The procedure of using quantitative real time polymerase chain reaction (Q-PCR) to quantify Pseudomonas fluorescens (P. fluorescens) from soil samples were developed. This allowed us to answer three questions:
1. What is the effect of field treatments on the amount of P. fluorescens;
2. What is the effect of P. fluorescens on disease levels?
3. What is the change of amount of P. fluorescens through out the years of transition?
It is found that both the “cropping system” as the fixed effect for whole plot factor and “organic amendment” as the fixed effect for sub-plot factor did not have significant effect on the amount of P. fluorescens.
Also, the effect of P. fluorescens on disease levels of both greenhouse and field results is inconclusive with the statistical analysis we performed so far.
However, the amount of P. fluorescens increased significantly after transition, which indicate that the transition does have impact on soil microbes.
Organic matter amendments to soil, in the form of preceding crop residues, cover crop residues, or direct organic matter applications, have been shown to affect levels of root and foliar diseases in several crops. Suppression of soilborne plant pathogens has been observed following additions of certain types of organic matter to soils. In some cases the mechanism of suppression in these systems has been found to be associated with increased microbial activity resulting from the influx of carbon and nitrogen supplied by the incorporated organic matter. Specific cropping systems have been shown to alter the associated soil microbial communities, and in some cases the population levels of known biological control agents have been enhanced. Foliar disease levels have been shown to be affected by applications of soil orgniac matter, even for diseases caused by pathogens that do not have a soilborne phase in their disease cycles. Possible mechanisms suggested for this type of disease suppression include changes in plant’s nutrient status and the phenomenon known as systemic acquired resistance or induced systemic resistance.
Based on this information, it seemed likely that differences in the disease suppressiveness of plots in the organic transition study would vary as a result of the cropping system and organic amendment treatments. We evaluated the diseases suppressiveness of the soil to soybean root diseases in greenhouse bioassays on soil samples taken over the course of the study, and we monitored disease development on the crops in the plots to evaluate the impact of the treatments on disease development in the field. In 2007, natural-occurring diseases, bacterial pustule and wild fire, were evaluated by percentage of leaf area infected in mid-August. These bacterial diseases were confirmed using the ooze-test and isolated on nutrient agar.
Soil samples were taken and assayed in the greenhouse with infestation of R. solani and F. solani on soybean.
Pseudomonas population was quantified with non-culturing method, real-time polymerase chain reaction (PCR). The working hypothesis is field treatments had an effect on Pseudomonas population and disease suppression.
Our short-term goal was to characterize and compare the effect of selected cropping systems and organic amendments in transitional farming systems on soil Pseudomonas population, and understand the links between plant disease suppression and Pseudomonas population.
Our intermediate-term goal was to develop relevant, accessible outreach and educational products for organic producers.
The long-term goal was to establish an interdisciplinary, cross-institutional organic farming systems research and education program, guided by an active partnership between organic producers, researchers, and extension educators, that improves the performance of organic farming systems and enhances the ability of North central region organic producers to meet the growing local and regional demand for organic produce.
Population levels of fluorescent species of Pseudomonas were used as indicators for disease suppressiveness of soils resulting from three cropping system and three organic amendment treatments as described in the project proposal. The soil samples used for Pseudomonas fluorescens quantification were collected using a 4.8 cm diameter soil corer inserted into the soil to a depth of 30 cm. Each core was fractionated into two depths, 0-15 cm and 15-30 cm. Two soil cores were collected from:
1) planting beds (high intensity system) or
2) approximately the 6th row from the edge of the plot (the intermediate intensity system) and
3) approximately 1.83–2.44 m into the plot, about the same distance into the plot as was sampled in the other two systems (low intensity system).
Soil cores from the same split-plot were combined into a composite sample. Sieved, moist soil samples were stored at -20 oC until further process. The 0-15 cm depth samples were used for P fluorescens quantification. The dates for samples used in this study were sampled on June-7-04, May-11-05, June-05-06.
DNA was extracted from 1.5 grams of soil samples using a FastDNA® Spin kit following the manufacturer’s procedure, and then cleaned up with hexadecyltrimethylammonium bromide (CTAB) (Ausubel et al., 1995) as mentioned in Riesenfeld et al. (2004). Briefly, adjust the NaCl concentration of DNA extract to 0.7 M, then add 0.1 volume of warm CTAB. Extract with 1 volume of chloroform:isoamyl alcohol (24:1). Precipitate with 2 volumes of cold 100% alcohol, and then washed with 70% alcohol. Resuspended in 100 ml of sterile double distilled water (ddH2O). The DNA concentration then was measure by Nanodrop ND-1000 Spectrophotometer.
QPCR standards were made from a pure culture of Pseudomonas fluorescens, pf-5, provided by Dr. Linda Thomashow. Fresh single colony of pf-5 grown on a luria broth plate (LB plate) was transfered to luria broth and allowed to incubate overnight. The culture broth was centrifuged to pellet the cells, which was then washed in PBS. The pellet was resuspended in 750 ml TE buffer, and transfer to bead beating. Following cell disruption in the bead beating apparatus, the resulting solution was mixed using a vortex mixer on the highest speed for 2 min.
Extract with equal volume of phenol/chloroform/isoamyl alcohol, then extract again with equal volume of chloroform/isoamyl alcohol. Precipitate DNA with 200 ml volume of 10.5 M sodium acetate) and 900 ml of isopropanol. Let stand 10 minutes at room temperature, then 2 hours at -80 oC. Centrifuge 30 min at 12000 rpm. Wash pellet with 500 ml of 70% ethanol. Resuspend in 200 ml TE buffer. The DNA extract was diluted ten times and measured by Nanodrop to be 140 ng/ml after dilution. 1.5 ml of 2×108 CFU/ml yields 200 ml of 1400 ng/ml.
