Kaare Johnsen

Pesticide effects on bacterial diversity in agricultural soils – a review

Abstract. According to guidelines for the approval of pesticides, side-effects on soil microorganisms should be determined by studying functional parameters such as carbon or nitrogen mineralisation. However, the microbial diversity may have been markedly changed following pesticide use despite unaltered metabolism, and such changes may affect soil fertility. This review evaluates new methods for measuring pesticide effects on bacterial diversity, and discusses how sampling should take temporal and spatial heterogeneity into account. Future research on pesticide approval protocols should establish the relationships between mineralisation assays and new and rapid bacterial diversity profiling methods, and should include the possible ecological implications of altered bacterial diversity for soil fertility.

Population dynamics of the fast-growing sub-populations of Pseudomonas and total bacteria, and their protozoan grazers, revealed by fenpropimorph treatment

The population dynamics of indigenous soil bacteria and protozoa on decaying barley roots were followed by using litter bags buried in laboratory-incubated soil. The soil was either non-treated or treated with the fungicide fenpropimorph (in the formulation Corbel) at concentrations corresponding to the recommended and at 10 times field dose (1.3 and 13 mg kg ˇ1 dry wt.). Number of total bacteria and number of Pseudomonas were detected, using both traditional plating and short-time incubations of ‘early’ colonies, to determine the fast-responding subpopulation of the culturable bacteria. The number of protozoa corresponding to the two subpopulations was followed. The results strongly indicate a predatory association between the protozoa and bacteria. This was shown by a tight temporal association, and by a stimulation of bacteria following predatory release when protozoa were inhibited by fenpropimorph. Thus, fenpropimorph disturbed population dynamics in concentrations, which can be reached in surface soils after distribution in the field. 7 2000 Elsevier Science Ltd. All rights reserved.

Interactions between saprotrophic fungi, bacteria and protozoa on decomposing wheat roots in soil influenced by the fungicide fenpropimorph (Corbel®): a field study.

Abstract Litterbags containing freshly harvested wheat roots with adhering rhizosphere soil were placed in their native soil. Four levels of the fungicide fenpropimorph were applied: 0, 1, 10 and 100 mg kg −1 soil. The experimental set-up was a randomised block design with five replicates. Litterbags were sampled six times over a 4-month period and microbial numbers determined. Bacteria (colony forming units, CFUs) and protozoa (most probable number) followed a similar pattern: a sudden decrease at the onset of the study period was followed by a fast recovery and increase. This ended at maximum mean values of ≈7.5 × 10 7 bacteria g −1 root litter by day 7 and 3 × 10 6 protozoa g −1 root litter by day 20, respectively. Fenpropimorph had no significant effect on either of these two groups, for which mean population sizes seemed to interact in a prey–predator manner. The fungi (stained by calcofluor white and fluorescein diacetate) were unaffected by the pesticide until day 20. Hereafter, fluorescein diacetate-active (FDA-active) hyphae were significantly inhibited in all fungicide treated plots, compared to the untreated plots 50 and 114 days after the application of fenpropimorph. Fungal colony forming units (fungal CFUs) on fenpropimorph-containing agar demonstrated a selection towards more fenpropimorph tolerant fungi in the 100 mg kg −1 fenpropimorph treated plots. The delayed effect of fenpropimorph on FDA-activity could be explained as a combined effect of the pesticide and the degradation product itself, and the fluctuating water stressing field conditions of this year. Our results, thus, demonstrated long term effects of fenpropimorph on the activity of saprotrophic fungi at recommended field dose, and that high dose fenpropimorph can select for fenpropimorph tolerant fungal populations.

