Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism VI. The Influence of the Epiphytic Bacteria on the Content of Extractable Auxin in the Plant

Sterile plants of maize, pea, and cucumber contain less auxin (extracted with methanol or ether) than nonsterile ones. The auxin content is restored within one day by reinfecting sterile plants (or only the shoots, with roots and culture medium remaining sterile) with epiphytic bacteria strains able to produce IAA or with soaking water of nonsterile seeds. Reinfection with bacteria, strains unable to produce IAA is ineffective. — The possibility of a bacterial auxin production during methanol extraction was excluded.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metaholism

During an ether extraction (20 h, 4°C), nonsterilc pea epicotyls deliver at least 5 times more auxin than sterile ones. A great part of the additional auxin originates from a bacterial auxin production during the extraction. Presence of chloramphenicol or streptomycin during the extraction lowers the auxin amount extractable from nonsterile epicotyls. In extraction conditions (i.e. covered with ether, 20 h, 4°C), epiphytic bacteria contained in homogenates of nonsterile plant parts produce IAA from added tryptophan. Furthermore, first results are presented underlining the fact that epiphytic bacteria already produce auxin at the surface of nonsterile plant parts (before an extraction).     

Epiphytic microorganisms and IAA synthesis

Summary Epiphytic microorganisms present on cotton plants synthesized 3-indoleacetic acid (IAA) from tryptophan. Microorganisms from the root zone synthesized 3 times the amount of IAA when compared with the shoot zone and the root zone contained a much higher number of microorganisms. IAA-synthesizing activity was eliminated when the tissues were treated with a weak solution of mercuric chloride. Various tests on the possible accumulation of IAA from external sources showed that IAA synthesized outside the plant does not accumulate in the plant. Although epiphytic microorganisms synthesize IAA in large amounts, they do not influence the IAA content of the plant due to (1) lack of available tryptophan, (2) destruction of the auxin by the microflora, and (3) the polar movement of the auxin.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Homogenates of epicotyls or roots of nonsterile pea plants incubated with tryptophan produce IAA within 1 to 4 hours, which was detected by means of the Avena curvature test and thin layer chromatography. Three results prove this short-term IAA production to be mainly caused by epiphytic bacteria: 1) Homogenates of sterile plant parts catalyze a conversion of tryptophan to IAA, a hundredfold lower. 2) Chloramphenicol or streptomycin very actively reduce the IAA gain obtained with nonsterile homogenates. 3) Washing solutions of nonsterile plant parts which do not contain plant enzymes but only epiphytic bacteria, produce IAA from tryptophan, too. IAA synthesis from tryptophan in vitro by enzymes of the pea plant occurs with lower intensity than hitherto known; possibly it is physiologically unimportant. It is discussed to what extent the hitherto existing research work about the IAA biogenesis in higher plants might be incriminated by disregarding tbe rôle of epiphytic bacteria.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

More “diffusible” auxin is received from nonsterile than from sterile corn coleoptile tips. An artificial reinfection of sterile coleoptiles with epiphytic, IAA-producing bacteria strains does, a superinfection of nonsterile coleoptiles does not increase the auxin amount. The difference between sterile and nonsterile tips persists if diffusion from the coleoptile surface is excluded by covering the surface with a paraffin layer. The greater the distance from the apex, the higher becomes the superiority of nonsterile tips. An artificial bacterial contamination of the contact face between tip and receiver agar block, or addition of glucose and tryptophan to the agar block, do not influence the received auxin amount. Consequently the additional, bacteria-produced auxin delivered by the nonsterile tip is not produced at the cut surface or in the agar but is present in the tissues of the coleoptile tip.     

