Study of mechanisms for plant growth promotion elicited by rhizobacteria in Arabidopsis thaliana

Plant growth-promoting rhizobacteria (PGPR) colonize plant roots and exert beneficial effects on plant health and development. We are investigating the mechanisms by which PGPR elicit plant growth promotion from the viewpoint of signal transduction pathways within plants. We report here our first study to determine if well-characterized PGPR strains, which previously demonstrated growth promotion of various other plants, also enhance plant growth in Arabidopsis thaliana. Eight different PGPR strains, including Bacillus subtilis GB03, B. amyloliquefaciens IN937a, B. pumilus SE-34, B. pumilus T4, B. pasteurii C9, Paenibacillus polymyxa E681, Pseudomonas fluorescens 89B-61, and Serratia marcescens 90-166, were evaluated for elicitation of growth promotion of wild-type and mutant Arabidopsis in vitro and in vivo. In vitro testing on MS medium indicated that all eight PGPR strains increased foliar fresh weight of Arabidopsis at distances of 2, 4, and 6 cm from the site of bacterial inoculation. Among the eight strains, IN937a and GB03 inhibited growth of Arabidopsis plants when the bacteria were inoculated 2 cm from the plants, while they significantly increased plant growth when inoculated 6 cm from the plants, suggesting that a bacterial metabolite that diffused into the agar accounted for growth promotion with this strain. In vivo, eight PGPR strains promoted foliar fresh weight under greenhouse conditions 4 weeks after sowing. To define signal transduction pathways associated with growth promotion elicited by PGPR, various plant-hormone mutants of Arabidopsis were evaluated in vitro and in vivo. Elicitation of growth promotion by PGPR strains in vitro involved signaling of brassinosteroid, IAA, salicylic acid, and gibberellins. In vivo testing indicated that ethylene signaling was involved in growth promotion. Results suggest that elicitation of growth promotion by PGPR in Arabidopsis is associated with several different signal transduction pathways and that such signaling may be different for plants grown in vitro vs. in vivo.     

Identification of rhizobacteria from maize and determination of their plant-growth promoting potential

During the growing season of 1986, the rhizobacteria (including organisms from the ectorhizosphere, the rhizoplane and endorhizosphere) of 20 different maize hybrids sampled from different locations in the Province of Quebec were inventoried by use of seven different selective media. Isolates were characterized by morphological and biochemical tests and identified using the API20E and API20B diagnostic strips.Pseudomonas spp. were the prominent bacteria found in the rhizoplane and in the ectorhizosphere.Bacillus spp. andSerratia spp. were also detected, but in smaller numbers. In the endorhizosphere,Bacillus spp. andPseudomonas spp. were detected in order of importance. Screening for plant growth-promoting rhizobacteria was carried out in three soils with different physical and chemical characteristics. The results depended on the soil used, but two isolates (Serratia liquefaciens andPseudomonas sp.) consistently caused a promotion of plant growth.Contribution no. 350 of the Research Station, Agriculture Canada, Sainte-Foy, Quebec.     

Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria

Although plant growth-promoting rhizobacteria (PGPR) have been reported to influence plant growth, yield and nutrient uptake by an array of mechanisms, the specific traits by which PGPR promote plant growth, yield and nutrient uptake were limited to the expression of one or more of the traits expressed at a given environment of plant–microbe interaction. We selected nine different isolates of PGPR from a pool of 233 rhizobacterial isolates obtained from the peanut rhizosphere on the basis of ACC-deaminase activity. The nine isolates were selected, initially, on the basis of germinating seed bioassay in which the root length of the seedling was enhanced significantly over the untreated control. All the nine isolates were identified as Pseudomonas spp. Four of these isolates, viz. PGPR1, PGPR2, PGPR4 and PGPR7 (all fluorescent pseudomonads), were the best in producing siderophore and indole acetic acid (IAA). In addition to IAA and siderophore-producing attributes, Pseudomonas fluorescens PGPR1 also possessed the characters like tri-calcium phosphate solubilization, ammonification and inhibited Aspergillus niger and A. flavus in vitro. P. fluorescens PGPR2 differed from PGPR1 in the sense that it did not show ammonification. In addition to the traits exhibited by PGPR1, PGPR4 showed strong in vitro inhibition to Sclerotium rolfsii. The performances of these selected plant growth-promoting rhizobacterial isolates were repeatedly evaluated for 3 years in pot and field trials. Seed inoculation of these three isolates, viz. PGPR1, PGPR2 and PGPR4, resulted in a significantly higher pod yield than the control, in pots, during rainy and post-rainy seasons. The contents of nitrogen and phosphorus in soil, shoot and kernel were also enhanced significantly in treatments inoculated with these rhizobacterial isolates in pots during both the seasons. In the field trials, however, there was wide variation in the performance of the PGPR isolates in enhancing the growth and yield of peanut in different years. Plant growth-promoting fluorescent pseudomonad isolates, viz. PGPR1, PGPR2 and PGPR4, significantly enhanced pod yield (23–26%, 24–28% and 18–24%, respectively), haulm yield and nodule dry weight over the control in 3 years. Other attributes like root length, pod number, 100-kernel mass, shelling out-turn and nodule number were also enhanced. Seed bacterization with plant growth-promoting P. fluorescens isolates, viz. PGPR1, PGPR2 and PGPR4, suppressed the soil-borne fungal diseases like collar rot of peanut caused by A. niger and PGPR4 also suppressed stem rot caused by S. rolfsii. Studies on the growth patterns of PGPR isolates utilizing the seed leachate as the sole source of C and N indicated that PGPR4 isolate was the best in utilizing the seed leachate of peanut, cultivar JL24. Studies on the rhizosphere competence of the PGPR isolates, evaluated on the basis of spontaneous rifampicin resistance, indicated that PGPR7 was the best rhizoplane colonizer and PGPR1 was the best rhizosphere colonizer. Although the presence of growth-promoting traits in vitro does not guarantee that an isolate will be plant growth promoting in nature, results suggested that besides ACC-deaminase activity of the PGPR isolates, expression of one or more of the traits like suppression of phytopathogens, solubilization of tri-calcium phosphate, production of siderophore and/or nodulation promotion might have contributed to the enhancement of growth, yield and nutrient uptake of peanut.     

含ACC脱氨酶的根际细菌提高植物抗盐性的研究进展

盐胁迫是抑制植物生长的主要非生物因素之一,高浓度的盐分不利于植物体的生长和发育,严重时会导致植物细胞及植物体死亡.已有大量实验结果显示含ACC脱氨酶的根际细菌可以缓解高盐对植物的危害.ACC脱氨酶可以降解乙烯的直接前体1-氨基环丙烷-1-羧酸(ACC),从而降低胁迫乙烯的合成量.胁迫乙烯是阻碍植物生长的主要原因.首先介...     

Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture

Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into α-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species.     

Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat

AIMS: Plant growth promoting rhizobacteria (PGPR) are commonly used as inoculants for improving the growth and yield of agricultural crops, however screening for the selection of effective PGPR strains is very critical. This study focuses on the screening of effective PGPR strains on the basis of their potential for in vitro auxin production and plant growth promoting activity under gnotobiotic conditions. METHODS AND RESULTS: A large number of bacteria were isolated from the rhizosphere soil of wheat plants grown at different sites. Thirty isolates showing prolific growth on agar medium were selected and evaluated for their potential to produce auxins in vitro. Colorimetric analysis showed variable amount of auxins (ranging from 1.1 to 12.1 mg l-1) produced by the rhizobacteria in vitro and amendment of the culture media with l-tryptophan (l-TRP), further stimulated auxin biosynthesis (ranging from 1.8 to 24.8 mg l-1). HPLC analysis confirmed the presence of indole acetic acid (IAA) and indole acetamide (IAM) as the major auxins in the culture filtrates of these rhizobacteria. A series of laboratory experiments conducted on two cv. of wheat under gnotobiotic (axenic) conditions demonstrated increases in root elongation (up to 17.3%), root dry weight (up to 13.5%), shoot elongation (up to 37.7%) and shoot dry weight (up to 36.3%) of inoculated wheat seedlings. Linear positive correlation (r = 0.99) between in vitro auxin production and increase in growth parameters of inoculated seeds was found. Based upon auxin biosynthesis and growth-promoting activity, four isolates were selected and designated as plant growth-promoting rhizobacteria (PGPR). Auxin biosynthesis in sterilized vs nonsterilized soil inoculated with selected PGPR was also monitored that revealed superiority of the selected PGPR over indigenous microflora. Peat-based seed inoculation with selected PGPR isolates exhibited stimulatory effects on grain yields of tested wheat cv. in pot (up to 14.7% increase over control) and field experiments (up to 27.5% increase over control); however, the response varied with cv. and PGPR strains. CONCLUSIONS: It was concluded that the strain, which produced the highest amount of auxins in nonsterilized soil, also caused maximum increase in growth and yield of both the wheat cv. SIGNIFICANCE AND IMPACT OF STUDY: This study suggested that potential for auxin biosynthesis by rhizobacteria could be used as a tool for the screening of effective PGPR strains.     

