DEVELOPMENT OF PASTEURIZED LIQUID SWINE MANURE AS A PLANT DISEASE CONTROL PRODUCT FOR HIGH-VALUE CROPS

Transcription

1 DEVELOPMENT OF PASTEURIZED LIQUID SWINE MANURE AS A PLANT DISEASE CONTROL PRODUCT FOR HIGH-VALUE CROPS Ontario Pork proposal #0426 Final Report (December 2006) G. Lazarovits, K.L. Conn, and E. Topp Southern Crop Protection and Food Research Centre Agriculture and Agri-Food Canada 1391 Sandford St. London, ON N5V 4T3 M. Tenuta Department of Soil Science University of Manitoba Winnipeg, MB R3T 2N2

2

3 1 G. Lazarovits 1, K.L. Conn 1, E. Topp 1, and M. Tenuta 2 1 SCPFRC, AAFC, 1391 Sandford St., London, ON N5V 4T3 2 Department of Soil Science, University of Manitoba, Winnipeg, MB, R3T 2N2 lazarovitsg@agr.gc.ca Phone: ext. 293 Fax: connk@agr.gc.ca Phone: ext. 285 Fax: toppe@agr.gc.ca Phone: ext. 235 Fax: tenutam@ms.umanitoba.ca Phone: Fax: INDEX Page Executive Summary... 2 Background... 4 Objectives... 7 Results and Discussion ) Efficiency of a continuous-flow composter to pasteurize LSM ) Temperature and time needed to kill Escherichia coli in LSM ) Isolation, identification, and determination of optimal growth conditions of the thermophylic bacteria ) Effect of LSM formulations on potato scab, verticillium wilt, and yield A) Addition of acidified LSM to a commercial potato field (site L1-2004) B) Addition of acidified LSM to soils from eight commercial potato fields in a micro-plot experiment C) Addition of VFA-free LSM plus formic acid to soils from two commercial potato fields in a micro-plot experiment D) Addition of acidified LSM to a commercial potato field (site L1-2005) ) Comparison of single verses double application of acidified LSM in the crop verses rotation year on potato scab, verticillium wilt, and yield ) Efficacy of ph-reduced LSM to reduce numbers of plant parasitic nematodes A) Addition of acidified LSM to soil from two commercial potato fields in a 53 micro-plot experiment in B) Addition of acidified LSM to soil from two commercial potato fields in a 57 micro-plot experiment in C) Addition of acidified LSM to a commercial potato fields (site L1-2005) D) Toxicity of acetic acid to the plant parasitic nematode Pratylenchus penetrans 61 Conclusions Acknowledgements Literature Cited Publications and presentations of the research in this project ( ) Budget ( )... 67

4 2 EXECUTIVE SUMMARY A continuous-flow composter for digestion of LSM was tested. The composter maintained a manure temperature of C for 3 months with manure continuously passing through it. The technology is inexpensive and the equipment is simple to maintain as it has no moving parts other than two pumps; one that circulates the manure and introduces air into the liquid, and one that pumps acid or molasses into the liquid to maintain ph at the desired level. The material coming out of the tank had no detectable E. coli or Salmonella spp. and it retained all of the starting nutrients. The liquid had no odour but if it was allowed to become anaerobic the odour returned. The liquid coming out of the composter should be considered as a liquid fertilizer/compost. We believe this compost is a safe and effective fertilizer. A manure temperature of at least 60 C is needed to kill Escherichia coli. At this temperature E. coli is killed within 15 minutes. To provide a margin of safety to make sure E. coli is killed, the manure should likely be kept in the composter for at least 30 minutes. Thermophylic bacteria were isolated from the digested manure to identify them to make sure none of them are potential animal or human pathogens, and determine optimum growth conditions. A special medium had to be developed in order for the thermophylic bacteria to grow outside of the composter. Four main groups of bacteria were isolated, but have not been identified yet. The optimum ph range for growth was ph Thus, the ideal manure ph in the composter would be 7.5 because at ph 8, some ammonia would be released. The bacteria could grow up to 65 C. Thus, this is the maximum temperature that the composter can be allowed to reach. LSMs containing volatile fatty acids are toxic to plant pathogenic microorganisms only in soils with a ph below 6. Addition of acidified LSM to commercial potato soils with a high ph soil reduced potato scab disease and plant parasitic nematodes to near zero. LSM in acid soils acts as a fumigant effectively killing most soil nematodes (beneficial and parasitic), with opportunistic nematodes rapidly filling the biological vacuum generated. The plant parasitic nematode Pratylenchus penetrans and the wilt fungus Verticillium dahliae are similarily sensitive to the dominant VFA (acetic acid) in LSM. These pathogens are synergistic in damaging potato, thus, effective control of both with LSM increases the probability of disease control. Reducing soil ph with sulfuric acid alone can result in significant reductions in potato scab

5 3 if the soil ph is made permanently acid for the growing season. Sulfuric acid alone had no effect on verticillium wilt. There is no long-term disease control by sulfuric acid alone if the soil ph returns to control levels. Tuber yield was doubled after addition of acidified LSM compared to the control treatment. Tuber yield was either not affected or reduced after addition of sulfuric acid alone. Application of acidified LSM in the year prior to a potato crop was just as effective at reducing disease as when applied in the year of the crop. Application of LSM in a rotation year will be safer with respect to potential contamination of the potatoes with potential human pathogens from the manure. Application of acidified LSM 2 years in a row reduced disease and increased tuber yield more than a single application. The sulfuric acid and LSM were applied using a manure spreader showing that a farmer could use this technology. Whether use of acidified LSM is economical for control of soilborne diseases depends upon such things as: (1) the VFA content of the manure; (2) ph and buffering capacity of the soil which affects how much sulfuric acid is needed to lower the ph. It may not be economical to acidify soils with ph above 6.5; (3) How far the soil ph is to be lowered. Reduction to ph 5 takes less acid than reduction to ph 4.5; (4) How far is scab severity decreased, plant parasitic numbers reduced, and yield increased. The scab severity and yield combine to determine the marketable yield; (5) How long the disease control lasts. If a single application of acidified LSM provides reduced disease for more than one potato crop, the treatment becomes more economical; and (6) Cost of sulfuric acid. At the moment there is a cheap source of sulfuric acid from Ethyl Canada Ltd., Sarnia, ON.

6 4 BACKGROUND Liquid swine manure (LSM) was shown in a previous study to reduce the incidence of verticillium wilt (caused by the fungus Verticillium dahliae), potato scab (caused by the bacterium Streptomyces scabies), as well as populations of plant parasitic nematodes (Conn & Lazarovits 1999). This however, occurred at only one (site B) of several commercial potato farms tested. At site B, LSM reduced disease incidence for 3 years after a single application. At another location (site M) a few kilometres away, the same LSM had little effect. The resting structures of V. dahliae, which initiate the disease, were found to be killed at site B, but not at site M. This was our first indication that LSM can control soilborne plant pathogens and that its effectiveness maybe soil specific. The results of this study also contradicted the advice of most text books on potato production that suggest that fresh manures not be applied to soil because they can make disease such as potato scab worse. Our results showed reductions or no change in disease in all cases. We proceeded to investigate how LSM may reduce disease, the reasons for the soil specificity, and to use this information to optimize disease control by LSM. Four mechanisms by which LSM can control plant pathogens in soil were identified. The first mechanism is toxicity due to the presence of volatile fatty acids (VFAs) in LSM (Conn et al. 2005, Tenuta et al. 2002). The main acid present is acetic acid (vinegar) but other related acids likely play a role. Since acidsat high ph exist as salts, the volatile fatty acids are only active in low ph soils. We have shown that LSM can also kill V. dahliae through the generation of ammonia or nitrous acid also following degradation of the manure by the microorganisms in the soil (Conn et al. 2005). In soils with high ph, ammonium is converted into ammonia, a gas toxic to V. dahliae. Ammonia cannot form in sufficient quantities to eradicate pathogens at ph levels below 8. During nitrification ammonium is converted to nitrite. This chemcial change results in a lowering of soil ph and when the ph is reduced to below 5, nitrite forms nitrous acid, a molecule which is also highly toxic to V. dahliae and other plant pathogens. There is an other mechanism by which LSM reduces disease and this is the stimulation of the growth of certain fungi in soil that exclude or interfere with growth of plant pathogens (Lazarovits et al. 2002). We have not yet examined this mechanism in detail. By identifying the mechanisms by which LSM controls plant pathogens we can now use the information to predict where these products will have the greatest benefit to producers of high-value crops.

