Three Enterobacter agglomerans strains which produce and excrete proteins with chitinolytic activity were found while screening soil-borne bacteria antagonistic to fungal plant pathogens. The chitinolytic activity was induced when the strains were grown in the presence of colloidal chitin as the sole carbon source. It was quantitated by using assays with chromogenic p-nitrophenyl analogs of disaccharide, trisaccharide, and tetrasaccharide derivatives of N-acetylglucosamine. A set of three fluorescent substrates with a 4-methylumbelliferyl group linked by -1,4 linkage to N-acetylglucosamine mono-or oligosaccharides were used to identify the chitinolytic activities of proteins which had been renatured following their separation by electrophoresis. This study provides the most complete evidence for the presence of a complex of chitinolytic enzymes in Enterobacter strains. Four enzymes were detected: two N-acetyl--D-glucosaminidases of 89 and 67 kDa, an endochitinase with an apparent molecular mass of 59 kDa, and a chitobiosidase of 50 kDa. The biocontrol ability of the chitinolytic strains was demonstrated under greenhouse conditions. The bacteria decreased the incidence of disease caused by Rhizoctonia solani in cotton by 64 to 86%. Two Tn5 mutants of one of the isolates, which were deficient in chitinolytic activity, were unable to protect plants against the disease.
The biocontrol activity of various fluorescent pseudomonads towards plant-pathogenic fungi is dependent upon the GacA/GacS-type two-component system of global regulators and the RpoS transcription sigma factor. In particular, these components are required for the production of antifungal antibiotics and exoenzymes. To investigate the effects of these global regulators on the expression of biocontrol factors by plant-associated bacteria other than Pseudomonas spp., gacA/gacS and rpoS homologues were cloned from biocontrol strain IC1270 of Serratia plymuthica, which produces a set of antifungal compounds, including chitinolytic enzymes and the antibiotic pyrrolnitrin. The nucleotide and deduced protein sequence alignments of the cloned gacA/gacS-like genes-tentatively designated grrA (global response regulation activator) and grrS (global response regulation sensor) and of the cloned rpoS gene revealed 64 to 93% identity with matching genes and proteins of the enteric bacteria Escherichia coli, Pectobacterium carotovora subsp. carotovora, and Serratia marcescens. grrA, grrS, and rpoS gene replacement mutants of strain IC1270 were deficient in the production of pyrrolnitrin, an exoprotease, and N-acylhomoserine lactone quorum-sensing signal molecules. However, neither mutant appeared to differ from the parental strain in the production of siderophores, and only grrA and grrS mutants were deficient in the production of a 58-kDa endochitinase, representing the involvement of other sigma factors in the regulation of strain IC1270's chitinolytic activity. Compared to the parental strain, the grrA, grrS, and rpoS mutants were markedly less capable of suppressing Rhizoctonia solani and Pythium aphanidermatum under greenhouse conditions, indicating the dependence of strain IC1270's biocontrol property on the GrrA/GrrS and RpoS global regulators.
The purpose of the present research was to study the diversity of bacterial endophytes inhabiting the halophytic plant black saxaul (Haloxylon aphyllum Minkw.). A total of 20 bacterial isolates were isolated from tissues of black saxaul and identified based on their 16S rRNA genes analysis and comparison with the closest relatives registered in GenBank nucleotide data bank from the National Centre for Biotechnology Information. The endophytes were checked for plant growth-promoting activity toward cucumber plants and the strains Bacillus amyloliquefaciens HAPH2, Priestia endophytica HAPH5, Bacillus subtilis HAPH7, Bacillus toyonensis HAPH8, Halomonas sulfidaeris HAPH11, Isoptericola halotolerans HAPH12, Planomicrobium soli HAPH15, and Pseudomonas kilonensis HAPH16 demonstrated high plant growth-promoting activity of cucumber in four soil salinity levels (0, 25, 50, and 100 mM) after seeds' inoculation. These bacterial endophytes were able to fix nitrogen, solubilize phosphates, and produce indole-3-acetic acid, 1-aminocyclopropane-1-carboxylate deaminase, and siderophores which are considered as the main plant growth-promoting traits. After field experiments, the best plant growth-promoters can be used as bioinoculants for plants' growth improvement in salinity conditions.
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