Endophytic bacterial communities in in vitro shoot cultures derived from embryonic tissue of hybrid walnut (Juglans × intermedia)

Abstract

A detailed picture of the endophytic bacterial community structure on phylum and genus level in in vitro shoot cultures of sixteen Juglans genotypes including Juglans regia, Juglans nigra and mainly Juglans hybrids was generated by using culture-dependent and culture-independent methods. A total of eight endophytic bacterial strains with a high identity score (97–100%) were isolated from tissue culture shoot material derived from embryonic tissue of Juglans hybrids by the culture-dependent approach. By sequence comparison of a 16S rRNA fragment to the NCBI database, they were identified as Acinetobacter, Brevundimonas, Moraxella, Pseudomonas, and Roseomonas species. The culture-independent approach involved Illumina MiSeq 16S rRNA amplicon sequencing. Five different phyla (Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria) were analyzed in more detail. The phylum Proteobacteria including genera such as Brevundimonas spp., Acinetobacter spp. and Moraxella spp. showed the highest relative abundance of endophytic bacteria. Thus, the results of the sequencing approach accorded well with those obtained in the culture-dependent approach. The abundance of Moraxella spp. was correlated with a good micropropagation in the Juglans genotypes under investigation, whereas, Brevundimonas spp. and Acinetobacter spp. were only detected in clones with medium propagation rates. Other bacterial endophytes known as plant growth promoting bacteria such as Pseudomonas spp., Erwinia spp., Burkholderia spp., Pelomonas spp., or Sphingomonas spp. were also found in in vitro cultures of a wide range of Juglans genotypes.