Effect of the plant probiotic bacteria on terpenoid indole alkaloid biosynthesis pathway gene expression profiling, vinblastine and vincristine content in the root of Catharanthus roseus.

BACKGROUND: Catharanthus roseus is the sole resource of vinblastine and vincristine, two TIAs of great interest for their powerful anticancer activities. Increasing the concentration of these alkaloids in various organs of the plant is one of the important goals in C. roseus breeding programs. Plant probiotic bacteria (PBB) act as biotic elicitors and can induce the synthesis of secondary products in plants. The purpose of this research is to study the effects of PBB on expression of the TIA biosynthetic pathway genes and the content of alkaloids in C. roseus. METHODS AND RESULTS: The individual and combined effects of P. fluorescens strains 169 and A. brasilense strains Ab-101 was studied for expression of the TIA biosynthetic pathway genes (G10H, DAT, T16H and CrPRX) using qRT-PCR and the content of vinblastine and vincristine using HPLC method in roots of C. roseus. P. fluorescens. This drastically increased the content of vinblastine and vincristine alkaloids, compared to the control in the roots, to 174 and 589 (µg/g), respectively. Molecular analysis showed bacterium significantly increased the expression of more genes in the TIA biosynthetic pathway compared to the control. P. fluorescens increased the expression of the final gene of the biosynthetic pathway (CrPRX) 47.9 times compared to the control. Our findings indicate the correlation between transcriptional and metabolic outcomes. The same was true for A. brasilense. CONCLUSIONS: It can be concluded that seed treatments and seedling root treatments composed of naturally occurring probiotic bacteria are likely to be widely applicable for inducing enhanced alkaloid contents in medicinal plants.

Evaluation of a new radiolabeled bombesin derivative with 99mTc as potential targeted tumor imaging agent.

Gastrin-releasing peptide (GRP) receptors are over-expressed in various human tumor including breast and prostate which can be targeted with bombesin for diagnosis and targeted therapy. High abdominal accumulation and the poor in vivo stability of radiolabeled bombesin analogues may represent a limitation for diagnostic imaging and targeted therapy. In this study a new bombesin derivative was labeled with 99mTc via HYNIC and tricine as a coligand and investigated further. The peptide HYNIC conjugate was synthesized on a solid phase using Fmoc strategy. Labeling with 99mTc was performed at 100 °C for 10 min and radiochemical analysis involved ITLC and HPLC methods. The stability of radiopeptide was checked in the presence of human serum at 37 °C up to 24 h. Internalization was studied with the human GRP receptor cell line PC-3. The Biodistribution was studied in mice. Labeling yield of >98 % was obtained to correspond a specific activity of ~80.9 GBq/µmol. Radioconjugate internalization into PC-3 cells was high and specific (15.6 ± 1.9 % at 4 h). A high and specific uptake in GRP-receptor-positive organs such as mouse tumor and pancreas (2.11 ± 0.18 and 1.78 ± 0.09 % ID/g after 1 h respectively) was also determined.

Selection of bacterial antagonists for the biological control of Botrytis cinerea in apple (Malus domestica) and in comparison with application of thiabendazole.

In this study fifteen strains of identified Pseudomonas fluorescens and Bacillus subtilis were investigated for biological control activity against Botrytis cinerea. P. fluorescens P-35 and B. subtilis B-16 showed the most inhibitory zone in dual culture assay against B. cinerea, In vitro. After ten days, P. fluorescens P-5 and B. subtilis B-3 showed the considerable results against B. cinerea on apple fruits and could reduce the grey mould from 100% to less than 35%. After twenty days, P. fluorescens P-5 and 8. subtilis B-16 decreased the disease from 100% to less than 65%. Also, application of thiabendazol at 1500 mg/litre was more effective and could reduce the disease from 100% to 30% and 60%, after 10 and 20 days respectively. Results indicated that there is no significant difference among the treatments (thiabendazol and bacterial strains). So, bacterial strains could not only control the disease but also be a reliable replacement instead of Thiabendazol.

Screening of Pseudomonas and Bacillus isolates for potential biocontrol of the damping-off of bean (Phaseolus coccineus).

