Virulence and indole-3-acetic acid (IAA) biosynthesis ability of Turkish Pseudomonas savastanoi pv. savastanoi isolates and susceptibility of some native olive genotypes

Aim of study: To evaluate the virulence and indole-3-acetic acid (IAA) biosynthesis ability of several Turkish P. savastanoi pv. savastanoi isolates and the susceptibility of some native genotypes to olive knot. Area of study: The Aegean, Marmara, and Mediterranean Regions of Turkey. Material and methods: 101 isolated bacteria were identified on the basis of biochemical, PCR for amplification of the bacterial iaaL gene, and pathogenicity tests. The virulence of the isolates was determined in a randomized experimental trial carried out by stem inoculation of pot-grown seedlings of olive (cv. ‘Manzanilla’) in the growing chamber. The amounts of IAA produced by the isolates were determined colorimetrically. The susceptibility of native olive genotypes was evaluated on 2-yr old plants inoculated with two distinct strains. Main results: Tested P. savastanoi pv. savastanoi isolates showed significant differences in virulence found to be associated with their geographical origin. The isolates produced IAA amounts varied from 148.67 to 0.3 μg mL-1. The geographical variation in IAA biosynthesis ability of the isolates was observed. No correlation (R=0.0225) was determined between virulence and IAA amounts of the isolates. Native olive genotypes indicated different susceptibility levels to the olive knot pathogen. No genotype tested had complete resistance. However, low susceptible genotypes (‘Memecik’, ‘Ayvalık’ and ‘Uslu’) were identified. Some genotypes had variable reactions depending on the isolate used. Research highlights: The results undergird the differences in the virulence and IAA production of the isolates within the area and also between geographical locations. Genotypes with low susceptibility can be used as genitors in further breeding studies.

Glycolate oxidase-dependent H2O2 production regulates IAA biosynthesis in rice

Background Glycolate oxidase (GLO) is not only a key enzyme in photorespiration but also a major engine for H2O2 production in plants. Catalase (CAT)-dependent H2O2 decomposition has been previously reported to be involved in the regulation of IAA biosynthesis. However, it is still not known which mechanism contributed to the H2O2 production in IAA regulation. Results In this study, we found that in glo mutants of rice, as H2O2 levels decreased IAA contents significantly increased, whereas high CO2 abolished the difference in H2O2 and IAA contents between glo mutants and WT. Further analyses showed that tryptophan (Trp, the precursor for IAA biosynthesis in the Trp-dependent biosynthetic pathway) also accumulated due to increased tryptophan synthetase β (TSB) activity. Moreover, expression of the genes involved in Trp-dependent IAA biosynthesis and IBA to IAA conversion were correspondingly up-regulated, further implicating that both pathways contribute to IAA biosynthesis as mediated by the GLO-dependent production of H2O2. Conclusion We investigated the function of GLO in IAA signaling in different levels from transcription, enzyme activities to metabolic levels. The results suggest that GLO-dependent H2O2 signaling, essentially via photorespiration, confers regulation over IAA biosynthesis in rice plants.

Caenorhabditis elegans Extracts Stimulate IAA Biosynthesis in Arthrobacter pascens ZZ21 via the Indole-3-pyruvic Acid Pathway

Inter-organismal metabolites play important roles in regulating organism behavior and the communication between organisms. Nematodes, the most abundant animals on earth, are crucial participants in soil ecosystems through their interactions with microbes. For example, bacterial-feeding nematodes increase the activity of indole-3-acetic acid (IAA)-producing bacteria and the IAA content in soil. However, the way in which these nematodes interact with bacteria and affect IAA biosynthesis is not well understood. Here, using the model nematode Caenorhabditis elegans and the plant-beneficial bacterium Arthrobacter pascens ZZ21, we examined the effects of nematode excretions or extracts on bacterial IAA biosynthesis. To explore the underlying regulatory mechanism in more detail, we performed transcriptome sequencing and metabolomic analysis. Our findings suggest that C. elegans extracts promote IAA biosynthesis in A. pascens ZZ21 by increasing the expression of genes and the abundance of intermediates involved in the indole-3-pyruvic acid (IPyA) pathway. C. elegans extracts also significantly influenced biosynthetic and metabolic activity in A. pascens ZZ21. Treatment with C. elegans extracts promoted pyruvate metabolism, the citrate cycle (TCA) cycle and the production of some TCA-cycle-related amino acids and inhibited oxidative phosphorylation, which induced the accumulation of reduced nicotinamide adenine dinucleotide (NADH). We propose that the extracts altered the metabolism of A. pascens ZZ21 to help the bacteria resist stress caused by their predator. Our findings indicate that bacterial-feeding nematodes mediate the interaction between nematodes and bacteria via their extracts, providing insights into the ecological function of C. elegans in soil.

