I started objectives 1 and 2 over the summer of 2016, including collecting soils from farmer-collaborators, and setting up first year of the manure experiment and collecting predator and plant damage data for objective 2. For objective 1, I set up 2 experiments to examine the effects of microbial community from different soils on herbivore performance. Through this experiment, we determined that sterilizing soils using steam treatments changed the nutrient content of the soil. We were unable to determine if effects on plant growth and herbivore performance were due to nutrient differences or the microbial community in the soil. Because of this, in 2017, we adjusted methods to better sterilize soils by using a filtration technique to separate microbial communities. When we applied this method to manipulate microbial communities, we found no differences between soils with and without microbial communities, and decided to focus on Objective 2 for the 2017 field season. Objective 2 was started in June 2016, earlier than anticipated to have another year of data. In 2016 and 2017 we collected predator community, early season pest damage data, herbivore performance data on pre-reproductive corn and silage yield data. We have also adjusted objective 2 by adding a cover crop treatment in addition to the manure treatment in a factorial design to isolate the effects of manure and cover crops on organic matter accumulation and soil health on insect pests/predator communities. This cover crop was planted in October 2016, and thus was one of the treatments in 2017. Overall, we have found that manure increased susceptibility to herbivores, contrary to our expectations, and are currently running a follow up greenhouse experiment to test the strength of this effect.
This proposed research is fundamental for understanding how soil fertility practices influence insect crop damage, and will provide insight about how management practices meant to improve soil quality can also alter risks from plant-feeding insects. Manure is used on Pennsylvania farms as a sustainable alternative to inorganic fertilizers, and understanding its influence is vital for integrated production systems typical of Pennsylvania. Manure tends to contain more phosphorus and micronutrients than inorganic fertilizers, possibly altering plant resistance to insect herbivores. These research will help clarify whether and why plants with manure treatments have more herbivores. By investigating protein/carbohydrate content, phytohormone induction, and elemental nutrient composition of experimental plants, my research will provide new mechanistic insights on how manure affects herbivores. If manure continues to increase plant susceptibility to herbivores compared to inorganic fertilizers, producers using manure may need to take further precautions against leaf-feeding insects.
OBJ 1 Methods:
Soils were collected at 4 paired farms in OH and PA in March and April of 2016. We collected soils from a farm practicing long term no-till and cover cropping and a nearby conventional farm on the same soil type. Each field was sampled at 15 points along a zigzag transect using a shovel. Four soils were collected from OH on paired farms on March 25, 2016. Soil DB1 was collected from a conventionally managed field. Soil DB2 was from a field that had been in no-till management for 45 years and was planted with a diverse cover crop mixture including rye, pearl millet, winter peas, cowpeas, sunn hemp, radishes, Ethiopian cabbage, rape and flax. Soil DB3 had been in no-till management for 3 years and was also planted with the diverse cover mixture. Soil DB4 was had been in no-till for 3 years and was planted with a rye cover crop. In PA we collected soils from a pair of farms on April 25, 2016. The ‘soil health’ soil, JH1, had been in no till for 40 to 50 years and were planted with a rye cover crop. Soil JH2 was from a neighboring conventional farm which was tilled and had not been planted with a cover crop. Soils were sieved with a 1 cm sieve and stored at 4˚C.
Soils were sent to the Pennsylvania State University Agricultural Analytical Service Laboratory (PSU AASL) for nutrient and organic matter testing. Soils were also sent to the Wood’s End Laboratory for soil health testing. We also tested the organic matter, aggregate stability, soil burst respiration, soil protein content, active carbon, nutrients and soil texture at the Cornell Soil Health Testing Laboratory.
Feeding assay with steam sterilized soils
The goal of this project was to determine how microbial communities in the soil influence plant susceptibility to herbivores. To manipulate the presence of microbes in each soil, we steam sterilized soils and compared plants grown in sterilized soil to plants grown in un-sterilized soils. To sterilize each soil, they were placed in perforated plastic bags and steamed for an hour, allowed to cool and sterilized again 24h later to account for microbes that may have survived the initial sterilization. All soils were then mixed 1:1 with play sand. Corn seeds (TA466) were surface sterilized for 30 mins using a 10% bleach solution and planted into these soils. Plants were allowed to grow for 5 weeks. Once the majority of the plants reached the 4-leaf stage, 4th instar black cutworms (Agrotis ipsilon) were allowed to feed in 5 cm plastic clip cages that were moved every two days to ensure a continuous supply of food. These larvae were allowed to feed for 6 days before they were weighed. Leaf damaged was estimated by scanning leaves and counting the number of 5x5mm squares were consumed. This experiment was first performed July 2016 with all collected soils, and was repeated September 2016 with only JH1 and JH2. Plant size was measured in September by measuring height to the last leaf collar from the soil surface.
