Document Type : Research Paper

Authors

• Marzieh Alinejad ¹
• Reza Talaei-Hassanloui ¹
• Seyedeh Masoomeh Zamani ²
• Hassan Askary ³
• Jamasb Nozari ¹

¹ Department of Plant Protection, Faculty of Agriculture, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

² Research Institute of Forests and Rangelands, Tehran, Iran

³ Iranian Research Institute of Plant Protection, Tehran, Iran

Abstract

Keywords

• Cucumber powdery mildew
• Microbial formulations
• Phenolic compounds
• Defense mechanism

Extended Abstract

Introduction

 Entomopathogenic fungi are important biological control agents that offer a promising alternative to synthetic chemical pesticides. Their effectiveness depends on producing large quantities of viable conidia and maintaining their quality during storage. Solid-state fermentation (SSF) using agricultural products is a common method for large-scale conidial production. However, the type of substrate plays a major role in conidial yield, germination rate, and production cost. The present study aimed to evaluate the biological performance and economic efficiency of five solid substrates (rice husk, wheat, wheat bran, broken corn, and flaked barley) in the production of conidia for four fungal isolates: Beauveria bassiana B2, Metarhizium anisopliae M1, Purpureocillium lilacinum Ento1, and Lecanicillium lecanii V198499.

 Materials and Methods

The experiment was conducted under laboratory conditions at 25 °C. Four entomopathogenic fungal isolates were cultured in potato extract liquid medium on a shaker (150 rpm) for 10 days to prepare the inoculum. Five solid substrates were used, including rice husk, wheat, wheat bran, broken corn, and flaked barley. Each substrate (200 g) was autoclaved, moistened with 100 ml of sterile water, and inoculated with 10 ml of the fungal suspension. The inoculated substrates were incubated at 25 °C for 14 days and manually mixed every 48 hours to improve sporulation. After incubation, substrates were air-dried, and conidia were harvested by mixing 1 g of dry substrate with 9 ml of water-Tween 80 solution (0.01%). Conidial concentrations were determined using a hemocytometer. The germination percentage was assessed weekly for 8 weeks by plating samples on PDA medium and counting germinated conidia under a microscope. The cost of each substrate per 100 g was recorded to assess economic performance. Then, a cost index was calculated based on the average number of conidia produced per gram of substrate. This index, expressed in Tomans per 10⁹ conidia, allowed direct comparison of cost efficiency among substrates.

Results and Discussion

Results showed that conidial production varied among fungal isolates and substrates. B. bassiana B2 yielded the highest number of conidia on flaked barley (5.3 × 10⁹ conidia/g), followed by rice husk (3.5 × 10⁹ conidia/g), while the lowest production was recorded on wheat (1.2 × 10⁹ conidia/g). In contrast, M. anisopliae M1 had the highest yield on wheat (4.6 × 10⁹ conidia/g), with flaked barley ranking second (3.1 × 10⁹ conidia/g) and wheat bran yielding the lowest (6.0 × 10⁸ conidia/g). L. lecanii V198499 produced the highest conidia on flaked barley (3.6 × 10⁹ conidia/g) and the lowest on wheat bran (4.6 × 10⁸ conidia/g). In P. lilacinum Ento1, flaked barley yielded 2.8 × 10⁹ conidia/g, while the lowest production was observed on rice husk (1.5 × 10⁸ conidia/g). These findings highlight the significant influence of substrate type on sporulation, indicating that both nutrient composition and structural properties of the substrates strongly affect fungal growth and conidiation efficiency. Conidial germination rates were also affected by substrate and incubation time. Flaked barley supported the highest initial germination (98%) across all isolates and retained 60% after 56 days for B. bassiana. Wheat (97% to 55%) and rice husk (95% to 44%) followed. In contrast, wheat bran and rice husk caused sharp declines in L. lecanii germination, dropping to 40% and 38% after eight weeks. P. lilacinum retained 62% germination on flaked barley but only 44% on rice husk and 46% on wheat bran. These results demonstrate that substrate properties such as aeration, porosity, and moisture retention have a crucial role in sustaining conidial viability during storage. Economic analysis showed that the cost per gram of substrate varied significantly among substrates. Flaked barley was the most economical option across all tested isolates—B. bassiana B2, M. anisopliae M1, P. lilacinum Ento1, and L. lecanii V198499—with costs of 0.015–0.034 USD g⁻¹ of substrate. In contrast, wheat bran consistently incurred the highest costs and yielded the lowest biological performance among all substrates. Overall, flaked barley provided the best balance between biological efficiency and cost-effectiveness, supporting its suitability as a sustainable substrate for large-scale production of entomopathogenic fungi.

Conclusion

Overall, the results showed that substrate type significantly influenced conidial yield, germination rate, and production cost of entomopathogenic fungi. Flaked barley resulted in the highest conidial production and germination rate in B. bassiana, P. lilacinum, and L. lecanii, while M. anisopliae performed best on wheat. Economically, flaked barley had the lowest cost per 10⁹ conidia across all isolates. In contrast, wheat bran showed the lowest biological performance and cost in all cases. Therefore, flaked barley is recommended as the most suitable substrate for large-scale production of the tested fungi.

Author Contributions

Marzieh Alinejad: investigation, methodology, formal analysis, visualization, draft preparation; Reza Talaei-Hassanloui: methodology, formal analysis, supervision, project administration and funding acquisition, final review and edit; Seyede Masoomeh Zamani: methodology, supervision, final review and edit; Hassan Askary: final review and edit; Jamasb Nozari: final review and edit.