Biological control of root rot and wilt diseases of cucumber using certain bioagents and biocides under greenhouse conditions

1 Journal of PhytoPathology and Disease Management Print ISSN: 3009-6111 Online ISSN: 3009-6170 Volume 12, Issue 1, 2025, Pages 1–7 Article Biological control of root rot and wilt diseases of cucumber using certain bioagents and biocides under greenhouse conditions Hesham K. Ghaleb1 | Mohamed M. El-Sheikh Aly1 | Moshref M. Shamrokh1 1Agricultural Botany Department, Faculty of Agriculture, Al-Azhar University, Assiut, Egypt DOI: 10.5281/zenodo.18012239 ARK: ark:/24629/PPDJ.v12i1.266 Received: 8 April 2025 Accepted: 15 July 2025 Published online: 20 July 2025 Correspondence: Mohamed M. El-Sheikh Aly Agricultural Botany Department, Faculty of Agriculture, Al-Azhar University, Assiut, Egypt. Email: [email protected] Abstract: Ten fungal isolates belonging to Rhizoctonia solani (4 isolates), Fusarium semitectum, F. solani, Macrophomina phaseolina, F. oxysporum f.sp. cucumerinum (2 isolates), and Sclerotium rolfsii were recovered from infected cucumber roots collected from various localities during the fall (2020) and spring (2021) growing seasons. Pathogenicity tests revealed that all isolates were capable of infecting cucumber plants, causing root rot and wilt diseases. Rhizoctonia solani isolate No. 3 was identified as the most destructive, resulting in the highest disease severity. Similarly, F. oxysporum f.sp. cucumerinum isolate No. 8 was the most virulent among the wilt pathogens. Significant variations were observed among the tested cucumber genotypes regarding their susceptibility to preand post-emergence damping-off. In vitro studies demonstrated that Trichoderma asperellum, T. harzianum, T. album, and T34 exhibited varying degrees of antagonistic activity against the pathogenic fungi. Additionally, different bacterial bioagents were evaluated under laboratory and greenhouse conditions; Pseudomonas fluorescens followed by Bacillus megaterium showed the highest efficacy in inhibiting the mycelial growth of R. solani and F. oxysporum f.sp. cucumerinum, whereas Bacillus subtilis exhibited moderate effects, and Paenibacillus polymyxa was the least effective. Furthermore, commercial biocides (Bio-Arc, Plant Guard, and Rizo-N) were evaluated during the fall (2022) and spring (2023) seasons under greenhouse conditions. Plant Guard was the most effective treatment at all tested concentrations in reducing disease severity caused by R. solani and F. oxysporum f.sp. cucumerinum. Moreover, Bio-Arc, followed by Rizo-N at the highest concentration, resulted in a significant reduction in the incidence of root rot and wilt diseases. Keywords: Cucumber, Biological control, Root rot, Rhizoctonia solani, Fusarium oxysporum. Article | Ghaleb et al. 2 | Journal of Plant Pathology and Disease Management | Vol. 12, No. 1 | 1. Introduction Cucumber (Cucumis sativus L.) is considered one of the major summer vegetable crops in commercial fields in Egypt. In recent decades, efforts have been concentrated on cultivating the crop in protected systems (greenhouses) during the autumn and winter seasons. Consequently, the cultivated area of cucumber in Egypt is expanding at a relatively fast rate, particularly in newly reclaimed desert lands. However, cucumber plants are attacked by several fungal diseases during various growth stages, causing considerable yield losses under both field and greenhouse conditions. Among these, soil-borne diseases are particularly destructive. Root rot and wilt, primarily caused by Rhizoctonia solani and Fusarium oxysporum f.sp. cucumerinum, are the most common diseases affecting cucumber plants, leading to damping-off and significant economic damage (Al-Tuwaijri, 2015; Martinez et al., 2003). Sabbagh et al. (2017) emphasized that damping-off and root rot are serious threats to cucumber at both seedling and adult stages under protected cultivation. Furthermore, Aljawasim et al. (2020) reported that several