the glasshouse simultaneously: Comparison of the efficacy of BCAs/PRIs with full dose applied one day before inoculation Dose comparison trials with three levels of BCA applied: half, full (normal) and double dose. Timing comparison trials with four application timings: 1) onetime application 1 day before inoculation; 2) one time application 1 day after inoculation; 3) 1 day before inoculation; 3) 1 day before inoculation with a 5-day interval; and 4) 1 day before inoculation with a 7-day interval. Glasshouse trials were carried out in Estonia and Denmark for the above-mentioned trials (1–3). The moderately resistant cv. Kuras and susceptible cv. Bintje were used in the WPA. Each cultivar was planted in 1 L plastic pots. The 1 L plastic pots were arranged on greenhouse benches in a randomized design and filled with a commercial limed peat substrate (Biolan) with pH 6.0 and with nutrients in the following amounts 1 L per mixture: 100 mg N, 60 mg P and 200 mg K. Sections of certified seed tubers containing an eye were planted in pots and maintained under controlled conditions at 23°C with a 12 hour photoperiod. The plants were grown for 6 weeks in the greenhouse before products were applied. Based on the results of the DLAs (Figure S1), TC 4, TC 6, Nsv 2, Polyversum and ChiProPlant were selected as promising candidates for the WPA and in agro trials. The products and full dose rates used in the trials are shown in Table 2 . Revus Top (250 g/l mandipropamid + 250 g/l difenoconazole) as a chemical reference and an untreated control were included for both cultivars. Each treatment combination was replicated four times. Each plant was sprayed until it ran off (approximately 5ml of foliar application of the fungicide or BCA products per plant) for treatments as well as for inoculation with P.infestans . Late blight progression was monitored visually five times during the experiment (1, 4-, 6-, 8-, and 11-days post-inoculation). The plants were assessed for late blight severity as a percentage of the blighted area. The disease severity data taken over time was used to calculate the area under the disease progress (AUDPC) (Shaner & Finney, 1977). rAUDPC and efficacy were then calculated using methods described in DLA. 2.5 In agro trials In agro trials were carried out in 2022 in Denmark (DK, cultivars ‘Kuras’ (susceptible) and ‘Nofy’ (Resistant)), Estonia (EE, cultivars ‘Kuras’ (resistant)and ‘Triton’ (susceptible)), Finland (FI, cultivars Kuras (resistant) and Mungo (susceptible)), Germany (DE, Kuras (susceptible) and Nofy (resistant)) and Scotland (SC, cultivars Maris Piper (moderately resistant) and Gatsby (resistant)). The experiment was conducted in a randomized complete block design with four replicates and a plot size of approximately 25 m 2 . The same BCA’s and PRI tested in WPA were included in the in agro trials at the same dosages (i.e., full dose rates), with exception for trials carried out in Finland where the Trichoderma treatments were not included. An untreated control treatment and a fungicide treatment were also included. All the fungicide treatments and dosages used in each country are given in S2. Table 2 List of biological control agents (BCA) and plant-resistant inducers (PRI) tested in glasshouse and in agro trials Product Active ingredient Type Dosage Water volume (l/ha) Polyversum 1x10 6 oospore/g Pythium oligandrum M1 BCA 200 g/ha 300 Nvs 2 Bacillus amyloliquefaciens BCA 5 kg/ha** 500 TC 4* Trichoderma atroviride* BCA 1,2x10 12 cfu/ha 300 TC 6* T. harzanium* BCA 1,2x10 12 cfu/ha 300 ChiProPlant Chitosan (80%) hydrochloride (20%) PRI 300 kg/ha 300 * The pure organisms, not formulated product were used; **In Finnish trial, the Nvs 2 dosage was 1 kg/ha, water volume 300 l/ha. The trials were inoculated by inoculating spreader/infection rows with 1 x 10 4 sporangia/mL of P. infestans . The same isolates as in glasshouse trials were also used to inoculate the field trials. In the Finnish field trials, there was natural infection. Treatment applications were started at the first occurrence of a late blight risk period at the individual trial sites, as determined by the local decision support systems (Blight Manager – in Denmark and Finland; Hutton Criteria – in Scotland; BliteCcast – in Estonia; Phytophthoramodel-Weihenstephan in Germany), and continued at weekly intervals for 8–12 weeks. Disease assessments were made as a percentage of infected foliage at a 7-day interval on all plots starting from the onset of late blight and continuing until 2 weeks before harvest. 2.6 Statistical analysis The R programming language (version 4.3.1) was used for statistical analyses (R Core Team, 2022). The DLA efficacy data was subjected to Gaussian linear models using the ‘lm’ function in the stats R package (R Core Team, 2022). The effect of factors and their interactions was determined via F-test using ANOVA. All WPA and in agro datasets were checked for normality (Shapiro-Wilk test) and for homogeneity of variances (Levene’s test). In case of equal variances, one-way analysis of variance (ANOVA) was performed, followed by Tukey HSD test to identify differences between the treatment variants. For non-normally distributed data, the Kruskal-Walli’s test was used. Post hoc analysis for treatment differences was conducted using Dunn's multiple comparisons test with Bonferroni correction. P-values < 0.05 were considered statistically significant. 3. Results 3.1 Detached leaf assay Seventeen BCAs and PRIs were tested in detached leaves in Petri dishes for selection more effective ones in controlling potato late blight with aim to use them in glasshouse and in agro studies. No infection was recorded in the resistant cultivars (i.e., ‘Sarpo Mira’ and ‘Nofy’), so these cultivars were excluded from the analyses. Infection was recorded in the untreated susceptible (‘Bintje’ and ‘Folva’) and moderately susceptible (Kuras’) cultivars, with the first symptoms occurring 3 days post-inoculation. Leaves treated with fungicide had no disease symptoms for the entire duration of the experiment. An ANOVA test to analyse results from all cultivars showed that all individual factors, product type, potato cultivar, and timing of treatments, had a significant effect on product efficacy ( p < 0.05). However, potato cultivar had the most pronounced effect on product efficacy to reduce incidence of disease (F = 946, df = 4, p < 0.001), followed by product type (F = 54, df = 21, p < 0.001), and thereafter timing of treatments (F = 4.5, df = 1, p = 0.033). Results also showed a significant effect of all two-ways interactions between the three experimental factors (p 0.001), followed by potato cultivar (F = 7.4, df = 2, p < 0.001). As the previous analysis included both the fungicide treated control and the untreated control, a further analysis was conducted without these treatments. This was done to make a direct comparison of the BCA and PRI treatments. The results showed a strong effect of all three single factors and their two-way interactions (p < 0.05), whereas the three-way interaction between potato cultivar, product type and timing of treatment was insignificant (F = 1, df = 38, p = 0.4). Due to the strong effect of potato cultivar, product type and timing of treatments, we separated the results to avoid biased comparisons. Fungicide treatments consistently showed the highest efficacy in disease reduction. Among tested BCAs and PRIs, potassium phosphite (Resistim) and chitosan hydrochloride (ChiProPlant) were the most effective in disease reduction (Fig. 1 ). Moreover, the highest efficacy in disease reduction was observed when treatments were applied one day before inoculation. In general, the treatment efficacy in disease reduction was higher in cv. Kuras compared to cv. Folva and cv. Bintje. Composite ranking A composite rank value was calculated to rank the performance if the BCAs and PRIs. Results from the untreated leaves and those treated with chemical fungicides were excluded before the analyses of composite ranking of the treatments across the cultivars. The highest-ranked product for late blight control was Resistim, followed by ChiProPlant at both application times. TC 1, TC 2, TC 3, TC 5, TC 6, TC 7, TC 8, Nsv 1, Nsv 2, Nsv 3, Nsv 6 and Polyversum performed better when applied 1 day before inoculation than 7 days before inoculation. Therefore, the composite ranking of the − 1-day treatment was used. Based on the results of the composite ranking, 4 BCAs (Nvs 2, TC 4, TC 6, Polyversum) and one PRI (ChiProPlant) were selected for subsequent experimentation. Resistim, though the most effective product, was not included because this product is considered a conventional fungicide in some countries. 3.2 Glasshouse experiments Five best performing treatments (Polyversum [ Pythium oligandrum ], Nvs 2 ( B. amyloliquefaciens ], TC 4 [ T. atroviride ], TC 6 [ T. harzianum ], and ChiProPlant [Chitosan hydrochloride]) selected according to results of DLA were tested in glasshouse grown potato plants to get more reliable results on efficacy of used treatments and to assess the effect of dose and timing on their efficacy in potato late blight control. Data from tests carried out in Estonia and Denmark on cv. Bintje and cv. Kuras were analysed. Infection was successful on bothcv. 'Bintje' and cv. 'Kuras', though in the latter, none of treatments with BCAs or PRIs resulted in significant disease reduction compared with the untreated control. Plants treated with fungicide had no late blight symptoms for the entire duration of the experiment in both cultivars, therefore were excluded from the statistical analyses. The first late blight symptoms occurred 4 days post-inoculation on untreated controls. 3.2.1 Effect of products According to the Kruskal-Walli's test performed on results of whole plant trials, product type had a significant effect on disease control efficacy across both potato cultivars and trials (χ² = 14.8, df = 5, p = 0.011). Furthermore, post hoc analysis revealed that the application of ChiProPlant and TC 4 resulted in significantly lower late blight severity compared with the untreated control (p = 0.006; p = 0.021). Furthermore, the Wilcoxon rank sum test indicated a significant difference in disease severity between the cv. Bintje and cv. Kuras (W = 1975.5, p = 0.018) across both countries. In analysis of data separately for each cultivar, the product type had a marked effect on disease control efficacy in cv. Bintje (χ² = 14.203, df = 5, p = 0.014), but not on cv. Kuras (χ² = 5.572, df = 5, p = 0.35) (Fig. 2 ). Post hoc analysis revealed that ChiProPlant ( p = 0.004), TC 4 ( p = 0.049), and TC 6 ( p = 0.037) demonstrated significantly higher disease control compared with the untreated control in the cv. Bintje trial (Fig. 3 ). 3.2.2 Effect of dose No dose-response effect was found in the glasshouse trial when analyzing data from both countries and both cultivars across all treatments (χ² = 2.904, df = 3, p = 0.406). The data for each cultivar was also analyzed separately. No statistically significant differences were found between treatment doses and the untreated control, although the Kruskal-Wallis test revealed a significant effect of dose on disease control efficacy in cv. Kuras (χ² = 37.06, df = 5, p < 0.001). We observed a significant product x dose interaction on disease control in cv. Bintje (F = 4.39, p < 0.001). Half, full and double dose of ChiProPlant showed significant disease reduction compared with the untreated control ( p < 0.001; p < 0.01 and p < 0.01 respectively), but there were no significant differences in disease reduction between the different ChiProPlant doses (Fig. 4 ). While no differences were observed between the different doses for TC4 in the overall analysis, we observed a significant increase in efficacy with dose when TC4 was analyzed alone where use of double dose resulted in significant disease reduction compared with half dose and untreated control (p < 0.01 and p < 0.05, respectively). The product x dose interaction on disease reduction was also significant in cv. Kuras (χ²= 51.301, df = 15, p = 0.001), but no statistically significant differences were observed between the treatment variants and the untreated control. 