Survival ofalginate-ecapsulated Pseudomonas fluorescens cells in soil

Alginate-encapsulated and unencapsulated cells of Pseudomonas fluorescens Rsf were introduced into soil microcosms with and without wheat plants to evaluate bacterial survival and colonization of the rhizoplane and rhizosphere. Encapsualtion of cells in alginate amended with skim milk or with skim milk plus bentonite clay significantly enchanced long-term survival of the cells. There was a negligible effect on long-term bacterial survival when cells were encapsulated in alginate amended with TY medium or soil extract, as compared to water. Drying of beads resulted in a significant reduction in bacterial viability. After addition to soil, cells in dried beads increased in numbers and exhibited stable population densities, whereas cells added in moist beads showed stable dynamics at a higher level. Cells encapsulated in dried beads or fresh beads survived better than unencapsulated cells added to soil. Both cells in moist and dried alginate beads also survide a dry/wet cycle in soil, whereas unencapsulated cells were sensitive to these moisture fluctuations. Shortly after inoculation and 63 days after this, cells from moist beads colonized wheat roots at significantly higher levels than unencapsulated cells, whereas cells in dried beads did so at levels similat to unencapsulated cells. Cells in beads initially placed at different distance from developing root mat were able to move towards and colonize the rhizosphere, at levels of roughly 10⁴ to 10⁶ colony-forming units fo P. fluorescens R2f per gram of dry soil. Correspondence to: J. T. Trevors or J. D. van Elsas     

Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives

Summary Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli -galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac⁺ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.Abbreviations bp base pairs - Nm neomycin - Km kanamycin - Sm streptomycin - Sp spectinomycin - Gm gentamycin - Tc tetracycline - Tp trimethoprim - Ot oxytetracycline - Rf rifampicin - Xgal 5-bromo-4-chloro-3-indolyl--d-galactoside     

Effect of Sodium Salicylate on the Population Dynamics of the Rhizobacterium Pseudomonas aureofaciens in the Wheat Rhizoplane and Adjacent Soil

The effect of sodium salicylate on the population dynamics of the rhizobacterium Pseudomonas aureofaciens BS1393 and its variant bearing the naphthalene biodegradation plasmid pBS216 was studied in the wheat rhizoplane and adjacent soil. Optimum salicylate concentration for the maintenance of the plasmid-bearing strain and for the normal growth of wheat was found to be 250 g/g soil. When the soil was supplemented with salicylate, the population of P. aureofaciens BS1393(pBS216) in the wheat rhizoplane and adjacent soil was, respectively, 4- and 20-fold higher than that of the parent strain lacking the plasmid.     

Competitive abilities of Tn5 Tox- mutants of a rhizobacterium inhibitory to wheat growth

Antibiosis has been thought to impart a competitive advantage to soil microorganisms. A rhizobacterium of the genus Pseudomonas produces a toxin that inhibits the growth of other microorganisms and winter wheat (Triticum aestivum L.). The bacterium was mutagenized with the Tn5 transposon to obtain toxin-negative (Tox^-) mutants or was selected for its spontaneous resistance to rifampicin. Tox^- mutants were used to determine the role of the toxin in wheat root inhibition, root colonization, and rhizosphere competitiveness. Four Tox^- (loss of inhibition of both E. coli and wheat root growth) and four partial Tox⁺ (partial loss of inhibition of E. coli and wheat root growth) Tn5 mutants were isolated. Seven of the mutants had different Tn5 chromosomal insertions, which suggests that toxin production is the result of several gene loci. Competitive root-colonization abilities of the Tox^- isolates were studied in winter wheat rhizospheres using varied population levels in autoclaved and nonautoclaved soil. Toxin production did not affect the competitive abilities of these organisms with native soil microflora. Results here indicate that toxin production by these organisms is not the primary mechanism of their competitive advantage in root colonization. Thus, opportunities exist for biological control of plant-suppressive bacteria using these Tox^- strains.     