The effect of plant growth-promoting rhizobacteria on the phytoextraction of Cd and Zn by Brassica napus L.

The test strains Bacteroidetes bacterium (Ba), Pseudomonas fluorescens (Pf) and Variovorax sp. (Va) were selected in advance for their in vitro capability for growth promotion of rapeseed in the presence of increased concentrations of Cd, Cu, Pb and Zn in the medium. In the pot experiment, the strains were used for single Ba, Pf, Va or combined Ba + Pf, Ba + Va, Pf + Va, and Ba + Pf + Va inoculation of B. napus growing in contaminated soil from alluvial deposits. The positive effect of bacterial strains on plant growth was observed in vitro, but was not confirmed in situ in the contaminated soil, where the tested strains inhibited biomass production, rather than stimulating it. However, single inoculation with Ba significantly increased the chlorophyll content and K⁺ concentration in the leaves. The inoculation of rapeseed with Ba and Va strains was indicated to be the most promising combination for phytoextraction of Cd and Zn from contaminated soil. Combined inoculation with Pf+Va and Pf + Ba+Va significantly decreased the concentration of heavy metals in the roots of rapeseed. We conclude that suitable combinations of PGPR can control the metal uptake of B. napus, selectively increasing either metal extraction or metal stabilization in the rhizosphere and offering promising applications in soil remediation.     

Plant growth promoting rhizobacteria accelerate nodulation and increase nitrogen fixation activity by field grown soybean [Glycine max (L.) Merr.] under short season conditions

A 3 × 2 × 2 factorial field experiment, organized in a randomized complete block split-plot with four replications, was conducted in 1994 to evaluate the effect of two plant growth-promoting rhizobacteria (PGPR) strains (Serratia liquefaciens 2-68 or Serratia proteamaculans 1-102) on nodulation, nitrogen fixation, and total nitrogen yield by two soybean cultivars in a short season area. The experiments were conducted at the Emile A. Lods Research Centre, McGill University, Macdonald Campus, Montreal, Canada, and performed at two adjacent sites. One site was fumigated with methyl bromide (50 g m^-2). Another site was kept unfumigated. Co-inoculation of soybean with B. japonicum and PGPR increased soybean nodulation and hastened the onset of nitrogen fixation, when the soils were still cool. Total fixed N, fixed N as a percentage of total plant N, and protein and N yield were also increased by PGPR inoculation. AC Bravor tended to be more responsive to both PGPR treatments for total fixed N and N yields than Maple Glen, suggesting that inoculation with PGPR was more effective for cultivars with higher yield potentials.     

Plant growth-promoting rhizobacteria reduce adverse effects of salinity and osmotic stress by regulating phytohormones and antioxidants in Cucumis sativus

We assessed the role of plant growth-promoting rhizobacteria (PGPR) strains viz. Burkholdera cepacia SE4, Promicromonospora sp. SE188 and Acinetobacter calcoaceticus SE370 in counteracting salinity and drought stress to cucumber plants. The control plants had stunted growth, while PGPR-treated plants had significantly higher biomass and chlorophyll contents under salinity and drought stress. The ameliorative effects of PGPR-application were also evidenced by the increased water potential and decreased electrolytic leakage. The PGPR-applied plants had reduced sodium ion concentration, while the potassium and phosphorus were abundantly present as compared to control under stress. Oxidative stress was mitigated by PGPR through reduced activities of catalase, peroxidase, polyphenol oxidase, and total polyphenol as compared to control. The control plants showed up-regulation of stress-responsive abscisic acid as compared to PGPR application, while salicylic acid and gibberellin 4 were significantly higher in PGPR. In conclusion, the PGPR application might be used in marginalized agricultural lands to increase crop productivity.     