The influence of epiphytic bacteriae on auxin metabolism

Summary Plants are settled by epiphytic bacteriae able to convert tryptophan to IAA. This bacterial activity is abolished by chloramphenicol and streptomycin but not by penicillin. Tryptophan conversion to IAA by plant parts or enzyme preparations is far more intensive in non-sterile conditions than in sterile ones. This is true for all investigated objects: Helianthus annuus, Phaseolus vulgaris, Pisum sativum, Triticum vulgare, Zea mays, Enteromorpha compressa, Fucus vesiculosus, Furcellaria fastigiata. From pea plants, 58 strains of IAA producing bacteriae were isolated and partly identified.While non-sterile plants (Pisum, Zea) contain considerable amounts of IAA (extraction, thin layer chromatography, biotest), hardly any traceable auxin can be extracted of sterile plants. But sterile plants re-infected with mixtures or single strains of suitable bacteriae again contain considerable amounts of extractable IAA.

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Zusammenfassung Pflanzen sind von epiphytischen Bakterien besiedelt, die Tryptophan zu IES umsetzen können. Diese bakterielle Aktivität wird unterbunden durch Chloramphenicol und Streptomycin, aber nicht durch Penicillin. Der Tryptophanumsatz zu IES durch Pflanzenteile oder Enzympräparate ist unter unsterilen Bedingungen weit intensiver als unter sterilen. Das trifft für alle untersuchten Objekte zu: Helianthus annuus, Phaseolus vulgaris, Pisum sativum, Triticum vulgare, Zea mays, Enteromorpha compressa, Fucus vesiculosus, Furcellaria fastigiata. Von Erbsenpflanzen konnten 58 Stämme IES-produzierender Bakterien isoliert und zum Teil identifiziert werden.Während unsterile Pflanzen (Pisum, Zea) beträchtliche IES-Mengen enthalten (Extraktion, Dünnschichtchromatographie, Biotest), kann aus sterilen Pflanzen kaum nachweisbares Auxin extrahiert werden. Aber sterile, mit Mischungen oder einzelnen Stämmen geeigneter Bakterien re-infizierte Pflanzen enthalten wieder beträchtliche Menge extrahierbarer IES.

     

The influence of gamma radiation on the biosynthesis of indoles and gibberellins in barley the action of zinc on the restitution of growth substance level in irradiated plants

Investigations were made on the effect of exposing barley seeds to gamma-radiation (5–40 kR), alone and in combination with the application of zinc (soaking the seeds in solutions containing 5.10^?5–5.10^?1% Zn for 12 hours before sowing) on growth and on the content of tryptophan, indole auxins and gibberellin-like substances in seven-day plants. Radiation decreased both growth and the content of tryptophan (e.g. by about 53% at 30 kR), of indole auxins (by about 60% auxin in the zone of IAA on the chromatogram at 30 kR), and also the content of gibberellin-like substances (by about 67% gibberellin content in the zone of GA₃ on the chromatogram) of plants. The irradiation of standard samples of tryptophan, indolyl-acetic acid and gibberellic acid alone with many times greater doses (up to 1000 kR) did not lead to marked radiochemical degradation of these substances. It can be assumed that radiation damages the enzyme systems “synthesizing” natural growth substances in plants. The damaging effect of radiation on auxins is already displayed in the synthesis of tryptophan, which is inhibited. Zinc interacts with the damaging effect of radiation on growth. Optimum concentrations of zinc (5.10^?3% Zn) counteract the effect of radiation, up to doses of about 12 kR, on the growth in height in 7-day plants so that it is equal to the controls. Normal content of tryptophan and auxin in the position of indolecetic acid on chromatograms can only be reached by the addition of zinc when the dose of radiation was not greater than about 8 kR, which is less than the influence exerted by zinc on the restitution of growth. On the other hand, the biosynthesis of gibberellin-like substances at the position of gibberellic acid on chromatograms can be restored by zinc to their original level to doses of up to 30 kR. The increased biosynthesis of auxins and gibberellins caused by zinc in irradiated plants is explained by the activation of the remaining and non—damaged enzyme systems carrying out this biosynthesis. The activation of the biosynthesis of growth substances by zinc will also contribute to the restitution effect of zinc on the growth of plants from irradiated seeds.     