3.2.3 Effect of application timing According to Kruskal-Walli's test, application timing significantly affected disease control efficacy in cv. Bintje (χ² = 27.96, df = 5, p < 0.001). However, post hoc analysis revealed that a statistically significant difference was observed only between the fungicide treatment and the untreated control ( p = 0.001). When excluding fungicide treatment from the analysis, pre-treatment of plants with the repeated application of BCAs/PRI 5 and 1 day before the inoculation demonstrated significantly higher efficacy in disease control compared with the untreated control ( p = 0.019). The BCA/PRI treatment of potato plants after pathogen inoculation did not have an effect on disease. Disease occurrence was significantly affected by product type x timing of BCA/PRI inoculation interaction in cv. Bintje (χ 2 = 63.50, df = 20, p < 0.001). Pre-treatment with ChiProPlant 7 and 1 day before the inoculation with P. infestans provided comparable efficacy to that of the chemical fungicide Revus Top. Both treatments resulted in significantly less disease compared with the untreated control ( p = 0.018) (Fig. 5 ). A significant effect of product type x timing of BCA/PRI inoculation interaction on disease control efficacy was also observed in the cv. Kuras (W = 73.22, df = 21, p < 0.001), but post hoc analysis revealed no significant differences between the BCA/PRI treatments and the untreated control. 3.3 In agro trials Nine field trials were established in five countries to assess the efficacy of BCAs and PRI in variable environmental conditions. However, only five trials out of nine had sufficient infection to assess the efficacy of disease control. No disease was observed in the Estonian trials, so data from these trials were excluded from the subsequent analysis. Likewise, data from trials with cv. Nofy in Denmark and Germany were excluded from subsequent analyses due to minimal to no disease symptoms. Data from the Danish and German trials for the cv. Kuras, as well as from the Scottish trials for the cv. Maris Piper (moderately resistant) and cv. Gatsby (resistant), were analyzed separately and in combined analysis. Due to the lower number of products tested in the Finnish trial, the data were not included in the combined analysis. Product type had a significant effect on disease control in the cv. Kuras in the German field trial (χ² = 21.39, df = 6, p = 0.002). ChiProPlant ( p = 0.008) and fungicide ( p < 0.001) provided significantly higher disease control compared with the untreated control (S3). In the Danish trials, although there was a significant effect of product type on disease severity in cv. Kuras, (χ² = 14.53, df = 6, p = 0.024), only the fungicide treatment showed significant disease reduction ( p = 0.002) (S4). In the trial conducted in Scotland with the moderately blight resistant cv. Maris Piper, there was a significant effect of product on disease control (χ = 21.30, df = 7, p = 0.003); however, only the fungicide treatment resulted in a significant disease reduction ( p = 0.003) (S5). Further statistical analysis omitting the fungicide treatment showed that ChiProPlant ( p = 0.03) and Polyversum ( p = 0.005) significantly reduced disease compared with the untreated control. ANOVA showed a significant effect of product type on disease control on cv. Gatsby (F = 10.09, p < 0.002), however this was only true for the fungicide treatment ( p < 0.001) (S6). Similarly, in the Finnish trials, there was also a significant effect of product on disease control in cv. Kuras (F = 3.58, p = 0.031) with only the fungicide treatment significantly reducing disease ( p = 0.038) (S7). A similar trend was observed in cv. Mungo (χ = 10.70, df = 4, p = 0.030), but post hoc analysis revealed no significant differences between product treatments and the untreated control (S8). When comparing data from all experiments, there was a significant effect of product on disease control overall (χ² = 62.746, df = 7, p < 0.001), as shown in Fig. 6 . Notably, treatments with Polyversum ( p = 0.01), Nsv 2 ( p = 0.016), ChiProPlant ( p = 0.002), and fungicide ( p < 0.001) resulted in less disease compared with the untreated control (Fig. 6 ). However, none of the treatments were as effective as the fungicide. 4. Discussion Through a series of DLA, glasshouse, and in-agro experiments, we demonstrate the efficacy of BCAs and PRIs against late blight across different agro-climatic areas with varying P. infestans populations. We also examine factors such as dose and timing of application, and their influence on the efficacy against late blight. Detached leaf and whole plant assays conducted in Denmark and Estonia revealed that across both trials, TC 4, TC 6 and ChiProPlant were the most effective products in controlling late blight in the susceptible cv. Bintje. There have been numerous reports of Trichoderma showing an inhibitory effect on P. infestans from previous studies. For example, Trichoderma strain HNA14 significantly reduced late blight in both glasshouse assays and field trials conducted by Yao et al. ( 2016 ). In addition, Mollah and Hasan (2023) showed that the T. harzanium based biological fungicide “Lycomax” was effective in controlling late blight under field conditions. These authors found that Lycomax reduced late blight severity by more than 90% compared to the untreated control, matching the performance of chemical fungicide Agrizeb (Mollah and Hassan, 2023 ). The biopolymer chitosan has also been found to exhibit antifungal properties against P. infestans (Yarullina et al. 2024 ), with its biocontrol properties being attributed to inducing plant defense responses in potato (Zheng et al. 2020), pathogen growth inhibition as well as having a synergistic effect on synthetic fungicides (Huang et al. 2020). However, the efficacy of the aforementioned products was not as high as that of synthetic fungicides in our trials, which consistently achieved 100% efficacy. This is consistent with previous studies in the literature, which have shown that synthetic fungicides generally provide stronger and more consistent control of late blight pathogen (Caulier et al. 2018 ; Dorn et al. 2011). We also observed a significant effect of cultivar on product efficacy in DLA and glasshouse trials. Cultivar is known to play a significant role in disease control (Carolan et al. 2017 ). In contrast to the cv. Bintje where treatments with ChiProPlant, TC4 and TC6 resulted in disease reduction, there was no significant reduction in late blight symptoms in the moderately resistant cv. Kuras. The resistance of the cv. Kuras to late blight, led to lower disease severity in the untreated control and absence of differences between the treatments. Conversely to our results, in a Swedish study involving the plant resistance inducer β-aminobutyric acid (BABA), it was observed that the effect of BABA on late blight symptoms was greater on the partially resistant cultivar than on the more susceptible cv. Bintje in greenhouse and field experiments (Liljeroth et al. 2010 ). Nevertheless, these findings suggest that it’s important to consider the level of resistance of the cultivar when evaluating the effectiveness of biocontrol products. Furthermore, evaluating product efficacy can be challenging when disease levels are extremely high or very low, as this limits observable differences between treatments. Our experiments showed a significant dose-response relationship only for treatments with TC 4 in the susceptible cv. Bintje, where treatment with double dose was superior compared with half dose. All treatments of ChiProPlant resulted in significant disease reduction but there were no statistically significant differences between the efficacy of used doses. In contrast, Liljeroth et al. ( 2010 ) reported that a higher concentration of PRI β-aminobutyric acid gave a stronger protective effect. Similarly, Liljeroth et al. ( 2016 ) observed a clear dose-response effect of phosphite on control of late blight in Swedish field trials, noting that higher doses resulted in increased efficacy. Our data indicate that product application timing is an important factor in the glasshouse assay. Pre-treatment with ChiProPlant one and seven days before inoculation yielded results comparable to that of fungicide treatment, achieving nearly 100% efficacy. Other pre-treatments with Nsv 2, Polyversum, TC 4 and TC 6 were less effective. On the contrary, post-inoculation treatment of potato plants had no effect on disease severity and may have even increased disease. This highlights the importance of adequate application timing of a biocontrol product. Better efficacy of a bio fungicide is more likely when the antagonist is applied early enough to prevent the pathogen from establishing itself and causing infection. To effectively use PRIs for disease control, they must be applied preventively, before disease establishment, to induce the plant's defense mechanisms (Altamiranda et al. 2008 ). In a glasshouse experiment, Savchuk and Fernando (2006) observed that applying antagonistic bacteria before, or at the same time as S. sclerotiorum inoculation led to complete disease inhibition by day 14, whereas applying the bacteria 24 or 48 hours later resulted in only minimal disease suppression. Studying the effects of biocontrol agents against P. infestans , Stephan et al. ( 2005 ) found that applying these products 24 hours before inoculation with the pathogen resulted in greater efficacy compared to applications made 1 hour prior to inoculation. Moreover, despite various treatment applications, none were found to have a curative effect on the disease (Stephan et al. 2005 ). In our field experiments, a clear effect of product on disease control was found across all trials. Treatments with Polyversum, Nsv2, ChiProPlant, and fungicide resulted in significant disease reduction compared with the untreated control, although none of the biocontrol products were as effective as the fungicide alone. In the trial conducted in Germany, ChiProPlant and fungicide treatment had a greater effect on disease reduction compared with the untreated control, but in the Danish trial, only the fungicide treatment was significantly more effective compared with the untreated variant. The same trend was observed in separate trials conducted in Scotland with the cvs. M. Piper and Gatsby. This is consistent with the findings of Caulier et al. ( 2018 ), who demonstrated that treatments with bacterial antagonists were significantly less effective in reducing disease severity in Belgian field studies compared to the synthetic fungicides. The limited effect of biocontrol products in field trials might be attributed to the variability in environmental conditions. Biocontrol agents are less likely to achieve successful control at the field level compared to laboratory or glasshouse settings, where temperature and relative humidity can be controlled. Kumbar et al. ( 2019 ) and Islam et al. ( 2022 ) demonstrated that various bacterial and fungal bioagents effectively reduced late blight disease severity in field trials; however, given the tropical climate, these results may differ from what could be expected in temperate regions. A study by Stridh et al. ( 2022 ) reported that the effectiveness of biocontrol products on potato early blight in greenhouse trials did not extend to field-scale trials. In contrast to our studies on potato late blight, none of the tested products