The effect of plant growth-promoting rhizobacteria on the growth,physiology, and Cd uptake of Arundo donax L.

In this study, plant growth-promoting potential isolates from rhizosphere of 10 weed species grown in heavy metal-contaminated areas were identified and their effect on growth, antioxidant enzymes, and cadmium (Cd) uptake in Arundo donax L. was explored. Plant growth-promoting traits of isolates were also analyzed. These isolates were found to produce siderophores and enzymes such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and aid in solubilization of mineral nutrients and modulate plant growth and development. Based on the presence of multiple plant growth-promoting traits, isolates were selected for molecular characterization and inoculation studies. Altogether, 58 isolates were obtained and 20% of them were able to tolerate Cd up to 400 ppm. The sequence analysis of the 16S rRNA genes indicates that the isolates belong to the phylum Firmicutes. Bacillus sp. along with mycorrhizae inoculation significantly improves the growth, the activity of antioxidants enzymes, and the Cd uptake in A. donax than Bacillus alone. Highly significant correlations were observed between Cd uptake, enzymatic activities, and plant growth characteristics at 1% level of significance. The synergistic interaction effect between these organisms helps to alleviate Cd effects on soil. Heavy metal-tolerant isolate along with arbuscular mycorrhizae (AM) could be used to improve the phytoremedial potential of plants.     

Application of plant growth-promoting rhizobacteria to soybean (Glycine max [L.] Merr.) increases protein and dry matter yield under short-season conditions

We previously reported that application of plant growth-promoting rhizobacteria (PGPR) increased soybean growth and development and, specifically, increased nodulation and nitrogen fixation over a range of root zone temperatures (RZTs) in controlled environment studies. In order to expand on the previous studies, field experiments were conducted on two adjacent sites, one fumigated with methyl bromide and one nonfumigated, in 1994. Two experiments were conducted at each site, one involving combinations of two soybean cultivars and two PGPR strains, the other involving the same factors, but also in combination with two strains Bradyrhizobium japonicum. Soybean grain yield and protein yield were measured. The results of these experiments indicated that co-inoculation of soybean with B. japonicum and Serratia liquefaciens 2-68 or Serratia proteamaculans 1-102 increased soybean grain yield, protein yield, and total plant protein production, compared to the nontreated controls, in an area with low spring soil temperatures. Interactions existed between PGPR application and soybean cultivar, suggesting that PGPRs applied to cultivars with higher yield potentials were more effective. PGPRs applied to the rhizosphere without addition of B. japonicum also increased only leaf area and seed number at the fumigated site. Overall, inoculation of soybean plants with PGPRs in the presence of B. japonicum increased soybean grain yield, grain protein yield, and total plant protein production under short season conditions.     

Salt-tolerant and plant growth-promoting bacteria isolated from Zn/Cd contaminated soil: identification and effect on rice under saline conditions

The bacteria of PDMCd0501, PDMCd2007, and PDMZnCd2003 were isolated from a Zn/Cd contaminated soil. They were classified as salt-tolerant bacteria in this experiment. The bacteria had indole-3-acetic acids (IAA) production, nitrogen fixation, and phosphate solubilization, under 8% (w/v) NaCl condition. Biochemical test (API 20E) and 16S rDNA sequencing identified PDMCd2007 and PDMCd0501 as Serratia sp. and PDMZnCd2003 was Pseudomonas sp. The effect of Pseudomonas sp. PDMZnCd2003 on the germination and seedlings of Oryza sativa L.cv. RD6 was determined under a salinity of 0–16 dS/m. The salinity levels of 4–16 dS/m affected to decrease germination and seedlings of rice. Comparison between uninoculated and inoculated system, however, Pseudomonas sp. PDMZnCd2003 had a negative impact on the rice growth. This unexpected effect was a case that should be concerned and studied further before application as a plant growth-promoting bacteria (PGPB).     