Feeding assay with soils slurries
Steam sterilization of the soils increased phosphorus availability and decreased organic matter content of the soils (PSU AASL test). To isolate the effect of microbes from changes in nutrient content, we performed the same feeding assay with JH1 and JH2, but used microbial slurries to manipulate the microbial community. Microbes were extracted from the soil by shaking sieved soil (2mm sieve) for 1 hour with half concentration Hoagland solution made using sterile DI water. These were allowed to settle for 1 hour and the liquid was centrifuged for 15 mins at 4000 rpm to separate soil particles from the microbial slurry. To account for the increased nutrient content from the microbial slurry for the no-microbe treatment, the slurry was passed through a 22µm vacuum filter. These filtered and unfiltered slurries were applied to steam sterilized soils. Sterilized corn seeds were planted into these soils in January 2017. Slurries were applied every week for 5 weeks. The feeding assay was performed as before.
OBJ 2 Methods:
2016: The goal of objective 2 was to compare plant susceptibility under two fertilizer treatments: manure and inorganic fertilizer. We applied manure before planting at a rate of 13.1 ton/ acre on 5/27/2016 (47.9 % solids, total N = 19.76 lb/ton, ammonium 3.35 lb/ton, phosphate 11.81 lb/ton, potash 11.86 lb/ton). Inorganic fertilizer (10:30:10 NPK starter at a rate of 174 lbs/acre) was applied at planting. These treatments were applied on eight 50ft by 100ft blocks in a one-acre field. Corn was planted 5/31/16, with a 30,000 seeds/acre population on 30 in rows (Master’s Choice, variety MC5250).
Corn was harvested for silage on 9/19/2016. Rows 9 and 10 were harvested to measure yield by weighing the end product. This was subsampled for moisture content. Subsamples (2/plot) were dried in drying oven at 55C for 1 week and weighed to determine moisture content.
2017: A cover crop of winter wheat was planted 10/19/2016, treated with Chlorpyrifos methyl and Cyfuthrin insecticides (Strocides) in split-split plots in the previously manure or inorganic fertilizer plots. There were 4 plots/treatment, and 16 plots total. As in 2016, 13.1 tons of manure was applied 5/23/2017. We planted the same variety as 2016 (Master’s Choice, Variety MC5250), using a 6-row planter. Population was 30,000. Manure was collected from the ground for testing at the soil analytics lab (6/9/2017). Analysis found that manure on the ground after 2.5 weeks was 65.9 % solids, 45.40 lb/ton total nitrogen, 0.39lb/ton ammonium n, organic N 45.01lb/ton, potash 21.42 lb/ton. Starter fertilizers were applied at a rate of 150 lbs/acre of MicroEssentials 12-40 – 0 10S 1Zn in the inorganic treatment. To balance manure and inorganic treatments,421 lbs/acre of 19:19:19 NPK were applied by hand on 7/12/2017. We weighed out the amount of fertilizer required in 2 rows/plot and applied it evenly to the roots of the plants in the row. Plot was harvested for silage on 10/13/2017, and final weight was calculated as in 2016.
2017: Nine samples were taken/plot using an oakfield soil corer (2cm in diameter) to a depth of 20cm on 5/10/2017. These were submitted to the PSU AASL for standard analysis and organic matter content.
Cover crop assessment and weed biomass
2017: We collected cover crop and weed above-ground biomass in 2 0.25 m2 randomly placed quadrats from each plot and took photographs to assess percent cover using imageJ. Weed and cover-crop biomass was assessed separately. Samples were dried in a drying oven at 60˚C for 4 days, and weighed.
Pitfall Traps and Sentinel prey:
2016: Pitfall traps were 1-quart deli containers, dug into the soil surface so they were level with the soil, filled with ½ cup of 50% propylene glycol, and covered by a plastic plate that served as a rain cover. Pitfall traps were collected after 48 hours. Pitfall traps were opened once a month on 6/20/2016, 7/13/2016, 8/15/2016, 9/18/2016. We identified important arthropod predators and decomposers including spiders, millipedes, centipedes and harvestmen, ants and beetles were counted from pitfall traps samples. Carabid beetles were identified to species where possible.
