A detached leaflet assay to determine the pathogenicity of diverse Botrytis spp. causing chocolate spot disease in faba bean (Vicia faba L.)

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Maniruzzaman, Hamid Khazaei, Minna Haapalainen, Frederick L. Stoddard This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6784656/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chocolate spot (CS) disease, caused by certain Botrytis species, is one of the most destructive diseases of faba bean worldwide. This research set out to determine whether different Botrytis species cause different responses in faba bean genotypes that display varied levels of CS susceptibility. Three faba bean inbred lines – Kontu, Mélodie/2, and ILB 938/2 – identified as highly susceptible, moderately susceptible, and moderately resistant, respectively, were chosen as hosts, and 21 Botrytis isolates, representing six species ( B. fabae , B. fabiopsis , B. cinerea , B. pseudocinerea , B. medusae , and B. euroamericana ), were used as disease agents. A PCR assay using primers targeting the 10 NEP protein genes was used to genotype Botrytis isolates. The Botrytis isolates were grown on half-strength PDA medium and treated with near-UV light to induce sporulation. Spore suspensions were inoculated onto detached, fully expanded leaflets. Results revealed a significant difference in virulence reactions among the inbred lines, isolates and their interaction. All the 21 Botrytis species/isolates tested induced CS symptoms. B. fabae 19B053-4 was the most virulent, causing the largest lesions, followed by B. fabiopsis 19B175, B. fabae 17B4, B. fabiopsis 17B24, and B. cinerea 19B048. At the other extreme, lesions developed most slowly with B. cinerea 17B10-1, B. fabiopsis 19B024, B. fabae 17B28, and B. cinerea 19B014. The DNA analysis revealed two main clusters and four sub-clusters among the Botrytis species, showing species-level groupings. The detached leaflet assay enabled a rapid and effective comparison of the pathogenicity of the studied Botrytis isolates. Detached leaflet assay chocolate spot disease screening Botrytis species moderate resistant isolate-genotype interaction Figures Figure 1 Figure 2 Introduction Faba bean ( Vicia faba L.) is a cool-season grain legume crop with great potential to contribute to sustainable agriculture and protein security globally. Its nitrogen fixation ability is considered the strongest among cool-season grain legumes, which leads the crop to produce protein-rich seeds (Klippenstein et al., 2022 ). The crop is an attractive solution for providing sustainable plant-based protein for food and feed, while avoiding the growing global demand for chemical nitrogen fertilizer. Nevertheless, faba bean is considered to be very sensitive to chocolate spot (CS) disease, which is one of the most devastating diseases affecting faba bean globally (Rubiales and Khazaei, 2022 ), as it can cause yield losses of up to 90% in susceptible cultivars (Gorfu and Yaynu, 2001 ; Stoddard et al., 2010 ; Beyene et al., 2018 ; Barilli et al., 2025 ). CS disease is caused by several necrotrophic fungi in the genus Botrytis . It is a polycyclic disease, with conidia serving as the primary source of inoculum, typically produced on residues from previous crops (Fan et al., 2015 ). These conidia are dispersed by wind and deposited on faba bean leaves, stems, flowers, and pods. Under favorable environmental conditions (~ 20°C, 98% RH) the conidia germinate, initiating infection and enabling rapid disease spread across the field. Infection is facilitated by the production of phytotoxins and cell wall-degrading enzymes such as polygalacturonases (Leisen et al., 2022 ). Symptoms of CS disease include oblong to elliptical lesions ranging in color from reddish to chocolate brown, often with a darker margin and concentric ring patterns (El-Metwally et al., 2010 ). These symptoms typically appear on the upper surface of leaves, increasing in number and size, and may coalesce. In severe cases, extensive blackening occurs, leading to the death of the entire plant. Botrytis fabae Sard. is the primary pathogen responsible for CS in faba bean. B. cinerea Pers. has also been reported to cause CS disease, either independently or as part of a pathogen complex (e.g., Harrison, 1988 ; Bilkiss et al., 2019 ). While B. fabae is host-specific, infecting only Vicia species, B. cinerea has a broad host range. Zhang et al. ( 2010 ) reported a third species, B. fabiopsis , from faba bean in Hubei Province, China, which produces symptoms similar to those caused by B. fabae and B. cinerea . B. fabiopsis may co-occur with B. fabae and B. cinerea , potentially contributing to complex infection dynamics (Teshome and Tagegn, 2013). The fourth species, B. pseudocinerea , closely related to B. cinerea (Plesken et al., 2015 ), has also been reported to infect faba bean (Bankina et al., 2021 ). Two further Botrytis species infect various legumes; B. euroamericana caused CS-like symptoms on faba bean, chickpea, field pea, lentil and soybean, while B. medusae infected narrow-leafed lupin, field pea and soybean (Brauna-Morževska et al., 2023 ). Recentl y, B. eucalypti has been reported to cause CS in faba bean in Qinghai Province, China (Ran et al., 2024 ). Only a few sources of moderate genetic resistance to CS disease have been identified in faba bean (e.g., Villegas-Fernández et al., 2009 ; Maalouf et al., 2016 ), and resistant germplasm remains scarce. While screening large faba bean germplasm collections and genetic mapping populations for CS pathology and genetic studies are essential, the screening process is labor-intensive and typically requires large space in greenhouses or semi-controlled field environments. Indoor or outdoor screening of large germplasm collections for CS is often unreliable due to uneven pathogen infection. Detached leaves are often used for large-scale screening, and a detached-leaflet assay offers an even more efficient alternative, requiring less space and resources (Isenegger et al., 2011 ). This method enables rapid and effective comparison of the pathogenicity of a large number of Botrytis isolates, allowing the selection of candidates for further detailed studies. Hence, we set up an experiment using three faba bean inbred lines with proven response to CS disease: one highly susceptible line (selected from cv Kontu, Gela et al., 2022 ; Feyissa, 2022 ), one moderately susceptible line (Mélodie/2; Feyissa, 2022 ), and one moderately resistant line (ILB 938/2, Khazaei et al., 2018), to evaluate the virulence and pathogenicity of a set of 21 Botrytis isolates. This study further dissects the molecular diversity of Botrytis species by analyzing necrosis- and ethylene-inducing protein (NEP) gene markers, which play a key role in inducing necrosis and cell death during host infection and disease progression. Materials and methods Development of Botrytis isolates A set of 21 isolates spanning six Botrytis species was obtained from Prof. Biruta Bankina at the Latvian University of Life Sciences and Technologies, Jelgava, Latvia. The isolates were cultured on half-strength PDA and maintained in darkness at room temperature (20°C) for 7 days, then put under near-ultraviolet light for 7 days to induce sporulation. Thereafter, the cultured plates were washed with sterile water and scraped with a glass rod to dislodge the spores. The suspension was filtered through two layers of cheesecloth into a 250 ml conical flask, then 5 ml of Tween 20 (0.03% v/v) solution was added to keep the spores in suspension (Terefe et al., 2015 ). The concentration of spores was determined using a haemocytometer and adjusted to 5×10 6 spores per milliliter. Plant material and growing conditions Three faba bean inbred lines were chosen for this work. ILB 938/2 exhibits moderate resistance and Mélodie/2 is moderately susceptible to CS disease. These two inbred lines serve as the parental lines of a well-characterized recombinant inbred line population used to study CS disease response (Gela et al., 2022 ). The Finnish cultivar Kontu is highly susceptible to CS disease (Feyissa, 2022 ). Seeds were surface sterilized with 70% ethanol for 3 min and rinsed three times with sterile distilled water. Sets of three seeds of each inbred line were planted in 2.5 L plastic pots filled with autoclaved peat soil (Kekkilä Coarse potting mix WR8494, Kekkilä-BVB OY, Vantaa, Finland). Plants were grown for 6–8 weeks in a controlled-environment growth chamber (Weiss-2000, Weiss Technik GmbH, Reiskirchen-Lindsruth, Germany). The temperature was set to 22°C during the day and 20°C at night, with a 12-hour photoperiod under visible light (150 µmol/m²/s photosynthetic photon flux density, PPFD). Inoculation Fully expanded leaves of six- to eight-week-old plants of the three inbred lines were collected from four to eight nodes two hours prior to inoculation and kept in sterile moist napkin paper. Leaves were washed with sterile distilled water, blotted dry, and placed in pairs on two layers of sterile filter paper in 90 mm petri dishes, which were arranged in a completely randomized factorial design with three replicates (five leaflets in each replicate per inbred line). Five ml of sterile water was applied in each petri dish to moisten the filter paper and to maintain high humidity within the dish. A piece of cotton moistened with sterile water was placed onto the petiole end to maintain the turgor pressure and prevent dryness. An experimental unit comprised three petri dishes containing 5 leaflets, inoculated with 20 µl suspension culture and a control was treated with sterile water in each replication for each accession. The petri dishes were organized in a plastic box (37.5 × 25.5 × 9.5 cm) and incubated in a growth chamber at 22°C and 12 h photoperiod with 170–188 µmol/m²/s PPFD. The fungal infection and necrosis were monitored every day to observe the first visible symptom, and after 5 days, leaflets were photographed against a measuring scale for image analysis. Molecular characterization of Botrytis isolates using NEP protein gene markers Botrytis isolates were genotyped using specific primer pairs for necrosis and ethylene-inducing proteins (NEP) ( Table S1 ). The Botrytis isolates were cultured on PDA containing a GVS Cellophane film (Diameter 76 mm, 0.1 µm; GVS, Bologna, Italy) on the medium and incubated in darkness at room temperature (20°C) for 14 days to produce enough mycelium. Mycelium was harvested using a sterile scalpel and put into a 1.5 ml Eppendorf tube; the lids of the tubes were opened for a few hours to allow the water from the mycelium to dry at room temperature. Subsequently, the air-dried mycelium was frozen into liquid nitrogen and grounded into powder using a plant DNA isolation kit (DNeasy® Plant Mini Kit, Qiagen). The extracted DNA was dissolved in 50 µl of TE buffer, quantified using a Nanodrop spectrophotometer (Thermo Scientific), and stored at -20°C. PCR amplification was carried out in a 20 µl reaction solution, having 4 µl template (6 ng/µl) of fungal DNA, 10 µl of DreamTaq Green PCR Master Mix (2X, contains Dream Taq DNA polymerase, DreamTaq Green buffer, MgCl 2 and dNTPs, supplemented with two tracking dyes and a density reagent that allows for direct loading of the PCR products on a gel; ThermoFisher Scientific, Waltham, MA, USA), 0.8 µl of each primer, and 4.4 µl of sterile water. PCR reactions were conducted in a thermocycler (Eppendorf, Germany), using the amplification program: 1 cycle of 2 min at 94°C, 35 cycles of 30 sec at 94°C, and 30 sec at 50°C or 58°C for annealing temperature, 1 min at 72°C; and 10 min at 72°C for final extension. A PCR mixture without fungal DNA template was used as a negative control. The PCR products were separated and analyzed by electrophoresis using a 1% agarose gel and 1x TBE buffer, staining with 0.5 µg/ml ethidium bromide and visualized in a Molecular Imager, Gel Doc (Bio-Rad Laboratories, Hercules, CA, USA) for the presence or absence of the expected size of band. Statistical analysis The infected area of the leaves was determined using ImageJ software. The scale was fixed at 1 cm and the estimated distance was 50 pixels (Rha et al., 2015 ). The mean infected area of the 5 infected leaves in an experimental unit was calculated. The data were transformed to log (area + 1) to normalize the variance, and used in the analysis of variance (ANOVA) to determine the effects of isolate, inbred line and their interaction. Means were separated using Tukey-HSD multiple pairwise comparisons using the JMP Pro SAS statistical computing package (Grayson et al., 2015 ). Pairwise genetic distances were estimated using Nei’s coefficient (Nei, 1978 ), and a dendrogram was constructed using the unweighted pair-group method with arithmetic mean (UPGMA) algorithm on Darwin v. 6.0.021. Results All 21 isolates caused CS symptoms. On average, Kontu showed the most susceptible reaction, ILB 938/2 the most resistant, and Mélodie/2 an intermediate reaction ( Figure S1 ). The first characteristic water-soaked symptoms of CS disease were found using B. fabae 19B053-4 on Kontu after 24 h, on Mélodie/2 after 36 h and on ILB 938/2 after 72 h. In contrast, symptoms induced by B. fabae 17B4 were observed after 36 h on Mélodie/2 and Kontu and after 96 h on ILB 938/2. By 120 h after inoculation, symptoms were obvious and consistent blackish lesions on most combinations of isolate by inbred line (Table 1 ). The ANOVA results showed significant variation among the Botrytis isolates, the faba bean inbred lines, and the isolate × inbred line interactions ( P < 0.0001, Table 2 ). Table 1 Time (h) to first visible symptoms induced by the Botrytis species on three inbred lines of faba bean. Sterile distilled water was used as a control. Botrytis species (isolate) Kontu Mélodie/2 ILB 938/2 Hours to first symptom B. fabae (19B053-4) 24 36 72 B. fabae (17B4) 36 36 96 B. fabae (17B28) 96 120 144 B. fabiopsis (19B175) 36 48 72 B. fabiopsis (17B24) 36 48 120 B. fabiopsis (17F9) 48 60 96 B. fabiopsis (18B16) 60 72 96 B. fabiopsis (17F4) 72 96 132 B. fabiopsis (19B024) 96 120 132 B. cinerea (19B048) 48 60 96 B. cinerea (19B026) 48 60 96 B. cinerea (17B10-1) 96 120 144 B. cinerea (B05.10) 60 96 96 B. cinerea (19B009) 72 96 132 B. cinerea (19B009) 96 120 144 B. cinerea (18B3) 48 48 60 B. pseudocinerea (18B11) 48 60 110 B. pseudocinerea (18B2-3) 72 96 132 B. pseudocinerea (18B6) 48 48 60 B. euroamerican a (19B053-3) 72 96 132 B. medusae (19B047) 60 60 72 Table 2 Analysis of variance showing the effects of fungal isolate, host inbred line, and isolate×inbred line on the areas of chocolate spot disease lesions. Source df Sum of Squares F Ratio Prob > F Isolate 20 144.16 24.58 < 0.0001 Inbred line 2 152.40 259.86 < 0.0001 Isolate × Inbred line 40 39.42 3.36 < 0.0001 Error 124 36.36 0.29 df, degrees of freedom. B. fabae 19B053-4 was the most virulent isolate on two of the lines, developing the largest lesions on Kontu (10.10 cm 2 necrosis) and Mélodie/2 (9.30 cm 2 necrosis), and the second-largest on ILB 938/2 (1.92 cm 2 ) (Fig. 1 ). B. fabiopsis 19B175 was the most virulent on ILB938/2 (2.16 cm 2 ) and the second on both Kontu (7.70 cm 2 ) and Mélodie/2 (5.87 cm 2 ). Isolate 19B048 was the most virulent from B. cinerea and the fifth most virulent overall (5.73 cm 2 on Kontu, 2.42 cm 2 on Mélodie/2 and 0.53 cm 2 on ILB 938/2), and isolate 18B11 was the most virulent from B. pseudocinerea (4.40 cm 2 on Kontu, 0.84 cm 2 on Mélodie/2 and 0.15 cm 2 on ILB 938/2). The smallest lesions were induced by B. cinerea 17B10-1 on Kontu (0.43 cm 2 ), Mélodie/2 (0.35 cm 2 ), and ILB 938/2 (0.08 cm 2 ), followed by B. fabiopsis 19B024 and B. fabae 17B28. In every case except B. fabae 17B4, the lesion on Kontu was larger than that on Mélodie/2. Similarly, in every case except B. fabiopsis 19B024 and B. euroamericana 19B053-3, the lesion on Mélodie/2 was larger than that on ILB 938/2. Nevertheless, in these cases the apparent crossover was much smaller than that required for statistical significance, and the overall significance of the isolate × inbred line term in the Analysis of Variance is attributable to the lack of discrimination between inbred lines by some isolates while others clearly affected one more than another. The NEP DNA marker analysis exhibited two main groups and four sub-clusters, with a few variations inside each group (Fig. 2 ). All B. cinerea clustered together with B. fabae. B. medusae and B. euroamericana isolates were grouped together. B. fabiopsis isolates and B. pseudocinerea mostly clustered together with some minor deviations ( B. fabiopsis isolate 19B175 and B. pseudocinerea isolate 18B6). Discussion The Botrytis spp. used in this study showed varying reactions on detached leaves of ILB 938/2, Kontu, and Mélodie/2 in an in vitro assay using spore suspension cultures. Our results revealed a significant difference among the isolates, inbred lines, and isolate × inbred line interactions. ILB 938/2 showed moderate resistance while Kontu performed completely susceptible and Mélodie/2 was moderately susceptible. These findings agreed with previous assessments of the quantitative CS resistance of these genotypes (Khazaei et al., 2018; Gela et al., 2022 ; Feyissa, 2022 ). In our study, the first visible water-soaked CS symptoms appeared within 24 hours, and as the