In this study fifteen isolates of identified Pseudomonas fluorescens and Bacillus subtilis were investigated for control of bean damping-off disease caused by Rhizoctonia solani. In vitro, P. fluorescens P-6 and B. subtilis B-3 showed the most inhibitory zone in dual culture assay against R. solani. The growth of P-6 (4.5 x 10(8) cfu/ml) was significantly higher than in the other treatments. In greenhouse condition, all of the isolates effectively controlled damping-off on bean. P. fluorescens P-5 and P-6 showed the considerable results against R. solani and could reduce the damping-off disease from 100% to less than 30%. P-5, P-6 (P. fluorescens) and 8-16 (8. subtilis) strains had the highest effect on fresh weight of bean.

Interaction of media on production and biocontrol efficacy of Pseudomonas fluorescens and Bacillus subtilis against grey mould of apple.

The medium has a profound effect on biocontrol agents, including ability to grow and effectiveness in disease control. In this study, growth and antagonistic efficacy of strains P-5 and P-35 (P. fluorescens), B-3 and B-16 (B. subtilis) were evaluated in combinations of two carbon (sucrose and molasses) and two nitrogen (urea and yeast extract) sources to optimize control of Botrytis cinerea on apple. All of the strains were grown in different liquid media (pH = 6.9) including: sucrose + yeast extract, molasses of sugar beet + yeast extract in 2:1 and 1:1 w/w ratios, molasses of sugar beet + urea, molasses, malt extract and nutrient broth. Apples (Golden Delicious) were inoculated by a 25-microl suspension of 10(6) spores of B. cinerea per ml, wounding each fruit (in two sites separately). Then a 25-microl suspension of each strain, containing 2 x 10(8) cfu ml(-1) grown in each of the above culture media, was applied to each wound. Results indicated that Molasses + Yeast extract (1:1 w/w) medium supported rapid growth in all of the strains. The final growth of B. subtilis B-16 in Molasses + Yeast extract (1:1 w/w) medium was 5 x 10(9) cfu ml(-1). After ten days, all of the strains significantly inhibited pathogenicity of B. cinerea on apples. The biocontrol efficacy of B. subtilis B-3 in Molasses + Yeast extract (1:1 w/w) medium reduced the severity of grey mould from 100% (inoculated control) to less than 26.9%. After 20 days, Strain B-3 showed a considerable biocontrol efficacy in Molasses medium and reduced the severity of grey mould from 100% (inoculated control) to less than 38.2%. The results obtained in this study could be used to provide a reliable basis for the increase of population of biocontrol agents in fermentation process.

Powder formulation of Burkholderia cepacia for control of rape seed damping-off caused by Rhizoctonia solani.

Talc-based formulation of Burkholderia cepaci strain Bu1 was tested as seed and soil drenchs separately for its ability to control Rhizoctonia soloni the causal agent of rape seed damping-off in greenhouse and field trials. In general, the formulated bacteria was more effective to suppress the disease than the suspension of bacteria cells in carboxymethylcellulose solution (1% w/v), in both greenhouse and field trials. The formulation of strain Bul as soil and seed treatments had the greatest effect on reducing the rape seed damping-off in greenhouse and field trials (66.7, 53.3, 64.4 and 40% respectively). The formulation of strain Bu1 as soil and seed treatments were the most effective treatments to increase the root dry weights in the infected soil in greenhouse. The formulation of strain Bul as soil drench had the greatest effect on enhancement of the fresh weight of roots and stem fresh and dry weights. The formulation of strain Bu1 stored at 4 degrees C exhibited better shelf Life and efficacy in vitro than it's counterpart stored at 25 degrees C.

Biocontrol of Phytophthora cactorum the causal agent of root and crown rot on apple (Malus domestica) by formulated Pseudomonas fluorescens.