2017: Two pitfall traps/plot in rows 5 and 10 were opened 7/24/2017-7/26/2017, 8/26/2017-8/28/2017 and 9/25/2017-9/27/2017. Additionally, predation rates were assessed using sentinel prey wax worms (Galleria mellonella). Two wax worms in each of 3 cages were put out in each plot on 7/20, 8/23, and 9/25 and assessed for predation after 24 hours.
2016: Damaged was assessed in 5 ft sections along in 4 locations in each plot (row 4 @ 70ft, row 8 @ 79ft, row 12 @ 28, row 16 @ 18ft) 3.5 weeks after planting (6/24/2017). Damage was categorized on an ordinal scale where 0 was no damage, 1 was ~1-10% leaf area gone, 2 was 10-25% damage, 3 was 25% – 60% damage and 4 was >60% leaf area gone. Identity of damage was also determined (particularly slug damage). Stand was assessed as well at this time by counting the number of plants in row six of each plot.
2017: Damaged was assessed in 5 ft sections along in 2 random locations in each plot 3 weeks after planting when the plants reached the V4 stage (6/29/2017). Damage was categorized as it was in 2016. Damage was primarily due to slugs, grasshoppers, black cutworm, grasshoppers and Japanese beetles. Cereal leaf beetles were also present. Stand was assessed at this time by counting the number of plants in 5ft sections.
2016: In late July, leaves were collected from the field and the most recent fully expanded lea was collected and wrapped around a dampened cotton ball. The V stage of each plant was recorded for each plant as well. Leaves were placed in a 3 oz diet cup. 3rd – 4th instar FAW larvae were weighed and allowed to feed on leaves for 5 days before final weight was measured. There was a high rate of mortality, probably due to high humidity inside the cup. Data were analyzed using ANCOVA with initial weight and developmental stage of the plant as covariates.
2017: Plant tissue was collected on 8/11/17 from 7 plants in rows 5 and 15 in each plot, rolled into a paper bag, and brought back to the lab. Between 1.1-1.2 g of this tissue was excised (around the center vein), rolled into a 1 oz diet cup containing a plaster base (~2mL of base to absorb moisture). To this tissue, 1 FAW caterpillar between 3rd and 4th instar was weighed and added to each diet cup with plant tissue. Plants 1-3 in each plot were started on 8/11/17, and Plants 4-7 from each plot were started 24 h later 8/12/17. Cups were covered with a perforated lid. A few mL of water were added to each diet cup plaster base to maintain humidity for the leaf tissue. After 5 days, caterpillars were weighed. Data were analyzed using ANCOVA with initial weight as a covariate.
Results of soil nutrient and health tests are reported in Table 1. Phosphorus content of soil was much higher in PA soils (JH1 and JH2) compared to OH soils. Solvita and Cornell soil health tests rank soils in almost the same way, and seem to be very closely related to the organic matter content of the soil.
Feeding assay with steam sterilized soils
The conventional soil DB1 did not support plant growth, and data are not included in any analyses. OH no-till soils DB2-DB4 had equivalent caterpillar growth (Fig 1; F=0.19, P=0.83) and none were affected by steam sterilization (F=0.94, P=0.34).
We analyzed both July and September experiments of PA soils, JH1 and JH2, together since there was no effect of date alone on BCW final weight (F=0.02, P=0.88). We found that steam sterilizing the “soil health” soil (JH1) increased BCW performance, while steam sterilizing the “conventional” soil (JH2) decreased BCW performance (Fig 2; interaction between soil and steaming: F=10.25, P=0.003). We hypothesize that some difference in the microbial communities in the soil health and conventional soils were responsible for this change in caterpillar performance.
In addition to the potential role for microbes in caterpillar performance, we observed a strong effect of phosphorus on BCW performance (Fig 3; Adj R2=0.62, F2,42=37.09, P<0.001). Phosphorus was the best predictor variable in model selection, with initial larval weight as a covariate. This is particularly problematic because steam sterilizing soils consistently increased the phosphorus content of the soils.
We also found a strong effect of plant height on final caterpillar weight, when initial larval weight is included as covariate (Fig 4; Adj R2=0.54, F2,13=9.667, P=0.003). This suggests an important link between the health of the plant and herbivore performance. Herbivore growth can be limited by plant nutritional quality and many herbivores benefit from fast growing plants (Price 1991).