incubation period progressed, the lesion size increased, following a linear pattern over time (Villegas-Fernández et al., 2012 ). Brauna-Morževska et al. ( 2023 ) also observed the first visible symptom within 24 h, but Taffa et al. ( 2013 ) noticed the first CS symptoms two days after inoculation with B. fabae . Other workers have detected the first CS disease symptom within six to eight hours of inoculation (Bouhassan et al., 2004 ). All the 21 Botrytis species/isolates used in this study caused CS symptoms. B. fabae isolates 19B053-4 and 17B4 were the quickest to cause symptoms and produced the largest lesions. These findings are in line with many previous studies showing that B. fabae is the main pathogen underlying CS (e.g., Bankina et al., 2021 ; Brauna-Morževska et al., 2023 ). The B. cinerea isolates were generally less virulent than the B. fabae isolates, with the exception of B. fabae 17B28, which was a very weak pathogen. This result aligns with the findings of Rhaiem ( 2020 ), who reported that B. fabae was generally more virulent than B. cinerea isolates when tested on both whole plants and detached leaves. In a detached leaflet assay of faba bean, B. fabae induced more progressive and larger necrotic lesions than B. cinerea (Omar et al., 1986 ), consistent with our findings. Brauna-Morževska et al. ( 2023 ) found that B. cinerea isolates were widely variable in pathogenicity across a range of legumes, and often appeared to be secondary pathogens or saprophytes on already infected tissues. B. pseudocinerea and B. fabiopsis have not been extensively studied to date. B. fabiopsis isolates 17B24 and 19B175 developed aggressive lesions on Kontu and Mélodie/2 in our study, whereas B. pseudocinerea 18B11 and 18B6 were more aggressive on Kontu. Our results confirm that these organisms are part of the complex of Botrytis species causing CS (Zhang et al., 2010 ; Brauna-Morževska et al., 2023 ). The individual isolates of B. medusae and B. euroamericana produced smaller lesions than the other species, suggesting that they may not be major contributors to the disease, in agreement with Brauna-Morževska et al. ( 2023 ). There was wide variation in pathogenicity within each species, with B. fabae 17B28, B. cinerea 17B10-1 and B. fabiopsis 19B024 all producing very small lesions. While previous comparisons have also found wide variation, these results are not well correlated with those of Brauna-Morževska et al. ( 2023 ), who found B. fabae 17B28 to be their most aggressive isolate on faba bean, although B. cinerea 17B10-1 was similarly weakly pathogenic in their study. Pathogens often lose virulence during culturing, and it may be that B. fabae 17B28 had done so, leaving the previously second most virulent isolate, 19B053-4, as the leader in the current experiment. NEP1-like proteins (NLPs) are phytotoxic agents that cause cell damage and death, and elicit defence responses in most dicotyledonous plants (Staats et al., 2007 ; Arenas et al., 2010 ). NLPs were first found in Fusarium oxysporum f. sp. erythroxyli . The oomycetes as well as phylogenetically a wide variety of microorganisms including true fungi, gram-positive and gram-negative bacteria, carry NLP genes. A differential expression pattern may be seen through the infection process by NLP-encoding genes (Arenas et al., 2010 ). Based on the NEP protein gene-based DNA markers we used in this study, B. cinerea , B. fabae and some B. pseudocinerea isolates showed genetic similarities. All B. fabiopsis isolates except the most virulent (19B175) clustered together. B. euroamericana and B. medusae also showed genetic similarities (Fig. 2 ). These findings are similar to those of Brauna-Morževska et al. ( 2023 ), who used RPB2, HSP60 and G3PDH gene sequences. Although B. cinerea and B. pseudocinerea are morphologically very similar, they are phylogenetically more distant from each other than B. cinerea is from B. fabae , and B. fabiopsis is very distant from B. fabae (Hyde et al., 2014 ). Our results are largely consistent with previous reports, with a few exceptions. These deviations might be due to the different DNA marker system we employed or growing conditions, mutation within long-lived somatic lineages, intense recombination and large population size of the Botrytis species (Fillinger and Elad, 2016 ). Detached leaflet assays have been widely used to study the response of faba bean germplasm to B. fabae (e.g., Hutson and Mansfield, 1980 ; Bouhassan et al., 2004 ; Villegas-Fernández et al., 2012 ; Brauna-Morževska et al., 2023 ). Although these assays allow screening of germplasm under uniform growing conditions, they may have negative effects, with senescence triggered by leaflet detachment (Liu et al., 2007 ). Detached leaflet assays have shown moderate and positive correlations with whole-plant field phenotyping (Bouhassan et al., 2004 ; Feyissa, 2022 ). Our study, which evaluated the response of three known faba bean inbred lines to B. fabae under both greenhouse and field conditions (Gela et al., 2022 ; Khazaei et al., 2018; Feyissa, 2022 ), demonstrated the reliability of our developed detached leaflet protocol. Conclusions The detached leaflet assay provided a rapid and effective method for comparing the pathogenicity of the 21 Botrytis isolates. We employed faba bean inbred lines with known response to B. fabae as checks to validate the assay. The data showed interactions between inbred lines and Botrytis isolates, with the CS-susceptible lines displaying varying responses to different species. The detached leaflet assay is space- and resource-efficient, making it a practical tool for high-throughput, preliminary screening for CS disease. Here, it has allowed identification of pathogenicity extremes for use in further experiments. In the future, it could allow rapid screening in genetic mapping analysis, leading to the identification of genome regions associated with disease resistance that can be followed up with more precise, hypothesis-driven experiments. We are now adapting this detached leaflet assay for screening a large faba bean genetic population for genetic studies. Declarations Acknowledgment We thank Prof. Biruta Bankina, Latvia University of Life Sciences and Technologies, Jelgava, Latvia, for the kind donation of the 21 isolates. We thank Markku Tykkyläinen and Eero Kuisma, technical assistants of the glasshouse of the University of Helsinki for their kind assistance during the experiments. We acknowledge Marjo Kilpinen, Eija Takala, Katrin Artola, Dr. Teng Zhang, and Dr. Sylvain Poque for their assistance during the lab work and statistical analysis. Authorship contribution Maniruzzaman : Investigation, Formal analysis, Writing – original draft. Hamid Khazaei : Writing – review & editing, Formal analysis, Resources. Minna Haapalainen : Supervision, Writing - review & editing. Frederick L. Stoddard : Supervision, Writing – review & editing, Investigation, Conceptualization, Funding acquisition, Resources. Funding The work was part of the project “Fabanova – Climate-ready faba beans for the Nordic and Baltic region” funded under the NordForsk call “Sustainable agriculture and climate change”. H.K. was supported by the Research Council of Finland, Academy projects, funding decision 363375 (Fabagen). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. 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C., Hallett, S. G., and Watson, A. K. (2000). Characterization of sporulation of Alternaria alternata f. sp. sphenocleae . Biocontrol Science and Technology , 10 : 385-398. Nei, M. (1978). Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics , 89: 583-590. Omar, S. A. M., Bailiss, K. W., and Chapman, G. P. (1986). Virus‐induced changes in the response of faba bean to infection by Botrytis . Plant Pathology , 35 : 86-92. Plesken, C., Weber, R. W., Rupp, S., Leroch, M., and Hahn, M. (2015). Botrytis pseudocinerea is a significant pathogen of several crop plants but susceptible to displacement by fungicide-resistant B. cinerea strains. Applied and Environmental Microbiology , 81 : 7048-7056. Ran, Y., Yu, M., Hou, L., and Liu, Y. (2024). First Report of chocolate spot caused by Botrytis eucalypti on faba bean in China. Plant Disease, 108: 1884. Rha, E. Y., Kim, J. M., and Yoo, G. (2015). Volume measurement of various tissues using the image J software. Journal of Craniofacial Surgery , 26 : 505-506. Rhaiem, A. (2020). Pathogenicity of Botrytis sp. isolates on Vicia faba based on two different methods. Journal of New Sciences , 76: 4452-4460. Rubiales, D., and Khazaei, H. (2022). Advances in disease and pest resistance in faba bean. Theoretical and Applied Genetics, 135: 3735-3756. Siewers, V., Viaud, M., Jimenez-Teja, D., Collado, I.G., Gronover, C. S., Pradier, J. M., Tudzynsk, B., and Tudzynski, P. (2005). Functional analysis of the cytochrome P450 monooxygenase gene bcbot1 of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor. Molecular Plant-Microbe Interactions , 18 : 602-612. Staats, M., van Baarlen, P., Schouten, A., van Kan, J. A., and Bakker, F. T. (2007). Positive selection in phytotoxic protein-encoding genes of Botrytis species. Fungal Genetics and Biology , 44 : 52-63. Staats, M., van Baarlen, P. and van Kan, J.A. (2005). Molecular phylogeny of the plant pathogenic genus Botrytis and the evolution of host specificity. Molecular biology and Evolution , 22 : 333-346. Stoddard, F., Nicholas, A., Rubiales, D., Thomas, J., and Villegas-Fernandez, A. M. (2010). Integrated pest management in faba bean. Field Crops Research , 115: 308-318. Taffa, E. T., Gurmessa, C. F., and Mariam, S. S. W. (2013). In vivo assay for antagonistic potential of fungal isolates against faba bean ( Vicia faba L.) chocolate spot ( Botrytis fabae Sard.). Jordan Journal of Biological Sciences , 6 : 183-189. Terefe, H., Fininsa, C., Sahile, S., and Tesfaye, K. (2015). Effect of temperature on growth and sporulation of Botrytis fabae , and resistance reactions of faba bean against the pathogen. Journal of Plant Pathology and Microbiology, 6 : 285. Villegas-Fernández, A. M., Sillero, J. C., and Rubiales, D. (2012). Screening faba bean for chocolate spot resistance: evaluation methods and effects of age of host tissue and temperature. European Journal of Plant Pathology , 132 : 443-453. Villegas-Fernández, A. M., Sillero, J. C., Emeran, A. A., Winkler, J., Raffiot, B., Tay, J., Flores, F., and Rubiales, D. (2009). Identification and multi-environment validation of resistance to Botrytis fabae in Vicia faba . Field Crops Research, 114: 84-90 Zhang, J., Wu, M. D., Li, G. Q., Yang, L., Yu, L., Jiang, D. H., Huang, H. C., and Zhuang, W. Y. (2010). Botrytis fabiopsis , a new species causing chocolate spot of broad bean in central China. Mycologia , 102 : 1114-1126. Additional Declarations The authors declare no competing interests. Supplementary Files FigureS1andTableS1.docx Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6784656","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":464172041,"identity":"65c74e85-abd4-4afe-bb85-a03dfc4cdc17","order_by":0,"name":"Maniruzzaman","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4999-5200","institution":"University of Helsinki","correspondingAuthor":true,"prefix":"","firstName":"","middleName":"","lastName":"Maniruzzaman","suffix":""},{"id":464172531,"identity":"153ec86e-c618-4ff8-9403-9d7517bc135d","order_by":1,"name":"Hamid Khazaei","email":"","orcid":"https://orcid.org/0000-0002-5202-8764","institution":"Luke","correspondingAuthor":false,"prefix":"","firstName":"Hamid","middleName":"","lastName":"Khazaei","suffix":""},{"id":464172734,"identity":"e2526ee7-50cf-4268-b48d-115642842d99","order_by":2,"name":"Minna Haapalainen","email":"","orcid":"","institution":"Luke","correspondingAuthor":false,"prefix":"","firstName":"Minna","middleName":"","lastName":"Haapalainen","suffix":""},{"id":464172735,"identity":"21ce53c5-7263-4763-9eb7-596901d4eddc","order_by":3,"name":"Frederick L. Stoddard","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Frederick","middleName":"L.","lastName":"Stoddard","suffix":""}],"badges":[],"createdAt":"2025-05-30 12:20:44","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6784656/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6784656/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83745051,"identity":"087ca380-6898-4b29-b478-00f8c84f08f7","added_by":"auto","created_at":"2025-06-02 03:47:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34586,"visible":true,"origin":"","legend":"\u003cp\u003eMean lesion size of 21 isolates of \u003cem\u003eBotrytis \u003c/em\u003espp. on faba bean inbred lines Kontu, Mélodie/2, and ILB 938/2. Values are back-transformed from the analysed natural logarithms, ± RMSE standard error.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6784656/v1/303bde4c41f44a7c74a633a8.png"},{"id":83744964,"identity":"d8b183a9-66df-4f3f-84ca-5e11659abf68","added_by":"auto","created_at":"2025-06-02 03:39:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54224,"visible":true,"origin":"","legend":"\u003cp\u003eUnrooted dendrogram of the 21 \u003cem\u003eBotrytis\u003c/em\u003e spp. isolates analyzed by 10 NEP DNA markers.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6784656/v1/aacf42adebc00ff3b3fa4108.png"},{"id":83745355,"identity":"faa2ca25-98b4-458c-b41d-ecb6e0a23119","added_by":"auto","created_at":"2025-06-02 03:55:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":844727,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6784656/v1/bd82fd71-acec-4d67-9d5c-8f80fe3bc6ed.pdf"},{"id":83744963,"identity":"33bc7cdc-8693-4c95-9bfb-8f70d97c9666","added_by":"auto","created_at":"2025-06-02 03:39:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1068105,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1andTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6784656/v1/38ad1a80c082f28ac1d3d905.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eA detached leaflet assay to determine the pathogenicity of diverse \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBotrytis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003espp. causing chocolate spot disease in faba bean (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVicia faba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e L.)\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFaba bean (\u003cem\u003eVicia faba\u003c/em\u003e L.) is a cool-season grain legume crop with great potential to contribute to sustainable agriculture and protein security globally. Its nitrogen fixation ability is considered the strongest among cool-season grain legumes, which leads the crop to produce protein-rich seeds (Klippenstein et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The crop is an attractive solution for providing sustainable plant-based protein for food and feed, while avoiding the growing global demand for chemical nitrogen fertilizer. Nevertheless, faba bean is considered to be very sensitive to chocolate spot (CS) disease, which is one of the most devastating diseases affecting faba bean globally (Rubiales and Khazaei, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), as it can cause yield losses of up to 90% in susceptible cultivars (Gorfu and Yaynu, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Stoddard et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Beyene et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Barilli et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCS disease is caused by several necrotrophic fungi in the genus \u003cem\u003eBotrytis\u003c/em\u003e. It is a polycyclic disease, with conidia serving as the primary source of inoculum, typically produced on residues from previous crops (Fan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These conidia are dispersed by wind and deposited on faba bean leaves, stems, flowers, and pods. Under favorable environmental conditions (~\u0026thinsp;20\u0026deg;C, 98% RH) the conidia germinate, initiating infection and enabling rapid disease spread across the field. Infection is facilitated by the production of phytotoxins and cell wall-degrading enzymes such as polygalacturonases (Leisen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Symptoms of CS disease include oblong to elliptical lesions ranging in color from reddish to chocolate brown, often with a darker margin and concentric ring patterns (El-Metwally et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These symptoms typically appear on the upper surface of leaves, increasing in number and size, and may coalesce. In severe cases, extensive blackening occurs, leading to the death of the entire plant.