Effects of inoculation with plant growth promoting rhizobacteria (PGPRs) andSinorhizobium fredii on biological nitrogen fixation, nodulation and growth ofGlycine max cv. Osumi

We investigated the effects of three plant growth promoting rhizobacteria (PGPR), on Biological Nitrogen Fixation (BNF), nodulation and growth promotion by soybean (Glycine max) var. Osumi plants. The strains, Aur 6, Aur 9 and Cell 4, belong toPsedomonas fluorescens, Chryseobacterium balustinum andSerratia fonticola, respectively. Inoculation modes for the PGPRs andSinorhizobium fredii (carried out through irrigation), were examined. In the first mode, PGPRs andS. fredii were co-inoculated. In the second mode, we first inoculatedS. fredii and after the PGPRs, which were added 5 or 10 days later (each inoculation being an independent treatment). In the third mode, the PGPRs were inoculated first, and theS. fredii was inoculated 5 days later. We also included treatments inoculated with only the PGPRs (one PGPR per treatment) and only withS. fredii. Plants were maintained in a greenhouse under controlled environmental conditions, and were sampled 3 months after sowing. The results obtained showed the effects of the inoculation sequence. The most significant effects on growth parameters (stem plus leaf weight and fresh root weight) were found when inoculations with PGPR andS. fredii were at different times or when we inoculated only with PGPR and the plants were watered with nitrogen. Co-inoculation had no positive effects on any parameter, probably due to competition between the PGPR andS. fredii. Our results indicate that the inoculation modes with PGPR and rhizobia play a very important role in the effects produced. Thus, although plant growth promoting rhizobacteria may interact synergistically with root-nodulating rhizobia, plant growth promoting rhizobacteria selected for one crop should be assessed for potentially hazardous effects on other crops before being used as inoculants.     

Plant regeneration from mesophyll-protoplasts of four different ecotypes and two marker lines from Arabidopsis thaliana using a unique protocol

A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of four ecotypes (Col C24, Per-1, Bur-0, Landsberg erecta) and two marker lines (M4 and M10) of Ardbidopsis thaliana is described. The different lines showed plating efficiencies between 1.0 and 3.9% using Nitsch medium or this medium supplemented with coconut water. For the differentiation of callus into normal shoots a single shoot regeneration medium was applicable to all ecotypes, but depending on the line other regeneration media showed to be more suitable. The results indicated that the protoplast culture procedure is applicable, with minor modifications, to all tested genotypes but the most suitable shoot regeneration medium should be established for each A. thaliana line.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzyl-aminopurine - IAA indole-3-acetic acid - IPA isopentenyladenine - IPAR isopentenyladenosine - MES 2-[N Morpholino]ethanesulfonic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

Integration of chitosan and plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus

Papaya ringspot virus-W (PRSV-W) and Tomato chlorotic spot virus (TCSV) are common viruses infecting vegetables in south Florida. Application of plant growth-promoting rhizobacteria (PGPR) has emerged as a potential alternative of chemical pesticides to control these viruses. But, it is not sufficient to completely replace chemical control. This study aimed to investigate the synergistic effect of chitosan and PGPR to control PRSV-W and TCSV. The efficiency of PGPR to suppress PRSV-W and TCSV was significantly improved when they were accompanied with chitosan treatment. The highest reduction in disease severity of both PRSV-W and TCSV was achieved when chitosan treatment was accompanied with mixture of two PGPR (IN937a + SE34) or three PGPR strains (IN937a + SE34 + SE56). The results of this study proved that implementation of chitosan and PGPR could significantly restrict losses due to PRSV-W and TCSV in squash and tomato, in Florida and the United States.     

Application of plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus

Papaya ringspot virus (PRSV-W) and Tomato chlorotic spot virus (TCSV) are responsible for severe losses in cucurbits and tomato production in south Florida and other regions in the USA. Traditional chemicals are not effective to control these viruses. Using plant growth-promoting rhizobacteria (PGPR) may present an alternative to control these viruses. Results from this study demonstrated that applying mixtures of PGPR strains is more efficient to control PRSV-W and TCSV compared to individual PGPR strain only. The application method significantly affected the efficiency of PGPR to control PRSV-W and TCSV. The highest reduction in disease severity of both PRSV-W and TCSV occurred in case of soil drenching with PGPR, followed by root dipping and seed coating treatments. Application of PGPR mixtures of (IN937a & SE34) or (IN937a &, SE34 & T4) were the most efficient methods to control these viral diseases.