Document Type : Research Paper
Authors
1 Department of biological control, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran.
2 Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Evin, Tehran, Iran.
Abstract
Keywords
Extended Abstract
Introduction
Powdery mildew disease of cucumber caused by the fungus Podosphaera xanthii is one of the most important yield-limiting diseases of cucurbit crops all of the world, causing yield losses of 20-40%. Challenges with chemical control, particularly fungal resistance development, highlight the need for safe, sustainable alternatives like biological control agents (BCAs). Bacillus subtilis and Trichoderma harzianum are key BCAs known to boost plant disease resistance by inducing phenolic compounds and activating plant defense pathways. The primary objective of this study was to investigate the changes in total phenol and flavonoid contents as well as specific phenolic compounds in P. xanthii-infected cucumber leaves treated with two BCAs (B. subtilis B2, T. harzianum K18) versus the chemical fungicide Domark. This research specifically aimed to evaluate the role of induced phenolic compound production as a key mechanism of these biological agents in controlling cucumber powdery mildew.
Materials and methods
This study examined changes in phenolic compounds in cucumber leaves infected with P. xanthii under the influence of two microbial formulations (B. subtilis B2 and T. harzianum K18) and the chemical fungicide Domark. Experiments were conducted in a factorial design in greenhouses in West Azerbaijan province. At the 3-4 leaf stage (fifth week), plants were treated by foliar spraying with microbial formulations at 2% concentration. Domark fungicide at 0.4% concentration and sterile distilled water were used as controls. Twenty-four hours post-treatment, pathogen inoculation was performed using a spore suspension. Each treatment had three replicates, with each replicate consisting of ten cucumber plants of the relatively susceptible cultivar "Nagin." At 24, 48, and 72 hours after infection, total phenol content was assayed using the Folin-Ciocalteu method, and total flavonoids were measured via the aluminum chloride method. Identification and quantification of seven specific phenolic compounds were performed using High-Performance Liquid Chromatography (HPLC). Disease symptoms were periodically assessed, and the disease severity index (DSI) was evaluated after 10 days using the Horsfall-Barratt 1-12 scale, with percent disease index (PDI) calculated. Data were analyzed using one-way ANOVA and LSD mean comparison test at 1% and 5% probability levels. Correlation between phenolic compound concentrations and disease severity was examined using Pearson’s correlation coefficient and linear regression.
Results and Discussion
Phenolic compound dynamics revealed the peak defensive response at 48 hours post-infection. B. subtilis B2 induced a 159% increase in total phenols and a 373% increase in total flavonoids. T. harzianum K18 showed a 129% increase in total phenols and 389% in flavonoids, with effects persisting until 72 hours. Caffeic acid was identified as the predominant phenolic compound in cucumber leaves, increasing by 154% under T. harzianum treatment. Rutin and ferulic acid increased by 103% and 205%, respectively, in the presence of T. harzianum. A strong negative correlation was observed between rutin concentration and disease severity. Disease suppression results indicated that B. subtilis B2 reduced disease severity by 30.84% (comparable to Domark fungicide), while T. harzianum K18 achieved a 39.16% reduction. Findings demonstrate that the two biocontrol agents, B. subtilis strain B2 and T. harzianum strain K18, induce significant accumulation of phenolic compounds during the critical plant response period (48 hours post-infection). These compounds contribute to disease control through multiple mechanisms: Rutin plays a pivotal role by neutralizing oxidative stress-derived free radicals and reinforcing cell walls, evidenced by its strong negative correlation with disease severity. Caffeic acid acts as a lignin precursor and inhibits key pathogen enzymes like cellulase and pectinase. Ferulic acid, with a 205% under Trichoderma, enhances lignification and cell wall fortification. Rosmarinic acid contributes through potent antioxidant and antimicrobial properties.
Conclusion
The results confirm the effectiveness of both biocontrol formulations in controlling cucumber powdery mildew through induced systemic resistance and increased production of essential defensive phenolics (notably rutin and caffeic acid). To achieve synergistic advantages, it is advisable to combine B. subtilis B2 (which induces stable flavonoids) with T. harzianum K18 (which induces wall-strengthening phenolic acids) as an immediate strategy. Future research should concentrate on: 1) Investigating the synergy between these agents under field conditions, 2) Assessing the expression of critical defense pathway genes (such as PAL), 3) Implementing large-scale field trials, and 4) Refining formulations. This strategy signifies a crucial step towards the development of sustainable methods for managing greenhouse crop diseases in Iran.
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