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBotrytis fabae\u003c/em\u003e Sard. is the primary pathogen responsible for CS in faba bean. \u003cem\u003eB. cinerea\u003c/em\u003e Pers. has also been reported to cause CS disease, either independently or as part of a pathogen complex (e.g., Harrison, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Bilkiss et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). While \u003cem\u003eB. fabae\u003c/em\u003e is host-specific, infecting only \u003cem\u003eVicia\u003c/em\u003e species, \u003cem\u003eB. cinerea\u003c/em\u003e has a broad host range. Zhang et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) reported a third species, \u003cem\u003eB. fabiopsis\u003c/em\u003e, from faba bean in Hubei Province, China, which produces symptoms similar to those caused by \u003cem\u003eB. fabae\u003c/em\u003e and \u003cem\u003eB. cinerea\u003c/em\u003e. \u003cem\u003eB. fabiopsis\u003c/em\u003e may co-occur with \u003cem\u003eB. fabae\u003c/em\u003e and \u003cem\u003eB. cinerea\u003c/em\u003e, potentially contributing to complex infection dynamics (Teshome and Tagegn, 2013). The fourth species, \u003cem\u003eB. pseudocinerea\u003c/em\u003e, closely related to \u003cem\u003eB. cinerea\u003c/em\u003e (Plesken et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), has also been reported to infect faba bean (Bankina et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Two further \u003cem\u003eBotrytis\u003c/em\u003e species infect various legumes; \u003cem\u003eB. euroamericana\u003c/em\u003e caused CS-like symptoms on faba bean, chickpea, field pea, lentil and soybean, while \u003cem\u003eB. medusae\u003c/em\u003e infected narrow-leafed lupin, field pea and soybean (Brauna-Morževska et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Recentl\u003cem\u003ey, B. eucalypti\u003c/em\u003e has been reported to cause CS in faba bean in Qinghai Province, China (Ran et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOnly a few sources of moderate genetic resistance to CS disease have been identified in faba bean (e.g., Villegas-Fern\u0026aacute;ndez et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Maalouf et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and resistant germplasm remains scarce. While screening large faba bean germplasm collections and genetic mapping populations for CS pathology and genetic studies are essential, the screening process is labor-intensive and typically requires large space in greenhouses or semi-controlled field environments. Indoor or outdoor screening of large germplasm collections for CS is often unreliable due to uneven pathogen infection. Detached leaves are often used for large-scale screening, and a detached-leaflet assay offers an even more efficient alternative, requiring less space and resources (Isenegger et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This method enables rapid and effective comparison of the pathogenicity of a large number of \u003cem\u003eBotrytis\u003c/em\u003e isolates, allowing the selection of candidates for further detailed studies. Hence, we set up an experiment using three faba bean inbred lines with proven response to CS disease: one highly susceptible line (selected from cv Kontu, Gela et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Feyissa, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), one moderately susceptible line (M\u0026eacute;lodie/2; Feyissa, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and one moderately resistant line (ILB 938/2, Khazaei et al., 2018), to evaluate the virulence and pathogenicity of a set of 21 \u003cem\u003eBotrytis\u003c/em\u003e isolates. This study further dissects the molecular diversity of \u003cem\u003eBotrytis\u003c/em\u003e species by analyzing necrosis- and ethylene-inducing protein (NEP) gene markers, which play a key role in inducing necrosis and cell death during host infection and disease progression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of Botrytis isolates\u003c/h2\u003e \u003cp\u003eA set of 21 isolates spanning six \u003cem\u003eBotrytis\u003c/em\u003e species was obtained from Prof. Biruta Bankina at the Latvian University of Life Sciences and Technologies, Jelgava, Latvia. The isolates were cultured on half-strength PDA and maintained in darkness at room temperature (20\u0026deg;C) for 7 days, then put under near-ultraviolet light for 7 days to induce sporulation. Thereafter, the cultured plates were washed with sterile water and scraped with a glass rod to dislodge the spores. The suspension was filtered through two layers of cheesecloth into a 250 ml conical flask, then 5 ml of Tween 20 (0.03% v/v) solution was added to keep the spores in suspension (Terefe et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The concentration of spores was determined using a haemocytometer and adjusted to 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e spores per milliliter.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlant material and growing conditions\u003c/h3\u003e\n\u003cp\u003eThree faba bean inbred lines were chosen for this work. ILB 938/2 exhibits moderate resistance and M\u0026eacute;lodie/2 is moderately susceptible to CS disease. These two inbred lines serve as the parental lines of a well-characterized recombinant inbred line population used to study CS disease response (Gela et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The Finnish cultivar Kontu is highly susceptible to CS disease (Feyissa, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeeds were surface sterilized with 70% ethanol for 3 min and rinsed three times with sterile distilled water. Sets of three seeds of each inbred line were planted in 2.5 L plastic pots filled with autoclaved peat soil (Kekkil\u0026auml; Coarse potting mix WR8494, Kekkil\u0026auml;-BVB OY, Vantaa, Finland). Plants were grown for 6\u0026ndash;8 weeks in a controlled-environment growth chamber (Weiss-2000, Weiss Technik GmbH, Reiskirchen-Lindsruth, Germany). The temperature was set to 22\u0026deg;C during the day and 20\u0026deg;C at night, with a 12-hour photoperiod under visible light (150 \u0026micro;mol/m\u0026sup2;/s photosynthetic photon flux density, PPFD).\u003c/p\u003e\n\u003ch3\u003eInoculation\u003c/h3\u003e\n\u003cp\u003eFully expanded leaves of six- to eight-week-old plants of the three inbred lines were collected from four to eight nodes two hours prior to inoculation and kept in sterile moist napkin paper. Leaves were washed with sterile distilled water, blotted dry, and placed in pairs on two layers of sterile filter paper in 90 mm petri dishes, which were arranged in a completely randomized factorial design with three replicates (five leaflets in each replicate per inbred line). Five ml of sterile water was applied in each petri dish to moisten the filter paper and to maintain high humidity within the dish. A piece of cotton moistened with sterile water was placed onto the petiole end to maintain the turgor pressure and prevent dryness. An experimental unit comprised three petri dishes containing 5 leaflets, inoculated with 20 \u0026micro;l suspension culture and a control was treated with sterile water in each replication for each accession. The petri dishes were organized in a plastic box (37.5 \u0026times; 25.5 \u0026times; 9.5 cm) and incubated in a growth chamber at 22\u0026deg;C and 12 h photoperiod with 170\u0026ndash;188 \u0026micro;mol/m\u0026sup2;/s PPFD. The fungal infection and necrosis were monitored every day to observe the first visible symptom, and after 5 days, leaflets were photographed against a measuring scale for image analysis.\u003c/p\u003e\n\u003ch3\u003eMolecular characterization of Botrytis isolates using NEP protein gene markers\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eBotrytis\u003c/em\u003e isolates were genotyped using specific primer pairs for necrosis and ethylene-inducing proteins (NEP) (\u003cb\u003eTable S1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBotrytis\u003c/em\u003e isolates were cultured on PDA containing a GVS Cellophane film (Diameter 76 mm, 0.1 \u0026micro;m; GVS, Bologna, Italy) on the medium and incubated in darkness at room temperature (20\u0026deg;C) for 14 days to produce enough mycelium. Mycelium was harvested using a sterile scalpel and put into a 1.5 ml Eppendorf tube; the lids of the tubes were opened for a few hours to allow the water from the mycelium to dry at room temperature. Subsequently, the air-dried mycelium was frozen into liquid nitrogen and grounded into powder using a plant DNA isolation kit (DNeasy\u0026reg; Plant Mini Kit, Qiagen). The extracted DNA was dissolved in 50 \u0026micro;l of TE buffer, quantified using a Nanodrop spectrophotometer (Thermo Scientific), and stored at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003ePCR amplification was carried out in a 20 \u0026micro;l reaction solution, having 4 \u0026micro;l template (6 ng/\u0026micro;l) of fungal DNA, 10 \u0026micro;l of DreamTaq Green PCR Master Mix (2X, contains Dream Taq DNA polymerase, DreamTaq Green buffer, MgCl\u003csub\u003e2\u003c/sub\u003e and dNTPs, supplemented with two tracking dyes and a density reagent that allows for direct loading of the PCR products on a gel; ThermoFisher Scientific, Waltham, MA, USA), 0.8 \u0026micro;l of each primer, and 4.4 \u0026micro;l of sterile water. PCR reactions were conducted in a thermocycler (Eppendorf, Germany), using the amplification program: 1 cycle of 2 min at 94\u0026deg;C, 35 cycles of 30 sec at 94\u0026deg;C, and 30 sec at 50\u0026deg;C or 58\u0026deg;C for annealing temperature, 1 min at 72\u0026deg;C; and 10 min at 72\u0026deg;C for final extension. A PCR mixture without fungal DNA template was used as a negative control. The PCR products were separated and analyzed by electrophoresis using a 1% agarose gel and 1x TBE buffer, staining with 0.5 \u0026micro;g/ml ethidium bromide and visualized in a Molecular Imager, Gel Doc (Bio-Rad Laboratories, Hercules, CA, USA) for the presence or absence of the expected size of band.