Role of Pseudomonas putida Indoleacetic Acid in Development of the Host Plant Root System

Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.     

The Potential of the Ni-Resistant TCE-Degrading Pseudomonas putida W619-TCE to Reduce Phytotoxicity and Improve Phytoremediation Efficiency of Poplar Cuttings on A Ni-TCE Co-Contamination

To examine the potential of Pseudomonas putida W619-TCE to improve phytoremediation of Ni-TCE co-contamination, the effects of inoculation of a Ni-resistant, TCE-degrading root endophyte on Ni-TCE phytotoxicity, Ni uptake and trichloroethylene (TCE) degradation of Ni-TCE-exposed poplar cuttings are evaluated.

After inoculation with P. putida W619-TCE, root weight of non-exposed poplar cuttings significantly increased. Further, inoculation induced a mitigation of the Ni-TCE phytotoxicity, which was illustrated by a diminished exposure-induced increase in activity of antioxidative enzymes. Considering phytoremediation efficiency, inoculation with P. putida W619-TCE resulted in a 45% increased Ni uptake in roots as well as a slightly significant reduction in TCE concentration in leaves and TCE evapotranspiration to the atmosphere.

These results indicate that endophytes equipped with the appropriate characteristics can assist their host plant to deal with co-contamination of toxic metals and organic contaminants during phytoremediation. Furthermore, as poplar is an excellent plant for biomass production as well as for phytoremediation, the obtained results can be exploited to produce biomass for energy and industrial feedstock applications in a highly productive manner on contaminated land that is not suited for normal agriculture. Exploiting this land for biomass production could contribute to diminish the conflict between food and bioenergy production.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Paenibacillus polymyxa Invades Plant Roots and Forms Biofilms

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa, 2003). Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a plasmid-borne gfp gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed.     

Colonization of Vitis vinifera by a Green Fluorescence Protein-Labeled, gfp-Marked Strain of Xylophilus ampelinus, the Causal Agent of Bacterial Necrosis of Grapevine

The dynamics of Xylophilus ampelinus were studied in Vitis vinifera cv. Ugni blanc using gfp-marked bacterial strains to evaluate the relative importance of epiphytic and endophytic phases of plant colonization in disease development. Currently, bacterial necrosis of grapevine is of economic importance in vineyards in three regions in France: the Cognac, Armagnac, and Die areas. This disease is responsible for progressive destruction of vine shoots, leading to their death. We constructed gfp-marked strains of the CFBP2098 strain of X. ampelinus for histological studies. We studied the colonization of young plants of V. vinifera cv. Ugni blanc by X. ampelinus after three types of artificial contamination in a growth chamber and in a greenhouse. (i) After wounding of the stem and inoculation, the bacteria progressed down to the crown through the xylem vessels, where they organized into biofilms. (ii) When the bacteria were forced into woody cuttings, they rarely colonized the emerging plantlets. Xylem vessels could play a key role in the multiplication and conservation of the bacteria, rather than being a route for plant colonization. (iii) When bacterial suspensions were sprayed onto the plants, bacteria progressed in two directions: both in emerging organs and down to the crown, thus displaying the importance of epiphytic colonization in disease development.     

Differential Rhizosphere Establishment and Cyanide Production by Alginate-Formulated Weed-Deleterious Rhizobacteria

The effects of Pseudomonas putida ATH2-1RI/9 and Acidovorax delafieldii ATH2-2RS/1 on rhizosphere colonization, cyanide production, and growth of velvetleaf and corn was examined. When formulated in alginate beads and inoculated onto velvetleaf and corn plants (10⁹ CFU/plant), only P. putida ATH2-1RI/9 consistently reduced velvetleaf growth. Neither isolate inhibited corn growth. Interestingly the levels of P. putida ATH2-1RI/9 in the velvetleaf rhizosphere were 1000-fold higher (7 × 10⁷ CFU/g root) than the A. delafieldii ATH2-2RS/1 populations. Cyanide (53–68 mM/g root) was recovered from the P. putida ATH2-1RI/9-inoculated velvetleaf plants. In contrast both A. delafieldii ATH2-2RS/1 and P. putida ATH2-1RI/9 colonized the corn rhizosphere to the same extent (1–5 × 10⁷ CFU/g root), producing 1 mM and 14 mM/g root respectively. These results suggest that bacterial formulation methods can influence the effectiveness of deleterious rhizobacteria in reducing weed growth.     

