Bacterial Biosynthesis of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase and Indole-3-Acetic Acid (IAA) as Endophytic Preferential Selection Traits by Rice Plant Seedlings

In this study, bacteria were isolated from the rhizosphere and inside the roots and nodules of berseem clover plants grown in the field in Iran. Two hundred isolates were obtained from the rhizosphere (120 isolates), interior roots (57 isolates), and nodules (23 isolates) of clover plants grown in rotation with rice plants. Production of chitinase, pectinase, cellulase, siderophore, salicylic acid, hydrogen cyanide, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, solubilization of phosphate, antifungal activity against various rice plant pathogen fungi, N₂ fixation, and colonization assay on rice seedlings by these strains was evaluated and compared (endophytic isolates vs. rhizosphere bacteria). The results showed both the number and the ability of plant growth-promoting (PGP) traits were different between endophytic and rhizosphere isolates. A higher percentage of endophytic isolates were positive for production of IAA, ACC deaminase, and siderophore than rhizosphere isolates. Therefore, it is suggested that clover plant may shape its own associated microbial community and act as filters for endophyte communities, and rhizosphere isolates with different (PGP) traits. We also studied the PGP effect of the most promising endophytic and rhizosphere isolates on rice seedlings. A significant relationship among IAA and ACC deaminase production, the size of root colonization, and plant growth (root elongation) in comparison with siderophore production and phosphate solubilization for the isolates was observed. The best bacterial isolates (one endophytic isolate and one rhizosphere isolate), based on their ability to promote rice growth and colonize rice roots, were identified. Based on 16S rDNA sequence analysis, the endophytic isolate CEN7 and the rhizosphere isolate CEN8 were closely related to Pseudomonas putida and Pseudomonas fluorescens, respectively. It seems that PGP trait production (such as IAA, ACC deaminase) may be required for endophytic and rhizosphere competence as compared to other PGP traits in rice seedlings under constant flooded conditions. The study also shows that the presence of diverse rhizobacteria with effective growth-promoting traits associated with clover plants may be used for sustainable crop management under field conditions.     

Native bacterial endophytes promote host growth in a species-specific manner; phytohormone manipulations do not result in common growth responses

Background

All plants in nature harbor a diverse community of endophytic bacteria which can positively affect host plant growth. Changes in plant growth frequently reflect alterations in phytohormone homoeostasis by plant-growth-promoting (PGP) rhizobacteria which can decrease ethylene (ET) levels enzymatically by 1-aminocyclopropane-1-carboxylate (ACC) deaminase or produce indole acetic acid (IAA). Whether these common PGP mechanisms work similarly for different plant species has not been rigorously tested.

Methodology/ Principal Findings

We isolated bacterial endophytes from field-grown Solanum nigrum; characterized PGP traits (ACC deaminase activity, IAA production, phosphate solubilization and seedling colonization); and determined their effects on their host, S. nigrum, as well as on another Solanaceous native plant, Nicotiana attenuata. In S. nigrum, a majority of isolates that promoted root growth were associated with ACC deaminase activity and IAA production. However, in N. attenuata, IAA but not ACC deaminase activity was associated with root growth. Inoculating N. attenuata and S. nigrum with known PGP bacteria from a culture collection (DSMZ) reinforced the conclusion that the PGP effects are not highly conserved.

Conclusions/ Significance

We conclude that natural endophytic bacteria with PGP traits do not have general and predictable effects on the growth and fitness of all host plants, although the underlying mechanisms are conserved.     