7 5 We also examined numerous soil properties for their role in regulating the efficacy of LSM to control pathogens. Soil ph was found to be the most important factor for determining whether LSM was toxic to V. dahliae (Conn & Lazarovits 2000). ph explains why LSM was only toxic at site B and not in the other soils since examined. Site B soil had a ph of about 5 whereas all others were above ph 6. However, we also found excellent pathogen kill in a soil from Florida that had a ph above 8. Here it was the ammonia that was active. Delayed kill of pathogens (after 6 weeks) by LSM was found in several soils that were initially at ph 6 but were at ph 5 at this time. Since VFAs only persist for a few days, the mode of action was shown to be related to nitrous acid formation. Soil moisture, which dilutes the active ingredients involved in the toxicity of LSM, had a significant impact on the rates required to have an impact on pathogens. The drier the soil the more toxic the manure. Activity was better in warmer soils than cold ones but the differences were not large. Soil texture and organic carbon content did not influence the toxicity of LSM. After determining that the VFAs in LSM kill V. dahliae microsclerotia, we wanted to determine the concentrations of VFAs in manures collected from differnt farms. Between the years 1996 and 2000 we collected LSM from a number of farm operations and stored these as frozen samples. These manures were tested for their toxicity to microsclerotia in site B soil of ph 5.0. We found that out of 20 manure samples, only about 50% contained sufficient VFAs to kill V. dahliae (Lazarovits et al. 2001). Manures with a concentration of about 100 mm total VFAs or greater were toxic to microsclerotia in the soil at ph 5.0. However, we did not have detailed information about the swine operations that these manures came from. In a detailed survey of 15 farm operations was carried out in order to determine why there was variable amounts of VFAs in LSM. The survey revealed that the concentration of VFAs was on average 5.5x greater in LSM obtained from finishing hog operations than that from sow operations. The diet of the pigs, the antibiotics used, and the manure storage conditions did not significantly effect the VFA content of the manures (Lazarovits et al. 2004). The higher VFA content of manures from finishing pig operations was attributed to the higher ratio of manure to water used to clean the barns (Lazarovits et al. 2004). In sow operations the animal densities tend to be four-times lower than is finishing barns and a larger quantity of water is used to clean sow barns. This information allow us to now predict which manures might be useful and possibly provide information as to how to maximise VFA content.

8 6 Combining the knowledge we generated, we initiated tests on formulations of LSM that had the characteristics needed to improve efficacy of disease control at reduced rates. The objective was to reduce the ph of LSM by addition of sulfuric acid (available in large quantities for a low price) so that it temporarily reduced the ph of the soil and converted more of the VFAs to the active form. An experiment in a commercial potato field (site G, near Palmer Rapids, ON) was set up in 2000 where ph-reduced LSM was used in a soil of ph 6.2 to bring the ph down to 5.2, which returned to that of the control after 1 week. This resulted in reduced potato scab disease (Lazarovits et al. 2001, 2002, 2003) in a field where non-formulated LSM would have no effect. In 2001, an experiment was set up at the London research centre with soil from site G. To this soil, sulfuric acid, LSM, ph-reduced LSM, and a VFA mixture matching the manure were added. The soil ph in all the acid treatments returned to that of the control treatment within a few weeks. The ph-reduced LSM and the VFA mixture killed V. dahliae and reduced disease severity (Lazarovits et al. 2002). Treatments where acid was added alone resulted in no disease reduction. Thus, by manipulation of soil ph, LSM can be made more effective in a broader range of soils. One of the objectives of the current study was to explore this further. Preliminary studies have also demonstrated that VFA content of LSM can be increased by manipulating nutrients and/or microorganisms in LSM during storage (Lazarovits et al. 2004). Addition of a carbon source also resulted in a reduction of LSM ph. Both these factors improve the disease control efficacy of LSM. This is being explored further in the current study. In most of the oral presentations made on the use of LSM for plant disease control, questions arose as to the safety of manure usage. Thus, the potential for use of LSM as a disease control product is directly dependent on not only its efficacy, but also on its perceived safety. A rudimentary aerobic thermophylic composter was built by for us on contract. This equipment allowed for air to be blown into the manure and upon addition of a small amount of carbon source bacterial activity raised the temperature to about 60 C. At these temperatures all human pathogens are known to be killed within minutes. Preliminary tests of LSM in this system indicated that all Escherichia coli in the manure were eliminated without any significant loss of nutrients (Lazarovits et al. 2002). The process is inexpensive and can be completed in 3-4 days. One of the objectives of the current study was to continue the investigation into aerobic thermophylic digestion of LSM using a continuous-

9 7 flow composter. OBJECTIVE The overall objective of this project is to formulate liquid swine manure (LSM) into a safe and effective control product for soilborne fungal, bacterial, and nematode plant pathogens. Specific objectives: 1) Test the efficiency of a continuous-flow composter for eradication of all potentially harmful microorganisms in manure. 2) Develop formulations of LSM from the liquid compost obtained after thermophylic treatment that controls diverse soilborne pathogens in various soils. 3) Demonstrate in large scale trials that formulated LSM derivatives can provide significant financial returns to producers of high-value crops such as potatoes. RESULTS AND DISCUSSION 1) Efficiency of a continuous-flow composter to pasteurize LSM A continuous-flow composter was built by Nuhn Industries and was tested at SCPFRC, London during the summer of 2004, and again in the summer and fall of 2005 at SCPFRC, London. The composter consisted of three 300 gal tanks in a vertical stack which were surrounded by insulation and covered in white siding (Fig. 1). There were openings between each tank for manure to pass. Inside the top two tanks there was 200 feet of a 2 inch plastic tubing in a coil. Manure entered this coil from the an inlet near the top of the rear side of the composter and exited into the bottom tank. The manure was pumped from holding tanks via a progressive cavity pump. Addition of manure to the bottom tank pushed the manure up through the tanks and exited from an outlet valve near the top of the front of the composter. The manure was collected in a manure spreader. The manure in the bottom tank was mixed by pumping the manure through a pipe out the front of the tank and then back in (Fig. 2). This pipe also contained a venturi to inject air into the composter. The ph and temperature of the manure passing through this pipe was continuously measured by probes inserted into a bi-pass pipe off the top of this pipe (Fig. 2). This information went to a controller (Fig. 3). The controller controlled two pumps (Fig. 3) which pumped sulfuric acid and molasses into

10 8 the composter. The acid was automatically pumped into the composter to keep the manure ph from going above 8. Molasses was used as a carbon source to balance the nitrogen and carbon in LSM. Addition of molasses also reduced the ph of the manure in the digester. Digestion of the manure by bacteria pushes up the ph and if the ph rises to 8.5-9, the digestion stops. The digestion also produces foam at times which was vented from the top of the tank along with the pasteurized manure. For research purposes, there were valves place on the composter where manure could be sampled from inside the middle or bottom of the coil or from inside the tanks. To continuously monitor the temperature of the manure at all these locations, temperature probes were placed in these locations and temperature recorded on a data logger. Figure 1. Continuous-flow composter for aerobic thermophylic digestion of liquid swine manure. Bacteria involved in the digestion of the manure generate heat and when the temperature of the manure rises above 60 C, potential animal and human pathogens are killed. Manure entered the digester from the storage tanks on the right and exited into the manure spreader on the left.

