Constructed wetlands as a component of the agricultural landscape: mitigation of herbicides in simulated runoff from upland drainage areas.

Constructed wetlands are a recommended practice for buffering pollutant source areas and receiving waters. A wetland consisting of a sediment trap and two treatment cells was constructed in a Mississippi Delta lake watershed. A 3-h simulated runoff event was initiated (2003) to evaluate fate and transport of atrazine and fluometuron through the wetland. Water samples were collected during a runoff simulation and then afterward at selected intervals for 21d, and analyzed for the herbicides. Breakthrough patterns for herbicide concentrations in water samples during the first 20h after simulated runoff showed peak concentrations in the first 6h, with gradual tailing as the herbicide pulse was diluted in the second, excavated (deeper) cell. Atrazine and fluometuron concentrations in the first (shallower, non-excavated) cell averaged 12- and 20-fold greater, respectively, than those in the second cell following simulated runoff, indicating entrapment in the first cell. Atrazine and fluometuron concentrations in the shallower cell decreased 32% and 22%, respectively, 9d following simulated runoff, indicating either degradation or sorption to soil or wetland flora. In the excavated cell, concentrations were even lower, and atrazine declined more rapidly than fluometuron. Results indicate constructed wetlands can improve downstream water quality though sequestration or processing of pollutants.

Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize.

Biological control of toxigenic Aspergillus flavus in maize through competitive displacement by non-aflatoxigenic strains was evaluated in a series of field studies. Four sets of experiments were conducted between 2007 and 2009 to assess the competitiveness of non-aflatoxigenic strains when challenged against toxigenic strains using a pin-bar inoculation technique. In three sets of experiments the non-aflatoxigenic strain K49 effectively displaced toxigenic strains at various concentrations or combinations. The fourth study compared the relative competitiveness of three non-aflatoxigenic strains (K49, NRRL 21882 from Afla-Guard®, and AF36) when challenged on maize against two aflatoxin- and cyclopiazonic acid (CPA)-producing strains (K54 and F3W4). These studies indicate that K49 and NRRL 21882 are superior to AF36 in reducing total aflatoxin contamination. Neither K49 nor NRRL 21882 produce CPA and when challenged with K54 and F3W4, CPA and aflatoxins were reduced by 84-97% and 83-98%, respectively. In contrast, AF36 reduced aflatoxins by 20% with F3W4 and 93% with K54 and showed no reduction in CPA with F3W4 and only a 62% reduction in CPA with K54. Because AF36 produces CPA, high levels of CPA accumulate when maize is inoculated with AF36 alone or in combination with F3W4 or K54. These results indicate that K49 may be equally effective as NRRL 21882 in reducing both aflatoxins and CPA in maize.

Delivery systems for biological control agents to manage aflatoxin contamination of pre-harvest maize.

While soil application of a competitive non-toxigenic Aspergillus flavus strains is successful in reducing aflatoxin contamination in certain crops, direct application to aerial reproductive structures could be more effective for maize. A sprayable, clay-based water-dispersible granule formulation was developed to deliver non-toxigenic A. flavus strain K49 directly to maize ears. The efficacy of the K49 water-dispersible granule in mitigating aflatoxin in maize (Zea mays L.) was evaluated. Field studies were conducted to compare K49 colonization and effectiveness in reducing aflatoxin contamination when applied either as a soil inoculant or as a directed spray in plots infested with toxigenic strain F3W4. Fifty percent of non-toxigenic A. flavus was recovered from non-treated controls and from plots soil inoculated with K49 on wheat. In spray treatments with formulated or unformulated K49 conidia, over 90% of A. flavus recovered was non-toxigenic. Soil-applied K49 reduced aflatoxin contamination by 65% and spray applications reduced contamination by 97%. These findings suggest direct spray application of non-toxigenic A. flavus strains may be better than soil inoculation at controlling maize aflatoxin contamination and that a water-dispersible granule is a viable delivery system for maintaining viability and efficacy of the biological control agent, K49.

Aspergillus flavus aflatoxin occurrence and expression of aflatoxin biosynthesis genes in soil.