In this study 284 isolates were isolated of apple trees' rhizosphere from Iran and 128 isolates were obtained from the collection of Research Department of Biological Control of University of Tehran. Four strains (P60, P61, P96, and P97) of Pseudomonas fluorescens were selected for greenhouse trials. The results of greenhouse trials showed dipping the crown and root of apple seedlings (MM106) combined with soil drench was more effective than dipping the crown and root on reducing the disease. After 6 weeks, strain P60 in dipping method combined with soil drench with 70% control, exhibited greatest effect on reducing the crown and root rot and was more effective than the fungicide metalaxyl-mancozeb. After 12 weeks, strains P60 and P96 in dipping method combined with soil drench with 55.6% and 44.5% control respectively, exhibited greatest effects on reducing the diseases Study of media on growth rate populations of effective strains exhibited that the beet molasses yeast extract (1:1) had more effect than nutrient broth(NB) medium. The initial high populations of powder formulations of strains P60 and P96 decreased during the storage at 4 and 25 degrees C over a 150-day period. In addition, formulations of strains stored at 4 degrees C had longer shelf life than those stored at 25 degrees C. In glasshouse trials, after 6 weeks, formulation of strain P60 and unformulated P60, obtained from NB medium and formulated P60, obtained from molasses yeast extract medium, and metalaxyl-mancozeb had highest effect on reducing the disease on apple rootstocks. After 12 weeks, formulation of strain P60 and unformulated bacteria obtained from both media, and metalaxyl-mancozeb with 57.1% control showed greatest effect on reducing the disease.

Evaluation of production of some antifungal metabolites by fluorescent pseudomonads in vitro and their inhibitory effect on Penicillium digitatum in semi-commercial condition.

Epiphytic fluorescent Pseudomonads isolated from the surfaces of citrus leaves and fruits collected from the citrus orchards located in the north of Iran were screened for antagonistic activity against Penicillium digitatum caused agent of green mold of citrus fruit. 9 isolates revealed antagonistic effect in dual culture and then evaluated for production of antimicrobial metabolites such as production hydrogen cyanide on King's B medium containing glycine, production of siderophore on blue CAS-agar medium and production protease enzyme on Skim Milk Agar (SMA) medium. About 28% strains produced hydrogen cyanide, 83% strains produced siderophore and 70% of them produced protease. Antagonistic effects of the screened 9 isolates were studied by inoculating of the infected orange fruits with the bacterium at the concentration of 10(8) cells/ml under sterilized condition at 20 degrees C. The three final screened antagonist isolates were more studied in semi commercial trails in cold storage with dipping of the orange fruits in the bacterial suspension at the concentration of 10(8) cells/ml and then 5 weeks storage at 4 degrees C. The isolate P39 showed to be the most effective for controlling of this disease and decreased the damage by 77.28% respectively.

Effect of timing of application Pseudomonas fluorescens in suppression Sclerotinia sclerotiorum, the causal agent of white mold in canola.

Biological control of sclerotinia disease, as an important alternative to chemical control, has received considerable attention due to the lack of resistant varieties in most crop, and increasing concerns over fungicide resistance in population of Sclerotinia sclerotiorum, and fungicide residues in the environment. One biocontrol agent, Pseudomonas fluorescens PB-3, has been showed the antagonistic relationship between itself and S. sclerotiorum was investigated in this study. A petal infection technique was used to detect efficacy of timing of application strain PB-3 in the suppression of S. sclerotiorum on canola. Significant difference in disease severity (p<0.05) were found with respect to timing of ascospore applications in the control treatments (ascospores only). The superior competitive ability strain PB-3 was demonstrated by its complete suppression of disease severity when applied as a co-inoculation treatment or prior to ascospores inoculation. Analysis of effect of applying strain PB-3 after ascospores was indicated that treatment in which strain PB-3 was added to petals 48 or 24 h after ascospores, or when there were no bacteria present at all, had higher rates of disease progression. It would be appear that bacteria are able to significantly inhibit disease when applied before or even at the same time as ascospores. In a practical sense, this could mean that a field application of antagonist could be concurrent with infection by the pathogen.

Effect of carbon and nitrogen sources on growth and biological efficacy of Pseudomonas fluorescens and Bacillus subtilis against Rhizoctonia solani, the causal agent of bean damping-off.

