Biofilm-associated indole acetic acid producing bacteria and their impact in the proliferation of biofilm mats in solar salterns

Biofilm mats appear in salterns distinctively during the monsoon season when the salinity decreases below 12 percentile salinity units and within a short period cover the entire surface area of the saltern. A study was carried out in two salterns viz. Nerul and Curca to find a possible reason for the rapid proliferation of these solar biofilms. Out of the 125 bacteria isolated from these biofilms, 16 produced indole-3-acetic acid (IAA). Rapid in-situ assay with Salkowski reagent and HPLC analysis confirmed the IAA production. Four isolates consistently produced high IAA concentrations ranging from 9.5 to 14.2 μg/mL in the presence of 4 mg/mL tryptophan concentrations in the growth media. The IAA-producing bacteria were Aeromonas aquariorum (N2), Pseudomonas alcaliphila (N3), Vibrio diazotrophicus (N6) and Pseudomonas pachastrellae (C3). These four IAA-producing bacteria also produced other growth promoting factors like ammonia. Three isolates produced siderophores and were phosphate solubilizers. There was enhancement in the growth of Cicer arietinum (length of the shoot and root) under axenic conditions and of biofilm mats (r = 0.9, p < 0.001; r = 0.8, p < 0.05 and r = 0.946, p < 0.01, respectively). This is, according to our knowledge, the first report indicating IAA-producing bacteria isolated from biofilms enhancing the proliferation of these biofilm mats in the solar salterns.     

Isolation and characterization of KDML105 aromatic rice rhizobacteria producing indole-3-acetic acid: impact of organic and conventional paddy rice practices

Indole-3-acetic acid (IAA) synthesis is a major property of rhizosphere bacteria. The IAA-producing ability of rhizobacteria may be influenced by agricultural management. We therefore evaluated the IAA-producing potential of rhizobacteria isolated during organic rice farming (ORF) and conventional rice farming (CRF) in Thung Kula Rong Hai areas of Thailand. The results indicated that ORF gave a significantly higher percentage of IAA producers (95·8%) than CRF (69·9%). The average IAA values of the ORF isolates were around two times higher than those of the CRF isolates both in the absence (12·8 and 5·8 μg IAA ml⁻¹, respectively) and presence of L-tryptophan (L-Trp) (35·2 and 17·2 μg IAA ml⁻¹, respectively). The 16S rRNA gene sequence analysis indicated that the 23 selected isolates belonged to 8 different genera—Sinomonas sp., Micrococcus sp., Microbacterium sp., Fictibacillus sp., Bacillus sp., Burkholderia sp., Leclercia sp. and Enterobacter sp. Interestingly, only three ORF isolates, i.e. ORF15-20 (Micrococcus sp.), ORF15-21 (Sinomonas sp.) and ORF15-23 (Sinomonas sp.), exhibited high IAA production ability without L-Trp (128·5, 160·8 and 174·7 μg IAA ml⁻¹, respectively). Meanwhile, a slight decrease in IAA production with L-Trp was noticed, suggesting that the L-Trp was not used for the IAA synthesis of these isolates. Biopriming with rhizobacterial isolates significantly enhanced the rate of germination of KDML 105 rice seeds compared to the control.     

Biofilm Dispersal of Neisseria subflava and Other Phylogenetically Diverse Oral Bacteria

Polystyrene petri dishes containing liquid medium were inoculated with single-cell suspensions of a fresh clinical isolate of Neisseria subflava and were incubated under conditions of low vibration. N. subflava colonies grew firmly attached to the surface of the dish, while the broth remained clear. Growing colonies released cells into the medium, resulting in the appearance of 10² to 10⁴ small satellite colonies attached to the surface of the dish in an area adjacent to each mature colony after 24 h. Satellite colonies grew in patterns of streamers shaped like jets and flares emanating from mature colonies and pointing toward the center of the dish. This dispersal pattern evidently resulted from the surface translocation of detached biofilm cells by buoyancy-driven convection currents that were generated due to slight temperature gradients in the medium. Streamers of satellite colonies ranged from 2 to >40 mm in length. Satellite colonies in very long streamers were relatively uniform in size regardless of their distance from the mature colony, suggesting that mature colonies released single cells or small clusters of cells into the medium and that the detachment, surface translocation, and subsequent surface reattachment of released cells were a transitory process. Incubation of N. subflava single cells in a perfused biofilm fermentor resulted in a large spike of the number of CFU in the perfusate after 9.5 h of growth, consistent with a rapid release of cells into the medium. Biofilm colonies of several other phylogenetically diverse oral bacteria, including Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Streptococcus mitis, and a prevalent but previously uncultured oral Streptococcus sp., exhibited similar temperature-dependent dispersal patterns in broth culture. This in vitro spreading phenotype could be a useful tool for studying biofilm dispersal in these and other nonflagellated bacteria and may have physiological relevance to biofilm dispersal in the oral cavity.     