fungal pathogens, including R. solani, F. oxysporum, F. solani, Sclerotium rolfsii, and Macrophomina phaseolina, are responsible for dampingoff and root rot in cucumber and watermelon, causing severe losses in seed germination and plant survival. Although chemical fungicides have provided satisfactory control of these diseases, they are considered major contributors to environmental pollution and pose health risks. Therefore, to avoid the hazards associated with fungicides, alternative control methods have been investigated. Biological control using antagonistic microorganisms has proven to be a successful, effective, and eco-friendly strategy to manage various plant diseases and reduce crop damage (Fasusi et al., 2021; Wang et al., 2018). In recent years, Trichoderma species, beneficial bacteria, and commercial biocides have been extensively used to enhance plant growth and combat diseases (Awad and Fayyadh, 2018; Mahmoud, 2015). For instance, Thabet (2023) evaluated the inhibitory effect of fungal bioagents (T. harzianum, T. asperellum, T. album, and T34) and bacterial isolates against the linear growth of R. solani, F. oxysporum f.sp. cucumerinum, and Verticillium albo-atrum in vitro. Additionally, several commercial biocides have been tested for their efficacy in controlling cucumber root rot and wilt diseases under greenhouse conditions, offering a promising alternative to chemical treatments (Thabet, 2023). The present study aims to isolate and identify the causal pathogens of root rot and wilt diseases in cucumber. Furthermore, the investigation intends to evaluate the efficacy of certain antagonistic bioagents (fungal and bacterial) and selected commercial biocides in controlling these diseases under greenhouse conditions, providing a sustainable approach for disease management. 2. Materials and Methods This study was conducted during the growing seasons of 2021 and 2022 under laboratory and greenhouse conditions at the Department of Agricultural Botany, Faculty of Agriculture, Al-Azhar University (Assiut Branch), Egypt. 2.1 Isolation, Purification, and Identification of Pathogens Samples were collected from the roots of cucumber and watermelon plants exhibiting typical symptoms of root rot and wilt. These samples were obtained from various locations in Al-Buhaira and Menoufia governorates, Egypt during the autumn growing season of 2019. Diseased roots were washed thoroughly with tap water, cut into small pieces (0.3–0.5 cm), surface-sterilized with 70% ethyl alcohol for 2–3 minutes, and dried between sterile filter papers. The pieces were then transferred onto Potato Dextrose Agar (PDA) medium supplemented with streptomycin to prevent bacterial growth and incubated at 25–27°C for 5-7 days. Hyphal tips or single spores were transferred to PDA slants to obtain pure cultures (Cowan et al., 1999). The isolated fungi were identified based on morphological and microscopic characteristics according to taxonomic keys (Barnett and Hunter, 1986; Nelson et al., 1983; Sneh et al., 1991). Identification was confirmed by the Department of Agricultural Botany, Al-Azhar University, Assiut, Egypt. 2.2 Pathogenicity Tests Pathogenicity tests were conducted under greenhouse conditions during the spring season of 2020 using the cucumber hybrid 'Hayel'. Plastic pots (25 cm diameter) were filled with 4 kg of soil that had been previously sterilized with a 5% formalin solution and covered with a plastic sheet for 7 days. The soil was then aerated for four weeks to remove formaldehyde residues. The experiment included pots filled with sterilized soil and inoculated with pathogenic fungi, as well as control pots. Five seeds were sown in each pot, with Article | Ghaleb et al. 3 | Journal of Plant Pathology and Disease Management | Vol. 12, No. 1 | four replicates per treatment. Disease incidence (preand post-emergence damping-off) was recorded at 15 and 30 days after sowing, while plant survival was recorded after 45 days. Disease severity was assessed after 60 days using a 0–5 scale described by Liu et al. (1995). 