Enhancing the Establishment of a Diazotrophic Community in Sugarcane Stem Cutting through Heat–Biocide Treatment

The inoculation of diazotrophs in sugarcane presents a challenge due to the plant’s vegetative propagation, which hampers the accurate assessment of the contribution of a new bacterial community in the presence of natural inhabitants. To surmount this obstacle, a modification of short heat treatment (HT—52 °C for 30 min) is normally employed to control ratoon stunt disease, with the addition of a chemical step testing chloride (HCl—1%) and acetic acid (Ac—2%) as biocides. Four experiments were conducted involving two sugarcane cultivars (IACSP95-5000 and RB867515) with and without inoculation of five diazotrophs: G. diazotrophicus, H. seropedicae, H. rubrisubalbicans, N. amazonense, and P. tropica. The HT treatments consisted of control, HT (52 °C for 30 min) alone; HT reduced to 10 min (10HT), biocides: hydrochloric acid (HCl—1%) and acetic acid (Ac—2% at 52 °C for 10 min, and 10HT + 10Ac at room temperature as an additional step—10 AcRT). Plant and bacterial growth (based on two methods—most probable number and qPCR) and nitrogenase activity (acetylene reduction activity—ARA) were measured. Ac was selected as a biocide as germination was not reduced. Inoculation improved germination and sugarcane biomass accumulation, especially with 10 HT + 10 AcRT. The reduction in HT time by 20 min and the addition of Ac (additional step) effectively controlled the natural diazotrophic community, establishing the five diazotrophs, promoting growth, and increasing ARA activity by 82% compared to the traditional HT.