## 2 Materials and Methods
### Greenhouse Experiment  
To evaluate the changes produced by biochar and biofertilizer on cultivated soil, we carried out a greenhouse experiment with tomatillo, also known as husk tomato (_Physalis ixocarpa_), for two crop cycles. We also studied the nutrient use efficiency, comparing chemical fertilization at 100% (without biochar) against 50% and 20% with biochar on crop yield, plant height, fruit weight, and root length. Finally, we compared the combination of biochar and biofertilizer with the treatments mentioned earlier on the variables measured in the plant.  
_Physalis ixocarpa_ is a Solanaceae native from Mexico and Central America, which adapts to a variety of climates, and it is therefore produced throughout Mexico and from United States to Nicaragua. Its fruits are widely used in Mexican cuisine and the whole plant in traditional medicine [57]. In addition, it has recently been investigated due to its content of vitamins, minerals, and compounds with antibacterial, antioxidant, antitumor, and anti-inflammatory activity [58]. Since 1970 its demand has increased due to higher domestic consumption and exports to the United States, Canada, Japan, Costa Rica, the Netherlands, the United Kingdom, the United Arab Emirates, Spain, and Germany [59].  
Seeds of _Physalis ixocarpa_ (Vita\({}^{\text{TM}}\), V 954) were germinated, and when seedlings reached 12 cm in height were transplanted in bags containing 3 kg of soil or soil with biochar (0.2% \(w/w\)). To study the effect of biochar and biofertilizer as soil conditioners, four treatments were tested: control treatment (CT: soil + 100% chemical fertilization), two treatments with biochar and chemical fertilization (BT20: soil + biochar + 20% chemical fertilization, and BT50: soil + biochar + 50% chemical fertilization), and one treatment with biofertilization (BBfT: soil + biochar + biofertilizer). The biofertilizer was diluted in each cycle to provide the same amount of N as 100% of chemical fertilization. Each treatment was tested in triplicate in random blocks with six plants each. Figure 1 shows the experimental design. The doses of fertilization were calculated according to Formula (3),considering the nutritional requirements of _Physalis ixocarpa_, for which 100% of chemical fertilization dose corresponds to 200-100-200-80.  
\[FD=\frac{NR-NS}{FE} \tag{3}\]  
where \(FD\) is the fertilization dose, \(NR\) is the kilograms of nutrients required by the crop, \(NS\) is the content of nutrients in the soil, and FE is the efficiency of absorption of the fertilizer by the crop in percentage.  
For the chemical fertilization, the source of nitrogen was urea (46-00-00), that of phosphorus: phosphate rock (00-32-00), that of potassium and nitrogen: potassium nitrate (13.8-44-00), and for calcium and nitrogen: calcium nitrate (15-00-00-26). The fertilization doses of each treatment were divided into 14 portions that were applied weekly during the crop development, from August to November 2018 for the first crop cycle and from April to July 2019 for the second crop cycle.  
Once the fruits filled the calyx that covered them, the harvest was carried out. The variables measured in the plant were height, root length, fruit weight, and yield per treatment. After harvest, the soil of each treatment was mixed, and three samples were taken to analyze and evaluate the effect of the treatments. For the second cycle, the soil was mixed again and packed into the bags. Seeds of tomatillo were germinated and transplanted as it was made for the first cycle.