The effect of microorganisms and sorption on the protease activity of plant roots

The protease activity of sterile roots of wheat was zero or very low, so that the determined values did not exceed limits of the experimental error. Roots colonized by microorganisms had a significant protease activity. The activity of protease on seeds and roots of the plants growing in a medium inoculated with the soil microflora was higher than in cases when only the epiphytic microflora of seeds served as a source of microorganisms. Sterile roots inoculated with three different strains of bacteria isolated from the rhizosphere and producing protease exhibited a considerable protease activity. The protease activity of non-sterile roots of plants growing in the dark was higher than that of plants growing under normal illumination. Crystalline proteinase was adsorbed on sterile roots and the activity of the enzyme was decreased in this adsorbed state. The adsorption of the enzyme was only slightly higher in the presence of calcium ions. Treatment of roots with a sodum chloride solution, with dextran and ethanol increased the adsorption of the proteinase by roots.     

Bacteria associated with orchid roots and microbial production of auxin

Associative bacteria of terrestrial (Paphiopedilum appletonianum) and epiphytic (Pholidota articulata) tropical orchids were investigated. Microbial community of epiphytic plant differed from that of the terrestrial one. Streptomyces, Bacillus, Pseudomonas, Burkholderia, Erwinia and Nocardia strains populated Paphiopedilum roots, whereas Pseudomonas, Flavobacterium, Stenotrophomonas, Pantoea, Chryseobacterium, Bacillus, Agrobacterium, Erwinia, Burkholderia and Paracoccus strains colonized Pholidota roots. Endophytic bacteria populations were represented with less diversity: Streptomyces, Bacillus, Erwinia and Pseudomonas genera were isolated from P. appletonianum, and Pseudomonas, Bacillus, and Flavobacterium genera were isolated from Ph. articulata. Microorganisms produced indole-3-acetic acid (IAA). Variations in its biosynthesis among the strains of the same genus were also observed. The highest auxin level was detected during the stationary growth phase. Biological activity of microbial IAA was proved by treatment of kidney bean cuttings with bacterial supernatants, revealing considerable stimulation of root formation and growth.     

B. thuringiensis is a Poor Colonist of Leaf Surfaces

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10⁶ cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10⁷ cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.     

Production of auxin and related compounds by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita

Mass spectrometric analysis revealed the presence of auxin, mainly in conjugated form, in secretions of Heterodera schachtii and Meloidogyne incognita, with or without treatment with DMT or resorcinol. M. incognita showed the highest production rates, though treatment of M. incognita with resorcinol had a negative effect on auxin production. Analysis of auxin precursor molecules in lysates of H. schachtii, M. incognita and Caenorhabditis elegans suggested that auxin is most probably a degradation product of tryptophan and that auxin may be synthesized via several intermediates, including indole-3-acetamide which is an intermediate of a pathway so far only characterized in bacteria. Furthermore, high levels of anthranilate, a degradation product of tryptophan in animals, but possibly also a precursor for auxin were detected.     

Gibberellin Induced Changes in Diffusible Auxins from Savoy Cabbage

Diffusates from apices of young plants of savoy cabbage treated with gibberellie acid (GA) and apices of control plants have been examined with respect to their content of Indole auxins. Three indole Compounds were detected and identified on the basis of their chromatographic characteristics in several systems. These compounds were: glucoubrassicin, indole-3-acetic acid (IAA) and indole-3-acetonitrile (IAN). An effect of GA on the total auxin activity of the diffusate was noted 90 hours after treatment, while an increase in stem height occurred 48 hours later. This increase in auxin effect of the entire diffusates was shown bv chromogenic development and bioassay of chromatograms of diffusates to be a result of an increase in level of the IAA content. A concomitant decrease in I the glucobrassicin content was indicated. Since GA was found to have no effect on the enzymatic conversion of tryptophan or tryptamine to IAA, it is proposed that the effect of GA is on the conversion of glucobrassicin to IAA.     