The carcinogen aflatoxin B1 (AFB1) produced by Aspergillus flavus is a major food safety concern in crops. However, information on AFB1 occurrence in soil and crop residue is scarce. A series of experiments investigated the occurrence of AFB1 in soil and corn residues and ascertained the ecology of A. flavus in a Dundee silt loam soil. Samples of untilled soil (0-2 cm) and residues were collected in March 2007 from plots previously planted with a corn isoline containing the Bacillus thuringiensis (Bt) endotoxin gene or the parental non-Bt isoline. AFB1 levels were significantly different in various corn residues. The highest AFB1 levels were observed in cobs containing grain, with 145 and 275 ng.g-1 in Bt and non-Bt, respectively (P > or = F = 0.001). Aflatoxin levels averaged 3.3 and 9.6 ng.g-1 in leaves and (or) stalks and cobs without grain, respectively. All soils had AFB1 ranging from 0.6 to 5.5 ng.g-1 with similar levels in plots from Bt and non-Bt corn. Based on cultural methods, soil contained from log10 3.1 to 4.5 A. flavus cfu.g-1 with about 60% of isolates producing aflatoxin. Laboratory experiments demonstrated that AFB1 is rapidly degraded in soil at 28 degrees C (half-life < or = 5 days). The potential of the soil A. flavus to produce aflatoxins was confirmed by molecular methods. Transcription of 5 aflatoxin biosynthesis genes, including aflD, aflG, aflP, aflR, and aflS, were detected by reverse transcription - polymerase chain reaction analysis in soil. Although AFB1 appears to be transient in soils, it is clear that AFB1 is produced in surface soil in the presence of corn residues, as indicated by A. flavus cfu levels, AFB1 detection, and expression of aflatoxin biosynthetic genes.

Mycotoxin occurrence and Aspergillus flavus soil propagules in a corn and cotton glyphosate-resistant cropping systems.

The effects of cotton-corn rotation and glyphosate use on levels of soil-borne Aspergillus flavus, aflatoxin and fumonisin contamination in corn and cotton seed were determined during 2002-2005 in Stoneville, Mississippi (USA). There were four rotation systems (continuous cotton, continuous corn, cotton-corn and corn-cotton) for both glyphosate-resistant (GR) and non-GR cultivars-herbicide system arranged in a randomized complete block design with four replications. Aspergillus flavus populations in surface (5-cm depth) soil, sampled before planting (March/April), mid-season June) and after harvest (September), ranged from 1.47 to 2.99 log (10) cfu g(-1) soil in the four rotation systems. Propagules of A. flavus were higher in the continuous corn system compared to the continuous cotton system on three sample dates, and cotton rotated with corn decreased A. flavus propagules in three of nine sample dates. Propagules of A. flavus were significantly greater in plots with GR cultivars compared to non-GR cultivars in three samples. In cotton seed, aflatoxin and fumonisin levels were similar (< or = 4 microg kg(-1) and non-detectable, respectively) regardless of rotation and glyphosate. In corn grain, aflatoxin was above the regulatory level (> or = 20 microg kg(-1)) only in GR cultivar in 2004 and 2005. Fumonisin was higher in non-GR cultivar (4 mg kg(-1)) regardless of rotation in 2004; however, in 2002, 2003 and 2005, aflatoxin and fumonisin levels were similar regardless of rotation and glyphosate. These results indicate the potential for increased aflatoxin and fumonisin levels (1 of 4 years) in corn; however, climatic conditions encountered during this study did not allow for mycotoxin production. In laboratory incubation studies, fairly high concentrations of glyphosate were required to inhibit A. flavus growth; however no short-term effect of soil treatment with glyphosate on A. flavus populations were observed. These data suggest that altered populations of A. flavus or higher aflatoxin concentrations in corn grain were due to indirect effects of the GR cropping system.

Population ecology of Aspergillus flavus associated with Mississippi Delta soils.

Understanding the source of Aspergillus flavus is required to manage aflatoxin contamination of maize (Zea mays L.). Studies assessed A. flavus propagules, Fusarium spp., and total fungi associated with Mississippi Delta soils. Soils from 12 and 15 sites were collected in 2000 and 2001, respectively. The propagule density of A. flavus ranged from log(10) 2.0 to 4.3 colony-forming units (cfu) g(-1) soil, while total fusaria ranged from log(10) 3.0 to 5.4 cfu g(-1) soil. The highest populations of A. flavus were associated with soils containing higher organic matter, especially in sites under a no-tillage management. The frequency of aflatoxin production in isolates ranged from 13 to 81% depending on soil. In 2001, there was a highly significant correlation between A. flavus and the history of maize cultivation. Soil fertility factors such as organic matter content, nitrate and extractable phosphorus correlated with the density of Aspergillus, Fusarium spp., and total fungi. The relationship between soil parameters and Aspergillus populations may be useful in predicting the contribution of soil microflora to aflatoxin contamination.