11 9 Figure 2. Continuous-flow composter. View of pump, pipes, and venturi for mixing and aerating the manure. Manure was pumped out from the bottom and then back in. A by-pass pipe on top contained temperature and ph probes for continuous measurement of temperature and ph. Figure 3. Continuous-flow composter. View of controller and pumps for automatic addition of acid and molasses. Information from the ph probe (Fig. 2) was used to add acid to keep the ph of the manure in the composter from rising above 8.

12 10 The theory behind this design was that the manure would be heated up inside the coil as it passed through the hot manure in the tanks. By the time it exited the coil the manure would be pasteurized. It would then feed the digestion reaction. From the preliminary experiments with a batch composter it was learned that addition of a carbon source to the manure was needed to balance the nitrogen and carbon in the manure so that the bacterial activity could increase the temperature from C to 65 C (Lazarovits et al. 2002). It was theorized that once the continuous-flow composter was fully operational, addition of fresh manure may provide all or nearly all the nutrients needed for the digestion because the bacteria only have to raise the temperature by a few degrees. This prototype composter was first tested in the summer of 2004, during which a number of minor modifications had to be made to the composter to make some of the component parts work properly. Once the problems were solved, there was not enough time to fully test the prototype before the weather became to cool. In 2005, this composter was further tested. The results showed that in continuous flow mode it was able to maintain temperatures of LSM (5500 L) at C for the 3-month period (July-Oct., 2005) (Fig. 4). Figure 5 shows the outside air temperature and LSM

13 11 temperature in the composter for a 6-day period in September. The technology is inexpensive and the equipment is simple to maintain as it has no moving parts other than two pumps; one that circulates the manure and introduces air into the liquid, and one that pumps acid or molasses into the liquid to maintain ph at the desired level. The material coming out of the tank had no detectable E. coli or Salmonella spp. and it retained all of the starting nutrients. In fact, often the nutrient value increased slightly as particulate material became solubilized. The liquid had no odour but if it was allowed to become anaerobic the odour returned. The liquid coming out of the composter is no longer manure as it has changed significantly in chemistry and microbial composition. It should be considered as a liquid fertilizer/compost. We believe this compost is a safe and effective fertilizer. We tested a number of variables that may affect the maintenance of temperatures above 60 C including the effect of ph, carbon addition, rate of flow of the manure, aeration, etc. Several key elements were identified as being problematic; the most important being the potential of plugging up the digester by sedimentation of solids. Therefore, a third prototype has been designed and agreement has been reached with Nuhn Industries for its construction for research purposes at the companies cost. This prototype will have continuous-flow in a downward direction such that the sediments will move with the manure. Furthermore, the tanks will be compartmentalized in order to make them more accessible for cleaning. We will double the aeration and increase the coils for heat exchange of the manure emptying into the chamber with manure exiting the chambers. This will allow for faster flow of manure through the composter. This tanker has been designed to fit into a barn of a swine producer. 2) Temperature and time needed to kill Escherichia coli in LSM To optimise the digestion process for killing E. coli and other potential animal and human pathogens in LSM, it is necessary to know the minimum temperature and time needed for the manure to remain in the digester. To find out this information, a laboratory experiment was set up in which E. coli was added to pre-heated (55, 60, 65, or 70 C) digested LSM and aliquots plated onto solid medium after various times to determine survival of E. coli. It was found that a minimum temperature of 60 C was needed to kill E. coli (Fig. 6). At 60 C, all the E. coli was killed within 15 min. At 70 C, the E. coli was almost instantly killed (Fig. 6). Thus, manure in the digester needs

14 12 to be at a temperature of at least 60 C and remain in the composter for at least 15 minutes to ensure kill of E. coli. To provide a margin of safety, the manure should likely be kept in the composter for at least 30 minutes.

15 13 3) Isolation, identification, and determination of optimal growth conditions of the thermophylic bacteria Aerobic thermophylic digestion of manure is the result of the growth of thermophylic bacteria which cause the temperature of the manure to rise. Determining the optimum growth conditions for these bacteria is necessary in order to maximise the temperature in the composter. Also, it is important to identify the thermophylic bacteria in the composter to make sure that they are not potential animal or human pathogens. A special LSM medium had to be developed in order to get the thermophylic bacteria to grow outside of the composter. Once this medium was developed, four main types of bacteria were isolated from digested LSM. Identification of these bacteria is underway. The first growth factor to be examined was ph. The bacteria were grown on solid manure medium (ph 7.0, 7.5, 8.0, 8.5, and 9.0) at 55 C for 4 days in order to find the optimum ph range for growth. The optimum ph range for all four bacteria was ph (Fig. 7). The bacteria grew slower at ph above or below this range. Thus, the ph of the manure inside the composter should be kept at ph 7.5 for optimal growth. While they grow equally well at ph 8, this ph is not desirable because some ammonia will be lost at ph 8, but very little at ph 7.5. The second growth factor examined was temperature. The bacteria were grown on solid manure medium (ph 7.5) at 55, 60, 65, or 70 C for 4 days. Two of the bacteria (R3-1 and R3-3) grew equally well at 55, 60, and 65 C but not at 70 C (Fig. 8). One bacterium (R4-1) grew best at 55 C and had no growth by 65 C (Fig. 8). Isolate R3-8 grew best at 65 C, although did not grow at 70 C (Fig. 8). These results demonstrate that some of the thermophylic

16 14 bacteria can survive a temperature of 65 C. This also means that the temperature of the composter cannot be allowed to reach 70 C because the thermophylic bacteria would die, shutting down the process.