Transfer of plasmid RP4 between pseudomonads after introduction into soil; influence of spatial and temporal aspects of inoculation

Abstract Transfer of plasmid RP4 between introduced strains of Pseudomonas fluorescens was studied in 2 soils, Ede loamy sand and Guelph loam, in non-rhizosphere and rhizosphere soil using soil chambers and microcosm systems. Short-term organism survival was generally at high levels (> 10⁶/g dry soil), in both soils, whereas long-term survival was poorer, particularly in the loamy sand. Amendment of this soil with bentonite clay improved bacterial survival. Plasmid transfer between donor and recipient strains freshly introduced into separate portions of Ede loamy sand, which were subsequently mixed, was only detected in the vicinity of growing wheat roots, suggesting roots stimulate bacterial migration and/or growth. However, no transfer was detected between resident donor and recipient cell populations (introduced 48 days previously), due to poor organism survival. Plasmid transfer was detected in the rhizosphere between established, resident donor cell populations, and newly-introduced recipients, and vice-versa, in both soils. These data suggested that plant roots enhance the frequency of bacterial matings not only between organisms present in the same niches, but also between organisms from different niches, or in different conditions of stress, probably by stimulating bacterial migration and/or growth, or by providing additional surfaces for cell-to-cell contact.     

Characterization of a <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> strain from tomato rhizosphere and its use for integrated management of tomato damping-off

A highly antagonistic Pseudomonas fluorescens strain was isolated from tomato rhizosphere and characterized for its in vitro and in vivo biocontrol potential against Pythium aphanidermatum. The identified Pseudomonas fluorescens strain (PfT-8) was capable of producing high levels of chitinase, β-1,3-glucanase, cellulase, fungitoxic metabolites and siderophores. Seven different carrier formulations including a talc-based powder, lignite-based powder, peat-based powder, lignite + fly ash-based powder, wettable powder, bentonite-paste and polyethylene glycol (PEG) paste were prepared utilizing PfT-8. Shelf life was evaluated for up to 6 months of storage at ambient room temperature (28 °C). Biocontrol efficacy of formulations was studied under greenhouse and field conditions. The formulations were stable up to at least 2 months of storage at ambient room temperature. Among the formulations, peat, lignite, lignite+fly-ash and bentonite paste based formulations maintained higher propagule number than others and also showed greater biocontrol potential. However, propagule number gradually decreased with time. Several organic amendments including farm yard manure (FYM), leaf-compost, poultry manure, press mud, vermi-compost and neem cake were incorporated into soil to study their influence on P. fluorescens colonization in the rhizosphere and on potential disease control. Soil incorporation of organic amendments and specifically poultry manure and FYM, significantly reduced damping-off incidence and also augmented the rhizosphere population of the marked␣P.␣fluorescens strain that was resistant to streptomycin and rifampicin. An integrated␣approach of damping-off management employing dual inoculation of PfT-8 in seed and soil coupled with soil application of organic amendments including poultry manure or␣FYM was evaluated under field conditions. Under these conditions, damping-off incidence substantially reduced by up to 90% and further the healthy plant stand, plant biomass and plant rhizosphere population of P. fluorescens increased significantly.     

Wheat Cultivar-Specific Selection of 2,4-Diacetylphloroglucinol-Producing Fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> Species from Resident Soil Populations