One of the most important environmental factors that regulate the growth and antagonistic efficacy of biocontrol agents is the medium. The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strains P-5 and P-6 (Pseudomonas fluorescens), B-3 and B-16 (Bacillus subtilis) and minimum cost of media, whilst maintaining biocontrol efficacy. All of the strains were grown in seven liquid media (pH=6.9) including: sucrose + yeast extract, molasses of sugar beet + yeast extract in 2:1 and 1:1 w/w ratios, molasses of sugar beet + urea, nutrient broth, molasses and malt extract, at an initial inoculation of 1 x 10(5) CFU ml(-1). Cells from over night cultures used to inoculate soil at 1 x 10(9) CFU cm(-3) soil. At the same time, fungal inoculum (infected millet seed with Rhizoctonia solani) was added to soil at the rate of 2 g kg(-1) soil. Results indicated that growth of P-6, B-3 and B-16 in molasses + yeast extract (1:1 w/w) medium was significantly higher than in the other media. Molasses + yeast extract (1:1 and 2:1 w/w) media supported rapid growth and high cell yields in P-5. In greenhouse condition, results indicated that the influence of the media on the biocontrol efficacy of P-5, P-6, B-3 and B-16 was the same and Pseudomonas fluorescens P-5 in molasses and malt extract media reduced the severity of disease up to 72.8 percent. On the other hand, there were observed significant differences on bean growth after one month in greenhouse. P-5 in molasses + yeast extract (1:1 w/w) medium had the most effects on bean growth promotion. In this study molasses media showed good yield efficacy in all of the strains. The high sucrose concentration in molasses justifies the high biomass in all of the strains. Also, the low cost of molasses allows its concentration to be increased in media. On the other hand, yeast extract was the best organic nitrogen source for antagonist bacteria but it is expensive for an industrial process. So it should be replaced by another industrial product instead of yeast extract, which confirm by an economic and technological study. The results obtained in this study could be used to provide a reliable basis to increase the population of biocontrol agents in fermentation process.

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mold, by Pseudomonas species on canola petals.

Sclerotinia sclerotiorum is an important pathogen on canola. Due to the public concern over pesticide use, alternative methods of disease control, such as biological control, should be considered. Several bacterial strains were isolated from canola and soja plants. Inhibition of S. sclerotiorum by bacterial strains in vitro was assayed on PDA medium in dual culture test. Eight Pseudomonas sp. strains (PB-3, PB-4, PB-5, PB-6, PB-7, PB-8, PB-10 and PB-11) caused inhibition zone against 5. sclerotiorum hyphal growth. The biocontrol potential of the bacteria was tested in a plant assay. Disease suppression was investigated using a petal inoculation technique. Canola petals were pretreated with bacteria, and then inoculated with 5. sclerotiorum ascospores 24 h later. Greenhouse experiment showed that application of Pseudomonas sp. strains (1 x 10(8) cfu ml(-1)) effectively suppressed S. sclerotiorum (1 x 10(5) ascospores ml(-1)) on petals and all of them achieved significant (P<0.01) disease suppression. Fourteen days after inoculation, strain PB-3 had 88/7% disease control and strain PB-4 had 69/9% disease control. Result from all studies indicates PB-3 to be effective biocontrol against S. sclerotiorum of canola. PB-3, PB-4, PB-7, PB-8, PB-10 and PB-11 were identified as Pseudomonas fluorescens biovar III. PB-5 and PB-6 was identified as Pseudomonas fluorescens biovar II. Strains PB-3, PB-4, PB-6, PB-10 and PB-11 produced protease and HCN. Strain PB-5 produce protease; no HCN.

Powder formulations of two strains of Bacillus subtilis for control of rape seed damping-off caused by Rhizoctonia solani.

Talc-based formulations of Bacillus subtilis strains B1 and B2 were tested as seed and soil treatments separately for their ability to control Rhizoctonia solani, the causal agent of rape seed damping-off, in greenhouse and field trials. In general, the formulated bacteria was more effective to suppress the disease than the suspension of bacterial cells in carboxymethylcellulose solution (1%, w/v), in both greenhouse and field trials. The formulations of strain B1 as soil treatment and strain B2 as seed treatment in greenhouse, and the formulations of strain B2 as seed and soil treatments in field trials had the greatest effect on reducing the rape seed damping-off (66.7%, 73.3%, 41.3%, and 42.4%, respectively). The formulations of strain B1 as soil treatment and strain B2 as seed treatment were the most effective treatments to increase the root dry weights in the infected soil in greenhouse. The formulation of strain B2 as soil treatment had the greatest effect on enhancement of the fresh weight of roots and stem fresh and dry weights. The formulations of strains B1 and B2 stored at 4 degrees C exhibited better shelf life and efficacy in vitro than their counterparts stored at 25 degrees C. Long-term stability of the formulation of strain B1 was found to be better.