Indole-3-Acetic Acid Is Synthesized by the Endophyte Cyanodermella asteris via a Tryptophan-Dependent and -Independent Way and Mediates the Interaction with a Non-Host Plant

The plant hormone indole-3-acetic acid (IAA) is one of the main signals playing a role in the communication between host and endophytes. Endophytes can synthesize IAA de novo to influence the IAA homeostasis in plants. Although much is known about IAA biosynthesis in microorganisms, there is still less known about the pathway by which IAA is synthesized in fungal endophytes. The aim of this study is to examine a possible IAA biosynthesis pathway in Cyanodermella asteris. In vitro cultures of C. asteris were incubated with the IAA precursors tryptophan (Trp) and indole, as well as possible intermediates, and they were additionally treated with IAA biosynthesis inhibitors (2-mercaptobenzimidazole and yucasin DF) to elucidate possible IAA biosynthesis pathways. It was shown that (a) C. asteris synthesized IAA without adding precursors; (b) indole-3-acetonitrile (IAN), indole-3-acetamide (IAM), and indole-3-acetaldehyde (IAD) increased IAA biosynthesis; and (c) C. asteris synthesized IAA also by a Trp-independent pathway. Together with the genome information of C. asteris, the possible IAA biosynthesis pathways found can improve the understanding of IAA biosynthesis in fungal endophytes. The uptake of fungal IAA into Arabidopsis thaliana is necessary for the induction of lateral roots and other fungus-related growth phenotypes, since the application of the influx inhibitor 2-naphthoxyacetic acid (NOA) but not the efflux inhibitor N-1-naphtylphthalamic acid (NPA) were altering these parameters. In addition, the root phenotype of the mutation in an influx carrier, aux1, was partially rescued by C. asteris.

Characterization and synthesis of indole-3-acetic acid in plant growth promoting Enterobacter sp.

The strains have remarkable IAA-producing capabilities. Genomic analysis and intermediate assay indicated the involvement of the indole-3-pyruvic acid pathway of IAA biosynthesis. These microbes significantly promoted the growth of maize.

Deciphering Operation of Tryptophan Independent Pathway in High Indole -3-Acetic Acid (IAA) producing Micrococcus aloeverae DCB-20

Genus Micrococcus is considered a high IAA producer. However, interestingly, there is no report on the tryptophan independent pathway operation in this genus. Consequently, the present study was undertaken to evaluate high IAA production by Micrococcus aloeverae DCB-20 and generate reasonable evidence for the occurrence of the tryptophan-independent pathway. Strain DCB-20 produced a high quantity of 880.51 µM or 154.3 µg ml−1 IAA in LB broth supplemented with L-tryptophan. The tryptophan independent pathway operation was supported by IAA production in Tris-minimal broth (TM broth) medium supplemented with acid hydrolyzed casein, which lacks tryptophan. The HPLC analysis showed the absence of tryptophan either from exogenous or endogenous sources in TM broth in the presence of casein acid hydrolysate inoculated with M. aloeverae DCB-20. The absence of tryptophan was further confirmed by the appearance of non-pigmented colonies of Chromobacterium violaceum TRFM-24 on Tris-minimal agar (TM agar) containing acid-hydrolyzed casein. This is probably the first report on IAA biosynthesis by M. aloeverae DCB-20 employing tryptophan independent pathway. This simple technique can also be adapted to detect operation of the tryptophan independent pathway in other bacteria.