successfully controlled another potato foliar disease - early blight in field settings (Stridh et al. 2022 ). There are only a few investigations on the efficacy of biocontrol agents against late blight in temperate climate zone in Europe, to the best of our knowledge. In the study by Dorn et al. ( 2007 ), various natural products, including products containing microorganisms from the genera Bacillus , Trichoderma , and Pythium , which had previously shown some efficacy in vivo assays, were examined in field trials in Switzerland. The efficacy of used natural compounds and microbial preparations was limited in field settings. The authors of this study speculated that the poor performance of natural products could be attributed to their lack of stability in the existing environmental conditions rather than their inherent ineffectiveness (Dorn et al. 2007 ). On the other hand, Kurzawinska and Mazur (2009), reported the potential use of Polyversum ( P. oligandrum ) and Biochikol 020 PC (chitosan) in protecting potatoes against late blight based on three-year field experiments. Both products effectively reduced the mean disease index and also the incidence of tuber infection caused by P. infestans . It was noted that disease development was influenced by the weather conditions during the vegetative period (Kurzawinska & Mazur). Humidity and cool temperature are critical factors that contribute to the rapid development of P. infestans infection (Fry 2015). Additionally, in the field experiments conducted by Caulier et al. ( 2018 ), treatment with Bacillus subtilis strain 30B-B6 was shown to significantly reduce late blight severity throughout the crop season. The results of our study demonstrate that certain biological agents have potential to control P. infestans under glasshouse as well as in field conditions. However, further field trials in temperate regions are necessary for a comprehensive evaluation of biocontrol agents and their potential integration into an effective integrated pest management system. Cultivar resistance, timing of application, and environmental conditions must be taken into account when integrating biocontrol products to late blight management strategies. Declarations Acknowledgements This study was conducted as part of 'ECOSOL - Eco-friendly solutions for the integrated management of late and early blight of potatoes' a project carried out under the H2020 SusCrop - ERA-NET Cofund Action on Sustainable Crop Production. Partners in ECOSOL received national funding from The Green Development and Demonstrations Program (Denmark), Estonian Science Foundation (Estonia), Ministry of Agriculture and Forestry of Finland (Finland), German Research Foundation (Germany), SLU Grogrund - The Centre for breeding of food crops and the Swedish Research Council Formas (Sweden) and Department for Environment, Food & Rural Affairs (UK). Novonesis A/S is highly appreciated for kindly providing BCA products for using in the studies. We would also like to thank Prof. J. 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International Journal of Biological Macromolecules, 182, 1670–1680. https://doi.org/10.1016/j.ijbiomac.2021.05.097 Supplementary Files Supplementaryfiles.docx Cite Share Download PDF Status: Published Journal Publication published 30 Jul, 2025 Read the published version in Potato Research → Version 1 posted Reviewers agreed at journal 31 Mar, 2025 Reviewers invited by journal 28 Mar, 2025 Editor invited by journal 28 Feb, 2025 Editor assigned by journal 27 Feb, 2025 First submitted to journal 25 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Dotson","email":"","orcid":"","institution":"Swedish University of Agricultural Sciences: Sveriges lantbruksuniversitet","correspondingAuthor":false,"prefix":"","firstName":"Bradley","middleName":"","lastName":"Dotson","suffix":""},{"id":435225644,"identity":"9fb148de-1906-45fb-b344-92c5ba3e3f3f","order_by":14,"name":"Sabine Ravnskov","email":"","orcid":"","institution":"Aarhus University: Aarhus Universitet","correspondingAuthor":false,"prefix":"","firstName":"Sabine","middleName":"","lastName":"Ravnskov","suffix":""},{"id":435225645,"identity":"9e64bcae-042e-478f-b223-e937c78fe7cd","order_by":15,"name":"Alison Lees","email":"","orcid":"","institution":"The James Hutton Institute","correspondingAuthor":false,"prefix":"","firstName":"Alison","middleName":"","lastName":"Lees","suffix":""}],"badges":[],"createdAt":"2025-02-25 11:04:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6104485/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6104485/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11540-025-09899-1","type":"published","date":"2025-07-30T16:29:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79836453,"identity":"c7da9256-70c8-4f83-afb2-24c42fdaac65","added_by":"auto","created_at":"2025-04-03 11:24:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25459,"visible":true,"origin":"","legend":"\u003cp\u003eComposite ranking of biological control agents (BCAs) and plant resistant inducers (PRIs) applied as repeated pre-treatments at 1 (a), and 7 (b) days before inoculation of potato leaves with \u003cem\u003ePhytophthora infestans\u003c/em\u003e. The lower the rank score, the higher the efficacy of the product.\u003csup\u003e. \u003c/sup\u003eThe products contain the following active ingredient or organism: Resistim (Potassium phosphite), ChiProPlant (Chitosan hydrochloride), Nvs 1 (\u003cem\u003eBacillus velezensis\u003c/em\u003e), Nvs 2 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e), Nvs 3 (\u003cem\u003eBacillus velezensis \u003c/em\u003eand \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e), Nvs 3 (\u003cem\u003eBacillus velezensis\u003c/em\u003e, liquid formulation), Nvs 4 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e, liquid formulation), Polyversum (\u003cem\u003ePythium oligandrum), \u003c/em\u003eSerenade (\u003cem\u003eBacillus amyloliquefaciens)\u003c/em\u003e, TC 1 (\u003cem\u003eTrichoderma harzianum), \u003c/em\u003eTC\u003cem\u003e \u003c/em\u003e2\u003cem\u003e (Trichoderma capillare BP-B2-1), \u003c/em\u003eTC 3 (\u003cem\u003eTrichoderma virens \u003c/em\u003eGv-29), TC 4 (\u003cem\u003eTrichoderma atroviride \u003c/em\u003eJWB), TC 5 (\u003cem\u003eTrichoderma atroviride)\u003c/em\u003e, TC 6 (\u003cem\u003eTrichoderma harzianum)\u003c/em\u003e, TC 7 (\u003cem\u003eTrichoderma. tomentosum)\u003c/em\u003e, TC 8 (\u003cem\u003eTrichoderma reesei \u003c/em\u003eTMW4.2103), and TC 9 (\u003cem\u003eTrichoderma afroharzianum \u003c/em\u003eT-22).\u003cbr\u003e\n\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6104485/v1/dce16702935dc1249d8271ab.png"},{"id":79836457,"identity":"fb6bca3a-3156-4e5c-a8f6-15556090d3c5","added_by":"auto","created_at":"2025-04-03 11:24:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52996,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of biological control agents and plant resistant inducers against late blight on potato plants in whole plant assay (\u003cem\u003ein planta\u003c/em\u003e) in cv. Kuras when applied one day before inoculation. Efficacy of the product is expressed as percentage of the relative area under the disease progress curve (rAUDPC) relative to rAUDPC of the untreated control. All differences between the treatments were not statistically significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). The tested products are Polyversum (\u003cem\u003ePythium oligandrum\u003c/em\u003e), ChiProPlant (Chitosan hydrochloride), TC 4 (\u003cem\u003eTrichoderma atroviride\u003c/em\u003e), TC 6 (\u003cem\u003eT. harzianum\u003c/em\u003e), and Nvs 2 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6104485/v1/e5a5026acfb4aaaa17207d99.png"},{"id":79836769,"identity":"e668afe2-5058-4bbb-a0ad-ae38ba048b34","added_by":"auto","created_at":"2025-04-03 11:32:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66465,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of different products (of biological control agents, plant resistant inducers, and fungicide) at different doses in controlling late blight in cv. Bintje when applied one day before inoculation. Efficacy of the product is expressed as percentage of relative area under the disease progress curve (rAUDPC) relative to rAUDPC of the untreated control. Asterisks indicate statistically significant differences (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The tested products are Polyversum (\u003cem\u003ePythium oligandrum\u003c/em\u003e), ChiProPlant (Chitosan hydrochloride), TC 4 (\u003cem\u003eTrichoderma atroviride\u003c/em\u003e), TC 6 (\u003cem\u003eT. harzianum\u003c/em\u003e), and Nvs 2 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6104485/v1/74408fcc5947afd604590640.png"},{"id":79836456,"identity":"312779b7-60b5-404e-b7c7-5c75e1febbab","added_by":"auto","created_at":"2025-04-03 11:24:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45488,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of different products (of biological control agents, plant resistant inducers, and fungicide) at different doses (half, normal or full, and double) in controlling late blight in cv. Bintje when applied one day before inoculation. Efficacy of the product dose is expressed as percentage of relative area under the disease progress curve (rAUDPC) relative to rAUDPC of the untreated control. Asterisks indicate statistically significant differences (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001). The tested products are Polyversum (\u003cem\u003ePythium oligandrum\u003c/em\u003e), ChiProPlant (Chitosan hydrochloride), TC 4 (\u003cem\u003eTrichoderma atroviride\u003c/em\u003e), TC 6 (\u003cem\u003eT. harzianum\u003c/em\u003e), and Nvs 2 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6104485/v1/c61e65d7494e2a93cfec48af.png"},{"id":79836461,"identity":"ada66af4-99cc-454b-bb56-4fcaff0827c8","added_by":"auto","created_at":"2025-04-03 11:24:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85336,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of different products (of biological control agents, plant resistant inducers, and fungicide) at different application timings (1 day before inoculation with \u003cem\u003ePhytophthora infestans\u003c/em\u003e, 1 day before inoculation with \u003cem\u003eP. infestans\u003c/em\u003e and continued at 7 days interval, 1 day before inoculation with \u003cem\u003eP. infestans\u003c/em\u003e and continued at 5 days interval, 1 after inoculation with \u003cem\u003eP. infestans\u003c/em\u003e) on potato late blight in cv. Bintje. Efficacy of the treatment timing is expressed as percentage of rAUDPC relative to relative area under the disease progress curve (rAUDPC) of the untreated control. Asterisks indicate statistically significant differences (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u0026nbsp;The tested products are represented by the following abbreviations PV (Polyversum, containing \u003cem\u003ePythium oligandrum\u003c/em\u003e), CP (ChiProPlant, containing chitosan hydrochloride), TC 4 (\u003cem\u003eTrichoderma atroviride\u003c/em\u003e), TC 6 (\u003cem\u003eT. harzianum\u003c/em\u003e), Nvs 2 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e), and fungicide.