Suppression of seed rot and preemergence of chickpea by seed treatments with fluorescent pseudomonads in Iran.

Species of Pythium isolated from rotted chickpea seeds and damped-off seedlings and chickpea soils at experimental field of Agriculture faculty of Tehran University in Karaj area that caused seed rot and preemergence damping-off of chickpea were Pyhium ultimum var. ultimum. One of the most important of soilborne fungal pathogens of the chickpea in Iran is seed rot and preemergence damping-off caused by Pythium ultimum Trow. Consequently, growers can expect as much as > 80% reduction in stand and yield if measures are not taken to control Pythium. Currently, most commercial seeds of chickpea are treated with pesticides. Fluorescent pseudomonads applied to seed are known to reduce soilborne diseases of chickpea caused by Pythium spp. In this study rotted chickpea seeds and diseased seedlings and soil samples were collected from experimental field in Karaj. Soils and roots used as sources of bacteria were collected from field. Fluorescent pseudomonads were isolated by plating samples on S1 and King's Medium B (KMB). Bacteria were preserved in 0.1 M MgSO4 for long-term storage; and NAG (containing 2% glucose) slants and plates at 4 degrees C short-term storage. Of 20 fluorescent pseudomonads isolated on S1 medium, 2 isolates selected for next tests. All strains significantly increased emergence as compared to the infested control in greenhouse trial; isolate Pf-4 consistently provided the best protection against Pythium. Seedling emergence from all bacteria seed treatments was statistically lower than the chemical treatments. All strains significantly increased fresh weight of chickpea as compared to the infested control in greenhouse trial. Seed treatment with metalaxyl were statistically better than captan in sterilized soil. In nonsterilized soil collected from the field artificially infested with P. ultimum, all strains significantly increased fresh weight of chickpea as compared to the infested control in greenhouse trial. Seedling emergence from seed treatment with isolate Pf-1 was the least effective treatment on fresh weight of chickpea in nonsterilized soil infested with P. ultimum. Seed treatment with captan was statistically better than metalaxyl in nonsterilized soil.

Biocontrol of Rhizoctonia solani, the causal agent of bean damping-off by fluorescent pseudomonads.

Rhizosphere bacteria belonging to the fluorescent pseudomonads are receiving increasing attention for the protection of plants against soil-borne fungal pathogens. Among these pathogens, Rhizoctonia solani, the causal agent of bean damping- off is very important in bean fields of Iran. In this study, the antagonistic activity of 46 isolates of fluorescent pseudomonads (isolated from different area of Iran) and Pseudomonas fluorescens strain CHA0 investigated against one isolate of R. solani. About 64% of isolates revealed antagonistic activity against R. solani. Production of antifungal metabolites such as HCN, siderophore and protease was evaluated. The results showed that 97.8%, 17% and 78% of isolates produced siderophore, HCN and protease respectively. There was no significant correlation between antagonistic activity and production of these metabolites. Isolates P-5, P-10 and P-32 with strain CHA0 were selected in order to investigate involvement of siderophore, volatile metabolites (HCN), and non-volatile metabolites in reducing mycelial growth of R. olani. Isolate P-5 showed much more inhibitory effect by production of volatile metabolites and siderophore. Non-volatile metabolites in isolates P-32 and P-5 completely inhibited mycelial growth of the fungus. After the primary labrotory tests, isolates P-14, P-35, P-30, P-5 and strain CHA0 were selected for in vivo experiments. These selected isolates with benomyl fungicide were used as seed coating and soil drenching in sterile soil under greenhouse condition. The result indicated that in seed treatment method, isolates P-30 by 66% had the most effect in disease reduction while in soil treatment method, strain CHAO by 60% had the most effect, such that this two isolates showed significant differences in comparison with plants inoculated with R. solani inoculums.