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe infected area of the leaves was determined using ImageJ software. The scale was fixed at 1 cm and the estimated distance was 50 pixels (Rha et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The mean infected area of the 5 infected leaves in an experimental unit was calculated. The data were transformed to log (area\u0026thinsp;+\u0026thinsp;1) to normalize the variance, and used in the analysis of variance (ANOVA) to determine the effects of isolate, inbred line and their interaction. Means were separated using Tukey-HSD multiple pairwise comparisons using the JMP Pro SAS statistical computing package (Grayson et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Pairwise genetic distances were estimated using Nei\u0026rsquo;s coefficient (Nei, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1978\u003c/span\u003e), and a dendrogram was constructed using the unweighted pair-group method with arithmetic mean (UPGMA) algorithm on Darwin v. 6.0.021.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAll 21 isolates caused CS symptoms. On average, Kontu showed the most susceptible reaction, ILB 938/2 the most resistant, and M\u0026eacute;lodie/2 an intermediate reaction (\u003cb\u003eFigure S1\u003c/b\u003e). The first characteristic water-soaked symptoms of CS disease were found using \u003cem\u003eB. fabae\u003c/em\u003e 19B053-4 on Kontu after 24 h, on M\u0026eacute;lodie/2 after 36 h and on ILB 938/2 after 72 h. In contrast, symptoms induced by \u003cem\u003eB. fabae\u003c/em\u003e 17B4 were observed after 36 h on M\u0026eacute;lodie/2 and Kontu and after 96 h on ILB 938/2. By 120 h after inoculation, symptoms were obvious and consistent blackish lesions on most combinations of isolate by inbred line (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The ANOVA results showed significant variation among the \u003cem\u003eBotrytis\u003c/em\u003e isolates, the faba bean inbred lines, and the isolate \u0026times; inbred line interactions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\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\u003eTime (h) to first visible symptoms induced by the \u003cem\u003eBotrytis\u003c/em\u003e species on three inbred lines of faba bean. Sterile distilled water was used as a control.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBotrytis\u003c/em\u003e species (isolate)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKontu\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u0026eacute;lodie/2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eILB 938/2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eHours to first symptom\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabae\u003c/em\u003e (19B053-4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabae\u003c/em\u003e (17B4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabae\u003c/em\u003e (17B28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabiopsis\u003c/em\u003e (19B175)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabiopsis\u003c/em\u003e (17B24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabiopsis\u003c/em\u003e (17F9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabiopsis\u003c/em\u003e (18B16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabiopsis\u003c/em\u003e (17F4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. fabiopsis\u003c/em\u003e (19B024)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e (19B048)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e (19B026)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e (17B10-1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e (B05.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e (19B009)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e (19B009)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e (18B3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. pseudocinerea\u003c/em\u003e (18B11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. pseudocinerea\u003c/em\u003e (18B2-3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. pseudocinerea\u003c/em\u003e (18B6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. euroamerican\u003c/em\u003ea (19B053-3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. medusae\u003c/em\u003e (19B047)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72\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 \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance showing the effects of fungal isolate, host inbred line, and isolate\u0026times;inbred line on the areas of chocolate spot disease lesions.\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSum of Squares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF Ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProb\u0026thinsp;\u0026gt;\u0026thinsp;F\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e144.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInbred line\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e152.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e259.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate \u0026times; Inbred line\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eError\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003edf, degrees of freedom.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eB. fabae\u003c/em\u003e 19B053-4 was the most virulent isolate on two of the lines, developing the largest lesions on Kontu (10.10 cm\u003csup\u003e2\u003c/sup\u003e necrosis) and M\u0026eacute;lodie/2 (9.30 cm\u003csup\u003e2\u003c/sup\u003e necrosis), and the second-largest on ILB 938/2 (1.92 cm\u003csup\u003e2\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eB. fabiopsis\u003c/em\u003e 19B175 was the most virulent on ILB938/2 (2.16 cm\u003csup\u003e2\u003c/sup\u003e) and the second on both Kontu (7.70 cm\u003csup\u003e2\u003c/sup\u003e) and M\u0026eacute;lodie/2 (5.87 cm\u003csup\u003e2\u003c/sup\u003e). Isolate 19B048 was the most virulent from \u003cem\u003eB. cinerea\u003c/em\u003e and the fifth most virulent overall (5.73 cm\u003csup\u003e2\u003c/sup\u003e on Kontu, 2.42 cm\u003csup\u003e2\u003c/sup\u003e on M\u0026eacute;lodie/2 and 0.53 cm\u003csup\u003e2\u003c/sup\u003e on ILB 938/2), and isolate 18B11 was the most virulent from \u003cem\u003eB. pseudocinerea\u003c/em\u003e (4.40 cm\u003csup\u003e2\u003c/sup\u003e on Kontu, 0.84 cm\u003csup\u003e2\u003c/sup\u003e on M\u0026eacute;lodie/2 and 0.15 cm\u003csup\u003e2\u003c/sup\u003e on ILB 938/2). The smallest lesions were induced by \u003cem\u003eB. cinerea\u003c/em\u003e 17B10-1 on Kontu (0.43 cm\u003csup\u003e2\u003c/sup\u003e), M\u0026eacute;lodie/2 (0.35 cm\u003csup\u003e2\u003c/sup\u003e), and ILB 938/2 (0.08 cm\u003csup\u003e2\u003c/sup\u003e), followed by \u003cem\u003eB. fabiopsis\u003c/em\u003e 19B024 and \u003cem\u003eB. fabae\u003c/em\u003e 17B28.\u003c/p\u003e\u003cp\u003eIn every case except \u003cem\u003eB. fabae\u003c/em\u003e 17B4, the lesion on Kontu was larger than that on M\u0026eacute;lodie/2. Similarly, in every case except \u003cem\u003eB. fabiopsis\u003c/em\u003e 19B024 and \u003cem\u003eB. euroamericana\u003c/em\u003e 19B053-3, the lesion on M\u0026eacute;lodie/2 was larger than that on ILB 938/2. Nevertheless, in these cases the apparent crossover was much smaller than that required for statistical significance, and the overall significance of the isolate \u0026times; inbred line term in the Analysis of Variance is attributable to the lack of discrimination between inbred lines by some isolates while others clearly affected one more than another.\u003c/p\u003e \u003cp\u003eThe NEP DNA marker analysis exhibited two main groups and four sub-clusters, with a few variations inside each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All \u003cem\u003eB. cinerea\u003c/em\u003e clustered together with \u003cem\u003eB. fabae. B. medusae\u003c/em\u003e and \u003cem\u003eB. euroamericana\u003c/em\u003e isolates were grouped together. \u003cem\u003eB. fabiopsis\u003c/em\u003e isolates and \u003cem\u003eB. pseudocinerea\u003c/em\u003e mostly clustered together with some minor deviations (\u003cem\u003eB. fabiopsis\u003c/em\u003e isolate 19B175 and \u003cem\u003eB. pseudocinerea\u003c/em\u003e isolate 18B6).