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6104485/v1/ab195aa7c9436c80c8682708.png"},{"id":79836459,"identity":"72f25d05-1e7b-4b2b-808b-23bf09254c8d","added_by":"auto","created_at":"2025-04-03 11:24:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":54605,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of different products (biological control agents, plant resistant inducers, and fungicide) on late blight\u003cem\u003e \u003c/em\u003eunder field conditions in trials conducted in 3 countries (Denmark, Scotland, and Germany) with a range of cultivars (Kuras, Maris Piper, and Mungo). Efficacy of the product is expressed as percentage of relative area under the disease progress curve (rAUDPC) relative to rAUDPC of the untreated control. Asterisks indicate statistically significant differences (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The tested products are represented by the following abbreviations PV (Polyversum, containing \u003cem\u003ePythium oligandrum\u003c/em\u003e), CP (ChiProPlant, containing Chitosan hydrochloride), TC 4 (\u003cem\u003eTrichoderma atroviride\u003c/em\u003e), TC 6 (\u003cem\u003eT. harzianum\u003c/em\u003e), Nvs 2 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e), and fungicide.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6104485/v1/34fd9368477b5c2ba98e063e.png"},{"id":88268480,"identity":"7112219f-2258-4589-a7cc-cd77113cb3af","added_by":"auto","created_at":"2025-08-04 16:52:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1307122,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6104485/v1/fa1d4322-93f7-4820-887b-62e1f6f4ff22.pdf"},{"id":79836451,"identity":"72b1ab1d-f4a8-488e-8ef4-3e2ac0e42ef3","added_by":"auto","created_at":"2025-04-03 11:24:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":376470,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-6104485/v1/287cb1aa373a5a7ce38a6166.docx"}],"financialInterests":"","formattedTitle":"Use of Biocontrol Agents and Plant Resistance Inducers for the control of potato late blight","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePotato late blight, caused by the oomycete \u003cem\u003ePhytophthora infestans\u003c/em\u003e (Mont.) de Bary, is a significant disease worldwide, posing a substantial threat to potato production (Haverkort et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The disease can lead to complete crop failure under conducive environmental conditions (Dowley et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Current management strategies for late blight are dependent on the intensive use of synthetic pesticides (Cooke et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which has resulted in the emergence of fungicide-resistant strains of the pathogen (Schepers et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Abuley et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and which poses a potential risk to the environment and human health (EFSA 2020). To promote Integrated Pest Management (IPM) strategies and reduce reliance on chemical inputs in the agricultural sector, several registered synthetic fungicides have been delisted through restrictions imposed by The Sustainable Use of Pesticides Directive (SUD) by the European Commission (European Parliament \u0026amp; Council, 2009). To promote ecologically sustainable disease control measures, a search for alternative approaches, such as biological control for late blight management, is of utmost importance.\u003c/p\u003e \u003cp\u003eMicrobial biocontrol agents (BCAs) and plant resistance inducers (PRIs) could be environmentally sustainable alternatives to combat agriculturally relevant plant diseases (Alexandersson et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Several beneficial bacterial, oomycetes and fungal strains have demonstrated biocontrol against \u003cem\u003eP. infestans in vitro\u003c/em\u003e and under glasshouse conditions, with a few studies also investigating these effects in the field (\u003cem\u003ein agro\u003c/em\u003e) (as reviewed in Hashemi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Most of the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein planta\u003c/em\u003e (glasshouse) studies have focused on the biocontrol potential of antagonistic bacteria, oomycetes and fungi from the genera \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePseudomonas, Streptomyces, Lysobacter, Pythium, Chaetomium\u003c/em\u003e and \u003cem\u003eTrichoderma\u003c/em\u003e (Caulier et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; De Vrieze et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lazazzara et al. 2017; Kurzawińska \u0026amp; Mazur, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Shanthiyaa et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mollah \u0026amp; Hassan, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, biologically active compounds including chitosan have also been shown to successfully control late blight. Huang et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported a pronounced antimicrobial effect of chitosan against potato late blight in \u003cem\u003ein vitro\u003c/em\u003e assays and Cooke and Little (2001) demonstrated that foliar sprays of phosphonic acid reduced late blight infection of potato tubers in glasshouse and field experiments.\u003c/p\u003e \u003cp\u003eHowever, a universal challenge in selecting and employing BCAs for disease control is their susceptibility to various environmental conditions. Microbial agents demonstrating effectiveness \u003cem\u003ein vitro\u003c/em\u003e often fail to control plant pathogens under field conditions (Dorn et al. 2011). Furthermore, determining the optimal dosage and timing of application can contribute significantly to the ability of biocontrol agents to suppress target pathogens (Raziq and Fox, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReliable data demonstrating efficacy of BCAs under field conditions are necessary for the successful integration of biological control into potato IPM strategies. To the best of our knowledge, no previous large-scale field studies evaluating biocontrol agents against \u003cem\u003eP. infestans\u003c/em\u003e across multiple regions of Europe have been conducted to date.\u003c/p\u003e \u003cp\u003eIn the present study, biological agents (BCAs) and plant resistance inducers (PRIs) were tested for their ability to control late blight in laboratory, glasshouse and field trials on potato cultivars with varying levels of host resistance. The study was aimed at selection of effective BCAs and PRIs in controlling of potato late blight and determination of optimal dosages and timing of applications. The following hypothesis were studied: (1) timing of application is critical for the efficacy of BCAs, and that BCAs are more effective when applied before the onset/arrival of pathogen, (2) BCAs are cultivar dependent, with some BCAs showing more effect on certain cultivars than others.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Types of experiments\u003c/h2\u003e \u003cp\u003eThree types of experiment, including a detached leaf assay (DLA) in petri dishes, whole plant assay in the glasshouse (WPA), and field trials (\u003cem\u003ein agro\u003c/em\u003e) were carried out to study the effect of BCAs and PRIs on late blight potatoes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Products\u003c/h2\u003e \u003cp\u003eSeventeen biological control agents (BCAs) and three plant resistance inducers (PRIs) were tested for their ability to control late blight, using DLA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The five best-performing BCAs/PRIs from the DLA (TC 4, TC 6, Nvs 2, Polyversum, ChiProPlant) were then selected for testing using whole plant assay (WPA) and \u003cem\u003ein agro\u003c/em\u003e trials. All the treatments and dosages used are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003eList of biological control agents and plant-resistant inducers tested in detached leaf assay\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProduct\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive ingredient\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDosage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWater volume (l/ha)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNvs 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus velezensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5kg/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNvs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5kg/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNvs 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eB. velezensis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5kg/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNvs 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eB. velezensis\u003c/em\u003e (liquid formulation)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 l/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNvs 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e (liquid formulation)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 l/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNvs 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eB. velezensis\u003c/em\u003e and\u003c/p\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e (liquid formulation)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 l/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolyversum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1x10\u003csup\u003e6\u003c/sup\u003e oospore/g \u003cem\u003ePythium oligandrum\u003c/em\u003e M1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e200 g/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeranade ASO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1x10\u003csup\u003e11\u003c/sup\u003e cfu/l \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e QST 713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 l/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. harzianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 2*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. capillare\u003c/em\u003e BP-B2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 3*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. virens\u003c/em\u003e Gv-29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 4*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. atroviride\u003c/em\u003e JWB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. atroviride\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 6*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. harzianum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. tomentosum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. reesei\u003c/em\u003e TMW4.2103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 9*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. afroharzianum\u003c/em\u003e T-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e4\u003c/sup\u003ecfu/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResistim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePotassium phosphite: 6.8% phosphonate, 10.9% potassium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 l/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFytosol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.5 g/L COS-OGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 l/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChiProPlant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChitosan (80%) hydrochloride (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300 g/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRanman Top\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160 g/l cyazofamid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFungicide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80 g/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e*\u003c/sup\u003eThe pure organisms, not formulated product were used; 1The type of product were (1) biological control agent (BCA), Plant resistant inducer (PRI), and synthetic or conventional fungicide (Fungicide)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBacterial products Nvs 1\u0026ndash;6 were kindly provided by Novonesis A/S (Denmark). Spore suspensions of the \u003cem\u003eTrichoderma\u003c/em\u003e species, obtained from Laura Grenville-Briggs Didymus, Swedish Agricultural University, were cultured in Petri dishes