Feeding assay with soils slurries
Because of the confounding effects of nutrients and microbial content of the soils, we manipulated the soil microbial communities by re-introducing microbes from a slurry to steam sterilized soils. This method allowed us to maintain a consistent nutritional content of the soil but change the microbial community. In contrast to our previous experiments, we found that herbivore performance was not affected by the presence of microbes from the slurry (Fig 5; F=0.30, P=0.58), nor did we see any differences in caterpillar performance on these soils (F=1.41, P=0.24). These results suggest that either the microbial community in the slurry did not colonize the soil and roots of their treatments, or the effects we saw in the previous experiments were caused by changes in the nutrient content of the soils were not due to microbes.
OBJ 2 Results
Assessed predator activity using sentinel waxwork caterpillars 1/month July-Sept 2017. Predation increased over the season (Fig 6; F=7.46, P<0.001), but there was no effect of fertilizer treatments (F = 0.29, P=0.59) or cover cropping (F=1.19, P=0.28).
Predation was extremely high in the later half of the season regardless of treatment (most plots had 100% predation), suggesting that predator populations are active. Manure is thought to increase alternative prey to support predator populations by increasing detritivore populations (Purvis and Curry 1984; Halaj and Wise, 2002). We have found that even inorganic soils with lower decomposing cover can support high predation rates.
Damage assessment and feeding assay:
In 2016 we measured early season damage and found no difference between manure and inorganic treatments (Figure 7; Z=-1.55, P=0.12). In 2017, when we measured early season damage, we found that manure plants had more damage than inorganic plants (Z=2.80, P<0.001), and a marginal interaction between fertilizers and cover crops (Z=1.80, P=0.07).
We found similar results when we measured caterpillar performance in the lab. In 2016 we found that herbivores performed marginally better on manure-fertilized plants than inorganically fertilized ones (Figure 8: t=1.99, P=0.06). In 2017, we found no differences in caterpillar performance between manure and inorganic fertilizer treatments with and without cover crops (F=0.004, P=0.95).
The few studies that have compared manure and inorganic fertilizers have found that inorganic fertilizers often have higher pest abundances compared to manure treated plots. In the seven studies published in the last 30 years on manure and inorganic fertilizers which measured pest abundances, 7 out of 18 site-years had significantly higher pest abundances in inorganic plots compared to manure plots. Only 1 of the 18 site-years had significantly higher pest abundances in manure plots (Culliney and Pimentel 1986, Eigenbrode and Pimentel 1988, Costello and Altieri 1995, Kanost et al. 2004, Alyokhin et al. 2005, Garratt et al. 2010, Duchovskiene et al. 2012). However, nutrition, especially nitrogen is thought to influence herbivore performance strongly (Butler et al. 2012). In 2016, our manure treatments and inorganic fertilizer treatments were approximately equivalent. The manure treatment had 21 lbs/ac available N, 35 lbs/ac P2O5, and 36 lbs/ac K2O, while the inorganic fertilizer treatment had 17 lbs/ac N, 52 lbs ac P2O5 and 17 lbs/ac K2O. The increase in FAW performance on manure plants relative to inorganic fertilized plants may be due to a higher level of available N when applied, and more slow release N throughout the season that improved plant nutrition for herbivores. In 2017, manure plants had higher nutrition early in the season before we side-dressed with inorganic fertilizer to balance nutrients between treatments. Manured soils had 91 lbs/ac available N, 144 lbs/ac P2O5, and 87.8 lbs/ac K2O, while inorganic plots were fertilized with 18 lbs/ac N, 60 lbs/ac P2O5, and 0 lbs/ac K2O. In this case, early season damage was higher in manure plots than inorganic plots. Once we side-dressed with 80 lbs of 19:19:19 inorganic fertilizer to address this inequality, we found no differences in fall armyworm performance on excised leaves.
Alternatively, 2017 was a heavy slug year in PA. Slugs are particularly damaging to young corn plants and often shelter in areas with higher organic cover. Manure plots may have experienced higher damage in 2017 because of increased slug activity in these plots.
2016: Measured yield – manure had lower yields than inorganic, likely due to lower emergence early in season in manure plots (Figure 4; t=-2.22, P=0.07).
2017: Measured yield and found no effect of fertilizer (F=0.0, P=0.99) or cover crop (F=0.26, P=0.56) although there was a marginal interaction between cover crops and fertilizer (F=3.73, P=0.08).
The yield was likely different in manure and inorganic treatments in 2016 due to inconsistent planting into high manure residue in the manure plots. Seeds were missing from patches of rows in the manure plots when we investigated immediately after seedling emergence.
We did not find yield differences in 2017, suggesting that the higher early season insect damage in manure plots did not have a lasting effect on yields.