\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eThe \u003cem\u003eBotrytis\u003c/em\u003e spp. used in this study showed varying reactions on detached leaves of ILB 938/2, Kontu, and M\u0026eacute;lodie/2 in an \u003cem\u003ein vitro\u003c/em\u003e assay using spore suspension cultures. Our results revealed a significant difference among the isolates, inbred lines, and isolate \u0026times; inbred line interactions. ILB 938/2 showed moderate resistance while Kontu performed completely susceptible and M\u0026eacute;lodie/2 was moderately susceptible. These findings agreed with previous assessments of the quantitative CS resistance of these genotypes (Khazaei et al., 2018; Gela et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Feyissa, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, the first visible water-soaked CS symptoms appeared within 24 hours, and as the incubation period progressed, the lesion size increased, following a linear pattern over time (Villegas-Fern\u0026aacute;ndez et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Brauna-Morževska et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also observed the first visible symptom within 24 h, but Taffa et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) noticed the first CS symptoms two days after inoculation with \u003cem\u003eB. fabae\u003c/em\u003e. Other workers have detected the first CS disease symptom within six to eight hours of inoculation (Bouhassan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll the 21 \u003cem\u003eBotrytis\u003c/em\u003e species/isolates used in this study caused CS symptoms. \u003cem\u003eB. fabae\u003c/em\u003e isolates 19B053-4 and 17B4 were the quickest to cause symptoms and produced the largest lesions. These findings are in line with many previous studies showing that \u003cem\u003eB. fabae\u003c/em\u003e is the main pathogen underlying CS (e.g., Bankina et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Brauna-Morževska et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The \u003cem\u003eB. cinerea\u003c/em\u003e isolates were generally less virulent than the \u003cem\u003eB. fabae\u003c/em\u003e isolates, with the exception of \u003cem\u003eB. fabae\u003c/em\u003e 17B28, which was a very weak pathogen. This result aligns with the findings of Rhaiem (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who reported that \u003cem\u003eB. fabae\u003c/em\u003e was generally more virulent than \u003cem\u003eB. cinerea\u003c/em\u003e isolates when tested on both whole plants and detached leaves. In a detached leaflet assay of faba bean, \u003cem\u003eB. fabae\u003c/em\u003e induced more progressive and larger necrotic lesions than \u003cem\u003eB. cinerea\u003c/em\u003e (Omar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e), consistent with our findings. Brauna-Morževska et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that \u003cem\u003eB. cinerea\u003c/em\u003e isolates were widely variable in pathogenicity across a range of legumes, and often appeared to be secondary pathogens or saprophytes on already infected tissues.\u003c/p\u003e \u003cp\u003e \u003cem\u003eB. pseudocinerea\u003c/em\u003e and \u003cem\u003eB. fabiopsis\u003c/em\u003e have not been extensively studied to date. \u003cem\u003eB. fabiopsis\u003c/em\u003e isolates 17B24 and 19B175 developed aggressive lesions on Kontu and M\u0026eacute;lodie/2 in our study, whereas \u003cem\u003eB. pseudocinerea\u003c/em\u003e 18B11 and 18B6 were more aggressive on Kontu. Our results confirm that these organisms are part of the complex of \u003cem\u003eBotrytis\u003c/em\u003e species causing CS (Zhang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Brauna-Morževska et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The individual isolates of \u003cem\u003eB. medusae\u003c/em\u003e and \u003cem\u003eB. euroamericana\u003c/em\u003e produced smaller lesions than the other species, suggesting that they may not be major contributors to the disease, in agreement with Brauna-Morževska et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere was wide variation in pathogenicity within each species, with \u003cem\u003eB. fabae\u003c/em\u003e 17B28, \u003cem\u003eB. cinerea\u003c/em\u003e 17B10-1 and \u003cem\u003eB. fabiopsis\u003c/em\u003e 19B024 all producing very small lesions. While previous comparisons have also found wide variation, these results are not well correlated with those of Brauna-Morževska et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who found \u003cem\u003eB. fabae\u003c/em\u003e 17B28 to be their most aggressive isolate on faba bean, although \u003cem\u003eB. cinerea\u003c/em\u003e 17B10-1 was similarly weakly pathogenic in their study. Pathogens often lose virulence during culturing, and it may be that \u003cem\u003eB. fabae\u003c/em\u003e 17B28 had done so, leaving the previously second most virulent isolate, 19B053-4, as the leader in the current experiment.\u003c/p\u003e \u003cp\u003eNEP1-like proteins (NLPs) are phytotoxic agents that cause cell damage and death, and elicit defence responses in most dicotyledonous plants (Staats et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Arenas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). NLPs were first found in \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003eerythroxyli\u003c/em\u003e. The oomycetes as well as phylogenetically a wide variety of microorganisms including true fungi, gram-positive and gram-negative bacteria, carry NLP genes. A differential expression pattern may be seen through the infection process by NLP-encoding genes (Arenas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Based on the NEP protein gene-based DNA markers we used in this study, \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eB. fabae\u003c/em\u003e and some \u003cem\u003eB. pseudocinerea\u003c/em\u003e isolates showed genetic similarities. All \u003cem\u003eB. fabiopsis\u003c/em\u003e isolates except the most virulent (19B175) clustered together. \u003cem\u003eB. euroamericana\u003c/em\u003e and \u003cem\u003eB. medusae\u003c/em\u003e also showed genetic similarities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings are similar to those of Brauna-Morževska et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who used RPB2, HSP60 and G3PDH gene sequences. Although \u003cem\u003eB. cinerea\u003c/em\u003e and \u003cem\u003eB. pseudocinerea\u003c/em\u003e are morphologically very similar, they are phylogenetically more distant from each other than \u003cem\u003eB. cinerea\u003c/em\u003e is from \u003cem\u003eB. fabae\u003c/em\u003e, and \u003cem\u003eB. fabiopsis\u003c/em\u003e is very distant from \u003cem\u003eB. fabae\u003c/em\u003e (Hyde et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our results are largely consistent with previous reports, with a few exceptions. These deviations might be due to the different DNA marker system we employed or growing conditions, mutation within long-lived somatic lineages, intense recombination and large population size of the \u003cem\u003eBotrytis\u003c/em\u003e species (Fillinger and Elad, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDetached leaflet assays have been widely used to study the response of faba bean germplasm to \u003cem\u003eB. fabae\u003c/em\u003e (e.g., Hutson and Mansfield, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Bouhassan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Villegas-Fern\u0026aacute;ndez et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Brauna-Morževska et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although these assays allow screening of germplasm under uniform growing conditions, they may have negative effects, with senescence triggered by leaflet detachment (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Detached leaflet assays have shown moderate and positive correlations with whole-plant field phenotyping (Bouhassan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Feyissa, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our study, which evaluated the response of three known faba bean inbred lines to \u003cem\u003eB. fabae\u003c/em\u003e under both greenhouse and field conditions (Gela et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khazaei et al., 2018; Feyissa, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), demonstrated the reliability of our developed detached leaflet protocol.