Enterobacter cloacae, an Endophyte That Establishes a Nutrient-Transfer Symbiosis With Banana Plants and Protects Against the Black Sigatoka Pathogen

Banana (Musa spp.) is an important crop worldwide, but black Sigatoka disease caused by the fungus Pseudocercospora fijiensis threatens fruit production. In this work, we examined the potential of the endophytes of banana plants Enterobacter cloacae and Klebsiella pneumoniae, as antagonists of P. fijiensis and support plant growth in nutrient limited soils by N-transfer. The two bacterial isolates were identified by MALDI-TOF mass spectrometry and corroborated by 16S rRNA sequence analysis. Both bacteria were positive for beneficial traits such as N-fixation, indole acetic acid production, phosphate solubilization, negative for 1-aminocyclopropane 1-carboxylic acid deaminase and were antagonistic to P. fijiensis. To measure the effects on plant growth, the two plant bacteria and an E. coli strain (as non-endophyte), were inoculated weekly for 60 days as active cells (AC) and heat-killed cells (HKC) into plant microcosms without nutrients and compared to a water only treatment, and a mineral nutrients solution (MMN) treatment. Bacterial treatments increased growth parameters and prevented accelerated senescence, which was observed for water and mineral nutrients solution (MMN) treatments used as controls. Plants died after the first 20 days of being irrigated with water; irrigation with MMN enabled plants to develop some new leaves, but plants lost weight (−30%) during the same period. Plants treated with bacteria showed good growth, but E. cloacae AC treated plants had significantly greater biomass than the E. cloacae HKC. After 60 days, plants inoculated with E. cloacae AC showed intracellular bacteria within root cells, suggesting that a stable symbiosis was established. To evaluate the transference of organic N from bacteria into the plants, the 3 bacteria were grown with 15NH4Cl or Na15NO3 as the nitrogen source. The 15N transferred from bacteria to plant tissues was measured by pheophytin isotopomer abundance. The relative abundance of the isotopomers m/z 872.57, 873.57, 874.57, 875.57, 876.57 unequivocally demonstrated that plants acquired 15N atoms directly from bacterial cells, using them as a source of N, to support plant growth in restricted nutrient soils. E. cloacae might be a new alternative to promote growth and health of banana crops

Production of Lipopeptides by Fermentation Processes: Endophytic Bacteria, Fermentation Strategies and Easy Methods for Bacterial Selection

Lipopeptides constitute an important class of microbial secondary metabolites. Some lipopeptides have potent therapeutic activities such as antibacterial, antiviral, antifungal, antitumor and immunomodulator. Surfactin, iturin, fengycin, lichenysin and bacillomycin D from Bacillus species, daptomycin from Streptomyces roseosporus and rhamnolipids from Pseudomonas aeruginosa are among the most studied lipopeptides. These molecules are good candidates to replace those antibiotics and antifungals with no effect on pathogenic microorganisms. Microbial lipopeptides are produced via fermentation processes by bacteria, yeast and actinomycetes either on water miscible and immiscible substrates. However, the major bottlenecks in lipopeptide production are yield increase and cost reduction. Improving the bioindustrial production processes relies on many issues such as selecting hyperproducing strains and the appropriate extraction techniques; purification and identification by Polymerase Chain Reaction(PCR), High Performance Liquid Chromatography-Mass Spectrometry(HPLC-MS), Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry(MALDI-TOF-MS); the use of cheap raw materials and the optimization of medium-culture conditions. The purpose of this chapter is to orient the reader on the key elements in this field, including the selection of analytical strategies to get a good microbial strain as well as to show some examples of liquid and solid-state low-cost fermentation processes. Last, we introduce endophytic bacteria as lipopeptide-producer candidates