Detection and Characterization of ACC Deaminase in Plant Growth Promoting Rhizobacteria

The enzyme 1-aminocyclopropane-1-carboxylate deaminase converts ACC, the precursor of the plant hormone ethylene to α-ketobutyrate and ammonium. The enzyme has been identified in few soil bacteria, and is proposed to play a key role in plant growth promotion. In this study, the isolates of plant growth promoting rhizobacteria were screened for ACC deaminase activity based on their ability to grow on ACC as a sole nitrogen source. The selected isolates showed the presence of other plant growth promoting characteristics such as IAA production, phosphate solubilization and siderophore production. The role of ACC deaminase in lowering ethylene production under cadmium stress condition was also studied by measuring in vitro ethylene evolution by wheat seedlings treated with ACC deaminase positive isolates. Nucleic acid hybridization confirmed the presence of ACC deaminase gene (acdS) in the bacterial isolates.     

Bioprospecting in potato fields in the Central Andean Highlands: Screening of rhizobacteria for plant growth-promoting properties

The Central Andean Highlands are the center of origin of the potato plant (Solanum tuberosum). Ages of mutualism between potato plants and soil bacteria in this region support the hypothesis that Andean soils harbor interesting plant growth-promoting (PGP) bacteria. Therefore, the aim of this study was to isolate rhizobacteria from Andean ecosystems, and to identify those with PGP properties. A total of 585 bacterial isolates were obtained from eight potato fields in the Andes and they were screened for suppression of Phytophthora infestans and Rhizoctonia solani. Antagonistic mechanisms were determined and antagonistic isolates were further tested for phosphate solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and production of NH₃- and indole-3-acetic acid (IAA). PGP was studied in healthy and R. solani diseased plantlets under growth room conditions. Performance was compared to the commercial strain B. subtilis FZB24^® WG. Isolates were dereplicated with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), and identified with 16S rRNA gene sequencing and multi locus sequence analysis (MLSA). A total of 10% of the isolates were effective antagonists, of which many were able to solubilize phosphate, and produce IAA, ACC deaminase, NH₃ and hydrogen cyanide (HCN). During growth room experiments, 23 antagonistic isolates were associated with plant growth-promotion and/or disease suppression. Ten isolates had a statistically significant impact on test parameters compared to the uninoculated control. Three isolates significantly promoted plant growth in healthy plantlets compared to the commercial strain, and seven isolates outperformed the commercial strain in in vitro R. solani diseased plantlets.     

Isolation of ACC deaminase-producing habitat-adapted symbiotic bacteria associated with halophyte Limonium sinense (Girard) Kuntze and evaluating their plant growth-promoting activity under salt stress

Background and aims

Many plant growth-promoting endophytes (PGPE) possessing 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity can reduce the level of stress ethylene and assist their host plants cope with various biotic and abiotic stresses. However, information about the endophytic bacteria colonizing in the coastal halophytes is still very scarce. This study aims at isolating efficient ACC deaminase-producing plant growth-promoting (PGP) bacterial strains from the inner tissues of a traditional Chinese folk medicine Limonium sinense (Girard) Kuntze, a halophyte which has high economic and medicinal values grown in the coastal saline soils. Their PGP activity and effects on host seed germination and seedling growth under salinity stress were also evaluated.

Methods

A total of 126 isolates were obtained from the surface sterilized roots, stems and leaves of L. sinense (Girard) Kuntze. They were initially selected for their ability to produce ACC deaminase as well as other PGP properties such as production of indole-3-acetic acid (IAA), N₂-fixation, and phosphate-solubilizing activities and subsequently identified by the 16S rRNA gene sequencing. For selected strains, seed germination, seedling growth, and flavonoids production in axenically growth L. sinense (Girard) Kuntze seedlings at different NaCl concentrations (0–500 mM) were quantified.

Results

Thirteen isolates possessing ACC deaminase activity were obtained. The 16S rRNA gene sequencing analysis showed them to belong to eight genera: Bacillus, Pseudomonas, Klebsiella, Serratia, Arthrobacter, Streptomyces, Isoptericola, and Microbacterium. Inoculation with four of the selected ACC deaminase-producing strains not only stimulated the growth of the host plant but also influenced the flavonoids accumulation. All four strains could colonize and can be re-isolated from the host plant interior tissues.