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe detached leaflet assay provided a rapid and effective method for comparing the pathogenicity of the 21 \u003cem\u003eBotrytis\u003c/em\u003e isolates. We employed faba bean inbred lines with known response to \u003cem\u003eB. fabae\u003c/em\u003e as checks to validate the assay. The data showed interactions between inbred lines and \u003cem\u003eBotrytis\u003c/em\u003e isolates, with the CS-susceptible lines displaying varying responses to different species. The detached leaflet assay is space- and resource-efficient, making it a practical tool for high-throughput, preliminary screening for CS disease. Here, it has allowed identification of pathogenicity extremes for use in further experiments. In the future, it could allow rapid screening in genetic mapping analysis, leading to the identification of genome regions associated with disease resistance that can be followed up with more precise, hypothesis-driven experiments. We are now adapting this detached leaflet assay for screening a large faba bean genetic population for genetic studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Biruta Bankina, Latvia University of Life Sciences and Technologies, Jelgava, Latvia, for the kind donation of the 21 isolates. We thank Markku Tykkyl\u0026auml;inen and Eero Kuisma, technical assistants of the glasshouse of the University of Helsinki for their kind assistance during the experiments. We acknowledge Marjo Kilpinen, Eija Takala, Katrin Artola, Dr. Teng Zhang, and Dr. Sylvain Poque for their assistance during the lab work and statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManiruzzaman\u003c/strong\u003e: Investigation, Formal analysis, Writing \u0026ndash; original draft. \u003cstrong\u003eHamid Khazaei\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Formal analysis, Resources. \u003cstrong\u003eMinna Haapalainen\u003c/strong\u003e: Supervision, Writing - review \u0026amp; editing. \u003cstrong\u003eFrederick L. Stoddard\u003c/strong\u003e: Supervision, Writing \u0026ndash; review \u0026amp; editing, Investigation, Conceptualization, Funding acquisition, Resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was part of the project \u0026ldquo;Fabanova \u0026ndash; Climate-ready faba beans for the Nordic and Baltic region\u0026rdquo; funded under the NordForsk call \u0026ldquo;Sustainable agriculture and climate change\u0026rdquo;. H.K. was supported by the Research Council of Finland, Academy projects, funding decision 363375 (Fabagen).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArenas, Y. C., Kalkman, E. R., Schouten, A., Dieho, M., Vredenbregt, P., Uwumukiza, B., Ruiz, M. O., and van Kan, J. A. (2010). 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First Report of chocolate spot caused by \u003cem\u003eBotrytis eucalypti\u003c/em\u003e on faba bean in China. \u003cem\u003ePlant Disease, 108:\u003c/em\u003e 1884.\u003c/li\u003e\n\u003cli\u003eRha, E. Y., Kim, J. M., and Yoo, G. (2015). Volume measurement of various tissues using the image J software. \u003cem\u003eJournal of Craniofacial Surgery\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e: 505-506.\u003c/li\u003e\n\u003cli\u003eRhaiem, A. (2020). Pathogenicity of \u003cem\u003eBotrytis \u003c/em\u003esp. isolates on \u003cem\u003eVicia faba\u003c/em\u003e based on two different methods. \u003cem\u003eJournal of New Sciences\u003c/em\u003e, \u003cem\u003e76:\u003c/em\u003e 4452-4460.\u003c/li\u003e\n\u003cli\u003eRubiales, D., and Khazaei, H. (2022). 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Molecular phylogeny of the plant pathogenic genus Botrytis and the evolution of host specificity. \u003cem\u003eMolecular biology and Evolution\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e: 333-346.\u003c/li\u003e\n\u003cli\u003eStoddard, F., Nicholas, A., Rubiales, D., Thomas, J., and Villegas-Fernandez, A. M. (2010). Integrated pest management in faba bean. \u003cem\u003eField Crops Research\u003c/em\u003e, \u003cem\u003e115:\u003c/em\u003e 308-318.\u003c/li\u003e\n\u003cli\u003eTaffa, E. T., Gurmessa, C. F., and Mariam, S. S. W. (2013). \u003cem\u003eIn vivo\u003c/em\u003e assay for antagonistic potential of fungal isolates against faba bean (\u003cem\u003eVicia faba\u003c/em\u003e L.) chocolate spot (\u003cem\u003eBotrytis fabae\u003c/em\u003e Sard.). \u003cem\u003eJordan Journal of Biological Sciences\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e: 183-189.\u003c/li\u003e\n\u003cli\u003eTerefe, H., Fininsa, C., Sahile, S., and Tesfaye, K. (2015). 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Identification and multi-environment validation of resistance to \u003cem\u003eBotrytis fabae\u003c/em\u003e in \u003cem\u003eVicia faba\u003c/em\u003e.\u003cem\u003e Field Crops Research, 114: \u003c/em\u003e84-90\u003c/li\u003e\n\u003cli\u003eZhang, J., Wu, M. D., Li, G. Q., Yang, L., Yu, L., Jiang, D. H., Huang, H. C., and Zhuang, W. Y. (2010). \u003cem\u003eBotrytis fabiopsis\u003c/em\u003e, a new species causing chocolate spot of broad bean in central China. \u003cem\u003eMycologia\u003c/em\u003e, \u003cem\u003e102\u003c/em\u003e: 1114-1126.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Helsinki","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Detached leaflet assay, chocolate spot, disease screening, Botrytis species, moderate resistant, isolate-genotype interaction","lastPublishedDoi":"10.21203/rs.3.rs-6784656/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6784656/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChocolate spot (CS) disease, caused by certain \u003cem\u003eBotrytis\u003c/em\u003e species, is one of the most destructive diseases of faba bean worldwide. This research set out to determine whether different \u003cem\u003eBotrytis\u003c/em\u003e species cause different responses in faba bean genotypes that display varied levels of CS susceptibility. Three faba bean inbred lines \u0026ndash; Kontu, M\u0026eacute;lodie/2, and ILB 938/2 \u0026ndash; identified as highly susceptible, moderately susceptible, and moderately resistant, respectively, were chosen as hosts, and 21 \u003cem\u003eBotrytis\u003c/em\u003e isolates, representing six species (\u003cem\u003eB. fabae\u003c/em\u003e, \u003cem\u003eB. fabiopsis\u003c/em\u003e, \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eB. pseudocinerea\u003c/em\u003e, \u003cem\u003eB. medusae\u003c/em\u003e, and \u003cem\u003eB. euroamericana\u003c/em\u003e), were used as disease agents. A PCR assay using primers targeting the 10 NEP protein genes was used to genotype \u003cem\u003eBotrytis\u003c/em\u003e isolates. The \u003cem\u003eBotrytis\u003c/em\u003e isolates were grown on half-strength PDA medium and treated with near-UV light to induce sporulation. Spore suspensions were inoculated onto detached, fully expanded leaflets. Results revealed a significant difference in virulence reactions among the inbred lines, isolates and their interaction. All the 21 \u003cem\u003eBotrytis\u003c/em\u003e species/isolates tested induced CS symptoms. \u003cem\u003eB. fabae\u003c/em\u003e 19B053-4 was the most virulent, causing the largest lesions, followed by \u003cem\u003eB. fabiopsis\u003c/em\u003e 19B175, \u003cem\u003eB. fabae\u003c/em\u003e 17B4, \u003cem\u003eB. fabiopsis\u003c/em\u003e 17B24, and \u003cem\u003eB. cinerea\u003c/em\u003e 19B048. At the other extreme, lesions developed most slowly with \u003cem\u003eB. cinerea\u003c/em\u003e 17B10-1, \u003cem\u003eB. fabiopsis\u003c/em\u003e 19B024, \u003cem\u003eB. fabae\u003c/em\u003e 17B28, and \u003cem\u003eB. cinerea\u003c/em\u003e 19B014. The DNA analysis revealed two main clusters and four sub-clusters among the \u003cem\u003eBotrytis\u003c/em\u003e species, showing species-level groupings. The detached leaflet assay enabled a rapid and effective comparison of the pathogenicity of the studied \u003cem\u003eBotrytis\u003c/em\u003e isolates.\u003c/p\u003e","manuscriptTitle":"A detached leaflet assay to determine the pathogenicity of diverse Botrytis spp. causing chocolate spot disease in faba bean (Vicia faba L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-02 03:39:32","doi":"10.21203/rs.3.rs-6784656/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5d4a786b-cae1-427d-9655-9b78d5c09c69","owner":[],"postedDate":"June 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T03:39:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-02 03:39:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6784656","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6784656","identity":"rs-6784656","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}