Effect of fluorescent pseudomonades and Trichoderma sp. and their combination with two chemicals on Penicillium digitatum caused agent of citrus green mold.

Citrus green mold (Penicillium digitatum) causes economic losses. Chemical fungicides such as imazalil provide the primary means for controlling green mold decay of citrus fruits. Continuous use of fungicides has faced two major obstacles- increasing public concern regarding contamination of perishables with fungicidal residues, and proliferation of resistance in the pathogen populations. The aim of this research was to determine if the attacks of green mold on orange could be reduced by usage of biocontrol agent alone or in combination with low dosage of imazalil or sodium bicarbonate. Pseudomonas fluorescens isolate PN, P. fluorescens isolate PS and Trichoderma virens isolate TE were evaluated as potential biological agents for control of green mold of oranges caused by P. digitatum. Increasing concentration of SB decreased spore germination of P. digitatum. In laboratory tests, a cell suspension (10(8) cells per ml.) of bacterial strains reduced the incidence of green mold. On fruits surface biocontrol activity of antagonistic isolates was significantly increased when combined with low dosage of imazalil (500ppm) or sodium carbonate (5%). Effect of Trichoderma virens on controlling P. digitatum was better than others with or without these chemicals.

Composition and antifungal activity of essential oils of Mentha piperita and Lavendula angustifolia on post-harvest phytopathogens.

The general antifungal activity of essential oils is well documented. The advantage of essential oils is their bioactivity in the vapor phase, a characteristic that makes them attractive as possible fumigants for stored product protection. Essential oils of aerial parts of Mentha piperita and Lavendula angustifolia were obtained with hydrodistillation and oils composition identified with GC-MS. Menthanol (36.24%) and menthone (32.42%) were the major compounds of the M. piperata essential oil. The essential oil of L. angustifolia was rich in linalool (49.2%) , linalyl acetate (12.3%), Lavendul acetate (6.5%), 4-terpineol (5.9%). Fungal toxicity of the essential oils were evaluated against three pathogenic fungi (Rhizopus stolonifer, Botrytis cinerea and Aspergillus niger) in vitro. Plate assayes showed that the different concentrations of essential oils have antifungal activity against these fungi, and the essential oil of L. angustifolia showed stronger fungistatic activity. Lavendula oil exhibited complete growth inhibition of all pathogens at 1000 ppm and minimum EC50 (311.24 ppm) resulted on B. cinerea.

Chemical composition and antifungal activity of the essential oils of three species of Artemisia on some soil-borne phytopathogens.

Various species of the genus Artemisia are used for their pharmacological, antimicrobial, antioxidant activity. Three species of this genus, Artemisia scoparia, A. sieberi and A. aucheri are widely distributed in desert area of Iran. In order to identify the chemical composition, aerial parts of A. scoparia, A. sieberi and A. aucheri were collected from Bajestan (Khorasan province) at flowering stage. The essential oils were obtained by hydro-distillation of air-dried samples and their chemical composition identified by GC-MS. Oxygenated monoterpens were the major components of the oils of three species. alpha-thujone (81.7%), beta-thujone (14.5%) and 1,8-cineol (1.9%) were the major compounds in the essential oil of A. scoparia. The essential oil of A. aucheri was rich in linalool (44.1%), gernyl acetate (10.7%), (E)-citral (9.7%) and (Z)-citral (7.7%), and the essential oil of A. sieberi was rich in beta-thujone (19.8%), alpha-thujone (10.5%), camphor (19.5%), verbenol (9.7%), p-mentha-1,5-dien-8-ol (6.4) and davanone (5.8%). The essential oils of the three species were tested for their antifungal activity against some soil-borne pathogenic fungi. Results of bioassay showed that the oils of A. aucheri and A. sieberi has stronger antifungal activity. Minimum EC50 (41.406 microL/L), resulted from A. aucheri on Rhizoctonia solani. The oil of A. sieberi showed fungistatic activity against, Tiarosporella phaseolina (MIC = 1000 microL/L, EC50 = 203.419 microL/L), Fusarium moniliforme (MIC=750 microL/L, EC50 = 211.072 microL/L), Fusarium solani (MIC = 750 microL/L, EC50 = 188.134 microL/L) whereas against R. solani (MIC = 250 microL/L, EC50 = 121.798 microL/L) exhibited high fungicidal activity.