Conclusions

These results demonstrate that ACC deaminase-producing habitat-adapted symbiotic bacteria isolated from halophyte could enhance plant growth under saline stress conditions and the PGPE strains could be appropriate as bioinoculants to enhance soil fertility and protect the plants against salt stress.     

Molecular diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing PGPR from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) rhizosphere

Aims

The present study was planned to investigate the diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing bacteria from the rhizosphere of wheat plants and subsequent evaluation of selected PGPR on growth enhancement of wheat seedlings under drought and saline conditions.

Methods

ACC deaminase producing plant growth promoting rhizobacteria (PGPR) were isolated from the rhizosphere of wheat and identified using 16S rRNA gene sequence analysis. Isolates were evaluated for various direct and indirect plant growth promoting (PGP) traits. Plant inoculation experiment was conducted using isolates IG 19 and IG 22 in wheat to assess their plant growth promotion potential under salinity and drought stress.

Results

Thirty-eight ACC deaminase producing PGPR were isolated which belonged to 12 distinct genera and falling into four phyla γ-proteobacteria, β-proteobacteria, Flavobacteria and Firmicutes. Klebsiella sp. was the most abundant genera and followed by Enterobacter sp. The isolates exhibited ACC deaminase activities ranging from 0.106–0.980 μM α- ketobutyrate μg protein^?1 h^?1. The isolates showed multiple PGP traits such as IAA production, phosphate, zinc, potassium solubilization and siderophore production. Enterobacter cloacae (IG 19) and Citrobacter sp. (IG 22) inoculated wheat seedlings showed notable increases in fresh and dry biomass under non-stress as well as under stressed condition.

Conclusion

To the best of our knowledge this is the first report of presence of ACC deaminase activity and other PGP traits from the genus Citrobacter and Empedobacter. Our finding revealed that the γ-proteobacteria group dominated the wheat rhizosphere. Plant inoculation with PGPR could be a sustainable approach to alleviate abiotic stresses in wheat plants. These native PGPR isolates could be used as potential biofertilizers for sustainable agriculture.

     

Promotion of tomato (Lycopersicon esculentum Mill.) plant growth by rhizosphere competent 1-aminocyclopropane-1-carboxylic acid deaminase-producing streptomycete actinomycetes

The ability of streptomycete actinomycetes to promote growth of tomato through the production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase was evaluated under gnotobiotic and greenhouse conditions. To achieve this, 64 isolates of Streptomyces spp. obtained from a tomato rhizosphere in the United Arab Emirates were initially selected for their ability to produce ACC deaminase as well as indole-3-acetic acid (IAA) and subsequently for their rhizosphere competence as root colonizers. Of the two selected ACC deaminase-producing isolates showing exceptional rhizosphere competence, S. filipinensis no. 15 produced both ACC deaminase and IAA, whilst S. atrovirens no. 26 did not produce IAA. Under greenhouse conditions, the application of S. filipinensis no. 15 or S. atrovirens no. 26 resulted in the reduction of the endogenous levels of ACC, the immediate precursor of ethylene, in both roots and shoots and increased plant growth. Plant growth promotion was most pronounced in the presence of S. filipinensis no. 15 compared to S. atrovirens no. 26. This relative superiority in performance shows the advantage conferred to S. filipinensis no. 15 due to its ability to produce both IAA and ACC deaminase. In comparison, an ACC deaminase-producing isolate of S. albovinaceus no. 41 which was neither rhizosphere-competent nor capable of producing IAA, failed to promote plant growth compared to S. filipinensis no. 15 or S. atrovirens no. 26 although the growth promotion obtained by S. albovinaceus no. 41 was significant compared to control. The application of S. globosus no. 8, which was not rhizosphere-competent and did not produce detectable levels of ACC deaminase or IAA did not promote plant growth. These results indicate the importance of rhizosphere competence. In conclusion I report the production of ACC deaminase by streptomycete actinomycetes and its ability to enhance plant growth through reduction in the in planta levels of endogenous ACC and the consequent lowering of endogenous ethylene levels in plant tissues.     