悉生培养微缩体系食细菌线虫提高土壤激素含量的机制

研究土壤食细菌线虫与细菌的相互作用及其生态功能是土壤生态学的核心内容之一。食细菌线虫取食细菌可以促进土壤中氮素的矿化,提高氮素养分的供给,改善土壤的营养条件,从而促进植物的生长发育。土壤食细菌线虫促进植物根系生长的"养分作用机制"已得到确认,而"激素作用机制"还存在争议。从供试土壤中筛选获得一株高效产IAA细菌和两种不同cp值的食细菌线虫,通过设置简化的悉生培养系统,对这两种土著食细菌线虫与土著产IAA细菌之间的相互作用,及其对土壤中IAA含量变化的影响进行研究。结果表明:两种食细菌线虫的取食均能促进细菌数量和活性的增强,食细菌线虫与产IAA细菌相互作用也能显著增加土壤中IAA的含量;这些促进作用受到接种食细菌线虫的种类以及培养时间的影响:在培养第10天和第20天时,接种cp值为1的中杆属食细菌线虫显著增加了产IAA细菌的数量;在培养第10天和第30天时,相比较接种cp值为2的头叶属食细菌线虫,接种中杆属食细菌线虫显著提高了土壤中IAA的含量。     

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.)

Root nodule accommodates various non-nodulating bacteria at varying densities. Present study was planned to identify and characterize the non-nodulating bacteria from the pea plant. Ten fast growing bacteria were isolated from the root nodules of cultivated pea plants. These bacterial isolates were unable to nodulate pea plants in nodulation assay, which indicate the non-rhizobial nature of these bacteria. Bacterial isolates were tested in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, phosphate solubilization, root colonization and biofilm formation. Six isolates were able to produce IAA at varying level from 0.86 to 16.16 μg ml^?1, with the isolate MSP9 being most efficient. Only two isolates, MSP2 and MSP10, were able to fix nitrogen. All isolates were able to solubilize inorganic phosphorus ranging from 5.57 to 11.73 μg ml^?1, except MSP4. Bacterial isolates showed considerably better potential for colonization on pea roots. Isolates MSP9 and MSP10 were most efficient in biofilm formation on polyvinyl chloride, which indicated their potential to withstand various biotic and abiotic stresses, whereas the remaining isolates showed a very poor biofilm formation ability. The most efficient plant growth promoting agents, MSP9 and MSP10, were phylogenetically identified by 16S rRNA gene sequence analysis as Ochrobactrum and Enterobacter, respectively, with 99 % similarity. It is suggested the potential endophytic bacterial strains, Ochrobactrum sp. MSP9 and Enterobacter sp. MSP10, can be used as biofertilizers for various legume and non-legume crops after studying their interaction with the host crop and field evaluation.     

Functional whole-colony screening method to identify antimicrobial peptides

A high throughput method for screening cDNA libraries has been developed to identify putative antimicrobial peptides (AMPs). It is based on a rapid dye inclusion assay for assessing antagonism of bacterial viability. Colonies are grown on a membrane on a permissive medium until full colony size is reached. The membrane, supporting the array of colonies, is transferred onto an inductive medium containing a vital dye. Upon expression of any antagonizing peptides, the cell membrane becomes compromised allowing dye infusion to permit visual identification of deleterious peptides.Our approach was validated by screening a synthetic oligonucleotide library expressed in Escherichia coli. A random oligonucleotide library, containing inserts of up to 75 nucleotides in length was constructed and expressed in E. coli. From a potential pool of 100 000 peptides, in a single round of screening, three were found to be antimicrobial: L1, L3, and L8. Peptide L1 was shown to have a concentration-dependent bactericidal effect against Gram-negative E. coli and moderate biostatic activity against the Gram-positive bacteria Listeria monocytogenes. L8 was found to have bacteriostatic, and possibly bactericidal effect against E. coli, Pseudomonas aeruginosa and Salmonella typhimurium. These results validated this high throughput AMP identification assay based on filter bound colony array libraries and vital dye inclusion.     