2.3 Susceptibility of cucumber hybrids Six cucumber hybrids (Mashoor, HCU 096, Jannt, Crystal, Go, and Hayel) were evaluated for their susceptibility to Rhizoctonia solani and Fusarium oxysporum f.sp. cucumerinum during the fall 2020 season. The experimental design and disease assessment (preand post-emergence damping-off, survival percentage) were carried out as described in the pathogenicity test. The percentage of infection was calculated according to the formula by ElHelaly et al. (1970). 2.4 In Vitro Studies 2.4.1 Antagonistic activity of Trichoderma spp. The inhibitory effect of Trichoderma harzianum, T. asperellum, T. album, and T34 (obtained from the Biological Control Unit, ARC, Giza) was evaluated against R. solani and F. oxysporum f.sp. cucumerinum using dual culture technique. A mycelial disc (6 mm) of the pathogen was placed on one side of a PDA plate, and a disc of the antagonist was placed on the opposite side. Plates inoculated with the pathogen alone served as controls. Four replicates were used for each treatment, and plates were incubated at 25±2°C. The percentage of growth inhibition was calculated using the following formula: Inhibition(%)= 𝐶 − 𝑇 𝑇 ×100 Where: C = Radial growth of the pathogen in the control. T = Radial growth of the pathogen in the treatment. 2.4.2 Antagonistic activity of bacteria Four bacterial isolates (Bacillus subtilis, B. megaterium, Paenibacillus polymyxa, and Pseudomonas fluorescens), obtained from MERCIN (Faculty of Agriculture, Ain Shams University, Egypt), were tested. Bacterial isolates were streaked 2 cm from the edge of PDA plates, and a 6 mm pathogen disc was placed in the center (Abou-Aly, 2008; Landa et al., 1997). Plates were incubated at 25°C for 5 days. The inhibition zone was measured, and the percentage of inhibition was calculated as described above. 2.5 Efficacy of commercial biocides under greenhouse conditions Three commercial biocides (Bio-Arc, Rizo-N, and Plant Guard) were evaluated during the fall (2022) and spring (2023) seasons. Sterilized soil was infested with R. solani or F. oxysporum f.sp. cucumerinum at a rate of 1% (w/w). The biocides were applied to the infested soil at three rates (1, 2, and 3 g or cm³/kg soil) before sowing. Control pots contained infested soil without biocides. Each treatment consisted of 5 replicates (one pot per replicate) with 5 seeds of the hybrid 'Hayel' per pot. Preand post-emergence damping-off, plant survival, and disease severity were recorded as previously described. 2.6 Statistical analysis The obtained data were subjected to statistical analysis of variance (ANOVA). The Least Significant Difference (L.S.D.) test was used to compare treatment means at a probability level of 0.05, as described by Gomez and Gomez (1984). 3. Results and Discussion 3.1 Isolation and pathogenicity tests Ten fungal isolates were recovered from infected cucumber roots collected from different localities. The isolated fungi included Rhizoctonia solani (4 isolates), Fusarium oxysporum f.sp. cucumerinum (2 isolates), F. semitectum, F. solani, Macrophomina phaseolina, and Sclerotium rolfsii. Pathogenicity tests revealed that all fungal isolates were capable of infecting cucumber plants ('Hayel' hybrid), causing varying degrees of root rot and wilt symptoms. Data presented in Table (1) indicate that R. solani isolate No. 3 was the most destructive pathogen, recording the highest disease severity (88.12%), as well as high preand post-emergence damping-off. Among the wilt pathogens, F. oxysporum f.sp. cucumerinum isolate No. 8 was the most virulent, causing 77.15% disease severity. Significant differences were observed among the isolates compared to the control. M. phaseolina (isolate No. 7), F. semitectum (isolate No. 5), and S. rolfsii (isolate No. 10) exhibited lower disease severity, recording Article | Ghaleb et al. 4 | Journal of Plant Pathology and Disease Management | Vol. 12, No. 