17 15 4) Efficacy of LSM formulations to reduce potato scab Understanding the mechanism by which LSM reduces diseases allows for more informed usage and possibly for formulation to enhance its toxicity. Since the active ingredients, volatile fatty acids (VFAs), increase in toxicity as the ph is decreased, we tested the impact of adding sulfuric acid to soil and to the manure. Lowering the ph of LSM is necessary since it s normal range of ph is 6.8-8, and when it is added in large quantities it can raise the ph of some soils making the LSM less effective. A) Addition of acidified LSM to a commercial potato field (site L1-2004) An experiment was set up at a commercial potato farm near Aylmer, Ontario (referred to as site L1-2004) in the spring of 2004 to determine the efficacy, practicality, and financial return of using ph-reduced LSM to control potato scab under field conditions. Table 1 shows the treatments and the amount of acid and manure added. The target ph of the acid treatments was 4.3. Table 1. Amount of liquid swine manure (LSM) and sulfuric acid added to a commercial potato field (site L1-2004) in the spring of Potatoes were planted into this plot again in 2005 without further addition of manure or acid. Amount used (gal/ac) Treatment LSM 1 Sulfuric acid 2 Control none none Sulfuric acid none 290 LSM 5000 none Acidified LSM = The total VFA content of this LSM was 149 mm (acetic, 81 mm; propionic, 29 mm; isobutyric, 6.4 mm; n-valeric, 8.1 mm; isovaleric, 7.9 mm; and n-caproic, 17 mm). Amount used was 3% mass/mass soil which is equivalent to about 5000 gal/ac or 55,000 L/ha. 2 Sulfuric acid (98%) was added to the soil first followed by LSM. The acidified LSM treatment received 56 gal/ac more acid than the acid control treatment in order to prevent the LSM from raising soil ph. The amount of acid needed to bring the ph of the soil and the LSM down to the target of 4.3 was determined by titrating samples of the soil and LSM with acid before setting up the experiment. A ph of 4.3 was not always achieved in the field because of variability in soil ph across the plot and degree of mixing. Four replicate plots (30 x 12 ft) per treatment were set up in a randomized block design. For treatments requiring acid the acid was first spread over the soil surface. Application of LSM for the

18 16 acidified LSM treatments followed immediately. The plots were then cultivated to a depth of 6 inches. Application of the acid and LSM separately, rather than adding the acid to the LSM, avoided generation of foam which has been a major problem when acid is added directly to LSM. Potato tubers cv. Snowden were planted by the grower 2 weeks later. The farmer applied the usual fertilizer rates to all plots. The effect of manure on potato scab, soil ph, and yield were determined. Potatoes were planted in this plot again in 2005 without further addition of manure. The amount of disease and yield were determined. Both the sulfuric acid alone and acidified LSM treatments significantly reduced potato scab severity by about 8-fold compared to the control treatment in 2004 (Fig. 9A). This resulted in about an 8-fold increase in percent disease-free tubers for these treatments in 2004 (Fig. 9B). There was still significantly reduced disease in these treatments in 2005 (Fig. 9 D-F). LSM alone had no effect on scab. Reduction of scab by acidified LSM was expected, but we did not expect reduction by the addition of acid alone. The reduction observed, however, is attributed to the fact that the soil ph in this treatment remained highly acidic for both growing seasons (Fig. 10). While the soil ph did increase after day 0, by the end of the 2004 season it was still 1.6 ph units lower than the control. In earlier experiments addition of acid alone did not reduce scab severity but in those cases the soil ph returned to control levels within weeks of application (Lazarovits et al. 2002). Generally, scab severity increases as soil ph increases. Thus, since the acid treatment lowered soil ph for the whole growing season, this may explain the observed decrease in scab severity. We are following how long it takes the soil ph to return to control levels. By the end of the 2005 growing season, soil ph for all the treatments were between 4.9 and 5.4 (Fig. 10). The soil ph in a field where a potato crop is grown typically starts off high and decreases over the growing season by ph units. Thus, we think the optimum time to apply LSM is in the fall when the soil ph is naturally lower than in the spring.

19 17

20 18 Soil ph in the acidified LSM treatment also did not return to control levels (Fig. 10). Since acid alone reduced scab severity we were not able to differentiate from the contribution of the VFAs in the manure. We are now examining what happens to the disease incidence when the soil ph of the acid and acidified LSM treatments return to that of the control treatments. It is anticipated that scab severity will increase again for the acid treatment but not for the acidified LSM treatment. As well, the impact of these treatments on the S. scabies population in the soil is currently being carried out. This information will also help determine if these two treatments reduced scab by the same mechanism or not. None of the manure treatments affected tuber yield compared to the control treatment in either year (Fig. 11A and D). The acid treatment alone reduced yield in 2004 (Fig. 11A) likely due to phytotoxicity because of the low soil ph. The acidified LSM treatment significantly increased the marketable yield (0% surface scab) from 5 to 15 kg/plot while the LSM treatment increased it to 10 kg/plot in 2004 (Fig. 11B). This increase compared to the control was seen again in 2005 for these two treatments (Fig. 11E). The LSM treatment also doubled the marketable yield (up to 5% surface scab) in 2004 (Fig. 11C), and the acid and acidified LSM treatments resulted in a doubling of the yield in 2005 (Fig. 11F).

21 19

22 20 B) Addition of acidified LSM to soils from eight commercial potato fields in a microplot experiment Testing amendments in field plots is severely limited by the number of treatments that can be examined. In order to test a large number of treatments, we set up micro-plot experiments at the London research station. Drainage tiles (10 inch diameter, perforated) cut to 10 inch lengths are buried in soil to ground level. Soil is collected from commercial potato fields and brought to the station. All soil from each field is mixed to make it uniform with respect to soil chemistry and disease pressure. Various soil:amendment mixtures are then placed into the tiles (28 lbs per tile, six tiles per treatment in a randomized block design) and one potato tuber cv. Snowden planted in each tile 1 week after application of amendments. The soil was fertilized 1 week after planting with N/P/K at a rate of 230/170/170 lbs/ac, respectively. For the LSM treatments, the amount of N/P/K obtained from the manure was taken into account and the amount of inorganic fertilizer reduced accordingly so that the total fertilizer was about equal for all treatments. The effect of amendments on soilborne diseases and soil factors can then be determined. An experiment was set up in the spring of 2004 to duplicate the experiment set up at site L1 described in the previous section as well as test acidified LSM in soils from seven other commercial potato fields. The source of the soils and amounts of acid and manure added are shown in Table 2. The target ph for the acid treatments was 4.3. Potatoes cv. Snowden were planted 1 week after addition of the amendments. The effect of these treatments on potato scab, verticillium wilt, tuber yield, and soil ph was determined. Potatoes cv. Snowden were again planted into these treatments in 2005 and 2006 without further addition of amendments.

23 21 Table 2. Amount of liquid swine manure (LSM) and sulfuric acid added to soil from eight commercial potato fields in a micro-plot experiment to test the effect of acidified LSM on potato scab and verticillium wilt. Treatment (province Amount used (gal/ac) soil collected from) LSM 1 Sulfuric acid 2 Site B2 soil (ON) Control none none Sulfuric acid none 90 LSM 5000 none Acidified LSM = 146 Site GM soil (ON) Control none none Sulfuric acid none 26 LSM 5000 none Acidified LSM = 82 Site MS soil (PE) Control none none Sulfuric acid none 160 LSM 5000 none Acidified LSM = 216 Site BC soil (PE) Control none none Sulfuric acid none 260 LSM 5000 none Acidified LSM = 316 Site AF soil (NB) Control none none Sulfuric acid none 360 LSM 5000 none Acidified LSM = 416 Site BPF soil (PE) Control none none Sulfuric acid none 160 LSM 5000 none Acidified LSM = 216 Site L1 soil (ON) Control none none Sulfuric acid none 290 LSM 5000 none Acidified LSM = 346 Site HB soil (PE) Control none none Sulfuric acid none 290 LSM 5000 none