Isolation of ACC deaminase producing PGPR from rice rhizosphere and evaluating their plant growth promoting activity under salt stress

Aims

Bacteria possessing ACC deaminase activity reduce the level of stress ethylene conferring resistance and stimulating growth of plants under various biotic and abiotic stresses. The present study aims at isolating efficient ACC deaminase producing PGPR strains from the rhizosphere of rice plants grown in coastal saline soils and quantifying the effect of potent PGPR isolates on rice seed germination and seedling growth under salinity stress and ethylene production from rice seedlings inoculated with ACC deaminase containing PGPR.

Methods

Soils from root region of rice growing in coastal soils of varying salinity were used for isolating ACC deaminase producing bacteria and three bacterial isolates were identified following polyphasic taxonomy. Seed germination, root growth and stress ethylene production in rice seedlings following inoculation with selected PGPR under salt stress were quantified.

Results

Inoculation with selected PGPR isolates had considerable positive impacts on different growth parameters of rice including germination percentage, shoot and root growth and chlorophyll content as compared to uninoculated control. Inoculation with the ACC deaminase producing strains reduced ethylene production under salinity stress.

Conclusions

This study demonstrates the effectiveness of rhizobacteria containing ACC deaminase for enhancing salt tolerance and consequently improving the growth of rice plants under salt-stress conditions.     

Characterization of Plant Growth–Promoting Traits of Bacteria Isolated from Larval Guts of Diamondback Moth <Emphasis Type="Italic">Plutella xylostella</Emphasis> (Lepidoptera: Plutellidae)

Eight bacterial isolates from the larval guts of Diamondback moths (Plutella xylostella) were tested for their plant growth–promoting (PGP) traits and effects on early plant growth. All of the strains tested positive for nitrogen fixation and indole 3-acetic acid (IAA) and salicylic acid production but negative for hydrogen cyanide and pectinase production. In addition, five of the isolates exhibited significant levels of tricalcium phosphate and zinc oxide solubilization; six isolates were able to oxidize sulfur in growth media; and four isolates tested positive for chitinase and β-1,3-glucanase activities. Based on their IAA production, six strains including four that were 1-aminocyclopropane-1-carboxylate (ACC) deaminase positive and two that were ACC deaminase negative were tested for PGP activity on the early growth of canola and tomato seeds under gnotobiotic conditions. Acinetobacter sp. PSGB04 significantly increased root length (41%), seedling vigor, and dry biomass (30%) of the canola test plants, whereas Pseudomonas sp. PRGB06 inhibited the mycelial growth of Botrytis cinerea, Colletotrichum coccodes, C. gleospoiroides, Rhizoctonia solani, and Sclerotia sclerotiorum under in vitro conditions. A significant increase, greater than that of the control, was also noted for growth parameters of the tomato test plants when the seeds were treated with PRGB06. Therefore, the results of the present study suggest that bacteria associated with insect larval guts possess PGP traits and positively influence plant growth. Therefore, insect gut bacteria as effective PGP agents represent an unexplored niche and may broaden the spectrum of beneficial bacteria available for crop production.     

Enhancement of Festuca rubra L. germination and seedling growth by seed treatment with pathogenic Agrobacterium rhizogenes

The study concerned effects of two methods of red fescue seeds treating with wild type Agrobacterium rhizogenes strains (15834 and LBA 1334): soaking and matriconditioning with Micro-Cel E solid carrier. The effects were assessed from germination and seedlings growth rates. The bacterial indole-3-acetic acid (IAA) production and ACC deaminase activity were also determined. Both strains are able to produce the IAA and showed ACC deaminase activity in various amounts. The strains accelerated seeds germination, seedling emergence and development. The beneficial effect on those processes was visible when seeds were soaked for 1 h in bacterial suspension and, especially, when the bacteria were present during Micro-Cel E conditioning. The main effect observed upon inoculation of seeds during priming was increased growth of lateral roots and more complex architecture of the branching root system. Treating seeds with A. rhizogenes simultaneously with Micro-Cel E priming as a carrier is a promising method of enhancing grass germination and seedlings growth, particularly by improving the root system development.     