Transcriptomic profiling of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) seedlings in response to <Emphasis Type="Italic">Pseudomonas putida</Emphasis> stain FBKV2 inoculation under drought stress

Several mechanisms have been proposed for plant growth-promoting rhizobacteria (PGPR)-mediated drought stress tolerance in plants, but little is known about the molecular pathways involved in the drought tolerance promoted by PGPR. We, therefore, aim to study the differential gene response between Pseudomonas putida strain FBKV2 and maize interaction under drought stress using Illumina sequencing. RNA Seq libraries were generated from leaf tissue of maize seedlings with and without strain FBKV2 subjected to drought stress. The libraries were mapped with maize genome database for the identification of differentially expressed genes (DEGs). The expression studies confirmed the downregulation of ethylene biosynthesis (ET), abscisic acid (ABA) and auxin signaling, superoxide dismutase, catalase, and peroxidase in FBKV2-inoculated seedlings. On the other hand, genes involved in β-alanine and choline biosynthesis, heat shock proteins, and late embryogenesis abundant (LEA) proteins were upregulated, which could act as key elements in the drought tolerance conferred by P. putida strain FBKV2. Another remarkable expression was observed in genes encoding benzoxazinoid (BX) biosynthesis which act as the chemoattractant, which was further confirmed by gfp-labeled P. putida strain FBKV2 root colonization studies. Overall, these results indicate that secretion of BXs attracted P. putida strain FBKV2 resulted in root colonization and mediated drought tolerance by modulating metabolic, signaling, and stress-responsive genes.     

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.     

The Role of MPK6 as Mediator of Ethylene/Jasmonic Acid Signaling in <Emphasis Type="Italic">Serendipita indica</Emphasis>-Colonized Arabidopsis Roots

Serendipita indica is an axenically cultivable fungus, which colonizes a broad range of plant species including the model plant Arabidopsis thaliana. Root colonization by this endophyte leads to enhanced plant fitness and performance and promotes resistance against different biotic and abiotic stresses. The involvement of MPK6 in this mutualistic interaction had been previously shown with an mpk6 A. thaliana mutant, which failed to respond to S. indica colonization. Here, we demonstrate that mpk6 roots are significantly less colonized by S. indica compared to wild-type roots and the foliar application of plant hormones, ethylene, or jasmonic acid, restores the colonization rate at least to the wild-type level. Further, hormone-treated mpk6 plants show typical S. indica-induced growth promotion effects. Moreover, expression levels of several genes related to plant defense and hormone signaling are significantly changed at different colonization phases. Our results demonstrate that the successful root colonization by S. indica depends on efficient suppression of plant immune responses. In A. thaliana, this process relies on intact hormone signaling in which MPK6 seems to play a pivotal role.     

In situ localization of Paenibacillus polymyxa HKA-15 in roots and root nodules of soybean (Glycine max.L.)

Background and aims

Bacterial endophytes can colonize various plants and organs. However, endophytic bacteria (other than rhizobia) colonizing root nodules in legumes have been rarely analyzed. The present study aimed to examine the colonization and spread of gfp-tagged Paenibacillus polymyxa in soybean plants under gnobiotic conditions.

Methods

Inoculation with gfp-tagged Paenibacillus. polymyxa HKA ?15 alone and in combination with Bradyrhizobium japonicum were done on soybean seedlings. In situ localization was detected through confocal microscopy and PCR.

Results

Inoculation with P. polymyxa-gfp strain alone and in combination with B. japonicum DS-1 had a stimulatory effect on the plant growth. There was an increase in shoot (7.2 %) and root dry weights (14.5 %) when the two strains were co - inoculated over that of B. japonicum inoculation alone. In vivo simultaneous visualization using Confocal Laser Scanning Microscopy (CLSM) showed the localization of the gfp-tagged P. polymyxa cells in the root nodules and its spread in the root tissue, both tap as well as lateral roots. Systemic spread into aerial tissue did not occur as indicated by the absence of bacteria. CLSM observations of the presence of gfp-tagged P. polymyxa in the nodule and roots tissues was corroborated with PCR amplification of the gfp-gene from these tissues.

Conclusions

CLSM and PCR methods confirmed that P. polymyxa invades roots and root nodules of soybean, but the spread is restricted to root tissue only. The strain improves plant growth when inoculated singly or in combination with B. japonicum.     