Pseudomonas-specific primer pair, Primer I (5’-GAGTTTGATCCT-GGCTCAG-3’) and primer II (5’-CCTTCCTCCCAACTT-3’) were ordered from Integrated DNA Technologies, Inc. (Coralville, IA, USA). The primers target a 440 bp region of the 16S rRNA gene (Johnsen et al., 1999). Serial dilution of standards in 140 ng/ml, 27 ng/ml, 5.4 ng/ml, 1.08 ng/ml, 0.216 ng/ml, and 0.0432 ng/ml were used.
Real time PCR reaction for fluorescens Pseudomonads was performed by DNA engine OpticonTM using Qiagen QuantiTech SYBR green PCR kit following the manufacturer’s protocol. Reaction mixture (50 ml) consisted of 1 ml of serial dilution of standards, 12.5 ml of 2xQuantiTech SYBR green master mix, Primer I and Primer II with final concentration of 300 nM, and H2O to make up 25 ml in total for each reaction. PCR amplification program was set following the manufacturer’s protocol, except the following modification: 30 seconds at 50°C for annealing, and 30 seconds at 72°C for extension. The number of cycles was 41. At the end, the melting curve was generated from 60 – 95 oC read every 0.2 oC increment and hold 2 seconds. The melting curves were checked individually to make sure that the reactions were correct without error. The r2 threshold value of the standards is 98.5%.
Data of bioassays were compared by mixed models (PROC MIXED) and differences between means were tested with least-squares means (LSMEANS) using SAS 9.0 program (SAS Institute, Inc.). Within each year, the system is treated as split plot design that “block” is the random effect, “cropping system” is the fixed effect for whole plot factor and “organic amendment” is the fixed effect for sub-plot factor (Lochinkohl &Boehm, 2001).
To compare the effect of number of years since the beginning of transition on disease suppression, data from each year was normalized to avoid greenhouse condition fluctuation. Normalization was conducted by dividing the data with corresponding average derived from all the autoclaved soil samples at the same time the samples were assayed. The assumption is that bioassays in autoclaved soil provide a baseline for normalization of the greenhouse condition fluctuation. The same PROC MIXED procedure was used to analyze the data.
In the greenhouse bioassays, a general increase in disease suppressiveness was observed in the soils from all plots over the course of the study (2004 to 2006) for both Rhizoctonia root rot and sudden death syndrome of soybeans. However, no differences in soilborne disease suppression were detected as a result of the cropping system or amendment treatments. Differences in foliar/fruit disease levels resulting from the treatments were observed for some diseases on some crops in the field plots.
In the beginning of the transition (year 2003), no differences were seen in levels of diseases seen on tomatoes, soybeans, or pasture plants.
In 2004, levels of leaf rust on pasture grass were higher in plots receiving manure amendments, but no differences were seen in levels of wheat, cabbage, or broccoli diseases.
In 2005, higher levels on common rust were seen on corn in manure amended plots, and lower levels of powdery mildew were seen on squash plants in compost amended plots.
In 2006, the first assay year, Septoria leaf spot of tomatoes was found to be affected by both previous crop and amendment treatments, with lowest levels in the pasture plots and highest in the cash grain plots, and lower levels in the compost and manure amended plots than the non-amended plots. Incidence levels of bacterial spot of peppers were also highest in the cash grain plots and lowest in the vegetable plots.
In year 2008, all the archived soil samples were quantified for P. fluorescens:
1. The effect of treatments on P. fluorescens: Both the “cropping system” as the fixed effect for whole plot factor and “organic amendment” as the fixed effect for sub-plot factor did not have significant effect on the amount of P. fluorescens.
2. The effect of P. fluorescens on disease levels of both greenhouse and field results: In the field result of 2005, P. fluorescens has effect on the severity of Downey mildew of winter squash. In the greenhouse result, P. fluorescens has effect on root discoloration of SDS bioassay. P. fluorescens has effect on root volume of Rhizoctonia bioassay.
3. The amount of P. fluorescens increased significantly after transition. The normalized value in 2006 is significantly higher than that from both 2005 and 2004, whereas 2004 and 2005 had no difference.
Educational & Outreach Activities
Marzano, S. 2007 Assessment of disease suppression in farming systems in transition to organic production. Presentation at “Exploring the option: transition to organic”, University of Illinois Extension’s 2007 Organic Field Day series, July 26, Danforth, IL.
Marzano, S.L., and D. Eastburn. 2007. Presentation at the North Central Division of the American Phytopathological Society meetings June 19 – 21, Lafayette, IN.
Marzano, S.L., and D. Eastburn. 2007. Assessment of disease suppression in organic transition farming systems. Phytopathology 97:S71 (also a poster at the American Phytopathological Soc. and Soc. of Nematologists meeting July 28-Aug. 1, San Diego, CA).
1. The field treatments, cropping system and organic amendment, does not have significant effect on the amount of P. fluorescens.
2. The effect of P. fluorescens on disease levels of both greenhouse and field results was significant in some cases but the direction of effect was inconclusive with the statistical analysis we performed.
3. The amount of P. fluorescens increased significantly after transition.
Areas needing additional study
1. As we showed that the amount of P. fluorescens increased after transition, further study of the factors contributing to the increase would be interesting.
2. Our molecular, non-cultured based approach can be used to assess the effect of P. fluorescens population on disease levels by a small set of factorial designed experiment to elucidate the relationship between the two in the future study.