Evaluating the biochemical traits of novel Trichoderma-based biofilms for use as plant growth-promoting inoculants

Bacteria-mediated plant growth promotion is a well-established and complex phenomenon that is often achieved by the activities of more than one plant growth-promoting (PGP) trait, which may not always be present in a single organism. Biofilms developed using a combination of two organisms with useful plant growth-promoting rhizobacteria (PGPR) traits may provide a definite advantage. In this context, in vitro studies were conducted evaluating the PGP traits of novel biofilms developed using Trichoderma as matrix and agriculturally important bacteria (Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus subtilis) as partners. Such biofilms exhibited higher values for various biochemical attributes as compared to the individual organisms and dual cultures. Trichoderma–Bacillus and Trichoderma–Pseudomonas biofilms exhibited enhanced antifungal activity, ammonia, indole acetic acid (IAA) and siderophore production, as compared to the other treatments. Trichoderma–Azotobacter biofilm recorded the highest nitrogenase activity and 1-aminocyclopropane-1-carboxylic (ACC) deaminase activity. The synergism in terms of the PGP traits in the biofilms revealed their promise as superior PGP inoculants.     

Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings

Study of endophytic bacteria within plant seeds is very essential and meaningful on account of their heritability and versatility. This study investigated Bacillus bacterial communities within the seeds of four commercial tomato varieties, by 16S rRNA gene PCR-RFLP (restriction fragment length polymorphism). Phylogenetic analysis of 16S rRNA gene sequences indicated that the 22 representative isolates belonged to five species of genus Bacillus and the bacterial compositions showed remarkable differences among tomato varieties. Isolates exhibited multiple plant growth promoting (PGP) traits: 37 % of indole-3-acetic acid production; 37 % of phosphate solubilization; 24 % of siderophores production; 85 % of potential nitrogen fixation and 6 % of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Isolate HYT-12-1 was shown to have highest ACC deaminase activity (112.02 nmol α-ketobutyrate mg^?1 protein h^?1) among the five ACC deamiase producing strains. 16S rRNA gene sequencing indicated that the isolate HYT-12-1 shared the highest sequence similarity (100 %) with B. subtilis. PGP experiments under gnotobiotic and greenhouse conditions revealed the ability of strain HYT-12-1 to enhance the growth of tomato seedlings. This is the first study to describe endophytic Bacillus communities within tomato seeds, and the results suggest that B. subtilis strain HYT-12-1 would have a great potential for industrial application as biofertilizer in the future.     

Improved growth performance of the mangrove <Emphasis Type="Italic">Avicennia marina</Emphasis> seedlings using a 1-aminocyclopropane-1-carboxylic acid deaminase-producing isolate of <Emphasis Type="Italic">Pseudoalteromonas maricaloris</Emphasis>