Salkowski’s Reagent Test as a Primary Screening Index for Functionalities of Rhizobacteria Isolated from Wild Dipterocarp Saplings Growing Naturally on Medium-Strongly Acidic Tropical Peat Soil

Rhizobacteria isolated from wild dipterocarp saplings in Central Kalimantan, Indonesia, were subjected to Salkowski’s reagent test, which is often used in detecting indolic substances. Among 69 isolates grown in a low-nitrogen medium supplemented with L-tryptophan (TRP), culture fluids of 29 strains were positive to the test, in which 17 bacteria turned red and other 10 pink. All the red type rhizobacteria actively converted TRP into tryptophol (TOL), while some yielded indole-3-acetic acid (IAA) with TOL production. They also showed a capacity to decompose gallotannin into pyrogallol via gallic acid. On the other hand, an active IAA-producing Serratia sp. CK67, and three Fe-solubilizing Burkholderia spp. CK28, CK43, and Citrobacter sp. CK42, were all involved in pink type rhizobacteria, which were more effective, oxidative TRP-degraders than the red type rhizobacteria. Thus, Salkowski’s reagent test should be a useful primary index in the screening of functional rhizobacteria in peatland ecosystem.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

Culturable Heavy Metal-Resistant and Plant Growth Promoting Bacteria in V-Ti Magnetite Mine Tailing Soil from Panzhihua,China

To provide a basis for using indigenous bacteria for bioremediation of heavy metal contaminated soil, the heavy metal resistance and plant growth-promoting activity of 136 isolates from V-Ti magnetite mine tailing soil were systematically analyzed. Among the 13 identified bacterial genera, the most abundant genus was Bacillus (79 isolates) out of which 32 represented B. subtilis and 14 B. pumilus, followed by Rhizobium sp. (29 isolates) and Ochrobactrum intermedium (13 isolates). Altogether 93 isolates tolerated the highest concentration (1000 mg kg⁻¹) of at least one of the six tested heavy metals. Five strains were tolerant against all the tested heavy metals, 71 strains tolerated 1,000 mg kg⁻¹ cadmium whereas only one strain tolerated 1,000 mg kg⁻¹ cobalt. Altogether 67% of the bacteria produced indoleacetic acid (IAA), a plant growth-promoting phytohormone. The concentration of IAA produced by 53 isolates was higher than 20 µg ml⁻¹. In total 21% of the bacteria produced siderophore (5.50–167.67 µg ml⁻¹) with two Bacillus sp. producing more than 100 µg ml⁻¹. Eighteen isolates produced both IAA and siderophore. The results suggested that the indigenous bacteria in the soil have beneficial characteristics for remediating the contaminated mine tailing soil.     

Indole-3-acetic acid producing root-associated bacteria on growth of Brazil Pine (Araucaria angustifolia) and Slash Pine (Pinus elliottii)

Araucaria forests in Brazil today correspond to only 0.7 % of the original 200 km² of natural forest that covered a great part of the southern and southeastern area of the Atlantic Forest and, although Araucaria angustifolia is an endangered species, illegal exploitation is still going on. As an alternative to the use of hardwoods, Pinus elliottii presents rapid growth and high tolerance to climatic stress and low soil fertility or degraded areas. Thus, the objective of this study was to evaluate the effect of IAA-producing bacteria on the development of A. angustifolia and P. elliottii. We used five bacterial strains previously isolated from the rhizosphere of A. angustifolia, which produce quantities of IAA ranging from 3 to 126 μg mL^?1. Microbiolized seeds were sown in a new gnotobiotic system developed for this work, that allowed the quantification of the plant hormone IAA produced by bacteria, and the evaluation of its effect on seedling development. Also, it was shown that P. elliottii roots were almost as satisfactory as hosts for these IAA producers as A. angustifolia, while different magnitudes of mass increases were found for each species. Thus, we suggest that these microbial groups can be helpful for the development and reestablishment of already degraded forests and that PGPR isolated from Araucaria rhizosphere have the potential to be beneficial in seedling production of P. elliottii. Another finding is that our newly developed gnotobiotic system is highly satisfactory for the evaluation of this effect.     