An emerging body of evidence indicates a role for plant genotype as a determinant of the species and genetic composition of the saprophytic microbial community resident to the rhizosphere. In this study, experiments were conducted to determine the capacity of five different wheat cultivars to enhance resident populations and support introduced strains of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent pseudomonads, a group of bacteria known to provide biological control of several soilborne diseases. When soils were cropped with three successive 28-day growth cycles of wheat, the 2,4-DAPG-producing strains were consistently recovered from the rhizosphere of the cultivar Lewjain, and commonly were present at populations higher than those recovered from other wheat cultivars. Based on restriction fragment length polymorphism and sequence analyses of phlD, a key gene involved in 2,4-DAPG production, two previously undefined phlD+ genotypes, referred to as genotypes PfZ and PfY, were discovered. Wheat cultivar Lewjain was the primary source of genotype PfY while cultivar Penawawa yielded the majority of genotype PfZ. Based on 16S rDNA sequence analysis, both new phlD genotypes were classified as P. fluorescens. Comparison of the rhizosphere competence of 2,4-DAPG-producing P. fluorescens Q2-87 (genotype B) and P. fluorescens LR3-A28 (genotype PfY) showed that both strains persisted at similar populations in the rhizosphere of all cultivars tested over a 30 day period when introduced as a seed inoculant. However, when strain LR3-A28 was applied as a soil inoculant, this strain was recovered at higher populations from the rhizosphere of wheat cultivar Lewjain than from the rhizospheres of two other cultivars. No cultivar effects were shown for strain Q2-87. Collectively, these results add further to evidence indicating a degree of specificity in interactions between plant cultivars and specific members of the saprophytic microbial community. Furthermore, as 2,4-DAPG-producing fluorescent Pseudomonas spp. have a central role in the spontaneous reduction in severity of take-all disease of wheat in response to continuous wheat monoculture, we postulate that the use of specific cultivars, such as Lewjain, which possess a superior capacity to enhance resident soil populations of these bacteria may have potential to reduce the length of the monoculture period required to induce natural suppressiveness of soils toward this disease.     

Assessment of microbial diversity in the rhizosphere of Pinus roxburghii (Sarg.) and bio‐inoculant potential of selected pine bacterial isolates for wheat varieties based on cultureindependent and culture‐dependent techniques

• Evidence is lacking regarding compatibility of pine bacteria as bio‐inoculants for crops. The diversity and abundance of rhizosphere bacteria of Pinus roxburghii has never been investigated with simultaneous application of culture‐dependent and culture‐independent techniques. The present study was aimed to isolate, characterise, check the bio‐inoculant potential of pine bacteria and assess rhizosphere bacterial diversity using culture‐independent advanced approaches.
• Forty bacteria isolated from the rhizoplane of P. roxburghii growing in a cold climate at high altitude in Murree, were morphologically characterised; nine were identified by 16S rRNA sequence analyses and used in experiments. Diversity and abundance of the 16S rRNA gene and nif H gene in the rhizosphere was assessed by cloning, RFLP analysis, 454‐amplicon pyrosequencing and qPCR.
• The bacterial isolates significantly improved dry weight of shoot, root, root area, IAA and GA₃ content, number of grains plant^?1, weight of grains plant^?1 in wheat varieties Chakwal‐50 and Fareed‐06 under axenic and field conditions. The number of 16S rRNA sequences (2979) identified by pyrosequencing shared similarity with 13 phyla of bacteria and archaea.
• The results confirm the existence of diverse bacteria of agricultural and industrial importance in the rhizosphere and compatibility of rhizoplane bacteria as bio‐inoculants for wheat varieties.

     

Performance and persistence of phosphate solubilizingAzotobacter chroococcum in wheat rhizosphere

Survival and establishment of inoculant strains ofAzotobacter chroococcum NV 11 and its mutant NV 43 were assessed in sterilized soil and rhizosphere soil of wheat plants at 7 and 15 d interval, respectively, after sowing,i.e., up to 45 d under pot-house conditions. There was an apparent decrease in population of both strains in bulk soil but a steady increase was observed in root zones of the wheat plant. 10³ to 10⁴ introduced bacteria per g root were found sticking to roots. Further studies indicated that inoculant strains ofAzotobacter could survive, proliferate and establish well in root zones.     

Aluminum tolerance of wheat does not induce changes in dominant bacterial community composition or abundance in an acidic soil

Aims

Aluminum-tolerant wheat plants often produce more root exudates such as malate and phosphate than aluminum-sensitive ones under aluminum (Al) stress, which provides environmental differences for microorganism growth in their rhizosphere soils. This study investigated whether soil bacterial community composition and abundance can be affected by wheat plants with different Al tolerance.

Methods

Two wheat varieties, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive), were grown for 60 days in acidic soils amended with or without CaCO₃. Plant growth, soil pH, exchangeable Al content, bacterial community composition and abundance were investigated.