Laboratory assessment of atrazine and fluometuron degradation in soils from a constructed wetland.

Constructed wetlands offer promise for removal of nonpoint source contaminants such as herbicides from agricultural runoff. Laboratory studies assessed the potential of soils to degrade and sorb atrazine and fluometuron within a recently constructed wetland. The surface 3 cm of soil was sampled from two cells of a Mississippi Delta constructed wetland; one shallow area disturbed only hydrologically, and the second excavated to provide greater water-holding capacity. The excavated area was more acidic on average (pH 4.85 versus 5.21), but otherwise the physical properties and general microbial enzyme activities in the two areas were similar. Soils were treated with 84 and 68 microg kg(-1) soil (14)C-ring labeled atrazine and fluometuron, respectively, and incubated under either saturated (88% moisture, w:w) or flooded (1cm standing water) conditions. Soils were sampled over 32 days and extracted for herbicide and metabolite analysis. Under saturated conditions, fluometuron metabolized to desmethylfluometuron (DMF) with a half-life equal 25-27 days. However, under flooded conditions, the half-life of fluometuron was more than 175 days. Atrazine dissipated rapidly in saturated and flooded soil with a half-life of approximately 23 days, but only 10% of atrazine was mineralized to CO(2). The overall atrazine and fluometuron dissipation rates were similar between the two cells, but each area had a different pattern of metabolite accumulation. The major route of atrazine dissipation was incorporation of atrazine residues into methanol-nonextractable (soil-bound) components, with minimal extractable metabolite accumulation. A mixed-mode extractant (potassium phosphate:acetonitrile) recovered greater amounts of (14)C-residues from atrazine-treated soils, suggesting that hydrolysis of atrazine to hydroxylated metabolites was a major component of the bound residues. These studies indicate the potential for herbicide dissipation in wetland soils and a differential effect of flooding on the fate of these herbicides.

Comparison of cultural and analytical methods for determination of aflatoxin production by Mississippi Delta Aspergillus isolates.

This study compared cultural and analytical methods for detecting aflatoxin production by Aspergillus species. Aspergillus isolates were obtained from various Mississippi Delta crops (corn, peanut, rice, cotton) and soils. Most of the isolates (99%) were A. flavus and the remainder comprised A. parasiticus and A. nomius. The following three cultural methods were evaluated on potato dextrose agar: fluorescence (FL) on beta-cyclodextrin-containing media (CD), yellow pigment (YP) formation in mycelium and medium, and color change after ammonium hydroxide vapor exposure (AV). Aflatoxins in culture extracts were confirmed by thin-layer chromatography (TLC) and quantified by enzyme-linked immunosorbent assay (ELISA). Of the 517 isolates, 314 produced greater than 20 ng/g of total aflatoxin based on ELISA, and 180 produced greater than 10 000 ng/g of aflatoxin in the medium. Almost all the toxigenic isolates (97%) were confirmed by TLC as producers. Of the toxigenic isolates, as determined by ELISA, 93%, 73%, and 70% gave positive FL, YP, and AV responses, respectively. Of the 203 isolates producing less than 20 ng/g of aflatoxin, 20%, 6%, and 0% of respective FL, YP, and AV methods gave false-positive responses. The 9% false-positive results from TLC fall within this range. This study showed good agreement among all tested cultural methods. However, these cultural techniques did not detect aflatoxin in all cultures that were found to produce aflatoxins by ELISA, LC/MS, and TLC. The best results were obtained when the AV color change and CD fluorescence methods were used together, yielding an overall success rate comparable to TLC but without the need for chemical extraction and the time and expense of TLC.

Fluorescent Pseudomonas isolates from Mississippi Delta oxbow lakes: in vitro herbicide biotransformations.