Prevalence of the stx2 Gene in Coliform Populations from Aquatic Environments

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx₂ gene in coliform bacteria in sewage samples of different origins. The presence of the stx₂ gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10² genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx₂ gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx₂ gene in the wastewater studied.     

Indole-3-Acetic Acid Production by Endophytic Streptomyces sp. En-1 Isolated from Medicinal Plants

Plant-associated actinobacteria are rich sources of bioactive compounds including indole-derived molecules such as phytohormone indole-3-acetic acid (IAA). In view of few investigations concerning the biosynthesis of IAA by endophytic actinobacteria, this study evaluated the potential of IAA production in endophytic streptomycete isolates sourced from medicinal plant species Taxus chinensis and Artemisia annua. By HPLC analysis of IAA combined with molecular screening approach of iaaM, a genetic determinant of streptomycete IAA synthesis via indole-3-acetamide (IAM), our data showed the putative operation of IAM-mediated IAA biosynthesis in Streptomyces sp. En-1 endophytic to Taxus chinensis. Furthermore, using the co-cultivation system of model plant Arabidopsis thaliana and streptomycete, En-1 was found to be colonized intercellularly in the tissues of Arabidopsis, an alternative host, and the effects of endophytic En-1 inoculation on the model plant were also assayed. The phytostimulatory effects of En-1 inoculation suggest that IAA-producing Streptomyces sp. En-1 of endophytic origin could be a promising candidate for utilization in growth improvement of plants of economic and agricultural value.     

Studies of the cultivable flora of normal human feces

Highly diluted feces, obtained from healthy adult individuals, was plated on blood-agar plates which were incubated both aerobically and anaerobically. From the anaerobic plates containing 30 to 60 colonies, every colony was subcultured. Nearly all isolates were obtained in pure culture and partially characterized. It was found thatBacteroides species were the most predominant organisms, being present in numbers approximating 10¹⁰ per gram wet weight. Selected bacteria present in lower numbers were determined by plating appropriate dilutions of feces on selective media. It was found that coliforms, streptococci and lactobacilli were regularly present in concentrations of 10⁶ ? 10⁸ organisms per gram wet weight material, whileVeillonella, Streptococcus salivarius, Bacteroides melaninogenicus and staphylococci were present in lower numbers. Fusobacteria were only found in one sample, whileNeisseria were not detected in any of the samples. Wet mounts of fecal material, inspected by darkfield microscopy, did not reveal the presence of spirochetes. Anaerobes outnumbered facultative bacteria by a factor of 40, indicating that the human adult fecal flora is predominantly anaerobic. Total microscopic counts indicate that bacteria comprise approximately 30% of the mass of human feces.     

Association of non-heterocystous cyanobacteria with crop plants

Cyanobacteria have the ability to form associations with organisms from all domains of life, notably with plants, which they provide with fixed nitrogen, among other substances. This study was aimed at developing artificial associations between non-heterocystous cyanobacteria and selected crop plants. We isolated several non-heterocystous cyanobacteria from various rice fields. The cultures were tested for their capacity to produce the plant hormone indole-3-acetic acid (IAA), and the possible role of IAA in the association of cyanobacteria with seedling roots was evaluated. Axenic cultures were co-inoculated with 10-day-old plant seedlings of Triticum aestivum, Vigna radiata and Pisum sativum and incubated for 1 week. Cyanobacterial association with the roots of these seedlings was quantified by measuring chlorophyll-a. Cyanobacterial association with the roots was observed by light microscopy as well as by confocal laser scanning microscopy (CLSM). Based on sequence analysis of the 16S rRNA gene, the isolates were identified as Synechocystis sp., Chroococcidiopsis sp., Leptolyngbya sp., and Phormidium sp. CLSM observations revealed the intimate association of cyanobacteria with the seedling roots as well as invasion of the roots and root cells. Strains producing IAA were more efficient in the colonization of the roots than those that lacked this ability. IAA-producing cyanobacteria possess a tryptophan-dependent pathway, and these cyanobacteria showed IAA synthesis activity in the presence of roots in media lacking tryptophan. Based on the results of this study, we conclude that non-heterocystous cyanobacteria also have the potential for use in agriculture to improve the growth and yield of crop plants that do not naturally form associations with cyanobacteria.     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Apparent antimutagenic effect of ultraviolet irradiation