containing Potato Dextrose Agar (PDA). The plates were covered, sealed and incubated at 12h light (UV-A)/12 hr darkness at 20\u0026ndash;22\u0026deg;C. Once the cultures fully covered the plates and showed the characteristic green color of \u003cem\u003eTrichoderma\u003c/em\u003e spp., spore suspensions were prepared by flooding with distilled water and scraping the spores off into a beaker after filtering through a sterilized gauze. The solution was adjusted to 1x10\u003csup\u003e5\u003c/sup\u003e spores/ml with a haemocytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Inoculum preparation and inoculation\u003c/h2\u003e \u003cp\u003eInoculum for the DLA was prepared in three steps. \u003cem\u003ePhytophthora infestans\u003c/em\u003e isolates, including EU_43_A1, EU_41_A2 and one unique multilocus genotype (MLG) were grown on rye agar B media and incubated under fluorescent light at 17\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 14 days, as described in Abuley et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Detached leaves of the late blight susceptible cv. Bintje were inoculated with a sporangial suspension of the pathogen inoculum and incubated at 17\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 10 days. Sporangia that had formed on the leaves 10 days after inoculation were washed off with sterilized distilled water into a beaker and adjusted to a final concentration of 1x10\u003csup\u003e4\u003c/sup\u003e sporangia ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The suspension was incubated at 4\u003csup\u003e\u0026deg;\u003c/sup\u003eC for 4 h before the inoculation to promote zoospore release. Six droplets (10\u0026micro;l) of the sporangial suspension were then placed on the abaxial leaf surface in a humid chamber, covered with lids and placed in an incubation room at 17\u0026deg;C under 12 h/day of fluorescent light.\u003c/p\u003e \u003cp\u003eFor the WPA and \u003cem\u003ein agro\u003c/em\u003e experiments, \u003cem\u003eP. infestans\u003c/em\u003e isolates were grown on rye agar B at 18\u0026deg;C for 14 days. In the Estonian assays, two isolates with unique multilocus genotypes were used. In the Danish assay, three genotypes, including EU_43_A2, EU_41_A1 and one unique multilocus genotype were used. In Scotland, EU36 genotype was used. The sporangial suspension was prepared by flooding the plates with sterilized distilled water and washing the sporangia off into the beaker via a gauze. The suspension was adjusted to 1x10\u003csup\u003e5\u003c/sup\u003e sporangia/ml with a haemocytometer and incubated at 4\u003csup\u003e\u0026deg;\u003c/sup\u003eC for 4 h to promote zoospore release. Each plant was inoculated by 5 mL of suspension.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Detached leaf assay\u003c/h2\u003e \u003cp\u003eTwo late blight susceptible (\u0026lsquo;Folva\u0026rsquo; and \u0026lsquo;Bintje\u0026rsquo;), one moderately susceptible (\u0026lsquo;Kuras\u0026rsquo;), and two resistant (\u0026lsquo;Sarpo Mira\u0026rsquo; and \u0026lsquo;Nofy\u0026rsquo;) potato cultivars were used in the experiment. Seed tubers of each of the cultivars were planted in 5L plastic pots containing a peat-sandy/clay soil mixture in a ratio of 2:1 v/v. Nutrients were supplied in the following amounts per 100 L soil-peat mixture: 90 g KSO\u003csub\u003e4\u003c/sub\u003e, 60 g (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 33 g CaNO\u003csub\u003e3\u003c/sub\u003e, 7 g MgSO\u003csub\u003e4\u003c/sub\u003e, 7 g CuSO\u003csub\u003e4\u003c/sub\u003e, 133 g CaMg (CO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. The greenhouse was maintained at 20\u0026deg;C during the day and 18\u0026deg;C at night with a 16h photoperiod. Plants were drip-irrigated at a rate of 1mm/5min. For DLAs, leaves were harvested from 6-week-old potato plants and moistened cotton wool was wrapped around leaf petioles to keep the leaflets alive during the experiment. Leaves were placed in a Petri dish lined with moistened filter paper.\u003c/p\u003e \u003cp\u003eThe experiment was a 5x19x2 factorial completely randomized design with 5 levels of cultivars (Kuras\u0026rsquo;, \u0026lsquo;Bintje\u0026rsquo;, \u0026lsquo;Folva\u0026rsquo;, \u0026lsquo;Sarpo Mira\u0026rsquo;, and \u0026lsquo;Nofy\u0026rsquo;), 19 levels of products and 2 product timings (7 days before the inoculation with \u003cem\u003eP. infestans\u003c/em\u003e or 1 day before the inoculation). The BCAs and PRIs were applied according to the dosages shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In addition to the BCA products, a treatment with Ranman Top (160 g/l cyazofamid) was included as a fungicide reference and an untreated control. Applications were made until the run-off. Inoculation was done by dropping 6 10\u0026micro;l droplets of sporangial suspension on a leaflet. Four replications for each variant were included for each cultivar.\u003c/p\u003e \u003cp\u003eLeaves were monitored visually for late blight symptoms from 24 hours to 7 days after pathogen inoculation. Disease severity was evaluated daily as the percentage of leaf area covered with blight symptoms. The disease severity data taken over time was used to calculate the area under the disease progress curve (AUDPC) using the mid-point method (Shaner \u0026amp; Finney, 1977). The relative area under the disease progress curve (rAUDPC) was calculated by dividing the AUDPC by the maximum potential AUDPC as described by Fry \u003cem\u003eet al\u003c/em\u003e. 1978. The average rAUDPC value of untreated control was then used to calculate efficacy (i.e., the disease control offered by a product relative to an untreated control), using the method of Abuley and Nielsen (2017).\u003c/p\u003e \u003cp\u003eThe performance of products in controlling late blight was ranked based on their efficacy to reduce the severity of disease. Products with the same efficacy were given the same value. This calculation was done separately for each of the three cultivars and two application timings; thus, six values were obtained for a given product. The mean rank of the product across both application timing and all three cultivars was then calculated and assigned as the composite rank, giving a measure of average performance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Whole plant assays\u003c/h2\u003e \u003cp\u003eThree sub-experiments were carried out in the glasshouse simultaneously:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eComparison of the efficacy of BCAs/PRIs with full dose applied one day before inoculation\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eDose comparison trials with three levels of BCA applied: half, full (normal) and double dose.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTiming comparison trials with four application timings: 1) onetime application 1 day before inoculation; 2) one time application 1 day after inoculation; 3) 1 day before inoculation; 3) 1 day before inoculation with a 5-day interval; and 4) 1 day before inoculation with a 7-day interval.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eGlasshouse trials were carried out in Estonia and Denmark for the above-mentioned trials (1\u0026ndash;3). The moderately resistant cv. Kuras and susceptible cv. Bintje were used in the WPA. Each cultivar was planted in 1 L plastic pots. The 1 L plastic pots were arranged on greenhouse benches in a randomized design and filled with a commercial limed peat substrate (Biolan) with pH 6.0 and with nutrients in the following amounts 1 L per mixture: 100 mg N, 60 mg P and 200 mg K. Sections of certified seed tubers containing an eye were planted in pots and maintained under controlled conditions at 23\u0026deg;C with a 12 hour photoperiod. The plants were grown for 6 weeks in the greenhouse before products were applied.\u003c/p\u003e \u003cp\u003eBased on the results of the DLAs (Figure S1), TC 4, TC 6, Nsv 2, Polyversum and ChiProPlant were selected as promising candidates for the WPA and \u003cem\u003ein agro\u003c/em\u003e trials. The products and full dose rates used in the trials are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Revus Top (250 g/l mandipropamid\u0026thinsp;+\u0026thinsp;250 g/l difenoconazole) as a chemical reference and an untreated control were included for both cultivars. Each treatment combination was replicated four times. Each plant was sprayed until it ran off (approximately 5ml of foliar application of the fungicide or BCA products per plant) for treatments as well as for inoculation with \u003cem\u003eP.infestans\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eLate blight progression was monitored visually five times during the experiment (1, 4-, 6-, 8-, and 11-days post-inoculation). The plants were assessed for late blight severity as a percentage of the blighted area. The disease severity data taken over time was used to calculate the area under the disease progress (AUDPC) (Shaner \u0026amp; Finney, 1977). rAUDPC and efficacy were then calculated using methods described in DLA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 \u003cem\u003eIn agro\u003c/em\u003e trials\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn agro\u003c/em\u003e trials were carried out in 2022 in Denmark (DK, cultivars \u0026lsquo;Kuras\u0026rsquo; (susceptible) and \u0026lsquo;Nofy\u0026rsquo; (Resistant)), Estonia (EE, cultivars \u0026lsquo;Kuras\u0026rsquo; (resistant)and \u0026lsquo;Triton\u0026rsquo; (susceptible)), Finland (FI, cultivars Kuras (resistant) and Mungo (susceptible)), Germany (DE, Kuras (susceptible) and Nofy (resistant)) and Scotland (SC, cultivars Maris Piper (moderately resistant) and Gatsby (resistant)). The experiment was conducted in a randomized complete block design with four replicates and a plot size of approximately 25 m\u003csup\u003e2\u003c/sup\u003e. The same BCA\u0026rsquo;s and PRI tested in WPA were included in the \u003cem\u003ein agro\u003c/em\u003e trials at the same dosages (i.e., full dose rates), with exception for trials carried out in Finland where the \u003cem\u003eTrichoderma\u003c/em\u003e treatments were not included. An untreated control treatment and a fungicide treatment were also included. All the fungicide treatments and dosages used in each country are given in S2.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of biological control agents (BCA) and plant-resistant inducers (PRI) tested in glasshouse and \u003cem\u003ein agro\u003c/em\u003e trials\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProduct\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive ingredient\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDosage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWater volume (l/ha)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolyversum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1x10\u003csup\u003e6\u003c/sup\u003e oospore/g \u003cem\u003ePythium oligandrum\u003c/em\u003e M1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e200 g/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNvs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 kg/ha**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 4*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTrichoderma atroviride*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,2x10\u003csup\u003e12\u003c/sup\u003ecfu/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC 6*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. harzanium*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,2x10\u003csup\u003e12\u003c/sup\u003ecfu/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChiProPlant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChitosan (80%) hydrochloride (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300 kg/ha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e*\u003c/sup\u003eThe pure organisms, not formulated product were used; **In Finnish trial, the Nvs 2 dosage was 1 kg/ha, water volume 300 l/ha.