Fluorescent pseudomonads were a major component [log (10) 4.2-6.1 colony-forming units mL-1] of the culturable heterotrophic gram-negative bacterioplankton observed in three Mississippi Delta oxbow lakes in this study. Pure cultures of fluorescent pseudomonads were isolated from three Mississippi Delta oxbow lakes (18 per lake), using selective media S-1. Classical physiological tests and Biolog GN plates were used in criteria for taxonomic identification. Most isolates were identified as biotypes of Pseudomonas fluorescens 55% (II), 7% (III), and 25% (V). About 7% of the isolates were identified as P. putida and 7% as non-fluorescent Pseudomonas-like. Cell suspensions of these isolates were tested for their ability to metabolize/co-metabolize six 14C-radiolabeled herbicides (2,4-dichlorophenoxyacetic acid (2,4-D), cyanazine, fluometuron, metolachlor, propanil, and trifluralin) that are commonly used for crop production in this geographical area. Almost all (53 of 54) isolates transformed trifluralin via aromatic nitroreduction. Most isolates (70%) dechlorinated metolachlor to polar metabolites via glutathione conjugation. About 60% of the isolates hydrolyzed the amide bond of propanil (a rice herbicide) to dichloroaniline, with the highest frequency of propanil-hydrolyzing isolates observed in the lake from the watershed with rice cultivation. All propanil-hydrolyzing isolates were identified as P. fluorescens biotype II. No metabolism of cyanazine or fluometuron was observed by any isolates tested, indicating little or no potential for N-dealkylation among this group of bacterioplankton. No mineralization of 2,4-D labeled in either the carboxyl or ring position was observed. These results indicate that reductive and hydrolytic pathways for herbicide co-metabolism (aromatic nitroreduction, aryl acylamidase, and glutathione conjugation) are common in Mississippi Delta aquatic fluorescent pseudomonads; however, the potential for certain oxidative transformations (N-dealkylation, cyano group oxidation) may be rare in this group of bacterioplankton.

Effects of pH on chemical stability and de-esterification of fenoxaprop-ethyl by purified enzymes, bacterial extracts, and soils.

De-esterification is an initial step in the metabolism of certain herbicides, for example, fenoxaprop-ethyl [(+/-)-ethyl 2-[4-[(6-chloro-2-benzoxaolyl)oxy]phenoxy]propanoate] (FE). The ethyl-ester bond cleavage of FE to fenoxaprop acid (FA) by purified enzymes, crude bacterial enzyme preparations, and soils was investigated. In similar experiments fluorescein diacetate (FDA) was used as an alternative substrate. FE stability was pH sensitive in acidic buffered solutions; that is, below pH 4.6, rapid nonenzymatic hydrolysis of the benzoxazolyl-oxy-phenoxy ether linkage occurred, forming 6-chloro-2,3-dihydro-benzoxazol-2-one (CDHB) and ethyl 4-hydroxyphenoxypropanoate or 4-hydroxyphenoxypropanoate. With porcine esterase and cell-free Pseudomonas fluorescens extracts, activity on FE and FDA was most rapid at pH 7.6-8.6 but decreased 80-90% at pH 5.6. Yeast (Candida cylindrica) lipase-mediated de-esterification of FE and FDA was not as sensitive to pH; that is, activity at pH 4.6 was 70% of that at pH 7.6. Short-term incubations (20 h) were conducted in eight soils (pH 4.5-6.9) treated with (14)C-chlorophenyl ring-labeled FE (2 mg kg(-)(1)). In the most acidic soils (pH 4.4-4.5) 25% of the (14)C was recovered as FA, versus 30-40% in moderately acid soils (pH 5.0-5.6) and 55% in neutral soils (pH 6.8-6.9). There was a similar correlation between soil pH and FDA de-esterification. CDHB was formed in all acidic soils with levels 4-fold greater in pH 4.4-4.5 soils than in pH 5. 0-5.6 soils. CDHB was not formed in neutral soils. Results demonstrate some chemical hydrolysis (benzoxazolyl-oxy-phenoxy ether linkage) of FE in acid soils, the sensitivity of enzymatic de-esterification of FE to pH, and the potential of FDA as a colorimetric indicator for esterase hydrolysis of FE.

Degradation of 2,4-dinitrophenol and selected nitroaromatic compounds by Sphingomonas sp. UG30.

Sphingomonas strain UG30 mineralizes both p-nitrophenol (PNP) and pentachlorophenol (PCP). Our current studies showed that UG30 oxidatively metabolized certain other p-substituted nitrophenols, i.e., p-nitrocatechol, 2,4-dinitrophenol (2,4-DNP), and 4,6-dinitrocresol with liberation of nitrite. 2,6-DNP, o- or m-nitrophenol, picric acid, or the herbicide dinoseb were not metabolized. Studies using 14C-labelled 2,4-DNP indicated that in glucose-glutamate broth cultures of UG30, greater than 90% of 103 microM 2,4-DNP was transformed to other compounds, while 8-19% of the 2,4-DNP was mineralized within 5 days. A significant portion (20-50%) of the 2,4-DNP was metabolized to highly polar metabolite(s) with one major unidentified metabolite accumulating from 5 to 25% of the initial radioactivity. The amounts of 2,4-DNP mineralized and converted to polar metabolites was affected by glutamate concentration in the medium. Nitrophenolic compounds metabolized by UG30 were also suitable substrates for the UG30 PCP-4-monooxygenase (pcpB gene expressed in Escherichia coli) which is likely central to degradation of these compounds. The wide substrate range of UG30 could render this strain useful in bioremediation of some chemically contaminated soils.