Characterisation of root amino acid exudation in white clover (Trifolium repens L.)

Lateral roots are crucial for the plasticity of root responses to environmental conditions in soil. The bacterivorous microfauna has been shown to increase root branching and to foster auxin producing soil bacteria. However, information on modifications of plant internal auxin content by soil bacteria and bacterivores is missing. Therefore, the effects of a rhizosphere bacterial community and a common soil amoeba (Acanthamoeba castellanii) on root branching and on auxin (indole-3-acetic acid) metabolism in Lepidium sativum and Arabidopsis thaliana were investigated. In a first experimental series, bacteria increased conjugated auxin concentrations in L. sativum shoots, but did not alter free bioactive auxin content nor root branching. In contrast, in presence of soil bacteria plus amoebae free auxin concentrations in shoots and root branching increased, demonstrating that effects of bacteria on auxin metabolism in plants were strongly modified by the bacterivorous amoebae. In a second experiment, A. thaliana reporter plants for auxin (DR5) and cytokinin (ARR5) responded similarly with increased root branching in the presence of amoebae. Surprisingly, in reporter plants cytokinin but not auxin responses were detectable, accompanied by higher soil nitrate concentrations in the presence of amoebae. Likely, increased nitrate concentrations in the rhizosphere led to an accumulation of cytokinin and interactions with free auxin in plants and finally to increased root growth in the presence of amoebae. Altogether, the results show that mutual control mechanisms exist between plant hormone metabolism and microbial signalling, and that effects on hormonal concentrations of plants by free-living bacteria are strongly influenced by bacterial grazers like amoebae.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Proofs of different kind are presented of the existence of highly active bacteria producing IAA from tryptophan on plant surfaces and in plant homogenates. Both homogenates and washing solutions of nonsterile pea plant parts are active in producing IAA from tryptophan. Activity is much enhanced by the addition of glucose or by preincubating the preparations; it is abolished by sterile filtration, by some bactericidic and bacteriostatic substances, by chloramphenicol, streptomycin, and albucid (penicillin being only partly effective). Preparations of sterile plants do not produce IAA from tryptophan within the detection limit of the Salkowski test. The bacteria are even present on seed surfaces, in the air, and in aceton or ammonium sulfate precipitations of homogenates. Main products of the bacterial tryptophan conversion, as demonstrated by paper chromatography, are indolepyruvic acid, indoleacetic acid, indolecarboxaldehyde, and indolecarboxylic acid. In presence of glucose indolepyruvic acid is by far dominating. Many hitherto known results about tryptophan conversion to IAA by higher plants are likely to be falsified by epiphytic bacteria.     

Mitigation of growth retardation effect of plant defense activator,acibenzolar-<Emphasis Type="Italic">S</Emphasis>-methyl,in amaranthus plants by plant growth-promoting rhizobacteria

Four rhizobacterial strains and acibenzolar-S-methyl (ASM), a chemical activator, which suppressed foliar blight of amaranthus (Amaranthus tricolor L.) caused by Rhizoctonia solani Kühn were evaluated for their effect on plant growth. The experiments were performed both under sterile and non-sterile soil conditions, in the presence or absence of the pathogen. In all cases, plants treated with ASM showed significant reduction in growth, as determined by shoot length, and shoot and root dry weight when compared to other treatments. The growth retardation effect of ASM was more profound with respect to shoot length. Reduction in shoot length was least when plants were treated with a combination of the chemical activator and Pseudomonas putida 89B61 under non-sterile soil conditions in the absence of the pathogen. Both under sterile and non-sterile soil conditions, in the presence of the pathogen, reduction in shoot length due to application of ASM was diminished significantly when plants were treated with rhizobacterial strain Pseudomonas fluorescens PN026R. Combined use of plant growth-promoting rhizobacteria (PGPR) and ASM was found to be beneficial as the growth retardation effect of the plant defense activator was reduced by the growth-promoting ability of the rhizobacteria.     