Effect of plant growth promoting rhizobacteria,nematode parasitic fungi and root-nodule bacterium on root-knot nematodes Meloidogyne javanica and growth of chickpea

Effects of plant growth promoting rhizobacteria (Pseudomonas putida MTCC No. 3604 and Pseudomonas alcaligenes MTCC No. 493) and parasitic fungi (Pochonia chlamydosporia KIA and Paecilomyces lilacinus KIA) were studied, alone and together with Rhizobium sp. (charcoal commercial culture) on the growth of chickpea and multiplication of Meloidogyne javanica. Individually, P. putida 3604, P. alcaligenes 493 and Rhizobium caused a significant increase in the growth of chickpea in both nematode inoculated and uninoculated plants. Inoculation of Rhizobium with a parasitic fungus or with plant growth promoting rhizobaterium caused a greater increase in the growth of plants inoculated with nematodes than caused by either of them singly. Individually, P. lilacinus KIA caused a greater increase in the growth of nematode inoculated plants than caused by P. putida 3604 or P. alcaligenes 493. P. lilacinus KIA caused a greater reduction in galling and nematode multiplication followed by P. chlamydosporia KIA, P. putida 3604 and P. alcaligenes 493. Combined use of P. lilacinus KIA with Rhizobium was better in reducing galling and nematode multiplication than any other treatment. P. putida 3604 caused a greater colonization of root than P. alcaligenes 493 while P. lilacinus KIA was isolated from more nematodes than P. chlamydosporia KIA.     

The effects of herbicides and mycoparasites at different moisture levels on carpogenic germination in Sclerotinia sclerotiorum

Colonization of plant roots by fluorescent pseudomonads has been correlated with disease suppression. One mechanism may involve altered defense responses in the plant upon colonization. Altered defense responses were observed in bean (Phaseolus vulgaris) inoculated with fluorescent pseudomonads. Systemic effects of root inoculation by Pseudomonas putida isolate Corvallis, P. tolaasii (P9A) and P. aureofaciens REW1-I-1 were observed in bean leaves from 14-day-old plants. SDS- polyacrylamide gel electrophoresis demonstrated that levels of certain acid-soluble proteins increased in the leaf extracts of inoculated plants. Plants inoculated with REW1-I-1 produced more of a 57 M_r protein, and plants inoculated with isolates P9A and REW1-I-1 produced more of a 38 M_r protein. Northern hybridization revealed enhanced accumulation of mRNAs, that encode the pathogenesis-related protein PR1a, in leaves of plants inoculated with P. putida and REW1-I-1. Only REW1-I-1, but not P9A or P. putida induced symptoms of an hypersensitive response on tobacco leaves, bean cotyledons, and in bean suspension cultures. Phenolics and phytoalexins accumulated in bean cotyledons exposed to REW1-I-1 for 24 h but little change in levels of these compounds occurred in cotyledons inoculated with P9A and P. putida. Both suspension culture cells and roots treated with REW1-I-1 rapidly evolved more hydrogen peroxide than those exposed to P9A and P. putida. However, roots from 14-day-old plants colonized by P9A, P. putida or REW1-I-1 did not have higher levels of phenolics, phytoalexins or mRNAs for two enzymes involved in phenolic biosynthesis, phenylalanine-ammonia lyase and chalcone synthase. A selective induction of plant defense strategies upon root colonization by certain pseudomonads is apparent.     

The Effect of Pseudomonas putida Colonization on Root Surface Peroxidase

Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions.     

Suppression of Rhizoctonia solani diseases of sugar beet by antagonistic and plant growth-promoting yeasts

AIMS: Isolates of Candida valida, Rhodotorula glutinis and Trichosporon asahii from the rhizosphere of sugar beet in Egypt were examined for their ability to colonize roots, to promote plant growth and to protect sugar beet from Rhizoctonia solani AG-2-2 diseases, under glasshouse conditions. METHODS AND RESULTS: Root colonization abilities of the three yeast species were tested using the root colonization plate assay and the sand-tube method. In the root colonization plate assay, C. valida and T. asahii colonized 95% of roots after 6 days, whilst Rhod. glutinis colonized 90% of roots after 8 days. Root-colonization abilities of the three yeast species tested by the sand-tube method showed that roots and soils attached to roots of sugar beet seedlings were colonized to different degrees. Population densities showed that the three yeast species were found at all depths of the rhizosphere soil adhering to taproots up to 10 cm, but population densities were significantly (P < 0.05) greater in the first 4 cm of the root system compared with other root depths. The three yeast species, applied individually or in combination, significantly (P < 0.05) promoted plant growth and reduced damping off, crown and root rots of sugar beet in glasshouse trials. The combination of the three yeasts (which were not inhibitory to each other) resulted in significantly (P < 0.05) better biocontrol of diseases and plant growth promotion than plants exposed to individual species. CONCLUSIONS: Isolates of C. valida, Rhod. glutinis and T. asahii were capable of colonizing sugar beet roots, promoting growth of sugar beet and protecting the seedlings and mature plants from R. solani diseases. This is the first successful attempt to use yeasts as biocontrol agents against R. solani which causes root diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts were shown to provide significant protection to sugar beet roots against R. solani, a serious soil-borne root pathogen. Yeasts also have the potential to be used as biological fertilizers.     