24 22 Acidified LSM = The total VFA content of this LSM was 149 mm (acetic, 81 mm; propionic, 29 mm; isobutyric, 6.4 mm; n-valeric, 8.1 mm; isovaleric, 7.9 mm; and n-caproic, 17 mm). Amount used was 3% mass/mass soil which is equivalent to about 5000 gal/ac or 55,000 L/ha. 2 Sulfuric acid (98%) was added to the soil first followed by LSM. Different amounts of acid were needed for each soil because of the different ph and buffering capacities of the soils. The acidified LSM treatment received 56 gal/ac more acid than the acid control treatment in order to prevent the LSM from raising soil ph. The amounts of acid needed to bring the ph of the eight soils and the LSM down to the target of 4.3 was determined by titrating samples of the soils and LSM with acid before setting up the experiment. Addition of acidified LSM reduced potato scab to near zero levels in 2004 in all eight soils tested (Figs. 12A-D, 13A-D). This decrease was statistically different from the control treatments for all soils but one (site AF, Fig. 13A). Sulfuric acid alone also reduced scab compared to the control in 2004 (Figs. 12A-D, 13A-D), but not as much as acidified LSM did. LSM alone reduced scab in the two soils with the lowest ph in 2004 (Fig. 12A, B). LSM either had no effect or slightly increased scab in the other soils (Figs. 12C, D, 13A-D). Thus, as expected, LSM was effective in the low ph soils and acidified LSM was effective in all the soils in As discussed in the previous section, a possible explanation for why sulfuric acid alone reduced scab severity was because the soil ph in this treatment did not return to that of the control treatments. While the soil ph did increase after day 0, by the end of 2004 it was still 0.4 to 1.4 ph units lower than the control treatments, depending on the soil (Table 3). In 2005, scab severity in the acidified LSM treatment was still less the control treatment in six soils (Figs. 12G, H, 13E-H). Only potatoes from three of these soils treated with sulfuric acid alone still had less scab than the control treatments (Figs. 12H, 13G, H). LSM alone treatments had the same amount of scab as the control treatments (Figs. 12E-H, 13E-H). Thus, in the second year after application of amendments, the acidified LSM treatment was better than sulfuric acid alone at reducing scab severity. This difference was not due to a difference in soil ph between these treatments. Soil ph for sulfuric acid and acidified LSM in the fall of 2005 were similar for each of the eight soils (Table 3). In the soils which still had reduced scab in the acidified LSM treatment compared to the control, soil ph was still unit lower than the control treatments (Table 3). In 2006, scab severity in the acidified LSM treatment was still half that of the control

25 23 treatment in five soils (Figs. 12K, L, 13I, K, L). Only potatoes from three of these soils treated with sulfuric acid alone still had less scab than the control treatments (Figs. 12L, 13I, L). LSM alone treatments had the same amount of scab as the control treatments (Figs. 12I-L, 13I-L). Thus, in the third year after application of amendments, the acidified LSM treatment was better than sulfuric acid alone at reducing scab severity. In the soils which still had reduced scab in the acidified LSM treatment compared to the control, soil ph was still units lower than the control treatments (Table 3).

26 24

27 25

28 Table 3. Effect on soil ph of adding liquid swine manure (LSM) and sulfuric acid to soils from eight commercial potato fields in a micro-plot experiment. Treatment (province soil collected from) Pretreatment Time zero Fall 2004 Soil ph Spring 2005 Fall 2005 Fall 2006 Site B2 soil (ON) Control nd Sulfuric acid nd LSM nd Acidified LSM nd Site GM soil (ON) Control Sulfuric acid LSM Acidified LSM Site MS soil (PE) Control Sulfuric acid LSM Acidified LSM Site BC soil (PE) Control Sulfuric acid LSM Acidified LSM Site AF soil (NB) Control Sulfuric acid LSM Acidified LSM Site BPF soil (PE) Control Sulfuric acid LSM Acidified LSM Site L1 soil (ON) Control Sulfuric acid LSM Acidified LSM Site HB soil (PE) Control Sulfuric acid LSM

29 27 Acidified LSM nd = no data The amount of colonization of the potato plants by V. dahliae was reduced by 60-90% in the acidified LSM treatment compared to the control for potatoes grown in four soils in 2004 (Figs. 14C, D, 15A, D), six soils in 2005 (Figs. 14G, H, 15E-H), and one soil in 2006 (Fig. 14K). Due to variability in the data these differences were only statistically significant for one soil in 2004 (Fig. 14D), three soils in 2005 (Figs. 14H, 15G, H), and one soil in 2006 (Fig. 14K). This reduction in verticillium wilt was not due to just the reduced soil ph because sulfuric acid alone had very little effect. Also, the effect of acidified LSM on V. dahliae was more pronounced in the year after application of the manure than it was in the year of application. Thus, like with scab, the acidified LSM treatment was better than sulfuric acid alone at reducing verticillium wilt. Addition of acidified LSM significantly increased tuber yield over that of the control for six out of the eight soils in 2004 (Figs. 16B-D, 17A, B, D). The increase ranged from 1.7 to 2.6-fold (average of 2-fold) over the control treatments. There was no effect on tuber yield in 2005 (Figs. 16E-H, 17E-H) or 2006 (Figs. 16I-L, 17I-L). LSM alone significantly increased yield over that of the control for two soils in 2004 (Fig. 16B, C) but not in any other year. Sulfuric acid alone had no effect on yield compared to the control treatments in any year (Figs. 16, 17). These results indicate that acidified LSM has great potential for use in a diverse number of soils with disease issues. Potato scab was reduced for three seasons in some soils after a single application of acidified LSM. It will be necessary to learn to add just the right quantity of acid as to not permanently lower the soil ph such that reduced yield results. In addition, the increased amount of acid has a cost factor associated with it. In cases where the ph with the LSM was reduced for a short period, the yields in potatoes exceeded those of the control in several instances. This was never seen with acid alone.

30 28

31 29

32 30

33 31

34 32 C) Addition of VFA-free LSM plus formic acid to soils from two commercial potato fields in a micro-plot experiment LSM from sow operations has little or no VFAs (Lazarovits et al. 2004) and thus is not useful as a control product for soilborne plant pathogens. This could be changed if this LSM was formulated to contain VFAs. One way to do this is to add one or more VFAs to the manure. Formic acid would be the most promising VFA for this purpose because it was shown to be 7-times more toxic to the plant pathogen, Verticillium dahlliae, than acetic acid, the major VFA in LSM (Tenuta et al. 2002). LSM does not naturally contain any appreciable amount of formic acid (Tenuta et al. 2002). Since formic acid is more toxic than the VFAs found in LSM, less would be needed which would make it more economical to use. Thus, an experiment was set up to determine if addition of formic acid to LSM could make an effective disease control product. Results from this experiment will also provide information as to whether LSM plus formic acid provides better long-term disease control than formic acid by itself. The experiment was set up in the spring of 2004 in micro-plots using soil from sites L1 and GM. Table 4 shows the treatments for this experiment and the amounts of manure, formic acid, and sulfuric acid added. The LSM used in this experiment had practically no VFAs. The target ph for the acid treatments was 4.3. Potatoes cv. Snowden were planted 1 week after addition of the amendments. The effect of these treatments on potato scab, verticillium wilt, tuber yield, and soil ph was determined. Potatoes cv. Snowden were again planted into these treatments in 2005 and 2006 without further addition of amendments. Table 4. Amount of volatile fatty acid (VFA)-free liquid swine manure (LSM), formic acid (FA), and sulfuric acid added to soil from two commercial potato fields in a micro-plot experiment. Amount used (gal/ac) Treatment LSM 1 FA 2 Sulfuric acid 3 Site L1 soil Control none none none Sulfuric acid none none 290 FA none 39 none Acidified FA none LSM 5000 none none Acidified LSM 5000 none = 316 LSM + FA none