\u003c/p\u003e \u003cp\u003eThe trials were inoculated by inoculating spreader/infection rows with 1 x 10\u003csup\u003e4\u003c/sup\u003e sporangia/mL of \u003cem\u003eP. infestans\u003c/em\u003e. The same isolates as in glasshouse trials were also used to inoculate the field trials. In the Finnish field trials, there was natural infection. Treatment applications were started at the first occurrence of a late blight risk period at the individual trial sites, as determined by the local decision support systems (Blight Manager \u0026ndash; in Denmark and Finland; Hutton Criteria \u0026ndash; in Scotland; BliteCcast \u0026ndash; in Estonia; Phytophthoramodel-Weihenstephan in Germany), and continued at weekly intervals for 8\u0026ndash;12 weeks. Disease assessments were made as a percentage of infected foliage at a 7-day interval on all plots starting from the onset of late blight and continuing until 2 weeks before harvest.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe R programming language (version 4.3.1) was used for statistical analyses (R Core Team, 2022). The DLA efficacy data was subjected to Gaussian linear models using the \u0026lsquo;lm\u0026rsquo; function in the stats R package (R Core Team, 2022). The effect of factors and their interactions was determined via F-test using ANOVA.\u003c/p\u003e \u003cp\u003eAll WPA and \u003cem\u003ein agro\u003c/em\u003e datasets were checked for normality (Shapiro-Wilk test) and for homogeneity of variances (Levene\u0026rsquo;s test). In case of equal variances, one-way analysis of variance (ANOVA) was performed, followed by Tukey HSD test to identify differences between the treatment variants. For non-normally distributed data, the Kruskal-Walli\u0026rsquo;s test was used. \u003cem\u003ePost hoc\u003c/em\u003e analysis for treatment differences was conducted using Dunn's multiple comparisons test with Bonferroni correction. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Detached leaf assay\u003c/h2\u003e \u003cp\u003eSeventeen BCAs and PRIs were tested in detached leaves in Petri dishes for selection more effective ones in controlling potato late blight with aim to use them in glasshouse and \u003cem\u003ein agro\u003c/em\u003e studies. No infection was recorded in the resistant cultivars (i.e., \u0026lsquo;Sarpo Mira\u0026rsquo; and \u0026lsquo;Nofy\u0026rsquo;), so these cultivars were excluded from the analyses. Infection was recorded in the untreated susceptible (\u0026lsquo;Bintje\u0026rsquo; and \u0026lsquo;Folva\u0026rsquo;) and moderately susceptible (Kuras\u0026rsquo;) cultivars, with the first symptoms occurring 3 days post-inoculation. Leaves treated with fungicide had no disease symptoms for the entire duration of the experiment.\u003c/p\u003e \u003cp\u003eAn ANOVA test to analyse results from all cultivars showed that all individual factors, product type, potato cultivar, and timing of treatments, had a significant effect on product efficacy (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, potato cultivar had the most pronounced effect on product efficacy to reduce incidence of disease (F\u0026thinsp;=\u0026thinsp;946, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), followed by product type (F\u0026thinsp;=\u0026thinsp;54, df\u0026thinsp;=\u0026thinsp;21, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and thereafter timing of treatments (F\u0026thinsp;=\u0026thinsp;4.5, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.033).\u003c/p\u003e \u003cp\u003eResults also showed a significant effect of all two-ways interactions between the three experimental factors (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the three-way interaction between the three factors was non-significant (F\u0026thinsp;=\u0026thinsp;1, df\u0026thinsp;=\u0026thinsp;42, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.36). The product type was the factor with the most effect (F\u0026thinsp;=\u0026thinsp;62.7, df\u0026thinsp;=\u0026thinsp;21, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.001), followed by potato cultivar (F\u0026thinsp;=\u0026thinsp;7.4, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eAs the previous analysis included both the fungicide treated control and the untreated control, a further analysis was conducted without these treatments. This was done to make a direct comparison of the BCA and PRI treatments. The results showed a strong effect of all three single factors and their two-way interactions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the three-way interaction between potato cultivar, product type and timing of treatment was insignificant (F\u0026thinsp;=\u0026thinsp;1, df\u0026thinsp;=\u0026thinsp;38, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.4).\u003c/p\u003e \u003cp\u003eDue to the strong effect of potato cultivar, product type and timing of treatments, we separated the results to avoid biased comparisons. Fungicide treatments consistently showed the highest efficacy in disease reduction. Among tested BCAs and PRIs, potassium phosphite (Resistim) and chitosan hydrochloride (ChiProPlant) were the most effective in disease reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, the highest efficacy in disease reduction was observed when treatments were applied one day before inoculation. In general, the treatment efficacy in disease reduction was higher in cv. Kuras compared to cv. Folva and cv. Bintje.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eComposite ranking\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA composite rank value was calculated to rank the performance if the BCAs and PRIs. Results from the untreated leaves and those treated with chemical fungicides were excluded before the analyses of composite ranking of the treatments across the cultivars. The highest-ranked product for late blight control was Resistim, followed by ChiProPlant at both application times. TC 1, TC 2, TC 3, TC 5, TC 6, TC 7, TC 8, Nsv 1, Nsv 2, Nsv 3, Nsv 6 and Polyversum performed better when applied 1 day before inoculation than 7 days before inoculation. Therefore, the composite ranking of the \u0026minus;\u0026thinsp;1-day treatment was used. Based on the results of the composite ranking, 4 BCAs (Nvs 2, TC 4, TC 6, Polyversum) and one PRI (ChiProPlant) were selected for subsequent experimentation. Resistim, though the most effective product, was not included because this product is considered a conventional fungicide in some countries.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Glasshouse experiments\u003c/h2\u003e \u003cp\u003eFive best performing treatments (Polyversum [\u003cem\u003ePythium oligandrum\u003c/em\u003e], Nvs 2 (\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e], TC 4 [\u003cem\u003eT. atroviride\u003c/em\u003e], TC 6 [\u003cem\u003eT. harzianum\u003c/em\u003e], and ChiProPlant [Chitosan hydrochloride]) selected according to results of DLA were tested in glasshouse grown potato plants to get more reliable results on efficacy of used treatments and to assess the effect of dose and timing on their efficacy in potato late blight control. Data from tests carried out in Estonia and Denmark on cv. Bintje and cv. Kuras were analysed. Infection was successful on bothcv. 'Bintje' and cv. 'Kuras', though in the latter, none of treatments with BCAs or PRIs resulted in significant disease reduction compared with the untreated control. Plants treated with fungicide had no late blight symptoms for the entire duration of the experiment in both cultivars, therefore were excluded from the statistical analyses. The first late blight symptoms occurred 4 days post-inoculation on untreated controls.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Effect of products\u003c/h2\u003e \u003cp\u003eAccording to the Kruskal-Walli's test performed on results of whole plant trials, product type had a significant effect on disease control efficacy across both potato cultivars and trials (χ\u0026sup2; = 14.8, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011). Furthermore, \u003cem\u003epost hoc\u003c/em\u003e analysis revealed that the application of ChiProPlant and TC 4 resulted in significantly lower late blight severity compared with the untreated control (p\u0026thinsp;=\u0026thinsp;0.006; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021). Furthermore, the Wilcoxon rank sum test indicated a significant difference in disease severity between the cv. Bintje and cv. Kuras (W\u0026thinsp;=\u0026thinsp;1975.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018) across both countries.\u003c/p\u003e \u003cp\u003eIn analysis of data separately for each cultivar, the product type had a marked effect on disease control efficacy in cv. Bintje (χ\u0026sup2; = 14.203, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014), but not on cv. Kuras (χ\u0026sup2; = 5.572, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.35) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003ePost hoc\u003c/em\u003e analysis revealed that ChiProPlant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), TC 4 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049), and TC 6 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037) demonstrated significantly higher disease control compared with the untreated control in the cv. Bintje trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Effect of dose\u003c/h2\u003e \u003cp\u003eNo dose-response effect was found in the glasshouse trial when analyzing data from both countries and both cultivars across all treatments (χ\u0026sup2; = 2.904, df\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.406). The data for each cultivar was also analyzed separately. No statistically significant differences were found between treatment doses and the untreated control, although the Kruskal-Wallis test revealed a significant effect of dose on disease control efficacy in cv. Kuras (χ\u0026sup2; = 37.06, df\u0026thinsp;=\u0026thinsp;5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eWe observed a significant product x dose interaction on disease control in cv. Bintje (F\u0026thinsp;=\u0026thinsp;4.39, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Half, full and double dose of ChiProPlant showed significant disease reduction compared with the untreated control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 respectively), but there were no significant differences in disease reduction between the different ChiProPlant doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). While no differences were observed between the different doses for TC4 in the overall analysis, we observed a significant increase in efficacy with dose when TC4 was analyzed alone where use of double dose resulted in significant disease reduction compared with half dose and untreated control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe product x dose interaction on disease reduction was also significant in cv. Kuras (χ\u0026sup2;= 51.301, df\u0026thinsp;=\u0026thinsp;15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), but no statistically significant differences were observed between the treatment variants and the untreated control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Effect of application timing\u003c/h2\u003e \u003cp\u003eAccording to Kruskal-Walli's test, application timing significantly affected disease control efficacy in cv. Bintje (χ\u0026sup2; = 27.96, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, \u003cem\u003epost hoc\u003c/em\u003e analysis revealed that a statistically significant difference was observed only between the fungicide treatment and the untreated control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). When excluding fungicide treatment from the analysis, pre-treatment of plants with the repeated application of BCAs/PRI 5 and 1 day before the inoculation demonstrated significantly higher efficacy in disease control compared with the untreated control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019). The BCA/PRI treatment of potato plants after pathogen inoculation did not have an effect on disease.