Trichoderma is one of the most exploited biocontrol agents for the management of plant diseases. In biocontrol ecology, the critical factors are detection, and the monitoring and recovery of specific biocontrol agents either naturally present or deliberately released into the environment. Protoplast fusion is an appropriate tool for the improvement of biocontrol Trichoderma strains. Protoplast isolation from Trichoderma harzianum was achieved using 24 h culture age, 6.6 mg/ml Novazym L 1412 at 30°C which resulted the maximum protoplast yield of 5 × 10₈/ml. The self-fused protoplasts were regenerated and 12 fusants were selected based on their growth rate on 2% colloidal chitin medium. Next, a comparison was done for chitinase and antagonistic activity. Transcriptomic analysis based on quantitative real-time RT-PCR, demonstrated that T8-05 fusant expressed 1.5 fold of chit42 transcript as compared with the parental line. This fusant with 7.02±0.15U chitinase activity showed a higher growth inhibition rate (100%) than the parent strain, against Rhizoctonia solani. To obtain a genetically marked fusant that can be used as a biomonitor, the fusant was cotransformed with the gfp and amdS genes. The morphology and viability of selected cotransformant (FT8-7MK-05-2) was similar to the parent. Green fluorescing conidia were observed within the first 2 days of incubation in the soil, and this was followed by the formation of chlamydopores after 60 days. The colonisation of the gfp-tagged fusant was also monitored visually on R. solani sclerotia by scanning electron microscopy. Production of gfp-tagged fusant of Trichoderma spp. provides a potentially useful tool for monitoring hyphal growth patterns and the population of biocontrol agent isolates introduced into environmental systems.

Cyanide is used in many industries despite its toxicity. Cyanide biodegradation is affordable and eco-friendly. Sampling from cyanide-contaminated areas from the Muteh gold mine and isolation of 24 bacteria were performed successfully. The selected bacteria—‘Bacillus sp. M01’—showed maximum tolerance (15 mM) to cyanide and deposited in Persian Type Culture Collection by PTCC No.: 1908. In the primary experiments, effective factors were identified through the Plackett–Burman design. In order to attain the maximum degradation by Bacillus sp. M01 PTCC 1908, culture conditions were optimized by using response surface methodology. By optimizing the effective factor values and considering the interaction between them, the culture conditions were optimized. The degradation percentage was calculated using one-way ANOVA vs t test, and was found to have increased 2.35 times compared to pre-optimization. In all of the experiments, R2 was as high as 91%. The results of this study are strongly significant for cyanide biodegradation. This method enables the bacteria to degrade 86% of 10 mM cyanide in 48 h. This process has been patented in Iranian Intellectual Property Centre under Licence No: 90533.