Co-inoculation of Anabaena cylindrica and Azospirillum brasilense during initial growth and chloroplast pigments of corn

ABSTRACT Although the isolated effects of the cyanobacteria Anabaena cylindrica and the bacteria Azospirillum brasilense are well-known, the co-inoculation of both can promote other benefits to corn plants’ biological nitrogen fixation and growth. The aim was to evaluate the initial growth and chloroplast pigment levels of corn hybrids co-inoculated with A. brasilense and A. cylindrica. The experiment was conducted in greenhouse conditions using the completely randomized design in a factorial scheme 2 × 4 with two hybrids, Balu 184 and Balu 280 Pro, and four inoculation treatments: no inoculation, inoculation of A. cylindrica, inoculation of A. brasilense, and the co-inoculation of A. cylindrica + A. brasilense. Co-inoculation resulted in higher root growth and dry mass of the aerial part in both hybrids; however, the hybrid Balu 280 Pro presented higher responsivity. The co-inoculation of A. brasilense and A. cylindrica increased initial corn growth without altering the photosynthetic pigment levels.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

Microbiological quality analysis of inoculants based on Bradyrhizobium spp. and Azospirillum brasilense produced “on farm” reveals high contamination with non-target microorganisms

The use of inoculants carrying diazotrophic and other plant growth–promoting bacteria plays an essential role in the Brazilian agriculture, with a growing use of microorganism-based bioproducts. However, in the last few years, some farmers have multiplied microorganisms in the farm, known as “on farm” production, including inoculants of Bradyrhizobium spp. for soybean (Glycine max L. Merrill.) and Azospirillum brasilense for corn (Zea mays L.) or co-inoculation in soybean. The objective was to assess the microbiological quality of such inoculants concerning the target microorganisms and contaminants. In the laboratory, 18 samples taken in five states were serial diluted and spread on culture media for obtaining pure and morphologically distinct colonies of bacteria, totaling 85 isolates. Molecular analysis based on partial sequencing of the 16S rRNA gene revealed 25 genera of which 44% harbor species potentially pathogenic to humans; only one of the isolates was identified as Azospirillum brasilense, whereas no isolate was identified as Bradyrhizobium. Among 34 isolates belonging to genera harboring species potentially pathogenic to humans, 12 had no resistance to antibiotics, six presented intrinsic resistance, and 18 presented non-intrinsic resistance to at least one antibiotic. One of the samples analyzed with a shotgun-based metagenomics approach to check for the microbial diversity showed several genera of microorganisms, mainly Acetobacter (~ 32% of sequences) but not the target microorganism. The samples of inoculants produced on farm were highly contaminated with non-target microorganisms, some of them carrying multiple resistances to antibiotics.

Azospirillum Actinidiae sp. nov., a Nitrogen-Fixing Bacterium Isolated From The Roots of Kiwifruit Plants

A novel diazotrophic bacterium, designated CCTCC AB 2021101T, was isolated from fresh roots of kiwifruit. Cells of strain CCTCC AB 2021101T were Gram-negative, aerobic and rod-shaped, with motility provided by peritrichous flagella. The 16S rRNA analysis showed that strain CCTCC AB 2021101T belongs to the genus Azospirillum and is closely related to Azospirillum melinis (98.32%), Azospirillum oryzae (97.73%), Azospirillum lipoferum (96.98%), Azospirillum humicireducens (96.49%) and Azospirillum largimobile (96.01%) and lower sequence similarity (<96.0 %) to all other species of the genus Azospirillum. Strain CCTCC AB 2021101T was able to grow well at 35–40℃ and pH 6.0–7.0, and tolerated up to 3.0 % (w/v) NaCl. The major saturated fatty acids are C14:0, C16:0 and C18:0. C18:1 ω7c and C16:0 3-OH were the major unsaturated and hydroxylated fatty acid. The G+C content was 67.8 mol%. Strain CCTCC AB 2021101T gave positive amplification for dinitrogen reductase (nifH gene). Highest nifH gene sequence similarities were obtained with Azospirillum brasilense AWB14T(95.9%), Azospirillum zeae Gr24T(95.56%), Azospirillum picis DSM 19922T(96.79%), Azospirillum lipoferum B22T(94.88%) and Azospirillum oryzae COC8T(94.88%). The activity of the nitrogenase of the strain was further confirmed by acetylene-reduction assay, which was recorded as 81 nmol ethylene h-1. Based on these data, strain CCTCC AB 2021101T is considered to represent a novel endophytic diazotrophs species in the genus Azospirillum, for which the name Azospirillum actinidiae sp. nov. is proposed. The type strain is CCTCC AB 2021101T.