1 | 38.35%, 41.65%, and 42.25%, respectively. These results are in agreement with Martinez et al. (2003) and Al-Tuwaijri (2015), who identified R. solani and F. oxysporum as the most common and aggressive pathogens on cucumber. Consequently, the most aggressive isolates (R. solani No. 3 and F. oxysporum No. 8) were selected for further studies. Table 1: Disease severity of cucumber plants caused by the isolated fungi under greenhouse conditions during the fall 2022 growing season. Isolate No. The tested fungi Pre-emergence damping-off (%) Post-emergence damping-off (%) Survival (%) Disease Severity (%) 1 Rhizoctonia solani (1) 15.00 35.00 50.00 65.35 2 Rhizoctonia solani (2) 12.00 22.00 66.00 73.40 3 Rhizoctonia solani (3) 22.00 46.00 32.00 88.12 4 Rhizoctonia solani (4) 18.00 38.00 44.00 72.35 5 Fusarium semitectum 12.00 24.00 64.00 41.65 6 Fusarium solani 40.00 22.00 38.00 52.10 7 Macrophomina phaseolina 12.00 24.00 64.00 38.35 8 F. oxysporum f.sp. cucumerinum 20.00 55.00 25.00 77.15 9 Fusarium oxysporum 12.00 52.00 36.00 63.75 10 Sclerotium rolfsii 16.00 36.00 48.00 42.25 - Control (Uninfected) 0.00 0.00 100.00 0.00 L.S.D at 5% 4.18 5.36 6.65 7.15 3.2 Reaction of cucumber genotypes The susceptibility of six cucumber genotypes to the most aggressive isolates was evaluated under greenhouse conditions. Data in Table (2) show that all tested genotypes were susceptible to infection, though significant differences were observed. 'Jannt' and 'Go' were the most susceptible varieties to R. solani, recording disease severity of 65.40% and 55.10%, respectively. Conversely, 'Mashhor' and 'Hayel' were the least susceptible to R. solani. Regarding F. oxysporum f.sp. cucumerinum, 'Go' followed by 'Crystal' exhibited the highest disease severity (55.20% and 48.75%, respectively), while 'Mashhor' (28.45%) and 'Jannt' (32.40%) showed the lowest disease severity. These findings are consistent with reports by Al-Tuwaijri (2015) and Thabet (2023), who noted varietal differences in resistance to root rot and wilt pathogens. Table 2: Response of six cucumber genotypes to root rot and wilt diseases incited by the most aggressive Rhizoctonia solani and Fusarium oxysporum f.sp. cucumerinum isolates under greenhouse conditions. Cucumber genotype Rhizoctonia solani (Isolate 3) F. oxysporum f.sp. cucumerinum (Isolate 8) Pre % Post % Surv. % D.S. % Pre % Post % Surv. % D.S. % Hayel 25.00 20.00 55.00 35.25 10.00 15.00 75.00 45.15 HCU 096 15.00 20.00 65.00 45.75 10.00 10.00 80.00 38.75 Crystal 18.00 22.00 60.00 46.25 20.00 30.00 50.00 48.75 Go 22.00 28.00 50.00 55.10 15.00 20.00 65.00 55.20 Jannt 30.00 35.00 35.00 65.40 10.00 10.00 80.00 32.40 Mashhor 20.00 10.00 70.00 35.00 10.00 10.00 80.00 28.45 Control 0.00 0.00 100.00 0.00 0.00 0.00 100.00 0.00 L.S.D at 5% 10.50 8.37 5.15 12.11 8.75 7.15 9.75 10.65 Pre = Pre-emergence damping-off; Post = Post-emergence damping-off; Surv. = Survival; D.S. = Disease Severity. 3.3 In vitro biological control 3.3.1 Efficacy of Trichoderma spp. The antagonistic activity of Trichoderma species against the pathogenic fungi is presented in Table (3). Results indicated that all tested bioagents significantly inhibited the mycelial growth of the pathogens compared to the control. Trichoderma spp. grew rapidly over the mycelium of F. oxysporum and R. solani, preventing their development. T34 (biocontrol agent) exhibited the highest inhibitory effect, reducing the growth of R. solani by 86.33% and F. oxysporum by 88.65%. T. asperellum followed, showing 78.35% inhibition of R. solani. T. harzianum recorded the lowest inhibition percentages against both pathogens. These results align with El-Sheshtawy et al. (2009) and Malathi (2015), who reported the efficacy of Trichoderma spp. in hyperparasitizing soil-borne pathogens. Article | Ghaleb et al. 5 | Journal of Plant Pathology and Disease Management | Vol. 12, No. 1 | Table 3: Antagonistic activity of Trichoderma spp. on the mycelial growth inhibition of the pathogenic fungi in