Highly Efficient Biosynthesis of Indole-3-acetic acid by Enterobacter xiangfangensis BHW6

Background: Indole-3-acetic acid (IAA) plays an important role in the growth and development of plants. Various bacteria in the rhizosphere are capable to produce IAA that acts as a signaling molecule for the communication between plants and microbes to promote the plant growth. Due to the low IAA content and various interfering analogs, it is difficult to detect and isolate IAA from microbial secondary metabolites. Results: A predominant strain with a remarkable capability to secrete IAA was identified as Enterobacter xiangfangensis BHW6 based on 16S rRNA gene sequence, the determination of average nucleotide identity (ANI) and digital DDH (dDDH). The maximum IAA content (134-1129 μg/mL) was found with the addition of 0.2-15 g/L of L-tryptophan at pH 5 for 6 days, which was 4-40 fold higher than that in the absence of L-tryptophan. The highest yield of IAA was obtained at the stationary phase of bacterial growth. An acidic culture medium was preferred for the IAA biosynthesis of the strain. The strain was tolerant and stable to produce IAA in the presence 2.5%-5% (w/v) of NaCl. IAA was then isolated through column chromatography with a mobile phase of hexane/ethyl acetate (1/2, v/v) and characterized by 1H Nuclear Magnetic Resonance (1H NMR). Conclusions: A remarkable IAA production was obtained from E. xiangfangensis BHW6 that was tryptophan–dependent. According to genomic analysis, the ipdC gene coding for the key enzyme (indole-3-pyruvate decarboxylase) was identified indicating that IAA biosynthesis was mainly through the indole-3-pyruvia acid (IPyA) pathway, which was further confirmed by intermediate assay. E. xiangfangensis BHW6 with an important economic value has great prospect in agricultural and industrial application.

An Evolutionarily Primitive and Distinct Auxin Metabolism in the Lycophyte Selaginella moellendorffii

Auxin is a key regulator of plant growth and development. Indole-3-acetic acid (IAA), a plant auxin, is mainly produced from tryptophan via indole-3-pyruvate (IPA) in both bryophytes and angiosperms. Angiosperms have multiple, well-documented IAA inactivation pathways, involving conjugation to IAA-aspartate (IAA-Asp)/glutamate by the GH3 auxin-amido synthetases, and oxidation to 2-oxindole-3-acetic acid (oxIAA) by the DAO proteins. However, IAA biosynthesis and inactivation processes remain elusive in lycophytes, an early lineage of spore-producing vascular plants. In this article, we studied IAA biosynthesis and inactivation in the lycophyte Selaginella moellendorffii. We demonstrate that S. moellendorffii mainly produces IAA from the IPA pathway for the regulation of root growth and response to high temperature, similar to the angiosperm Arabidopsis. However, S. moellendorffii exhibits a unique IAA metabolite profile with high IAA-Asp and low oxIAA levels, distinct from Arabidopsis and the bryophyte Marchantia polymorpha, suggesting that the GH3 family is integral for IAA homeostasis in the lycophytes. The DAO homologs in S. moellendorffii share only limited similarity to the well-characterized rice and Arabidopsis DAO proteins. We therefore suggest that these enzymes may have a limited role in IAA homeostasis in S. moellendorffii compared to angiosperms. We provide new insights into the functional diversification of auxin metabolic genes in the evolution of land plants.

Metabolites of Bacterial-Feeding Nematodes Stimulate Bacterial Indole-3-Acetic Acid (IAA) Synthesis

Interorganismal metabolites play significant roles in regulating behaviors and communications between organisms. Nematodes are the most abundant animals on earth, and function well in soil ecosystem due to their interactions with microbes. Bacterial-feeding nematodes stimulate the activity of indole-3-acetic acid (IAA)-producing bacteria and increase the content of IAA in soil. However, we do not fully understand how bacterial-feeding nematodes interact with bacteria and affect IAA synthesis. In this study, the model nematode Caenorhabditis elegans and three species of soil-dwelling IAA-producing bacteria ( Bacillus amyloliquefaciens JX1, Arthrobacter pascens ZZ21 and A. chlorphenolicus L4) were employed to determine the effect of nematodes on the IAA biosynthesis of bacteria. Then the metabolites and extracts of C. elegans were tested the effect on three bacterial IAA synthesis (but only A. pascens ZZ21 for the extracts). Lastly, two soil-dwelling bacterial-feeding nematodes ( Mesorhabditis sp. and Acrobeloides sp.) and two IAA-producing bacteria ( B. amyloliquefaciens JX1 and A. pascens ZZ21) were subsequently used to explore the universality of this interaction. Our results showed that the metabolites or extracts of nematodes could promote the IAA biosynthesis of IAA-producing bacteria, and implied this stimulatory effect maybe widely spread in metabolites of bacterial-feeding nematodes and IAA-producing bacteria, but vary with nematodes and bacteria species. Our findings indicate that bacterial-feeding nematodes could mediate the interaction between nematodes and bacteria by their metabolites, except for their feeding behavior, and offer insights into the ecological function of the metabolites of nematodes.