Innovative sustainable bioreactor-in-a-granule formulation of Trichoderma asperelloides

Abstract

The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 10⁹ colony-forming units (CFU)/g within 14 days. Subsequently, G_Control, G_Lecithin, G_Break-Thru, G_Bentonite, and G_{Organic compost+Break-Thru} formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. G_Control, G_Break-Thru, G_Bentonite, and G_{Organic compost+Break-Thru} stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations G_Control and G_Bentonite remained consistent for > 3 months at ambient temperature, while in G_Bentonite and G_{Organic compost+Break-Thru} formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 10⁴ to 5 × 10⁶ CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life. KEY POINTS: • Innovative "bioreactor-in-a-granule" system for T. asperelloides is devised. • Dry granules of aerial conidia remain highly viable for 24 months at 4 °C. • Effective control of white-mold sclerotia via soil application of Trichoderma-based granules.

Keywords: Trichoderma; Biocontrol agent; Bioprotectant; Granule formulation; Solid-state fermentation; White mold.

Fig. 1

Pareto diagram (α = 0.05) demonstrating the significance of the moisture, inoculum density, substrate weight, fermentor type, and nitrogen content as predictors potentially affecting the viable spore production expressed in colony-forming units (CFUs) of Trichoderma asperelloides CMAA 1584 for medium optimization

Fig. 2

Shelf-life of Trichoderma asperelloides CMAA 1584 formulations stored in the fridge (4 °C) (A) and at ambient temperature (25 ± 2 °C) (B) for 24 months. Trends represent means (± standard error, n = 6)

Fig. 3

Soft and disintegrated Sclerotinia sclerotiorum sclerotia (A) and inhibition of myceliogenic germination (B) by Trichoderma asperelloides CMAA 1584 at different concentrations applied to the soil. Untreated group (control) is not shown as all sclerotia were healthy and germinated 100%. Bars indicate the mean (± standard error, n = 4) and lettering indicates significant differences (Tukey test, P < 0.05)