Trichoderma species are commonly used as biological control agents against soil-borne pathogens, such as Fusariumoxysporum f. sp. lycopersici (FoL), which causes tomato wilt. However, their effectiveness varies based on isolate traits and bioassay conditions. This research tested 46 native Tanzanian Trichoderma isolates, along with one commercial strain, against FoL strain PD- 87/91 using various in vitro bioassays. Dual culture tests were performed with both simultaneous and staggered inoculations, and pathogen growth inhibition (PGI) was analyzed via two-way ANOVA in R software. Significant impacts of the isolate, inoculation method, and their interaction on PGI were found (p < 0.001). Under simultaneous inoculation, PGI ranged from 62. 1% (Trichoderma erinaceum MG 3- 1) to 79. 9% (Trichodermaharzianum MG 7- 2), while under staggered inoculation, PGI varied from 47. 6% (Trichoderma virens NH 7- 3) to 58. 6% (Trichoderma harzianum SH 10- 2). Several native isolates performed better than the commercial control in both methods. Bell' s scale assessment showed mostly class 1 interactions during simultaneous inoculation, indicating complete pathogen suppression by Trichoderma, whereas class 2 interactions dominated during staggered inoculation. No class 4 or 5 interactions were detected. During interactions, yellow coloration was highly common (>80%) under both methods, though Pearson' s Chi-square test found no significant link between color and inoculation method (p = 0.892). VOC assays revealed significant differences among isolates (p < 0. 001), with SH 11- 2 (66. 22%) and KT 2- 2 (65. 92%) showing the highest VOC- mediated inhibition, outperforming the commercial strain (52. 23%). The findings indicate that Trichoderma' s antagonistic activity depends on both the specific isolate and the method, involving direct competition and VOC- based antibiosis. Combining PGI data, Bell' s scale, colour interaction, and VOC tests offers a comprehensive approach to selecting effective isolates for further research on biological control of tomato wilt in planta.

The effectiveness of Trichoderma -based biocontrol products depends on achieving high spore yields and maintaining viable propagules during storage. This study evaluated the effects of substrate type, isolate, and storage duration on spore production and viability of four Trichoderma harzianum isolates (BH10-2, KH5-1, MG7-2, and SH11-2) cultured on four cereal-based substrates: rice grain (RG), rice powder (RP), sorghum grain (SG), and sorghum powder (SP). The experiment was arranged in a factorial Completely Randomized Design (CRD) with three replications under laboratory conditions. Spore production was assessed 10 days after fermentation, while spore viability was determined after 30, 60, and 90 days of storage following a 7-day drying period. Colony-forming unit (CFU) data were log-transformed before analysis using two-way ANOVA for initial spore production and a linear mixed-effects model (LMM) for repeated measurements of spore viability during storage. Mean separation was performed using Tukey's HSD test at P ≤ 0.05. Initial spore production was significantly influenced by the interaction between isolate and substrate ( P < 0.05). Rice powder supported the highest spore production, with isolate BH10-2 producing the greatest spore yield (8.55 log CFU g ⁻¹ ), whereas isolate MG7-2 grown on sorghum grain recorded the lowest initial spore production (5.81 log CFU g ⁻¹ ). Spore viability was significantly affected by isolate, substrate, storage duration, and their interactions ( P < 0.05). Spore viability increased from Day 10 to Day 30, indicating continued sporulation during the early storage phase, before declining progressively through Days 60 and 90. Sorghum grain consistently maintained the highest spore viability during prolonged storage, particularly for isolate MG7-2, whereas rice powder exhibited the greatest reduction in viability after 90 days. Overall, the results demonstrate that substrate selection influences both initial sporulation and long-term storage stability of Trichoderma . The combination of isolate MG7-2 and sorghum grain showed the greatest potential for developing stable Trichoderma -based bioformulations suitable for sustainable biological control.