Morphological and physiological response of sour orange (Citrus aurantium L.) seedlings to the inoculation of taxonomically characterized bacterial endophytes

Entophytic bacteria (EBs) are very diverse and found in virtually all plant species studied. These natural EBs live insides the host plant and can be used to maximize crop and fruit yield by exploiting their potential. In this paper, EBs characterization from various citrus genotypes and their influence on the morphological and physiological functioning of sour orange (Citrus aurantium) seedlings are described. To assess the influence of 10 distinct EBs, three different techniques (injection, soil mix, and spray) were applied for single and mixed inoculation on sour orange (C. aurantium) seedlings. The selected strains were identified as firmicutes (Enterococcus faecalis, Bacillus safensis, Bacillus cereus, Bacillus megaterium, Brevibacillus borstelensis & Staphylococcus haemolyticus), and gamma Proteobacteria (Enterobacter hormachaei, Proteus mirabilis, Pseudomonas aeruginosa, & Pseudomonas sp.) by 16S rRNA gene sequencing. To investigate the influence of these EBs on host plant morphology, different parameters (morphometric) were recorded after five WOI (weeks of inoculation), including shoot/root length, shoot/root fresh and dry biomass, and biophysical analyses i.e., relative water content (RLWC). Physiological markers such as chlorophyll & carotenoid content, protein content, proline content, phenolics, and flavonoids were also analyzed to determine the influence of endophytes on sour orange seedlings. Five strains such as SM-34, SM-20, SM-36, SM-68, and SM-56 significantly improved the development and physiology of sour orange seedlings. Bacillus cereus and Pseudomonas aeruginosa produced the best outcomes in terms of plant growth. The relative quantification of bacterial inoculums was determined using real-time PCR. A rise in the number of bacterial cells in inoculated treatment suggests that bacterial strains survived and colonized successfully, and also shown their competitiveness with native bacterial community structure. As per the results of inoculation methods, soil mixing, and injection methods were determined to be effective for bacterial inoculation to plants but a variable trend was found for different parameters with test bacterial strains. After testing their impact on field conditions, these strains can be applied as fertilizers as an alternative to conventional chemical fertilizer, although in the context of mixed inoculation of bacterial strains, 5 M and 6 M performed best and enhanced plant growth-promoting activity.