vitro. Trichoderma Bioagents Mycelial Growth Inhibition (%) Against R. solani Against F. oxysporum f.sp. cucumerinum Trichoderma asperellum 78.35 82.66 Trichoderma harzianum 66.66 69.25 Trichoderma album 71.25 79.33 T34 (Biocontrol) 86.33 88.65 Control 0.00 0.00 L.S.D at 5% 1.39 2.15 3.3.2 Efficacy of bacterial bioagents Data in Table (4) demonstrate that all tested bacterial isolates significantly reduced the linear growth of R. solani and F. oxysporum. Pseudomonas fluorescens gave the highest inhibition of R. solani (81.75%) and F. oxysporum (86.67%), followed by Bacillus megaterium. Bacillus subtilis showed moderate efficacy, while Paenibacillus polymyxa was the least effective. The use of microbial antagonists offers an effective and eco-friendly strategy for controlling soil-borne pathogens, as supported by Gravel et al. (2004). 3.4 Efficacy of commercial biocides under greenhouse conditions The efficacy of commercial biocides (Bio-Arc, Plant Guard, and Rizo-N) in controlling root rot and wilt was evaluated during the fall (2022) and spring (2023) seasons (Table 5). Table 4: Effect of antagonistic bacteria on the mycelial growth inhibition of the pathogenic fungi in vitro. Antagonistic Bacteria Mycelial Growth Inhibition (%) Against R. solani Against F. oxysporum f.sp. cucumerinum Bacillus subtilis 72.33 81.66 Bacillus megaterium 79.15 84.33 Paenibacillus polymyxa 68.66 78.10 Pseudomonas fluorescens 81.75 86.67 Control 0.00 0.00 L.S.D at 5% 2.64 1.55 Table 5: Effect of commercial biocides on controlling cucumber root rot and wilt diseases under greenhouse conditions during the fall (2022) and spring (2023) growing seasons. Commercial Biocides Rate of Application Disease Severity (%) Fall (2022) Spring (2023) R. solani F. oxysporum R. solani F. oxysporum Bio-Arc 1 g 14.25 16.75 16.25 15.75 2 g 10.15 11.10 10.35 9.25 3 g 6.65 5.35 7.14 6.33 Plant Guard 1 cm³ 9.62 11.44 10.65 12.33 2 cm³ 6.66 8.54 7.85 7.66 3 cm³ 4.35 3.90 5.25 4.75 Rizo-N 1 g 16.65 14.75 16.40 17.75 2 g 12.25 10.25 11.50 12.66 3 g 9.15 6.65 6.67 7.33 Control -- 86.10 77.25 88.90 78.33 L.S.D at 5% 1.05 1.33 1.28 1.18 All tested biocides effectively reduced disease severity compared to the control. Plant Guard was the most effective treatment at all tested concentrations (1, 2, and 3 cm³/kg soil), significantly decreasing the severity of root rot caused by R. solani and wilt caused by F. oxysporum in both seasons. Bio-Arc, followed by Rizo-N, also provided significant disease control, particularly at the highest concentration (3 g/kg soil). These treatments resulted in the highest reduction of wilt disease compared to lower concentrations and the control. These findings are in accordance with El-Blasy (2006) and Thabet (2023), who confirmed the potential of these biocides in managing cucumber diseases. 4. Conclusion The present study highlights the significant threat posed by Rhizoctonia solani and Fusarium oxysporum f.sp. cucumerinum to cucumber production under greenhouse Article | Ghaleb et al. 6 | Journal of Plant Pathology and Disease Management | Vol. 12, No. 1 | conditions, as pathogenicity tests confirmed the susceptibility of various cucumber genotypes, particularly 'Jannt' and 'Go'. In vitro investigations demonstrated the high antagonistic potential of Trichoderma species, especially isolate T34, and bacterial bioagents like Pseudomonas fluorescens and Bacillus megaterium, while greenhouse experiments provided practical evidence that commercial biocides can effectively manage these diseases. 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Conflicts of Interest: The authors report no known financial or personal relationships that could have influenced the work presented in this article. How to cite this article: Ghaleb, H.K., El-Sheikh Aly, M.M., & Shamrokh, M.M. (2025). Biological control of root rot and wilt diseases of cucumber using certain bioagents and biocides under greenhouse conditions. Journal of PhytoPathology and Disease Management, 12(1), 1–8.