Genome Survey and Characterization of Endophytic Bacteria Exhibiting a Beneficial Effect on Growth and Development of Poplar Trees

The association of endophytic bacteria with their plant hosts has a beneficial effect for many different plant species. Our goal is to identify endophytic bacteria that improve the biomass production and the carbon sequestration potential of poplar trees (Populus spp.) when grown in marginal soil and to gain an insight in the mechanisms underlying plant growth promotion. Members of the Gammaproteobacteria dominated a collection of 78 bacterial endophytes isolated from poplar and willow trees. As representatives for the dominant genera of endophytic gammaproteobacteria, we selected Enterobacter sp. strain 638, Stenotrophomonas maltophilia R551-3, Pseudomonas putida W619, and Serratia proteamaculans 568 for genome sequencing and analysis of their plant growth-promoting effects, including root development. Derivatives of these endophytes, labeled with gfp, were also used to study the colonization of their poplar hosts. In greenhouse studies, poplar cuttings (Populus deltoides × Populus nigra DN-34) inoculated with Enterobacter sp. strain 638 repeatedly showed the highest increase in biomass production compared to cuttings of noninoculated control plants. Sequence data combined with the analysis of their metabolic properties resulted in the identification of many putative mechanisms, including carbon source utilization, that help these endophytes to thrive within a plant environment and to potentially affect the growth and development of their plant hosts. Understanding the interactions between endophytic bacteria and their host plants should ultimately result in the design of strategies for improved poplar biomass production on marginal soils as a feedstock for biofuels.Endophytic bacteria are bacteria that reside within the living tissue of their host plants without substantively harming it (19, 26). They are ubiquitous in most plant species, latently residing or actively colonizing the tissues. The diversity of cultivable bacterial endophytes is exhibited not only in the variety of plant species colonized but also in the many taxa involved, with most being members of common soil bacterial genera such as Enterobacter, Pseudomonas, Burkholderia, Bacillus, and Azospirillum (21, 23). Endophytic bacteria have several mechanisms by which they can promote plant growth and health. These mechanisms are of prime importance for the use of plants as feedstocks for biofuels and for carbon sequestration through biomass production. This is vital when considering the aim of improving biomass production of marginal soils, thus avoiding competition for agricultural resources, which is one of the critical socioeconomic issues of the increased use of biofuels.Like rhizosphere bacteria, endophytic bacteria have been shown to have plant growth-promoting activity that can be due to the production of phytohormones, enzymes involved in growth regulator metabolism, such as ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, auxins, indole-3-acetic acid (IAA), acetoin, 2,3-butanediol, cytokinins (3, 13-15, 20, 30), or combinations thereof. They can also improve plant growth via the fixation of nitrogen (diazotrophy) (9, 38).Typical examples of marginal soils include soils that have deteriorated due to the presence of heavy metals or organic contaminants. These are often soils with a history of industrial, military, or mining activities. Endophytic bacteria can assist their host plants in overcoming phytotoxic effects caused by environmental contamination (5, 11, 12, 36), which is of direct relevance for waste management and pollution control via phytoremediation technologies. When nonsterile poplar cuttings (Populus trichocarpa × deltoides cv. Hoogvorst) were inoculated with the endophyte Burkholderia cepacia VM1468, a derivative of B. cepacia Bu72 which possesses the pTOM-Bu61 plasmid coding for a constitutively expressed toluene degradation pathway, it was observed that in addition to decreasing the phytotoxicity and releasing toluene, strain VM1468 also considerably improved the growth of poplar trees in the absence of toluene (36). This observation, which was the first of its kind for poplar trees, prompted us to further study the poplar tree-associated beneficial endophytic bacteria in order to improve the overall performance of poplar trees, as it can enhance multiple applications, including biomass production, carbon sequestration, and phytoremediation. This was done by screening endophytic bacteria for their plant growth-promoting capabilities toward poplar trees by performing colonization studies with gfp-labeled strains, by examining their metabolic properties, and by initiating the genome sequencing of several strains.