Results

Atlas 66 showed better growth and lower rhizosphere soil pH than Scout 66 irrespective of CaCO₃ amendment or not, while there was no significant difference in the exchangeable Al content of rhizosphere soil between the two wheat lines. The dominant bacterial community composition and abundance in rhizosphere soils did not differ between Atlas 66 and Scout 66, although the bacterial abundance in rhizosphere soil of both wheat lines was significantly higher than that in bulk soil. Sphingobacteriales, Clostridiales, Burkholderiales and Acidobacteriales were the dominant bacteria phylotypes.

Conclusions

The difference in wheat Al tolerance does not induce the changes in the dominant bacterial community composition or abundance in the rhizosphere soils.     

Survival of lux-marked bacteria introduced into soil and the rhizosphere of bean (Phaseolus vulgaris L.)

The survival of lux-marked recombinants of Escherichia coli and Bacillus subtilis was studied in the rhizosphere of bean (Phaseolus vulgaris L.) and in bulk soil. The number of E. coli (pSB343) containing a complete lux operon did not differ significantly according to whether they were introduced into soil separately or together with a non-luminescent mutant Pseudomonas fluorescens R2fN. When genetically altered strains of E. coli and B. subtilis bearing a complete or an incomplete lux-reporter system were introduced into soil, the numbers of surviving cells were the same both in the rhizosphere and bulk soil. The insertion of lux genes into bacterial strains therefore does not affect their competitiveness and survival in the rhizosphere and bulk soil.The author is with the Department of Microbiology, University of Silesia, Jagielloska 28, 40-032 Katowice, Poland     

Assimilation of exogenous 2′-14C-indole-3-acetic acid and 3′-14C-tryptophan exposed to the roots of three wheat varieties

This study was conducted to determine if plants can assimilate indole-3-acetic acid (IAA) from rooting media and if exogenous L-tryptophan (L-TRP) can be assimilated and converted by plants into auxins. The addition of 2-¹⁴C-IAA (3.7 kBq plant^-1) to wheat (Triticum aestivum L.) seedlings of three varieties grown in nutrient solution resulted in the uptake (avg.=7.6%) of labelled IAA. Most of the label IAA was recovered in the shoot (avg.=7.2%) with little accumulation in the root (avg.=0.43%). A portion of the assimilated IAA-label in the plant was identified by co-chromatography and UV spectral confirmation as IAA-glycine and IAA-aspartic acid conjugates. Little of the assimilated IAA label was found as free IAA in the wheat plants. These same assimilation patterns were observed when 2-¹⁴C-IAA was added to wheat plants grown in sterile and nonsterile soil. In contrast, the wheat varieties assimilated considerably less (avg.=1.3%) of the added microbial IAA precursor, 3-¹⁴C-L-TRP (3.7 kBq plant^-1) and thus much lower amounts of IAA conjugates were detected. Glasshouse soil experiments revealed that 2 out of 3 wheat varieties had increased growth rates and increased yields when L-TRP (10^-5 and 10^-7 M) was added to the root zone. It is surmised that this positive response is a result of microbial auxin production within the rhizosphere upon the addition of the precursor, L-TRP. The amino acid composition of the root exudates plays a critical role in microbial production of auxins in the rhizosphere. This study showed that wheat roots can assimilate IAA from their rooting media, which will supplement the endogenous IAA levels in the shoot tissue and may positively influence plant growth and subsequent yield.     

Soil nitrogen transformations and nitrate utilization by Deschampsia flexuosa (L.) Trin. at two contrasting heathland sites

