In Vitro Eradication of Endophytic Bacteria to Enhance Micropropagation of L49 Guava (Psidium guajava']java L.)

Guava (Psidium guajava L.) is a globally important fruit species, and the development of effective micropropagation protocols for elite guava varieties is crucial for large-scale clonal propagation. However, endophytic bacterial contamination remains a significant challenge in guava micropropagation. This study aimed to detect and eliminate bacterial endophytes from nodal explants of L49 guava variety using an in vitro approach. Explants were collected from three different sources-Fruit Research Station, Dehgam (D), Forest Research Range, Basan (B), and nursery-grown plants (N)-which showed varying contamination rates of 95%, 50%, and 15%, respectively. The explants also exhibited higher browning (80%) and lower response rates (10%). After 20 days of culture inoculation, six bacterial endophytes were identified through 16S rRNA sequencing: Pseudomonas oryzihabitans, P. taiwanensis, P. qingdaonensis, Methylobacterium dankookense, M. radiotolerans, and Sphingopyxis soli. Antibiotic susceptibility testing identified eight effective antibiotics (tetracyclines, aminoglycosides, and fluoroquinolones), with gentamicin exhibiting the largest inhibition zones (3.5 cm). Gentamicin at concentrations of 300 and 400 ppm completely eliminated contamination, significantly outperforming the control (100% contamination), 100 ppm (65%), and 200 ppm (45%) concentrations. Based on these results, gentamicin (300 ppm) was selected for culture medium supplementation to control contamination effectively without any adverse effects on leaf and shoot growth of explants. This study is the first to report the successful in vitro eradication of endophytic bacteria in L49 guava through the identification and application of potent antibiotics, particularly gentamicin, thereby improving micropropagation protocols by addressing a critical contamination issue.