Quantification of the atrazine-degrading Pseudomonas sp. strain ADP in aquifer sediment by quantitative competitive polymerase chain reaction

The widely used herbicide atrazine and some of its degradation products are among the most commonly found xenobiotics in groundwater in Europe as well as in the USA. The bacterium Pseudomonas sp. strain ADP (P. ADP) possesses genes encoding atrazine mineralization on the self-transmissible plasmid pADP-1. In the present study, this ability of the strain to mineralize atrazine in aquifer sediment under both aerobic and denitrifying conditions at 10 degrees C was studied. P. ADP was able to mineralize more than 50% of 2.8 muM atrazine within 14 days under both growth conditions. Counts of degraders as colony forming units (CFU) on atrazine plates and counts of atzA gene copies as determined by quantitative competitive polymerase chain reaction (cPCR) were performed. The atzA gene encodes the enzyme which catalyzes the first step of atrazine mineralization by the strain. Quantification of the atzA gene gave rise to higher numbers than did counts of CFU. High nitrate concentrations inhibited atrazine mineralization and culturability on agar plates, but atzA copy numbers remained stable throughout the experiment. The results show a potential for bioaugmentation using P. ADP at both aerobic and denitrifying conditions and the use of cPCR as a tool for monitoring the bacteria independent of culturability.

Effects of fenpropimorph on bacteria and fungi during decomposition of barley roots

Abstract The study examined the effects of the fungicide fenpropimorph (in the formulation Corbel) on primary decomposer organisms in soil. Bacterial and fungal succession was followed on dead young barley roots buried in fungicide-treated or untreated soil. Fenpropimorph was added to the soil in a concentration realistically achieved in field topsoil when using the recommended dose. Over a 56-day period we measured the length of active fungal hyphae, the abundance of total culturable bacteria, the abundance of two culturable subgroups relevant to the soil environment (hyphae-forming actinomycetes and Pseudomonas), and the diversity of bacterial DNA. The soil fenpropimorph content followed first order disappearance concomitant with the first order formation of fenpropimorphic acid. Fenpropimorph inhibited the growth of active fungi during the first 10 days, when fungal activity peaked in both the treated and non-treated root samples. The number of total culturable bacteria was significantly lowered by fenpropimorph at day 17 and stimulated at day 56, indicating a possible indirect effect of the fungicide on the culturable bacteria as a whole. Nevertheless, culturable Pseudomonas and actinomycetes were not affected. The succession of the two bacterial subgroups differed considerably, so, whereas Pseudomonas peaked in the early decomposition stages, actinomycetes were most abundant after 2 months, and on the very first sampling day. Thus, the present findings suggest a possible effect of fenpropimorph on soil fertility. The diversity of total bacterial DNA measured by denaturing gradient gel electrophoresis (DGGE) was unaffected by fenpropimorph treatment, there being a clear and highly reproducible succession in bacterial diversity during decomposition of the barley roots. The diversity in the hotspot on the last sampling occasion at two months was similar to the diversity of the bulk soil, thus indicating a return to the level prior to addition of the roots.

Group-Specific PCR Primers to Amplify 24S a-Subunit rRNA Genes from Kinetoplastida (Protozoa) Used in Denaturing Gradient Gel Electrophoresis

We developed and tested a set of primers for amplification of a region of the 24S a-subunit rRNA genes (24S rDNA) specific to Kinetoplastida (Protozoa). The reverse primer was supplied with a GC rich region in the 5? end in order to make the PCR product suitable for analysis by denaturing gradient gel electrophoresis (DGGE). PCR product was obtained from all the kinetoplastids tested and no PCR product was obtained from any other Eukaryotes or Prokaryotes tested. It was possible to distinguish between all pure cultures of kinetoplastids by denaturing gradient gel electrophoresis in gels ranging from 20% to 60% denaturants. PCR-DGGE analysis of DNA purified from lake sediment revealed approximately 20 bands indicating high kinetoplastid diversity. Direct cloning and sequencing of 24S rDNA sequences retrieved from the lake sediment by PCR also showed high kinetoplastid diversity. Of 43 clones, 27 different sequences were found. Alignments and phylogenetic analysis showed that a majority of the sequences were most closely related to the Bodonidae. Four sequences were closer to the Trypanosomatidae, whereas three sequences fell outside both groups. The PCR-DGGE procedure developed in this study has been shown to be useful for distinguishing between different kinetoplastid species. Thus, it may be a useful tool for evaluating the genetic diversity of this group in environmental samples, e.g., as a result of perturbation. Another possible application of this method is in fast and accurate screening for the presence and identification of pathological parasitic Kinetoplastida from environmental samples and for diagnostics of human and animal infections.