Out of 62 bacterial isolates obtained from the mangrove Avicennia marina rhizosphere that grows along the Abu Dhabi coast, United Arab Emirates (UAE), an isolate of Pseudoalteromonas maricaloris (Wild type strain) (WT) produced relatively high levels of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase in vitro. Application of this WT strain under greenhouse conditions to A. marina seedlings significantly (P < 0.05), reduced endogenous levels of ACC in the roots and shoots, and significantly (P < 0.05) increased the levels of in planta endogenous plant growth regulators (PGRs) including indole-3-acetic acid (IAA), indole-3-pyruvic acid (IPYA), putrescine (Put), spermidine (Spd) and spermine (Spm) in roots and shoots compared with control mangrove seedlings. WT application has also significantly (P < 0.05) increased photosynthetic pigment contents, photosynthetic carbon assimilation, plant water use efficiency and promoted mangrove seedlings growth characteristics including increased dry weight and length of roots and shoots, total leaf area and the number of the side branches compared with control mangrove seedlings. In comparison, an ACC deaminase non-producing mutant strain (NPM) failed to reduce endogenous levels of ACC in the roots and shoots and also failed to increase endogenous PGRs and photosynthetic pigments and did not promote seedling growth. Both WT and NPM strains were incapable of producing in vitro detectable levels of IAA, IPYA, Gibberellic acid (GA₃), zeatin (Z), Put, Spd and Spm in the culture filtrates. This study demonstrated for the first time the ability of ACC deaminase-producing bacteria to promote mangrove growth under greenhouse conditions. P. maricaloris has potential as biological inoculants to promote the growth of mangrove seedlings in afforestation programs in nutrient impoverished sediments in hyper-saline coastal areas in the UAE.     

Inhibitory action of auxin on root elongation not mediated by ethylene

The inhibitory effects of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) on elongation growth of pea (Pisum sativum L.) seedling roots were investigated in relation to the effects of these compounds on ethylene production by the root tips. When added to the growth solution both compounds caused a progressively increasing inhibition of growth within the concentration range of 0.01 to 1 micromolar. However, only ACC increased ethylene production in root tips excised from the treated seedlings after 24 hours. High auxin concentrations caused a transitory increase of ethylene production during a few hours in the beginning of the treatment period, but even in 1 micromolar IAA this increase was too low to have any appreciable effect on growth. ACC, but not IAA, caused growth curvatures, typical of ethylene treatment, in the root tips. IAA caused conspicuous swelling of the root tips while ACC did not. Cobalt and silver ions reversed the growth inhibitory effects induced by ACC but did not counteract the inhibition of elongation or swelling caused by IAA. The growth effects caused by the ACC treatments were obviously due to ethylene production. We found no evidence to indicate that the growth inhibition or swelling caused by IAA is mediated by ethylene. It is concluded that the inhibitory action of IAA on root growth is caused by this auxin per se.     

Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing <Emphasis Type="Italic">Methylobacterium oryzae</Emphasis> and interactions with auxins and ACC regulation of ethylene in canola (<Emphasis Type="Italic">Brassica campestris</Emphasis>)

The possible interaction of the plant hormones auxin and ethylene and the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing bacteria on ethylene production in canola (Brassica campestris) in the presence of inhibitory concentrations of growth regulators were investigated. The effects of auxin (indole-3-acetic acid and 2,4-dichlorophenoxy acetic acid), auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid, ethylene precursor 1-aminocyclopropane-1-carboxylate and ethylene synthesis inhibitor l-α-(2-aminoethoxyvinyl)glycine hydrochloride on root elongation were concentration dependent. Exogenous addition of growth regulators influences the enzyme activities of ethylene production and we have presented here evidences that support the hypothesis that inhibitory effects of auxin on root elongation are independent of ethylene. Additionally, we have proved that inoculation of ACC deaminase containing Methylobacterium oryzae sequester ACC exuded from roots and hydrolyze them lowering the concentration of ACC in root exudates. However, the inhibitory actions of exogenous additions of auxins could not be ameliorated by bacterial inoculation that reduces ethylene concentration in canola seedlings.     