Strain SL3367 is a S. typhimurium LT2 hisG46 stock which spontaneously reverts to His⁺ at a high frequency. Plates of defined medium with 1% (v/v) nutrient broth inoculated with ca. 10⁸ washed SL3367 cells were incubated, untreated or after UV irradiation. After 2 days at 37°C, an average of 165 His⁺ colonies were obtained per control plate but significantly fewer, 105 His⁺ colonies, on plates irradiated at a fluence of 7 J/m². The dry weight of bacteria in washings from plates incubated 14 h (by which time growth of His^? cells had ceased) was the same for irradiated and non-irradiated plates but the yield of colony-forming units from irradiated plates was less than from control plates, by about the same factor as the reduction in yield of His⁺ colonies caused by the same fluence. Washings from incubated irradiated plates, but not those from control plates, contained long filaments as well as bacteria of normal size; on transfer to nutrient-agar slide cultures cells normal size grew into microcolonies but filaments did not grow. The reduced plateau yield of viable His^? cells caused by consumption of much of the growth-limiting supply of histidine by irradiated cells growing into non-viable filaments reduces the number of auxotrophic bacteria at risk for spontaneous reversion and so accounts for the apparent antimutagenic effect of UV irradiation. This effect was partly reversed by the presence of d,l-pantoyl lactone in the selection medium, and was also observed for yield of Trp⁺ colonies from trpE8 cultures with a high spontaneous reversion rate. Treatments not inducing cell filamentation did not result in the depression of spontaneous revertants and were detected as being mutagenic. The apparent antimutagenic effect may be expected for reversion of any auxotroph, unless masked by induced revertants and is particularly apparent in an auxotroph which reverts spontaneously at high frequency.     

Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots

We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammonium (39%), secrete siderophores (38%) and a limited part of them synthetized IAA and IAA-like molecules (5%). Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP) of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards.     

南方红豆杉产IAA内生芽胞杆菌的分离、鉴定及产脂肽类化合物研究

通过组织研磨法对南方红豆杉(Taxus wallichiana var. mairei)新鲜组织进行内生细菌的分离，利用16S rRNA测序技术对分离的内生细菌进行鉴定分析，使用高效液相色谱-高分辨飞行质谱测定菌液提取物IAA含量，利用HPLC-TOF-MS对提取物脂肽类化合物进行分离、鉴定，结合数据库筛选菌株产脂肽类化合物种类及含量差异。结果表明，从南方红豆杉根茎叶中共分离鉴定出内生细菌31株，筛选出产IAA菌株20株，其中编号为KLBMPTC10的芽胞杆菌属菌株产IAA含量高达210.3955 μg·L^-1。在对产IAA菌株所产脂肽类化合物的研究中发现，菌株KLBMPTC10是所有产IAA内生细菌中脂肽类化合物含量最丰富的，其产Iturin A2相对含量为26.06%，明显优于其他产IAA菌株。芽胞杆菌属菌株KLBMPTC10同时具备高产IAA和脂肽类化合物的能力，可作为研究菌株进一步开发。     

Contribution of Indole-3-Acetic Acid Production to the Epiphytic Fitness of Erwinia herbicola