Prey-predator dynamics in communities of culturable soil bacteria and protozoa: differential effects of mercury

We investigated whether the prey-predator dynamics of bacteria and protozoa were affected by inorganic mercury at concentrations of 0, 3.5 and 15 mg Hg(II) kg soil 21 . The amount of bioavailable Hg was estimated using a biosensor-assay based on the mer – lux gene fusion. The numbers of bacterial CFUs on the general medium 1/100 tryptic soy agar (TSA) were significantly decreased when the soil had been amended with Hg. In contrast, no effect was seen on the number of CFUs on the Pseudomonas-specific medium Gould’s S1 agar. Protozoan numbers estimated by the most probable number (MPN) method with 1/100 TSB as growth medium were also negatively affected by Hg. The different fractions of protozoa were affected to different degrees suggesting that amoebae were less sensitive than slow-growing flagellates, which again were less sensitive than the fast-growing flagellates. In contrast, Hg did not induce any detectable changes in the diversity of flagellate morphotypes. In the treatment with 15 mg Hg kg 21 a transiently increased number of bacteria was seen at day 6 probably concomitant with a decrease in the numbers of protozoa. This might indicate that Hg affected the prey-predator dynamics in communities of culturable bacteria and protozoa in soil. Furthermore, we showed that the number of Pseudomonas spp. was not affected by Hg whereas the number of bacteria growing on a general medium was. q 2003 Elsevier Ltd. All rights reserved.

The presence of embedded bacterial pure cultures in agar plates stimulate the culturability of soil bacteria

Traditional methods for bacterial cultivation recover only a small fraction of bacteria from all sorts of natural environments, and attempts have been made to improve the bacterial culturability. Here we describe the development of a cultivation method, based on the embedment of pure bacterial cultures in between two layers of agar. Plates containing either embedded Pseudomonas putida or Arthrobacter globiformis resulted in higher numbers of CFUs of soil bacteria (21% and 38%, respectively) after 833 h of incubation, compared to plates with no embedded strain. This indicates a stimulatory effect of the bacterial pure cultures on the cultivation of soil bacteria. Analysis of partial 16S rRNA gene sequences revealed a phylogenetical distribution of the soil isolates into 7 classes in 4 phyla. No difference was observed at the phylum or class level when comparing isolates grouped according to embedded strain. The number of isolates belonging to the same class as the embedded strain was reduced in comparison to that of plates with no embedded strain, indicating that intercellular signalling was unlikely to cause the observed stimulatory effect. Significantly higher fractions of isolates with less than 97% sequence homology to known sequenced isolates in GenBank were recovered from plates with embedded strains than from those without, which indicate a higher number of potential novel soil isolates. This approach for cultivation is therefore a feasible alternative or supplement to traditional cultivation on agar plates in order to enhance bacterial culturability.

Mercury Decreases Culturability of Pseudomonas frederiksbergensis JAJ 28 in Soil Microcosms

Mercury is a biologically potent heavy metal, which has been found to change the diversity of culturable bacteria. Therefore, we investigated whether Hg kills bacteria in soil or reduces culturability. Soil microcosms were inoculated with Pseudomonas frederiksbergensis JAJ 28 and were sampled regularly during 28 days. The total number of acridine orange-stained cells was relatively constant, and Hg reduced the number on only one sampling day. However, the fraction of culturable cells on 1/10 tryptic soy agar was lowered on days 6, 13, and 21. The number of microcolony forming units, which represents viable cells, was also affected by Hg, but this effect was delayed compared with the effects on CFUs. The amount of headspace CO2 per cell was overall increased by Hg, another indication of the toxic effects of Hg on the bacterial cells. Our results thus emphasize the need to take culturability into account when studying the effects of heavy metals on bacterial diversity. RID=”” ID=”” Correspondence to: K. Johnsen: email: kjo@vetinst.dk