The roots of the halophyte Salicornia brachiata are a source of new halotolerant diazotrophic bacteria with plant growth-promoting potential

Soil salinity is the major cause limiting plant productivity worldwide. Nitrogen-fixing bacteria were enriched and characterised from roots of Salicornia brachiata, an extreme halophyte which has substantial economic value as a bioresource of diverse and valuable products. Nitrogen-free semisolid NFb medium with malate as carbon source and up to 4% NaCl were used for enrichment and isolation of diazotrophic bacteria. The isolates were tested for plant growth-promoting traits and 16S rRNA, nifH and acdS genes were analysed. For selected strains, plant growth-promoting activities were tested in axenically grown Salicornia seedlings at different NaCl concentrations (0–0.5M). New halotolerant diazotrophic bacteria were isolated from roots of S. brachiata. The isolates were identified as Brachybacterium saurashtrense sp. nov., Zhihengliuella sp., Brevibacterium casei, Haererehalobacter sp., Halomonas sp., Vibrio sp., Cronobacter sakazakii, Pseudomonas spp., Rhizobium radiobacter, and Mesorhizobium sp. Nitrogen fixation as well as plant growth-promoting traits such as indole acetic acid (IAA) production, phosphate solubilisation, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity were demonstrated. For Brachybacterium saurashtrense and Pseudomonas sp., significant plant growth-promoting activities were observed in Salicornia in salt stress conditions. Salicornia brachiata is a useful source of new halotolerant diazotrophic bacteria with plant growth-promoting potential.     

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.     

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation

Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT^T) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 × 10⁻⁶. DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.     

Isolation and characterization of drought-tolerant ACC deaminase and exopolysaccharide-producing fluorescent Pseudomonas sp.

The enzyme 1-aminocyclopropane-1-carboxylate deaminase catalyzes the degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of the plant hormone ethylene, into α-ketobutyrate and ammonia. The enzyme has been detected in a limited number of bacteria and plays a significant role in sustaining plant growth and development under biotic and abiotic stress conditions by reducing stress-induced ethylene production in plants. We have screened 32 fluorescent Pseudomonas sp. isolated from rhizosphere and non-rhizosphere soils of different crop production systems for drought tolerance using polyethylene glycol 6000 (PEG 6000). Nine of these isolates were tolerant to a substrate metric potential of ?0.30 MPa (15 % PEG 6000) and therefore considered to be drought-tolerant. All of these drought-tolerant isolates were screened for ACC deaminase activity using ACC as the sole nitrogen source, and one (SorgP4) was found to be positive for ACC, producing 3.71?±?0.025 and 1.42?±?0.039 μM/mg protein/h of α-ketobutyrate under the non-stress and drought stress condition, respectively. The isolate SorgP4 also showed other plant growth-promoting traits, such as indole acetic acid production, phosphate solubilization, siderophore and hydrogen cyanide production. The ACC deaminase gene (acdS) from the isolate SorgP4 was amplified, and the nucleotide sequence alignment of the acdS gene showed significant homology with acdS genes of NCBI Genbank. The 16S rRNA gene sequencing analysis identified the isolate as Pseudomonas fluorescens. Both sequences have been submitted to the NCBI GenBank under the accession numbers JX885767 and KC192771 respectively.     

Infection of clover by plant growth promoting Pseudomonas fluorescens strain 267 and Rhizobium leguminosarum bv. trifolii studied by mTn5-gusA

Plant growth promoting Pseudomonas fluorescens strain 267, isolated from soil, produced pseudobactin A, 7-sulfonic acid derivatives of pseudobactin A and several B group vitamins. In coinoculation with Rhizobium leguminosarum bv. trifolii strain 24.1, strain 267 promoted clover growth and enhanced symbiotic nitrogen fixation under controlled conditions. To better understand the beneficial effect of P. fluorescens 267 on clover inoculated with rhizobia, the colonization of clover roots by mTn5-gusA marked bacteria was studied in single and mixed infections under controlled conditions. Histochemical assays combined with light and electron microscopy showed that P. fluorescens 267.4 (i) efficiently colonized clover root surface; (ii) was heterogeneously distributed along the roots without the preference to defined root zone; (iii) formed microcolonies on the surface of clover root epidermis; (iv) penetrated the first layer of the primary root cortex parenchyma and (v) colonized endophytically the inner root tissues of clover.