Glutathione-s-transferase activity and metabolism of glutathione conjugates by rhizosphere bacteria.

Glutathione-S-transferase (GST) activity was determined in 36 species of rhizosphere bacteria with the substrate 1-chloro-2,4-dinitrobenzene (CDNB) and in 18 strains with the herbicide alachlor. Highest levels of CDNB-GST activity (60 to 222 nmol (middot) h(sup-1) (middot) mg(sup-1)) were found in gram-negative bacteria: Enterobacter cloacae, Citrobacter diversus, Klebsiella planticola, Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, and Xanthomonas campestris. There was very low CDNB-GST activity in the gram-positive strains. Rapid metabolism of CDNB-glutathione conjugates, attributable to high levels of (gamma)-glutamyltranspeptidase, also occurred in the gram-negative bacteria, especially pseudomonads. Alachlor-GST activity detected in cell extracts and whole-cell suspensions of some strains of the families Enterobacteriaceae and Pseudomonaceae was 50- to 100-fold lower than CDNB-GST activity (0.5 to 2.5 nmol (middot) h(sup-1) (middot) mg(sup-1)) and was, for the most part, constitutive. The glutathione-alachlor conjugate was rarely detected. Cysteineglycine and/or cysteine conjugates were the major products of alachlor-GST metabolism. Whole-cell suspensions of certain Pseudomonas spp. dechlorinated from 20 to 75% of 100 (mu)M alachlor in 24 h. Results indicate that rhizosphere bacteria, especially fluorescent pseudomonads, may play an important role in the degradation of xenobiotics such as alachlor via GST-mediated reactions.

Evaluation of Diverse Antisera, Conjugates, and Support Media for Detecting Bradyrhizobium japonicum by Indirect Enzyme-Linked Immunosorbent Assay.

We evaluated three antisera and four enzyme conjugates for the detection of Bradyrhizobium japonicum by an indirect enzyme-linked immunosorbent assay in microtiter plates. Nitrocellulose membrane sheets were then evaluated as an alternative support medium by using some combinations. Partially purified immunoglobulin G (IgG) or unpurified antisera to strain USDA 110 raised in rabbits, goats, or sheep was reacted in microtiter plates with alkaline phosphatase conjugated to protein A, goat anti-rabbit (GAR), sheep anti-rabbit (SAR), or rabbit anti-goat (RAG) IgG. Cultures or nodules containing homologous rhizobia were detected with equal sensitivity when protein A, GAR, or SAR was reacted with 5 mug of protein IgG per ml or a 1:800 titer of antisera from rabbits, but not goats or sheep. RAG reacted with IgG or antisera from goats or sheep. The detection limit was 2 x 10 rhizobia per well. Rhizobia were spotted on nitrocellulose sheets as an alternative support medium, followed by soaking in 5 mug of protein per ml as IgG and 1:4,000 dilutions of protein A or GAR conjugate. Rhizobia in serogroup 110 were detected with the dye combination Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP), and rhizobia in serogroup 122 were detected with fast red-naphthol phosphate (FR-NP). At the conclusion of the 5-h assay, purple (NBT-BCIP) or red (FR-NP) spots were visible in positive reactions. The sensitivity of detection was about 1,000 rhizobial cells or 3 mug of nodules tissue.

Rapid Colored-Nodule Assay for Assessing Root Exudate-Enhanced Competitiveness of Bradyrhizobium japonicum.