Erwinia herbicola 299R produces large quantities of indole-3-acetic acid (IAA) in culture media supplemented with l-tryptophan. To assess the contribution of IAA production to epiphytic fitness, the population dynamics of the wild-type strain and an IAA-deficient mutant of this strain on leaves were studied. Strain 299XYLE, an isogenic IAA-deficient mutant of strain 299R, was constructed by insertional interruption of the indolepyruvate decarboxylase gene of strain 299R with the xylE gene, which encodes a 2,3-catechol dioxygenase from Pseudomonas putida mt-2. The xylE gene provided a useful marker for monitoring populations of the IAA-deficient mutant strain in mixed populations with the parental strain in ecological studies. A root bioassay for IAA, in which strain 299XYLE inhibited significantly less root elongation than strain 299R, provided evidence that E. herbicola produces IAA on plant surfaces in amounts sufficient to affect the physiology of its host and that IAA production in strain 299R is not solely an in vitro phenomenon. The epiphytic fitness of strains 299R and 299XYLE was evaluated in greenhouse and field studies by analysis of changes in the ratio of the population sizes of these two strains after inoculation as mixtures onto plants. Populations of the parental strain increased to approximately twice those of the IAA-deficient mutant strain after coinoculation in a proportion of 1:1 onto bean plants in the greenhouse and onto pear flowers in field studies. In all experiments, the ratio of the population sizes of strain 299R and 299XYLE increased during periods of active growth on plant tissue but not when population sizes were not increasing with time.

Many plant-associated bacteria have the ability to produce the plant growth regulator indole-3-acetic acid (IAA) (5, 9, 25, 33). IAA is involved in diseases caused by gall- and knot-forming bacterial species (33); however, its role in other bacteria remains undefined. It is unclear whether these bacteria produce IAA during colonization of plant surfaces and whether this metabolite is beneficial to the bacteria during their growth and survival in the phyllosphere. The production of IAA may enable bacteria to detoxify tryptophan analogues present on plant surfaces (15), to downregulate genes involved in plant defense responses (33), or to inhibit the development of the hypersensitive response by plants (26). We recently demonstrated that the ipdC gene, which encodes the indolepyruvate decarboxylase of Erwinia herbicola (Pantoea agglomerans) 299R and which is involved in the indolepyruvate pathway for IAA synthesis in this epiphytic strain (2), is osmoresponsive and plant inducible (3). We hypothesized that the secretion of IAA may modify the microhabitat of epiphytic bacteria by increasing nutrient leakage from plant cells; enhanced nutrient availability may better enable IAA-producing bacteria to colonize the phyllosphere and may contribute to their epiphytic fitness (1).Few studies have attempted to determine the ecological significance of IAA production in pathogenic bacteria. Varvaro and Martella (31) showed that IAA-deficient mutants of Pseudomonas syringae pv. savastanoi, obtained by selection for resistance to α-methyltryptophan, were reduced in their ability to colonize and survive on olive leaf surfaces. The survival of an α-methyltryptophan-resistant IAA-deficient mutant of P. syringae pv. savastanoi in knots also was affected, its population declining more rapidly than that of the parental strain when inoculated alone into oleander leaf tissue (28). The importance of IAA production in bacterial colonization of bean leaves was also tested with the brown spot pathogen P. syringae pv. syringae and an IAA-deficient mutant derived by insertional mutagenesis (21). Although no difference in the survival of the parental and mutant strains on bean leaves was observed in the greenhouse, a small difference in their behavior was apparent in experiments conducted in a mist chamber (21). There have been no studies of the role of IAA production in plant-associated bacteria that do not cause disease.IAA biosynthesis is not essential for bacterial growth and survival, since IAA-deficient mutants grow as well as their IAA-producing parental strain in vitro (2, 29). Large differences in the epiphytic behaviors of IAA-producing bacteria and isogenic IAA-deficient mutants consequently would not be expected. Even small contributions of IAA production to epiphytic fitness could account for the common presence of this phenotype in epiphytic bacteria (19). Measurements of changes in the ratio of two strains following coinoculation, a common approach in ecological studies, can allow the detection of even small differences in the competitive behaviors of two organisms. This approach can detect much smaller differences in behavior between closely related species than comparison of populations of these species when present singly in separate habitats (16). In this study, we tested the role of IAA in the epiphytic fitness of E. herbicola by comparing the relative changes in the population sizes of the parental and IAA-deficient mutant strains with time after their inoculation onto plants in both controlled and field environments.     

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K₂TeO₃ was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K₂TeO₃ concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K₂TeO₃-amended medium. The bona fide agrobacterium colonies growing on media amended with K₂TeO₃ were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10³ to 10⁴ agrobacteria · g of dry soil⁻¹ in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.