Two Dutch heathland sites Hoorneboeg (HB) and Ede, dominated by Deschampsia flexuosa and differing in nitrate production, were sampled for an entire growing season. A large number of soil and plant parameters were monitored in an attempt to assess the contribution of nitrate in the N supply and its assimilation by Deschampsia.Average NO₃ ^– and NH₄ ⁺ concentrations (mg kg^–1) in the top 10-cm depth were 0.03 and 2.2, respectively, for HB, and 2.1 and 6.7, respectively, for Ede. Laboratory incubations of intact cores and experiments with FH-layer suspensions showed significantly higher mineralization and nitrification rates for the Ede site during most of the season. Nitrification was largely controlled by the rate of net N-mineralization, which in turn was highly affected by soil moisture. Nitrate production was virtually zero at HB and accounted for 25% of the net N-mineralization at Ede.Shoot chemical composition showed no essential differences for the two sites, but mean in vivo (current) foliar NRA was almost 2-fold higher at Ede than at HB, indicating some utilization of nitrate at the former location. At the HB site with essentially no nitrate production, however, enzyme activities were clearly higher than basal constitutive levels in NH₄ ⁺-fed plants. Apparently, shoot NRA at the HB site became positively affected by factors other than nitrate availability and/or showed disproportional increases in response to atmospheric nitrate inputs. Root NRA displayed the same low basal level at the two sites. Nitrate fertilization (100 kg N ha^–1) yielded maximally induced foliar NRAs similar to levels found in hydroponic nitrate plants. Although no accumulation of free NO₃ ^– was observed in shoots from fertilized plots, increases in foliar concentrations of both organic N and carboxylates clearly indicated nitrate assimilation. Root NRA showed no response to nitrate addition.It is concluded that current NRA measurements in Deschampsia at heathland sites are of limited value only, especially when interpreted in isolation. A combined approach, using concurrently conducted soil and plant analyses, will allow the extent of nitrate utilization in the field to be best characterized.Publication 2013 of the Netherlands Institute of Ecology.FAX no corresponding author: +31 8306 23227     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

Variations in ergosterol content and ornithine decarboxylase activity of ectomycorrhizal root-soil systems

Influences of soil P fertilization on temporal changes in ergosterol content and ornithine decarboxylase (E.C. 4.1.1.17, ODC) activity were monitored in rhizosphere soil, non-rhizosphere soil and Pinus contorta roots ectomycorrhizal with Hebeloma crustuliniforme grown in a loamy sand. With addition of mycorrhizal inoculum to loamy sand, ODC activity mg^-1 root increased between 10% and 2 fold within 21 weeks of pianting. Inoculation also decreased root mass per seedling. Inoculation increased mycelia mass per root mass by up to 2 fold but no differences were observed for total seedling mass until 35 weeks. Intramatrical mycelia were detrimental to early plant growth, but inoculated seedlings had 1.7 times more root mass and 1.3 times more shoot mass at 35 weeks. Rhizosphere soil contained up to 5 times more mycelia and up to 6 times greater ODC activity than non-rhizosphere soil. Inoculation increased rhizophere metabolic activity and intramatrical mycelia mass. Their sensitivity to fungal inoculation, P fertilization and temporal trends may make the methods useful in studies of rhizosphere ecology and root-microbe relationships.     

Assessment of bacterial inoculant formulated with <Emphasis Type="Italic">Paraburkholderia tropica</Emphasis> to enhance wheat productivity

Paraburkholderia tropica is an endophytic nitrogen-fixing bacterium isolated from the rhizosphere, rhizoplane, and internal tissues of sugarcane and corn plants in different geographical regions. Other plant-growth-promoting abilities, such as phosphate solubilization and antifungal activity, have also been reported for this bacterium. With an aim at investigating the potential use of P. tropica as an inoculant for improving the performance of wheat crop, in this work we evaluated an experimental inoculant formulated with P. tropica MTo-293 with respect to root colonization, the practical aspects of its application, and the effects under field conditions when applied to wheat seeds. Bacterial colonization was monitored by culture dependent techniques and the wheat yield determined by quantifying the total grain production in two different seasons. Rhizoplane and endophytic colonization in wheat roots was achieved efficiently (on average, 8 and 4 log colony-forming units/g fresh weight, respectively) even at relatively low concentrations of viable bacteria in the inoculum under controlled conditions. P. tropica was compatible with a widely used fungicide, maintained viability for 48 h once applied to seeds, and was also able to colonize wheat roots efficiently. Furthermore, we were able to formulate an inoculant that maintained bacterial viability for relatively long time periods. Preliminary field assays were realized, and even though the average yields values for the inoculated treatments remained above the uninoculated ones, no significant effects of inoculation were detected with or without fertilization. The correct physiologic behavior of P. tropica suggests the necessity to continue with field experiments under different conditions.     