Rooting and establishment of Calabrian pine plantlets propagated in vitro: influence of growth substances, rooting medium and origin of explant

The effect of auxins and a cytokinin on induction of roots in cultured axillary shoots of Pinus brutia Ten, has been tested. Auxin was crucial for root initiation and the rooting response varied according to the type and concentration of auxin applied. Both auxin and cytokinin and the interactions between them affected the quantity and quality of the induced roots. Aerated non-sterile tap water was an effective rooting medium, comparable to agar. After planting out into soil, some of the influences of auxin and cytokinin could still be seen after six months. However, roots developed normally in the soil, displaying dichotomous lateral branches. The results draw attention to the need for care in the choice and application of the medium for the initial induction of roots. Results of greenhouse trials indicated that the most vigorous plants were obtained via the axillary shoots.     

Processes of plant colonization by Methylobacterium strains and some bacterial properties

The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacterium are indespensible inhabitants of the plant phyllosphere. Using maize Zea mays as a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied. A marked strain, Methylobacterium mesophilicum APR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves. Colonization of maize leaves by M. mesophilicum APR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface. In this case, the number of PPFMB cells on inoculated leaves increased with plant growth. During seed germination, no colonization of maize leaves with M. mesophilicum cells occurred immediately from the soil inoculated with the marked strain. Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air. PPFMB monocultures were not antagonistic to phytopathogenic bacteria. However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicum or M. extorquens did exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas camprestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense, and Agrobacterium tumifaciens). Neither epiphytic and soil strains of Methylobacterium extorquens, M. organophillum, M. mesophilicum, and M. fujisawaense catalyzed ice nucleation. Hence, they cause no frost injury to plants. Thus, the results indicate that the strains of the genus Methylobacterium can protect plants against adverse environmental factors.     

Effect of the culture medium and incubation time on auxins production by bacteria isolated from the roots of pine seedlings (Pinus silvestris l.).

Studies were performed on the effect of culture medium and incubation time on the production of auxins by bacteria. The bacteria studied produced more auxins in the mineral medium containing glucose and tryptophan than in that enriched with casamino acids and yeast extract. The amount of auxins elaborated depended both upon the strain and the age of the culture. Some strains produced the largest amounts of these substances after 7 days of incubation while others required a longer period. Most of the substances showing auxin activity were located on the chromatograms at Rf 0.3-0.4 and 0.8-1.0.     

Comparison of Diversity and Functions of Epiphytic Bacteria from Cultivated and Weed Plants in Agrocenoses

Diversity of epiphytic bacterial complexes from cultivated and weed plants was compared according to the frequency of predominance of bacterial taxa in these communities and using the principal component method. The presence of both common dominants on cultivated and weed plants and distinct differences in the composition of the epiphytic bacterial complexes of these plants was revealed. The representatives of chemolithotrophic bacteria capable of tetrathionate and thiosulfate assimilation were found only on weed plants. The antibiotic activity of bacteria isolated from weed plants was almost twice as high as that found for bacteria from cultivated plants. The obtained data indicate the positive effect of bacterial communities of weed plants, which make it possible to protect cultivated plants from phytopathogens.     

Growth substances in roots of tomato (Lycopersicon esculentum Mill.) infected with root-knot nematodes (Meloidogyne spp.)

Roots of tomato plants with galls caused by larvae of Meloidogyne spp. contained a similar concentration of auxin as uninfected roots, but a larger total amount because the roots of infected plants were heavier. The body contents and saliva or excretions of M. incognita larvae contained too little auxin to account for the increased amounts in infected roots. Roots with galls contained more bound auxin, released by alkaline hydrolysis or incubation after maceration, and more tryptophan and other amino acids, than uninfected roots. The larvae may hydrolyse the plant proteins to yield tryptophan, which may then react with the endogenous phenolic acids to produce auxin.