The effects of root exudate (RE) treatment on nodule occupancy by Bradyrhizobium japonicum were investigated by a rapid colored-nodule assay, which is based on the observation that B. japonicum L-110 and its antibiotically marked derivatives form dark-red nodules on certain soybean (Glycine max) cultivars, whereas other strains form beige nodules. The efficacy of the assay was confirmed by direct immunofluorescence and by antibiotic platings of nodule bacteria. Both logarithmic- and stationary-phase cultures of the reference strain, L-110Nal, were used in paired-competition studies with RE-treated or untreated cells of seven challenge strains. On the basis of field and greenhouse competition studies, these strains were placed into three competitiveness groups: high (AN-11, AN-16aStrRif, and AN-6), intermediate (AN-3 and 122SR), and low (I-110ARS and AN-18). Seedlings of G. max cv. Centennial were inoculated with two ratios of challenge to reference strain, 1:1 and 1:9, and nodule occupancy was determined after the V4 to V5 stage of ontogeny. Two of the strains showed increased occupancy in response to RE treatment at the 1:1 inoculation ratio. Logarithmic- and stationary-phase cultures of AN-6 showed increased occupancy, from 22 to 38% (P < 0.10) and from 23 59 39% (P < 0.05), respectively. While the maximum increase for stationary-phase cultures of AN-16aStrRif was from 34 to 47% (P < 0.05), logarithmic-phase cultures failed to respond to RE treatment. The results of these studies indicate that RE treatment increases the nodule occupancy of some, but not all, B. japonicum strains and that the colored-nodule assay could be rapidly and reliably used to determine the competitive ability of B. japonicum.

Improved Method of Typing Bradyrhizobium japonicum in Soybean Nodules.

An improved method for antibiotic resistance recovery of Bradyrhizobium japonicum from soybean (Glycine max (L.) Merr.) nodules that is simple, time saving, and economical was developed. This technique involves the use of two 96-well microtiter plates as a multinodule sterilization chamber and a template and a third plate as a 16-point replicator constructed with steel nails affixed to the plate with epoxy cement. With this system a team of four technicians could type 3,000 nodules per day. This method was useful in assessing strain establishment and interstrain competition when one or more uniquely labeled strains of B. japonicum were inoculated onto either growth-room- or field-grown soybeans. Contamination was low and reproducibility across replicates approached the theoretical upper limit. Simplicity in design and use made this recovery method especially adaptable for field studies in which large numbers of nodules were required to provide a representative statistical sample offering good precision.

Kinetics of denitrifying growth by fast-growing cowpea rhizobia.

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO(3), NO(2), and N(2)O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K(s)) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K(s), 3.4 and 3.8 muM; Y, 16.0 and 14.0 g . cells eq; mu(max), 0.41 and 0.33 h. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K(s) values were 41, 270, and 460 muM for nitrous oxide, nitrate, and nitrite, respectively; the K(s) for A28 grown on nitrate was 250 muM. The results are kinetically and thermodynamically consistent in explaining why O(2) is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (mu(max), 0.41 h) when A26 was grown with either O(2) or NO(3) as an electron acceptor and were only slightly reduced when A28 was grown with NO(3) (0.25 h) as opposed to O(2) (0.33 h).

New selective media for enumeration and recovery of fluorescent pseudomonads from various habitats.

New media (S1 and S2) were formulated that provide a high degree of selectivity and detection of fluorescent pseudomonads on initial plating. The selectivity of the S-type media was based on a detergent, sodium lauroyl sarcosine, and an antibiotic, trimethoprim. A total of five soils from different geographical locations and one sewage sludge sample were examined. On S1 medium, isolates from two soils with low fluorescent pseudomonad populations exhibited a high frequency of arginine dihydrolase (78%) and oxidase-positive (95%) phenotypes, but no fermentative isolates were recovered. Medium S2 was more defined and selective than S1, but lower numbers of fluorescent pseudomonads were recovered on S2. In soils in which fluorescent pseudomonads represent a small proportion of the total population, S1 medium consistently recovered high percentages of fluorescent phenotypes (82.5%).

Identity and behavior of xylem-residing bacteria in rough lemon roots of Florida citrus trees.

An aseptic vacuum extraction technique was used to obtain xylem fluid from the roots of rough lemon (Citrus jambhiri Lush.) rootstock of Florida citrus trees. Bacteria were consistently isolated from vascular fluid of both healthy and young tree decline-affected trees. Thirteen genera of bacteria were found, the most frequently occurring genera being Pseudomonas (40%), Enterobacter (18%), Bacillus, Corynebacterium, and other gram-positive bacteria (16%), and Serratia (6%). Xylem bacterial counts fluctuated seasonally. Bacterial populations ranged from 0.1 to 22 per mm of root tissue (about 10 to 2 x 10 bacteria per g of xylem) when bacterial counts were made on vascular fluid, but these numbers were 10- to 1,000-fold greater when aseptically homogenized xylem tissue was examined similarly. Some of the resident bacteria (4%) are potentially phytopathogenic. It is proposed that xylem bacteria have an important role in the physiology of citrus.