刺槐林魔芋健康高产的土壤微生态机制

通过对刺槐林和农田魔芋健株根区、根表及根外土壤微生物区系及养分含量比较,探索刺槐林魔芋健康高产的土壤微生态机制。结果表明:(1)刺槐林魔芋根外和根表土壤细菌数量分别较农田增加11.8%和588.9%,根区土壤真菌数量较农田显著减少74.4%。(2)刺槐林魔芋根区、根表及根外土壤中的有益优势微生物数量及其比例远高于农田魔芋,有害微生物数量远低于农田魔芋;在刺槐林魔芋根区、根表及根外土壤中,3种优势细菌为放射型根瘤菌(Rhizobium radiobacter)、苏云金芽孢杆菌(Bacillus thuringiensis)及摩氏假单胞菌(Pseudomonas mosselii),其中,根表土壤中放射型根瘤菌及苏云金芽孢杆菌数量分别为农田的25.7倍及13.0倍;2种优势真菌为黒附球菌(Epicoccum nigrum)和疣孢青霉(Penicillium verruculosum),1种优势放线菌为绿淀粉酶链霉菌(Streptomyces viridodiastaticus),其中刺槐林魔芋根表和根外土壤中黒附球菌数量分别较农田高159.2%和120.3%;大量存在于刺槐林下魔芋根外土壤中的疣孢青霉、以及根区、根表、根外土壤中的绿淀粉酶链霉菌在农田魔芋相应部位均未检出。(3)刺槐林下魔芋根外、根区土壤有机质含量分别较农田显著增加167.6%、39.6%,但速效P、K含量较农田分别显著降低85.6%~91.3%、12.4%~13.0%。研究认为,刺槐林魔芋健康高产与其根区、根表及根外土壤中特有的有益优势微生物数量多、有害微生物数量少以及土壤有机质含量高密切相关。     

Survival of Bacillus subtilis AF 1 in the bacterized peanut rhizosphere and its influence on native microflora and seedling growth

A mutant of Bacillus subtilis AF 1 (an antifungal strain with plant growth-promoting activity, resistant to 100 g streptomycin/ml) was isolated from pigeon pea rhizosphere and, when used in peanut bacterization, decreased fungal and bacterial numbers and increased the actinomycete population in the rhizosphere. Over 28 days, AF 1 declined by 0.9 log c.f.u. in sterilized soil and by 2.6 log c.f.u. in native soil. From the seedlings raised with peanut seeds bacterized with AF 1 at 6.5 log c.f.u./seed, 4.1 and 4.7 log c.f.u. of AF 1 were recovered from the rhizosphere and total underground parts, respectively. Bacterization with AF 1 had no significant growth-promoting or -inhibiting effect on the peanut.     

Biocontrol of Collar Rot Disease of Betelvine ( Piper betle L.) Caused by Sclerotium rolfsii by Using Rhizosphere-Competent Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N

Collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii is difficult to control by conventional means by use of chemicals; therefore, use of biocontrol agents is desirable. In the present study, 186 bacterial strains of different morphological types were screened for their biocontrol activity against S. rolfsii under in vitro conditions. Two strains, Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N, were selected for further studies because of their ability to inhibit the mycelial growth of the pathogen significantly. Spontaneous rifampicin-resistant (Rif^r) derivatives of P. fluorescens NBRI-N6 and P. fluorescens NBRI-N showing growth rate and membrane protein composition comparable to the wild type were selected to facilitate their monitoring in the rhizosphere. Field trials demonstrated that strain P. fluorescens NBRI-N6 was better than P. fluorescens NBRI-N in increasing the yield of betelvine significantly, whereas a consortium of the two strains controlled the disease more than either of the strains. The screening method should prove useful in identifying rhizosphere bacteria with the greatest potential for controlling diseases caused by phytopathogenic fungi. RID= ID= <E5>Correspondence to:</E5> C.S. Nautiyal; <E5>email:</E5> nautiyalnbri&commat;yahoo.com Received: 5 August 2002 / Accepted: 7 October 2002     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.