\u003c/p\u003e \u003cp\u003eDisease occurrence was significantly affected by product type x timing of BCA/PRI inoculation interaction in cv. Bintje (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;63.50, df\u0026thinsp;=\u0026thinsp;20, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pre-treatment with ChiProPlant 7 and 1 day before the inoculation with \u003cem\u003eP. infestans\u003c/em\u003e provided comparable efficacy to that of the chemical fungicide Revus Top. Both treatments resulted in significantly less disease compared with the untreated control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA significant effect of product type x timing of BCA/PRI inoculation interaction on disease control efficacy was also observed in the cv. Kuras (W\u0026thinsp;=\u0026thinsp;73.22, df\u0026thinsp;=\u0026thinsp;21, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but \u003cem\u003epost hoc\u003c/em\u003e analysis revealed no significant differences between the BCA/PRI treatments and the untreated control.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 \u003cem\u003eIn agro\u003c/em\u003e trials\u003c/h2\u003e \u003cp\u003eNine field trials were established in five countries to assess the efficacy of BCAs and PRI in variable environmental conditions. However, only five trials out of nine had sufficient infection to assess the efficacy of disease control. No disease was observed in the Estonian trials, so data from these trials were excluded from the subsequent analysis. Likewise, data from trials with cv. Nofy in Denmark and Germany were excluded from subsequent analyses due to minimal to no disease symptoms. Data from the Danish and German trials for the cv. Kuras, as well as from the Scottish trials for the cv. Maris Piper (moderately resistant) and cv. Gatsby (resistant), were analyzed separately and in combined analysis. Due to the lower number of products tested in the Finnish trial, the data were not included in the combined analysis.\u003c/p\u003e \u003cp\u003eProduct type had a significant effect on disease control in the cv. Kuras in the German field trial (χ\u0026sup2; = 21.39, df\u0026thinsp;=\u0026thinsp;6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). ChiProPlant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) and fungicide (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) provided significantly higher disease control compared with the untreated control (S3).\u003c/p\u003e \u003cp\u003eIn the Danish trials, although there was a significant effect of product type on disease severity in cv. Kuras, (χ\u0026sup2; = 14.53, df\u0026thinsp;=\u0026thinsp;6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024), only the fungicide treatment showed significant disease reduction (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (S4).\u003c/p\u003e \u003cp\u003eIn the trial conducted in Scotland with the moderately blight resistant cv. Maris Piper, there was a significant effect of product on disease control (χ\u0026thinsp;=\u0026thinsp;21.30, df\u0026thinsp;=\u0026thinsp;7, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003); however, only the fungicide treatment resulted in a significant disease reduction (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) (S5). Further statistical analysis omitting the fungicide treatment showed that ChiProPlant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) and Polyversum (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) significantly reduced disease compared with the untreated control. ANOVA showed a significant effect of product type on disease control on cv. Gatsby (F\u0026thinsp;=\u0026thinsp;10.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.002), however this was only true for the fungicide treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (S6).\u003c/p\u003e \u003cp\u003eSimilarly, in the Finnish trials, there was also a significant effect of product on disease control in cv. Kuras (F\u0026thinsp;=\u0026thinsp;3.58, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031) with only the fungicide treatment significantly reducing disease (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038) (S7). A similar trend was observed in cv. Mungo (χ\u0026thinsp;=\u0026thinsp;10.70, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030), but \u003cem\u003epost hoc\u003c/em\u003e analysis revealed no significant differences between product treatments and the untreated control (S8).\u003c/p\u003e \u003cp\u003eWhen comparing data from all experiments, there was a significant effect of product on disease control overall (χ\u0026sup2; = 62.746, df\u0026thinsp;=\u0026thinsp;7, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Notably, treatments with Polyversum (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01), Nsv 2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016), ChiProPlant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), and fungicide (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) resulted in less disease compared with the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, none of the treatments were as effective as the fungicide.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThrough a series of DLA, glasshouse, and in-agro experiments, we demonstrate the efficacy of BCAs and PRIs against late blight across different agro-climatic areas with varying \u003cem\u003eP. infestans\u003c/em\u003e populations. We also examine factors such as dose and timing of application, and their influence on the efficacy against late blight.\u003c/p\u003e \u003cp\u003eDetached leaf and whole plant assays conducted in Denmark and Estonia revealed that across both trials, TC 4, TC 6 and ChiProPlant were the most effective products in controlling late blight in the susceptible cv. Bintje. There have been numerous reports of \u003cem\u003eTrichoderma\u003c/em\u003e showing an inhibitory effect on \u003cem\u003eP. infestans\u003c/em\u003e from previous studies. For example, \u003cem\u003eTrichoderma\u003c/em\u003e strain HNA14 significantly reduced late blight in both glasshouse assays and field trials conducted by Yao et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, Mollah and Hasan (2023) showed that the \u003cem\u003eT. harzanium\u003c/em\u003e based biological fungicide \u0026ldquo;Lycomax\u0026rdquo; was effective in controlling late blight under field conditions. These authors found that Lycomax reduced late blight severity by more than 90% compared to the untreated control, matching the performance of chemical fungicide Agrizeb (Mollah and Hassan, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The biopolymer chitosan has also been found to exhibit antifungal properties against \u003cem\u003eP. infestans\u003c/em\u003e (Yarullina et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), with its biocontrol properties being attributed to inducing plant defense responses in potato (Zheng et al. 2020), pathogen growth inhibition as well as having a synergistic effect on synthetic fungicides (Huang et al. 2020). However, the efficacy of the aforementioned products was not as high as that of synthetic fungicides in our trials, which consistently achieved 100% efficacy. This is consistent with previous studies in the literature, which have shown that synthetic fungicides generally provide stronger and more consistent control of late blight pathogen (Caulier et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Dorn et al. 2011).\u003c/p\u003e \u003cp\u003eWe also observed a significant effect of cultivar on product efficacy in DLA and glasshouse trials. Cultivar is known to play a significant role in disease control (Carolan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast to the cv. Bintje where treatments with ChiProPlant, TC4 and TC6 resulted in disease reduction, there was no significant reduction in late blight symptoms in the moderately resistant cv. Kuras. The resistance of the cv. Kuras to late blight, led to lower disease severity in the untreated control and absence of differences between the treatments. Conversely to our results, in a Swedish study involving the plant resistance inducer β-aminobutyric acid (BABA), it was observed that the effect of BABA on late blight symptoms was greater on the partially resistant cultivar than on the more susceptible cv. Bintje in greenhouse and field experiments (Liljeroth et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Nevertheless, these findings suggest that it\u0026rsquo;s important to consider the level of resistance of the cultivar when evaluating the effectiveness of biocontrol products. Furthermore, evaluating product efficacy can be challenging when disease levels are extremely high or very low, as this limits observable differences between treatments.\u003c/p\u003e \u003cp\u003eOur experiments showed a significant dose-response relationship only for treatments with TC 4 in the susceptible cv. Bintje, where treatment with double dose was superior compared with half dose. All treatments of ChiProPlant resulted in significant disease reduction but there were no statistically significant differences between the efficacy of used doses. In contrast, Liljeroth et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) reported that a higher concentration of PRI β-aminobutyric acid gave a stronger protective effect. Similarly, Liljeroth et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) observed a clear dose-response effect of phosphite on control of late blight in Swedish field trials, noting that higher doses resulted in increased efficacy.\u003c/p\u003e \u003cp\u003eOur data indicate that product application timing is an important factor in the glasshouse assay. Pre-treatment with ChiProPlant one and seven days before inoculation yielded results comparable to that of fungicide treatment, achieving nearly 100% efficacy. Other pre-treatments with Nsv 2, Polyversum, TC 4 and TC 6 were less effective. On the contrary, post-inoculation treatment of potato plants had no effect on disease severity and may have even increased disease. This highlights the importance of adequate application timing of a biocontrol product. Better efficacy of a bio fungicide is more likely when the antagonist is applied early enough to prevent the pathogen from establishing itself and causing infection. To effectively use PRIs for disease control, they must be applied preventively, before disease establishment, to induce the plant's defense mechanisms (Altamiranda et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In a glasshouse experiment, Savchuk and Fernando (2006) observed that applying antagonistic bacteria before, or at the same time as \u003cem\u003eS. sclerotiorum\u003c/em\u003e inoculation led to complete disease inhibition by day 14, whereas applying the bacteria 24 or 48 hours later resulted in only minimal disease suppression. Studying the effects of biocontrol agents against \u003cem\u003eP. infestans\u003c/em\u003e, Stephan et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) found that applying these products 24 hours before inoculation with the pathogen resulted in greater efficacy compared to applications made 1 hour prior to inoculation. Moreover, despite various treatment applications, none were found to have a curative effect on the disease (Stephan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our field experiments, a clear effect of product on disease control was found across all trials. Treatments with Polyversum, Nsv2, ChiProPlant, and fungicide resulted in significant disease reduction compared with the untreated control, although none of the biocontrol products were as effective as the fungicide alone. In the trial conducted in Germany, ChiProPlant and fungicide treatment had a greater effect on disease reduction compared with the untreated control, but in the Danish trial, only the fungicide treatment was significantly more effective compared with the untreated variant. The same trend was observed in separate trials conducted in Scotland with the cvs. M. Piper and Gatsby. This is consistent with the findings of Caulier et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), who demonstrated that treatments with bacterial antagonists were significantly less effective in reducing disease severity in Belgian field studies compared to the synthetic fungicides.