35 Acidified LSM + FA = 316 Site GM soil Control none none none Sulfuric acid none none 26 FA none 39 none Acidified FA none LSM 5000 none none Acidified LSM 5000 none = 52 LSM + FA none Acidified LSM + FA = 52 1 This LSM contained almost no VFAs. Amount used was 3% mass/mass soil which is equivalent to about 5000 gal/ac or 55,000 L/ha. 2 Formic acid (85%) was added to LSM or to water before addition to soil. 3 Sulfuric acid (98%) was added to the soil first followed by LSM. Different amounts of acid were needed for each soil because of the different ph and buffering capacities of the soils. The acidified LSM treatment received 26 gal/ac more acid than the acid control treatment in order to prevent the LSM from raising soil ph. The amounts of acid needed to bring the ph of the two soils and the LSM down to the target of 4.3 was determined by titrating samples of the soils and LSM with acid before setting up the experiment. 33 The acidified LSM plus FA and the acidified FA treatments significantly reduced scab in site GM soil compared to the control in 2004 (Fig. 18A). No other treatments had any effect. In 2005 and 2006, these three treatments were not different from the control (Fig. 18B, C). Three treatments (FA, LSM, and LSM plus FA) had a scab severity twice that of the control in 2005 (Fig. 18B). Thus, in this soil adding sulfuric and formic acid to a VFA-free LSM resulted in reduced scab for 1 year while addition of LSM and/or FA resulted in higher scab in the second year. It is interesting that the ph of the soil in the four acidified treatments in the site GM soil was still 1 unit below the control treatment by the fall of 2005 (Table 5) but that this did not result in reduced scab. By 2006, the ph of the acidified treatments was back close to control levels (Table 5). In site L1 soil, all four acidified treatments reduced scab to near zero levels in 2004 (Fig. 18D) and levels remained greater than 75% lower than the control in 2005 (Fig. 18E). By 2006, only the acidified LSM and acidified LSM plus FA were still lower than the control (Fig. 18F). The reduced scab in three of these treatments (sulfuric acid, acidified LSM, and acidified LSM plus FA) may be partially attributed to the fact that the soil ph in these treatments was still units below the control by the fall of 2005 (Table 5). However, this was not true for the GM soil discussed above.

36 34 Also, the soil ph of the acidified FA treatment had returned to the control level by the fall of 2004 (Table 5) but had less scab than the control. This indicates that the reduced scab in 2005 seen in the acidifed FA treatment was due to something else other than suppressed soil ph.

37 Table 5. Effect on soil ph of adding volatile fatty acid (VFA)-free liquid swine manure (LSM), formic acid (FA), and sulfuric acid to soils from two commercial potato fields in a micro-plot experiment. Treatment (province soil collected from) Pretreatment Time zero Fall 2004 Soil ph Spring 2005 Fall 2005 Spring 2006 Fall 2006 Site L1 soil (ON) Control Sulfuric acid FA Acidified FA LSM Acidified LSM LSM + FA Acidified LSM + FA Site GM soil (ON) Control Sulfuric acid FA Acidified FA LSM Acidified LSM LSM + FA Acidified LSM + FA The amount of colonization of potatoes by V. dahliae was reduced by 60% in the acidified FA treatment compared to the control for the second year in site L1 soil (Fig. 19E). Verticillium wilt was also reduced by 50% in GM soil in 2004 for the acidified LSM and acidified LSM plus FA treatments (Fig. 19A), and in 2005 for the acidified LSM plus FA treatment (Fig. 19B), although these treatments were not statistically different from the control treatment. Thus, as with scab, the acidified LSM plus FA treatment was better than sulfuric acid alone. The only treatment to significantly increase tuber yield in 2004 was the acidified LSM treatment in both soils (Fig. 20A, D). In 2005, only the acidified LSM plus FA treatment in the GM soil had a higher yield than the control (Fig. 20B). In 2006, all treatments were the same as the controls except for the acidified FA in site L1 soil which was 50% lower than the control (Fig. 20C, F). Thus, LSM was the only treatment to increase yield.

38 36

39 37

40 38 D) Addition of acidified LSM to a commercial potato field (site L1-2005) A new experiment was set up at site L1 (referred to as site L1-2005) in the spring of 2005 to test the effect of acidified LSM (5000 gal/ac or 55,000 L/ha) on potato scab and tuber yield. This time, the soil ph was not reduced as much as in the experiment set up in The total VFA content of the LSM used in this experiment was 369 mm (acetic, 233 mm; propionic, 70 mm; isobutyric, 20 mm; n-valeric, 9.4 mm; isovaleric, 25 mm; and n-caproic, 9.7 mm). Sulfuric acid and LSM were applied using a liquid manure spreader. In the treatments receiving both sulfuric acid and LSM, the acid was applied first followed by LSM. Sulfuric acid (50%) was added at two rates, 300 and 580 gal/ac, to bring the soil ph down from 6.9 to 5.8 and 4.6, respectively. Two rates of acid, 350 and 425 gal/ac, were also used with the LSM to bring the soil ph down to 6.2 and 5.5, respectively. Thus, the treatments included a control, sulfuric acid treatments #1 and #2, LSM, acidified LSM treatments #1 and #2, and a non-amended treatment. The plan for this treatment was to apply acidified LSM in the fall of However, due to time constraints, this was not done. For the 2005 growing season, this treatment was essentially a second control treatment. Four replicate plots (50 x 18 ft) per treatment were set up in a randomized block design. There was a 30 ft border between the replicates. After the amendments were applied the plots were cultivated to a depth of 6 inches. Potato tubers cv. Snowden were planted by the grower 2 weeks later (six rows per plot). No extra fertilizer was applied to the LSM treatments. The other treatments received the typical amount of fertilizer. The effect of the treatments on potato scab, soil ph, and yield was determined. The middle two rows of each plot were dug and yield determined. 100 tubers were rated for scab severity. The rest of the plot was then dug and the tubers discarded. Two treatments, sulfuric acid (day 0 soil ph 4.6) and acidified LSM (day 0 soil ph 5.5) significantly reduced potato scab severity by 2.5-times compared to the control and non-amended treatments (Fig. 21A). The percent disease-free tubers and tubers with less than 5% scab were also increased with these two treatments (Fig. 21B, C). Enough of the VFAs in this acidified LSM treatment would have been in the toxic form to result in reduced disease. LSM alone and the acidified LSM (day 0 soil ph 6.2) did not reduce scab severity as expected because at ph above 6, the VFAs in the LSM would be 100% in the non-toxic form. The other acid treatment (day 0 soil ph 5.8) did not significantly reduce scab severity (Fig. 21A). Thus, adding acid alone to bring the soil

41 39 ph down to around 5.5 was not as effective as acid plus LSM. Only when enough acid is added to bring the ph down under 5, does acid alone reduce scab severity (Figs. 9A, 12, 13, and 18). Figure 22 shows the soil ph for the treatments over the growing season. Soil ph for the control and non-amended treatment declined over the growing season by 1.5 units (Fig. 22A) which is typical for soil in which a potato crop is grown. The soil phs for the acid treatments stayed more or less constant over the growing season (Fig. 22B). The soil phs for the LSM treatments increased over the first 2 weeks, then declined to lower ph levels by the end of the growing season (Fig. 22C). Thus, the ideal time to add acid and LSM would be in the fall immediately after the potato crop is harvested. This is because the soil ph is naturally lower than in the spring, which means less acid would be need to make the LSM effective. None of the treatments resulted in a significant increase in tuber yield (Fig. 21D-F). Since no additional fertilizer was applied to the LSM treatments, this demonstrates that this rate of LSM provided enough nutrients for the potatoes. This saved spending money on fertilizer. This site was not available in 2006 to plant potatoes into again so soil from all the treatments except the non-amended treatment was transferred to the micro-plots and potatoes planted without further treatment. Two treatments, sulfuric acid (day 0 soil ph 4.6) and acidified LSM (day 0 soil ph 5.5) still had reduced scab severity compared to the control (Fig. 23A). The other acid treatment (day 0 soil ph 5.8) had the same amount of scab as the control (Fig. 23A). Thus, like in 2005, use of acidified LSM and soil to ph 5.5 was effective against scab but sulfuric acid alone was not. Yield was about 50% higher in the two treatments with reduced scab in 2006, although it was not statistically significant (Fig. 23B).

