Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.

Evaluation and biochemical characterization of a distinctive pyoverdin from a pseudomonas isolated from chickpea rhizosphere

Microbial siderophores confiscate the available ferric ions around the roots and trigger a reaction resulting in plant growth promotion. In our study, a high level of siderophore production was observed from a newly isolated Pseudomonas sp. from the rhizosphere of Chickpea plants. Under an iron depleted condition in Standard Succinic acid medium a 1000 µgmL-1 of siderophore production was achieved. Increasing the concentration of iron showed an inverse relationship between growth and siderophore production. Fourier Transform Infrared Spectroscopy (FTIR) analysis of the purified crystals, its UV spectral analysis and High Pressure Liquid Chromatography (HPLC) revealed the identity of the siderophore as similar to that of pyoverdin with distinctive characters. Electron spray ionization mass spectroscopy (ESIMS) shows presence of abundance of A1 ions (419 m/z) and branching of amino acids from B1-B5. This pyoverdin contains a cyclic tetra peptide but Serine and Arginine are missing. Based on our analysis and deviations from the reported structure of pyoverdin it is suggested that this pseudomonas produces distinctly characterized pyoverdin siderophore.

Genetic diversity of siderophore-producing bacteria of tobacco rhizosphere / Diversidade genética de bactérias de rizosfera de tabaco produtoras de sideróforos

The genetic diversity of siderophore-producing bacteria of tobacco rhizosphere was studied by amplifiedribosomal DNA restriction analysis (ARDRA), 16S rRNA sequence homology and phylogenetics analysis methods. Studies demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium. A total of 28 ARDRA patterns were identified among the 299 siderophore-producing bacterial isolates.The 28 ARDRA patterns represented bacteria of 14 different genera belonging to six bacterial divisions, namely β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli, and Actinobacteria. Especially, γ- Proteobacteria consisting of Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia and Stenotrophomonas genus encountered 18 different ARDRA groups. Results also showed a greater siderophore-producing bacterial diversity than previous researches. For example, Sphingobacterium (isolates G-2-21-1 and G-2-27-2), Pseudomonas poae (isolate G-2-1-1), Enterobacter endosymbiont (isolates G-2-10-2 and N-5-10), Delftia acidovorans (isolate G-1-15), and Achromobacter xylosoxidans (isolates N-46-11HH and N-5-20) were reported to be able to produce siderophores under low-iron conditions for the first time. Gram-negative isolates were more frequently encountered, with more than 95% total frequency. For Gram-positive bacteria, the Bacillus and Rhodococcus were the only two genera, with 1.7% total frequency. Furthermore, the Pseudomonas and Enterobacter were dominant in this environment, with 44.5% and 24.7% total frequency, respectively. It was also found that 75 percent of the isolates that had the high percentages of siderophore units (% between 40 and 60) belonged to Pseudomonas. Pseudomonas sp. G-229-21 screened out in this study may have potential to apply to low-iron soil to prevent plant soil-borne fungal pathogen diseases.

A diversidade genética de bactérias de rizosfera de tabaco produtoras de sideróforos foi estudada por meio da técnica de análise de restrição do DNA ribossomal amplificado (ARDRA), homologia de seqüência de 16s rRNA e métodos de análise filogenética. Observou-se que 85% do total de 354 isolados produziram sideróforos em meio liquido com restrição de ferro.Entre os 299 isolados produtores de sideróforos identificou-se 28 padrões ARDRA, que representaram 14 gêneros bacterianos diferentes, pertencentes a seis divisões bacterianas: β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli e Actinobacteria. γ- Proteobacteria, consistindo de Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia e Stenotrophomonas, pertenceram a 18 grupos ARDRA. Os resultados também mostraram uma diversidade maior de bactérias produtoras de sideróforos doque a relatada em outros estudos. Por exemplo, Sphingobacterium (isolados G-2-21-1 e G-2-27-2), Pseudomonaspoae (isolado G-2-1-1), Enterobacter endosymbiont (isolados G-2-10-2 e N-5-10), Delftia acidovorans (isolado G-1-15) e Achromobacter xylosoxidans (isolados N-46-1HH e N-5-20), capazes de produzir sideróforos em condições de baixa disponibilidade de ferro, foram relatados pela primeira vez. Isolados Gram negativos foram encontrados com maior freqüência, correspondendo a mais de 95% da freqüência total. Entre as bactérias Gram positivas, foram encontrados apenas os gêneros Bacillus e Rhodococcus, com 1,7% da freqüência total. Além disso, neste ambiente houve predominância de Pseudomonas e Enterobacter, com 44,5% e 24,7% da freqüência total, respectivamente. Verificou-se também que 75% dos isolados com alta porcentagem de unidades de sideróforos (% entre 40 e 60) pertenceram a Pseudomonas. Pseudomonas sp. G- 229-21, selecionado neste estudo, apresenta potencial de aplicação em solos com baixo teor de ferro para prevenção dedoenças fúngicas em plantas.