Contribution of phlA and some metabolites of fluorescent pseudomonads to antifungal activity.

Fluorescent Pseudomonas species are an important group of PGPR that suppress fungal root and seedling disease by production of antifungal metabolites such as 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, pyrolinitrin, siderophores and HCN. The compound 2,4-DAPG is a major determinant in biocontrol of plant pathogens. A 7.2 kbp chromosomal DNA region, carrying DAPG biosynthetic genes (phlA, phlC, phlB, phlD, phIE and phlF). Detecting the ph1 genes make them an ideal marker gene for 2,4-DAPG-producing fluorescent pseudomonad's. In this study we detected ph1A gene (that convert MAPG to 2,4-DAPG) using PCR assay with primers phlA-1r and phlA- f that enabled amplification of phlA sequences from fluorescent pseudomonad's from ARDRA group 1 and 3. We could detect phlA gene in P. fluorescens strains CHAO, Pf-44, Pf-1, Pf-2, Pf-3, Pf-17, Pf-62 and Pf-64, native isolates of Iran. The efficacy of this method for rapid assay characterizing rhizosphere population of 2,4-DAPG producing bacteria from soil of different area of Iran is in progress. We used a collection of 48 fluorescent pseudomonas strains in vitro, with known biological control activity against some soil born phytopathogenic fungi such as, Macrophomina phaseoli, Rhizoctonia solani Vericillium dahlia, Phytophthora nicotiana, Pythium spp. and Fusarium spp. and the potential to produce known secondary metabolites such as protease. Strains Pf-1, Pf-2, Pf-3, Pf-17, Pf-33 and Pf-44 showed the best antifungal activity against all fungi used in this study. Thirty-eight of 48 strains produced protease. The ability to rapidly characterize populations of 2,4-DAPG producers will greatly enhance our understanding of their role in the suppression of root disease.

Heterogeneity of the memory CD4 T cell response: persisting effectors and resting memory T cells.

Defining the cellular composition of the memory T cell pool has been complicated by an inability to distinguish effector and memory T cells. We present here an activation profile assay, using anti-CD3 and antigenic stimuli, that clearly distinguishes effector and memory CD4 T cells and defines subsets of long-lived memory CD4 T cells based on CD62 ligand (CD62L) expression. The CD62L(low) memory subset functionally resembles effector cells, exhibiting hyper-responsiveness to antigenic and anti-CD3 mediated stimuli, high proliferative capacity, and rapid activation kinetics. The CD62L(high) memory subset functionally resembles resting memory cells, exhibiting hyporesponsiveness to anti-CD3 stimuli, lower proliferative capacity, and slower activation kinetics. Our results indicate that the memory CD4 T cell pool is heterogeneous, consisting of persisting effectors and resting memory T cells.

Effector CD4 T cells are biochemically distinct from the memory subset: evidence for long-term persistence of effectors in vivo.

Memory T cell responses are believed to be mediated by long-lived memory T cells that arise directly from a subset of short-lived, activated effector T cells that have reverted to the resting state. Although widely accepted, definitive proof that memory T cells arise from effectors is lacking because of the inability to reliably distinguish these subsets based on known phenotypic or functional parameters. We have used a biochemical approach to distinguish effector and memory CD4 T cell subsets and follow the differentiative fate of effector cells in vivo. When examined biochemically, effector and memory CD4 T cells are strikingly distinct and exhibit qualitative and quantitative differences in tyrosine phosphorylation. These effector-specific patterns were identical in effectors derived either from naive CD4 T cells (primary effectors) or memory CD4 T cells (memory effectors). To monitor the fate of effector cells in vivo, Ag-activated CD4+ TCR-transgenic T cells were transferred into irradiated BALB/c mice. These TCR-transgenic CD4 T cells persisted in adoptive hosts for several months, gave a recall response to Ag, yet exhibited effector-specific biochemical profiles. These results suggest that a subset of effector CD4 T cells can persist in vivo and contribute to long-term immunity by mediating secondary immune responses.