42 40

43 41

44 42

45 43 5) Effect of single verses double application of acidified LSM in the crop verses rotation year on potato scab, verticillium wilt, soil ph, and yield Tests so far have focussed predominantly on the effect of spirng application of LSM to fields with high incidence of potato scab. It may be more beneficial to apply LSM in a rotation year. To test this, a micro-plot experiment was set up in the spring of 2004 in which LSM, acidified LSM, formic acid (FA), and acidified FA were added to soils from sites L1 and GM (Table 6). Single and double LSM applications were included. All of these treatments were duplicated and potatoes cv. Snowden planted into one of each duplicate treatment in One set of duplicate treatments had nothing planted into them in In 2005 and 2006, potatoes were planted into all the treatments. Table 6. Amount of liquid swine manure (LSM), formic acid (FA), and sulfuric acid added to soil from two commercial potato fields in a micro-plot experiment to compare LSM application in the potato crop verses rotation year. Amount used (gal/ac) Treatment LSM 1 FA 2 Sulfuric acid 3 Potatoes grown in 2004, 2005, and 2006 Site L1 soil Control none none none Sulfuric acid none none 290 FA none 39 none Acidified FA none LSM 5000 none none Acidified LSM 5000 none = 346 LSM* 5000 none none Acidified LSM** 5000 none = 346 Site GM soil Control none none none Sulfuric acid none none 26 FA none 39 none Acidified FA none LSM 5000 none none Acidified LSM 5000 none = 82 LSM* 5000 none none Acidified LSM** 5000 none = 82 No potatoes grown in 2004, potatoes grown in 2005 and 2006 Site L1 soil Control none none none Sulfuric acid none none 290 FA none 39 none

46 Acidified FA none LSM 5000 none none Acidified LSM 5000 none = 346 LSM* 5000 none none Acidified LSM** 5000 none = 346 Site GM soil Control none none none Sulfuric acid none none 26 FA none 39 none Acidified FA none LSM 5000 none none Acidified LSM 5000 none = 82 LSM* 5000 none none Acidified LSM** 5000 none = 82 1 The total VFA content of this LSM was 149 mm (acetic, 81 mm; propionic, 29 mm; isobutyric, 6.4 mm; n-valeric, 8.1 mm; isovaleric, 7.9 mm; and n-caproic, 17 mm). Amount used was 3% mass/mass soil which is equivalent to about 5000 gal/ac or 55,000 L/ha. 2 Formic acid (85%) was added to LSM or to water before addition to soil. 3 Sulfuric acid (98%) was added to the soil first followed by LSM. Different amounts of acid were needed for each soil because of the different ph and buffering capacities of the soils. The acidified LSM treatment received 56 gal/ac more acid than the acid control treatment in order to prevent the LSM from raising soil ph. The amounts of acid needed to bring the ph of the two soils and the LSM down to the target of 4.3 was determined by titrating samples of the soils and LSM with acid before setting up the experiment. * LSM (5000 gal/ac rate) was applied to this treatment in The total VFA content of this LSM was 261 mm (acetic, 162 mm; propionic, 54 mm; isobutyric, 15 mm; n-valeric, 6.2 mm; isovaleric, 16 mm; and n-caproic, 7.8 mm). ** LSM (5000 gal/ac rate) was also applied to this treatment in There was no need to add acidified LSM because the soil ph was still low from the 2004 acidified LSM treatment. 44 All four acidified treatments reduced scab severity from a rating of 2 to near zero levels in site L1 soil in 2004 (Fig. 24A). The amount of scab for all the treatments was very similar for 2005 (Fig. 24B). Also, the amount of scab in 2005 for the treatments that did not have potatoes in 2004 (Fig. 24C) was almost identical to those that did have potatoes (Fig. 24B). A second application of LSM in 2005 did not have any effect on scab because scab was still low from the 2004 application (Fig. 24B, C). In 2006, all the acidified treatments except for sulfuric acid alone were still less than half that of the controls (Fig 24D, E). In site GM soil, the acidified LSM treatments caused the largest decrease (75%) in scab severity in 2004 (Fig. 25A). In 2005, the acidified LSM plus LSM treatment that had potatoes in both

47 45 years still had the lowest scab rating (Fig. 25B). This treatment and sulfuric acid alone had the lowest scab levels for the treatments that did not receive potatoes in 2004 (Fig. 25C). In 2006, only the acidified LSM plus LSM treatment still had lower scab severity than the control for the treatment that received potatoes in all 3 years (Fig. 25D). Only the acidified FA treatment was less than the control for the treatments that did not receive potatoes in 2004 (Fig. 25E). The initial soil ph of all the duplicate treatments for both soils were very similar but the fall 2004 values were higher for the treatments that did not have potatoes (Table 7). Soil ph for site L1 soil was 0.1 to 0.8 units higher and for site GM it was 0.4 to 1.1 units higher. Soil phs of the acidified treatments for both soils had returned to control levels by the fall of 2006 (Table 7). There was no reduction of V. dahliae infection in the potato plants by any treatments in site L1 soil in 2004 (Fig. 26A) In 2005, there was a 30-40% reduction for the acidified treatments compared to the control treatment for treatments with potatoes in both years (Fig. 26B) and potatoes only in 2005 (Fig. 26C). There was no effect in 2006 (Fig. 26 D, E). For site GM soil, there was no statistical difference in V. dahliae infection between treatments in any of the 3 years (Fig. 27). However, the LSM plus LSM treatment reduced infection by 75% in years 2004 and 2005 where two crops of potatoes were grown (Fig. 27A, B). The acidified LSM treatment reduced infection the most compared to the control treatment in the treatments with no potatoes in 2004 (Fig. 27C). The acidified LSM plus LSM treatment increased tuber yield by % compared to the control treatment for both soils and years 2004 and 2005 (Figs. 28, 29A-C) except in 2004 in L1 soil (Fig. 28A). The single application of acidifed LSM also increased yield by 130% compared to the control in site GM soil in 2004 (Fig. 29A) and in the 2005 treatment that did not have potatoes in 2004 (Fig. 29C). The acidified LSM treatment and LSM plus LSM treatment increased yield by 100% in both soils in the treatments without potatoes in 2004 (Figs. 28C, 29C). None of the treatments had significantly higher yields than the control treatments for either soil in 2006 (Figs. 28D, E, 29D, E). One treatment, acidified FA, had a lower yield than the control in site L1 soil in 2006 (Fig. 28D). Thus, applying LSM in the year of potatoes or the year prior to potatoes did not result in much difference in the amount of disease or tuber yield in this experiment. Applying LSM in the season prior to potatoes appears to be as effective as applying in the same year as potatoes. This

48 experiment also showed that application of acidified LSM 2 years in a row was better than a single application at reducing scab severity and increasing tuber yield. 46

