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

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, GControl, GLecithin, GBreak-Thru, GBentonite, and GOrganic 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. GControl, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations GControl and GBentonite remained consistent for > 3 months at ambient temperature, while in GBentonite and GOrganic 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.

Optimizing mass production of Trichoderma asperelloides by submerged liquid fermentation and its antagonism against Sclerotinia sclerotiorum.

Commercial products based on Trichoderma are obtained mainly from solid-state fermentation. Submerged liquid fermentation is the most appropriate method compared to the solid medium for large-scale production of Trichoderma spp. The present study aimed to optimize the combination of key variables that influence the liquid fermentation process of Trichoderma asperelloides LQC-96 for conidial production coupled with its efficiency in the control of Sclerotinia sclerotiorum. In addition, we verified whether the optimized culture conditions can be used for the conidial production of Trichoderma erinaceum T-12 and T-18 and Trichoderma harzianum T-15. Fermentation studies were performed in shake flasks following a planned experimental design to reduce the number of tests and consumable costs. The effect of temperature, pH, photoperiod, carbon:nitrogen ratio and water activity on conidial production were assessed, which of pH was the only meaningful factor contributing to increased conidial production of T. asperelloides LQC-96. From the five variables studied initially, pH and C:N ratio were further used in the second design (rotational central composite design-RCCD). Hence, the best conditions for the production of T. asperelloides LQC-96 conidia by liquid fermentation consisted of initial pH of 3.5, C:N ratio of 200:1 at 30 °C, without glycerol, and under 24 h photoperiod. The highest conidial concentration was observed after seven days of fermentation. Under these optimal conditions, T. erinaceum T-12 and T-18, and T. harzianum T-15 were also cultivated, but only LQC-96 efficiently parasitized S. sclerotiorum, precluding sclerotium myceliogenic germination. Our findings propose optimal fermentation conditions that maximize conidial production of T. asperelloides as a potential biofungicide against S. sclerotiorum.

Dual functionality of Trichoderma: Biocontrol of Sclerotinia sclerotiorum and biostimulant of cotton plants.

Microbial crop protection products based on Trichoderma have the ability to display multifunctional roles in plant protection, such as pathogen parasitism, enhance nutrient availability and stimulate plant growth, and these traits can be used to enhance the overall agronomic performance of a variety of crops. In the current study, we explored the multifunctional potential of two indigenous Brazilian strains of Trichoderma (T. asperelloides CMAA 1584 and T. lentiforme CMAA 1585) for their capability of controlling Sclerotinia sclerotiorum, a key plant pathogen of cotton, and for their ability of growth promotion in cotton plants (Gossypium hirsutum). Both strains were able to solubilize mineral phosphorus (CaHPO4), to release volatile organic compounds that impaired the mycelial growth of S. sclerotiorum, and to promote the growth of cotton plants under greenhouse conditions. In dual culture, Trichoderma strains reduced the growth rate and the number of sclerotia formed by S. sclerotiorum. By treating sclerotia with conidial suspensions of these Trichoderma strains, a strong inhibition of the myceliogenic germination was observed, as a result of the marked mycoparasitic activity exerted on the sclerotia. The parasitism over S. sclerotiorum was more effective with T. asperelloides CMAA 1584, whilst the biostimulant effects on cotton growth were more pronounced with T. lentiforme CMAA 1585, which also showed a higher capacity of phosphate solubilization. Thus, T. asperelloides CMAA 1584 displays higher efficiency in controlling S. sclerotiorum, while T. lentiforme CMAA 1585 is more suitable as a biostimulant due to its ability to promote growth in cotton plants. Overall, these Trichoderma strains may be used in mixture to provide both pathogen control and promotion of plant growth, and this strategy will support growers in minimizing the use of synthetic fertilizers and fungicides against white mold in cotton crops.