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

Gloria M Macedo-Raygoza,Katia R Prieto,Fernanda M Prado,Blondy B Canto-Canché,Monica Carrillo-Beltrán,Massuo J Kato,Miguel J Beltrán-García,Paolo Di Mascio,James F White,Lydia F Yamaguchi,Benjamín Valdez-Salas

doi:10.3389/fmicb.2019.00804

Abstract

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.

Highlights

Bananas (Musa spp.) are included in the top five main staple food crops in the world; approximately 100 million tons of bananas are produced annually in approximately 120 countries in tropical and subtropical regions (Ploetz et al, 2015)
Endophytic Bacteria Isolation and Characterization In a previous study, 150 bacterial strains were obtained as endophytes from different tissues of commercial banana Cavendish Grand Naine grown in two different banana-producing regions at central Pacific in Mexico
The bacterial strains were selected for their capacity to grow fast in mineral medium supplemented with colloidal chitin as carbon source; to grow on Norris medium as a way to probe of their N2 fixing capacity and grow satisfactorily in culture media based on extracts from different banana plant tissues

Summary

Introduction

Bananas (Musa spp.) are included in the top five main staple food crops in the world; approximately 100 million tons of bananas are produced annually in approximately 120 countries in tropical and subtropical regions (Ploetz et al, 2015). In Mexico and many other countries, the major threat to banana production is black Sigatoka disease caused by Pseudocercospora fijiensis (Previously: Mycosphaerella fijiensis), which often reduces yield by more than 50%. Endophytic bacteria are becoming increasingly recognized in crop production because of their potential as agents in plant growth promotion, stress alleviation and biological control as well as their role in making available organic nitrogen sources for plants (Beltran-Garcia et al, 2014b; Santoyo et al, 2016; Maksimov et al, 2018; White et al, 2018). We addressed the potential application of two endophytic bacteria that were isolated from leaves and roots of banana plants cv. ‘Cavendish Grand Naine,’ with capacities to support the growth of their host plant in extremely poor soil nutrient conditions and antagonistic activity against the black Sigatoka pathogen.

Methods

Results

Discussion

Conclusion