49 47

50 Table 7. Effect on soil ph of adding liquid swine manure (LSM), formic acid (FA), and sulfuric acid added to soil from two commercial potato fields in a micro-plot experiment to compare LSM application in the potato crop verses rotation year. Treatment (province soil collected from) Pretreatment Time zero Fall 2004 Soil ph Spring 2005 Fall 2005 Spring 2006 Fall 2006 Potatoes grown in 2004, 2005, and 2006 Site L1 soil (ON) Control Sulfuric acid FA Acidified FA LSM Acidified LSM LSM* ** Acidified LSM* ** Site GM soil (ON) Control Sulfuric acid FA Acidified FA LSM Acidified LSM LSM* ** Acidified LSM* ** No potatoes grown in 2004, potatoes grown in 2005 and 2006 Site L1 soil (ON) Control Sulfuric acid FA Acidified FA LSM Acidified LSM LSM* ** Acidified LSM* ** Site GM soil (ON) Control Sulfuric acid FA Acidified FA LSM Acidified LSM LSM* ** Acidified LSM* **

51 * Treatments received a second application of LSM in ** Soil ph after application of LSM in

52 50

53 51

54 52

55 53 6) Efficacy of acidified LSM to reduce numbers of plant parasitic nematodes A) Addition of acidified LSM to soil from two commercial potato fields in a micro-plot experiment in 2004 Soils from sites BPF and MS used in the micro-plot experiment described on page 19 contained high numbers of nematodes. Thus, these soils were selected to examine the effect of LSM and acidified LSM on plant parasitic nematodes. The treatments are shown in Table 8. Table 8. Amount of liquid swine manure (LSM) and sulfuric acid added to soil from two commercial potato fields in a micro-plot experiment in 2004 to test the effect of acidified LSM on plant parasitic nematodes. Treatment (province Amount used (gal/ac) soil collected from) LSM 1 Sulfuric acid 2 Site BPF soil (PE) Control none none Sulfuric acid none 160 LSM 5000 none Acidified LSM = 216 Site MS soil (PE) Control none none Sulfuric acid none 160 LSM 5000 none Acidified LSM = The total VFA content of this LSM was 149 mm (acetic, 81 mm; propionic, 29 mm; isobutyric, 6.4 mm; n-valeric, 8.1 mm; isovaleric, 7.9 mm; and n-caproic, 17 mm). Amount used was 3% mass/mass soil which is equivalent to about 5000 gal/ac or 55,000 L/ha. 2 Sulfuric acid (98%) was added to the soil first followed by LSM. Different amounts of acid were needed for each soil because of the different ph and buffering capacities of the soils. The acidified LSM treatment received 56 gal/ac more acid than the acid control treatment in order to prevent the LSM from raising soil ph. The amounts of acid needed to bring the ph of the eight soils and the LSM down to the target of 4.3 was determined by titrating samples of the soils and LSM with acid before setting up the experiment. The starting ph of these soils was 6.6 and 6.1 for BPF and MS, respectively (Table 9). The target soil ph of the acidified treatments was 4.3 and the actual values obtained are shown in Table 9. By the end of the growing season the ph of the soils in these treatments had increased by ph units but were still lower than the control treatments by ph units (Table 9).

56 54 Table 9. Effect on soil ph of adding liquid swine manure (LSM) and sulfuric acid to soils from two commercial potato fields in a micro-plot experiment in Treatment (province Soil ph soil collected from) Pre-treatment Time zero Fall 2004 Site BPF soil (PE) Control Sulfuric acid LSM Acidified LSM Site MS soil (PE) Control Sulfuric acid LSM Acidified LSM Samples of these soils were taken in the fall of 2004 and analysed for total and plant parasitic nematodes. Only acidified LSM significantly reduced the number of plant parasitic nematodes compared to the control treatment (Table 10). This was true for Pratylenchus, Tylenchorhynchus, and Criconema spp. Figure 30 shows a Pratylenchus spp. nematode isolated from the sulfuric acid treatment in the MS soil. Total plant parasitic nematode numbers were reduced 19 and 17-fold for the BPF and MS soils, respectively. The sulfuric acid treatment caused a 2-fold increase in parasitic nematodes compared to the control treatment for the BPF soil (Table 10). The acidified LSM also reduced the number of non-plant parasitic nematodes in the BPF soil by 2-fold but had no effect on the non-plant parasitic nematode numbers in the MS soil (Table 10). LSM had no effect on the total or plant parasitic nematode numbers (Table 10). Figure 30. A juvenile of the root lesion nematode (Pratylenchus spp.) extracted from the sulfuric acid treatment in the MS soil.

57 Table 10. Effect of liquid swine manure (LSM) and acidified LSM added to soils from two commercial potato fields (sites BPF and MS) on nematode populations. Treatment 1 Total 2 parasitic 3 Plant Site BPF soil Nematodes (#/kg soil) Non-plant parasitic Pratylenchus spp. Tylenchorhynchus spp. 55 Criconema spp. Control 15,500 a 4 3,880 b 11,600 a 2,660 b 911 b 286 a Sulfuric acid 13,300 ab 7,000 a 6,300 b 4,160 a 2,340 a 440 a LSM 14,000 ab 2,820 b 11,200 a 1,600 b 963 b 180 a Acidified LSM 7,500 b 200 c 7,300 b 154 c 0 c 0 a Site MS soil Control 16,800 a 5,580 a 11,200 a 3,660 a 1,480 a 120 a Sulfuric acid 16,100 a 6,020 a 10,100 a 4,050 a 2,070 a 100 a LSM 15,700 ab 3,620 a 12,100 a 2,480 a 1,010 a 32 a Acidified LSM 10,300 b 323 b 10,000 a 323 b 0 b 0 a 1 These soils were selected to determine the effect of these treatments on nematodes because they contained high nematode populations. Soil samples were taken at the end of the 2004 growing season. 2 Nematodes were extracted using Cobb sieving-decanting and then isolated from soil particles and debris using the Sugar-flotation method by the Soil Ecology Laboratory at the University of Manitoba, MB. 3 Included lesion nematode (Pratylenchus penetrans and P. crenatus based on molecular sequencing examination), stunt nematode (two Tylenchorhynchus spp. were observed though not identified to species), and ring nematode (Criconema spp.). 4 Means (n = 6) for each soil and column followed by a different letter are significantly different (P < 0.05, Student-Newman-Keuls Method). The nematode community resulting from the acidified manure treatment was dominated by opportunistic bacterivore (Rhabditidae) and fungivore nematodes (Aphelenchus and Aphelencoides spp.). This is characteristic of soil that has been exposed to a fumigant generally lethal to soil organisms. These nematodes are rapid growing and are able to colonize a vacuum and absence of other nematodes indicative of more complete sol food web. Thus, these results show that acidified LSM can dramatically reduce the numbers of plant parasitic nematodes to levels below threshold levels for causation of plant disease. Also, since the sulfuric acid treatment did not reduce the number of plant parasitic nematodes, the reduction seen

58 56 with the acidified LSM most likely was due to the presence of VFAs and not because the soil ph was lowered. An abundance of conidia of the fungus Alternaria were observed in the acidified LSM treatment compared to the control treatment. Many of the conidia were characteristic of A. solani (Fig. 31) which causes early blight disease of potatoes. If this fungus is A. solani, then this means that addition of acidified LSM to some soils could possibly make early blight disease worse. Figure 31. A conidium of the fungus Alternaria isolated from the soil of the acidified LSM treatment.