\u003c/p\u003e \u003cp\u003eThe limited effect of biocontrol products in field trials might be attributed to the variability in environmental conditions. Biocontrol agents are less likely to achieve successful control at the field level compared to laboratory or glasshouse settings, where temperature and relative humidity can be controlled. Kumbar et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Islam et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) demonstrated that various bacterial and fungal bioagents effectively reduced late blight disease severity in field trials; however, given the tropical climate, these results may differ from what could be expected in temperate regions. A study by Stridh et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that the effectiveness of biocontrol products on potato early blight in greenhouse trials did not extend to field-scale trials. In contrast to our studies on potato late blight, none of the tested products successfully controlled another potato foliar disease - early blight in field settings (Stridh et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are only a few investigations on the efficacy of biocontrol agents against late blight in temperate climate zone in Europe, to the best of our knowledge. In the study by Dorn et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), various natural products, including products containing microorganisms from the genera \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eTrichoderma\u003c/em\u003e, and \u003cem\u003ePythium\u003c/em\u003e, which had previously shown some efficacy in \u003cem\u003evivo\u003c/em\u003e assays, were examined in field trials in Switzerland. The efficacy of used natural compounds and microbial preparations was limited in field settings. The authors of this study speculated that the poor performance of natural products could be attributed to their lack of stability in the existing environmental conditions rather than their inherent ineffectiveness (Dorn et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, Kurzawinska and Mazur (2009), reported the potential use of Polyversum (\u003cem\u003eP. oligandrum\u003c/em\u003e) and Biochikol 020 PC (chitosan) in protecting potatoes against late blight based on three-year field experiments. Both products effectively reduced the mean disease index and also the incidence of tuber infection caused by \u003cem\u003eP. infestans\u003c/em\u003e. It was noted that disease development was influenced by the weather conditions during the vegetative period (Kurzawinska \u0026amp; Mazur). Humidity and cool temperature are critical factors that contribute to the rapid development of \u003cem\u003eP. infestans\u003c/em\u003e infection (Fry 2015). Additionally, in the field experiments conducted by Caulier et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), treatment with \u003cem\u003eBacillus subtilis\u003c/em\u003e strain 30B-B6 was shown to significantly reduce late blight severity throughout the crop season.\u003c/p\u003e \u003cp\u003eThe results of our study demonstrate that certain biological agents have potential to control \u003cem\u003eP. infestans\u003c/em\u003e under glasshouse as well as in field conditions. However, further field trials in temperate regions are necessary for a comprehensive evaluation of biocontrol agents and their potential integration into an effective integrated pest management system. Cultivar resistance, timing of application, and environmental conditions must be taken into account when integrating biocontrol products to late blight management strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted as part of \u0026apos;ECOSOL - Eco-friendly solutions for the integrated management of late and early blight of potatoes\u0026apos; a project carried out under the H2020 SusCrop - ERA-NET Cofund Action on Sustainable Crop Production. Partners in ECOSOL received national funding from The Green Development and Demonstrations Program (Denmark), Estonian Science Foundation (Estonia), Ministry of Agriculture and Forestry of Finland (Finland), German Research Foundation (Germany), SLU Grogrund - The Centre for breeding of food crops and the Swedish Research Council Formas (Sweden) and Department for Environment, Food \u0026amp; Rural Affairs (UK). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNovonesis A/S is highly appreciated for kindly providing BCA products for using in the studies.\u003c/p\u003e\n\u003cp\u003eWe would also like to thank Prof. J. Philipp Benz (Technical University of Munich) for providing \u003cem\u003eTrichoderma\u003c/em\u003e strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbuley, I. K., Lynott, J. S., Hansen, J. G., Cooke, D. E. L., \u0026amp; Lees, A. K. (2023). The EU43 genotype of Phytophthora infestans displays resistance to mandipropamid. Plant Pathology, 72(7), 1305\u0026ndash;1313. https://doi.org/10.1111/ppa.13737\u003c/li\u003e\n\u003cli\u003eAlexandersson, E., Mulugeta, T., Lankinen, \u0026Aring;., Liljeroth, E., \u0026amp; Andreasson, E. (2016). Plant Resistance Inducers against Pathogens in Solanaceae Species\u0026mdash;From Molecular Mechanisms to Field Application. International Journal of Molecular Sciences, 17(10), 1673. https://doi.org/10.3390/ijms17101673\u003c/li\u003e\n\u003cli\u003eAltamiranda, E. A. G., Andreu, A. B., Daleo, G. R., \u0026amp; Olivieri, F. P. (2008). Effect of \u0026beta;-aminobutyric acid (BABA) on protection against Phytophthora infestans throughout the potato crop cycle. Australasian Plant Pathology, 37(4), 421. https://doi.org/10.1071/AP08033\u003c/li\u003e\n\u003cli\u003eCarolan, K., Helps, J., Van Den Berg, F., Bain, R., Paveley, N., \u0026amp; Van Den Bosch, F. (2017). Extending the durability of cultivar resistance by limiting epidemic growth rates. Proceedings of the Royal Society B: Biological Sciences, 284(1863), 20170828. https://doi.org/10.1098/rspb.2017.0828\u003c/li\u003e\n\u003cli\u003eCaulier, S., Gillis, A., Colau, G., Licciardi, F., Li\u0026eacute;pin, M., Desoignies, N., Modrie, P., Legr\u0026egrave;ve, A., Mahillon, J., \u0026amp; Bragard, C. (2018). Versatile Antagonistic Activities of Soil-Borne Bacillus spp. And Pseudomonas spp. Against Phytophthora infestans and Other Potato Pathogens. Frontiers in Microbiology, 9, 143. https://doi.org/10.3389/fmicb.2018.00143\u003c/li\u003e\n\u003cli\u003eCooke, L. R., \u0026amp; Little, G. (2002). 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Microorganisms. 2021;9:1489. doi: 10.3390/microorganisms9071489.\u003c/li\u003e\n\u003cli\u003eYao, Y., Li, Y., Chen, Z., Zheng, B., Zhang, L., Niu, B., Meng, J., Li, A., Zhang, J., \u0026amp; Wang, Q. (2016). Biological Control of Potato Late Blight Using Isolates of Trichoderma. 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International Journal of Biological Macromolecules, 182, 1670\u0026ndash;1680. https://doi.org/10.1016/j.ijbiomac.2021.05.097\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"potato-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"potr","sideBox":"Learn more about [Potato Research](http://link.springer.com/journal/11540)","snPcode":"11540","submissionUrl":"https://www.editorialmanager.com/potr/default2.aspx","title":"Potato Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biological control agent; Phytophthora infestans; Solanum tuberosum, Trichoderma spp., Chitosan hydrochloride, Bacillus spp","lastPublishedDoi":"10.21203/rs.3.rs-6104485/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6104485/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLate blight (\u003cem\u003ePhytophthora infestans\u003c/em\u003e) is a major disease in potato cultivation, managed primarily with synthetic fungicides. This reliance makes potatoes one of the crops with the highest fungicide load. To reduce chemical dependence and promote Integrated Pest Management (IPM), alternative solutions are needed. This study explores biological control agents (BCAs) and plant resistance inducers (PRIs) as sustainable alternatives. We screened 17 BCAs and 3 PRIs in a detached leaf assay (DLA) and selected the most effective for further trials. The chosen BCAs \u0026mdash; Polyversum (\u003cem\u003ePythium oligandrum\u003c/em\u003e), Nvs 2 (\u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e), TC 4 (\u003cem\u003eTrichoderma atroviride\u003c/em\u003e), TC 6 (\u003cem\u003eT. harzianum\u003c/em\u003e)\u0026mdash;and the PRI ChiProPlant (Chitosan hydrochloride) were tested in greenhouse and field trials across five European countries. In greenhouse trials, ChiProPlant and \u003cem\u003eT. atroviride\u003c/em\u003e significantly controlled late blight. Dosage did not affect the efficacy of the BCAs and PRI, but application timing was crucial. Treatments applied 1 and 5 days before inoculation were most effective, while post-inoculation treatments were least effective. Field trials showed that weekly applications of ChiProPlant, Polyversum, and Nvs 2 significantly reduced late blight infection. However, none matched the efficacy of synthetic fungicides. This study highlights the potential of BCAs and PRIs in late blight management but underscores the need for further research to optimize their integration into IPM strategies.\u003c/p\u003e","manuscriptTitle":"Use of Biocontrol Agents and Plant Resistance Inducers for the control of potato late blight","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 11:24:28","doi":"10.21203/rs.3.rs-6104485/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-31T08:07:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-28T08:36:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Potato Research","date":"2025-02-28T08:04:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-28T04:22:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Potato Research","date":"2025-02-25T06:04:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"potato-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"potr","sideBox":"Learn more about [Potato Research](http://link.springer.com/journal/11540)","snPcode":"11540","submissionUrl":"https://www.editorialmanager.com/potr/default2.aspx","title":"Potato Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e8b9cf92-0da4-406f-be65-ad6a31d4c5a1","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:47:56+00:00","versionOfRecord":{"articleIdentity":"rs-6104485","link":"https://doi.org/10.1007/s11540-025-09899-1","journal":{"identity":"potato-research","isVorOnly":false,"title":"Potato Research"},"publishedOn":"2025-07-30 16:29:19","publishedOnDateReadable":"July 30th, 2025"},"versionCreatedAt":"2025-04-03 11:24:28","video":"","vorDoi":"10.1007/s11540-025-09899-1","vorDoiUrl":"https://doi.org/10.1007/s11540-025-09899-1","workflowStages":[]},"version":"v1","identity":"rs-6104485","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6104485","identity":"rs-6104485","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}