Evaluating the Effect of Potato Leafhopper Feeding on Biological Nitrogen Fixation in Alfalfa

Objective 1: Measure the effect of PLH feeding on nitrogen fixation and soil nitrogen uptake in a field setting.

To measure the effect of PLH feeding on nitrogen fixation, I conducted a field experiment using caged alfalfa plants with and without PLH. Comparing plants with and without PLH identifies the specific effect PLH feeding has on nitrogen fixation. To determine whether PLH disrupts nitrogen fixation, I measured natural nitrogen isotope ratios (15-N/14-N) of alfalfa tissue to determine the source of nitrogen (14-N atmospheric vs. 15-N soil). The 14-N isotope accounts for approximately 99% of atmospheric nitrogen, whereas 15-N is derived primarily from organic material. Looking at 15-N/14-N ratios reveals if plants predominantly receive nitrogen from the atmosphere or not. For another treatment, I amended soils with 15-N fertilizer, which discerned if alfalfa adjusts its nitrogen sources from atmosphere to soil when disturbed by PLH feeding.

In addition to using isotope ratios to answer my question, I also used two types of ‘Saranac’ alfalfa: one is effective (normal type) and the other is ineffective (non-nitrogen fixing). PLH feeding affects numerous physiological processes in alfalfa, and ineffective alfalfa allowed me to determine if PLH feeding alters nitrogen assimilation in ways other than disrupting nitrogen fixation. Ineffective alfalfa still forms root nodules but these nodules are non-functional, which is evident by their coloration. Functional nodules are pink (due to the presence of leghemoglobin) and non-functional nodules are grey. Preliminary tests compared two effective and four ineffective types of ‘Saranac’ alfalfa and found comparable germination rates in the selected effective and ineffective types. Ineffective alfalfa served as a control in my experiment to explore if PLH feeding disrupts nitrogen fixation.

My field study was conducted at one experimental site over one field season. The alfalfa for this study was planted in August, 2017, at the Western Maryland Research and Education Center in Keedysville, MD. All treatments were replicated four times and arranged in a randomized complete block split plot design. Each trial (harvest) consisted of four main- and two subplot treatments. Main plots consisted of: 1) Effective alfalfa supplemented with 15-N, 2) Effective alfalfa without 15-N, 3) Ineffective alfalfa supplemented with 15-N, and 4) Ineffective alfalfa without 15-N. Subplot treatments included cages with PLH or cages without PLH. Fine mesh cages (2.5 m x 2.5 m) containing PLH receive economic threshold levels of PLH based on plant height at 14 days after harvest. There were a total of 16 main plots, each 6 m × 6 m, and two subplots per main plot.

A spring harvest of the entire field occurred in mid-May, 2018. Following this harvest, I flagged subplots and used spray paint on the soil surface around flags to mark subplot locations. I then used an organic insecticide to remove any PLH from subplots. After establishing subplots, I applied 15-N at a rate of 2.4 kg/ha to designated subplots. 15-N can persist for extensive periods of time in soil and should remain prevalent throughout my study. I then erected field cages at the flagged subplot locations. 14 days after harvest, I aspirated PLH into designated cages. To collect PLH for aspiration, I used a D-Vac on a small strip of alfalfa, which I maintained throughout the growing season at Keedysville.

After 35 days, cages were removed and I dug up 5 alfalfa plants at 15 cm deep in each plot to measure N components of roots, crowns, and shoots in the lab. I then used a Carter mower to harvest the area under each cage (2.5 m x 2.5 m). Harvest samples were used to determine forage yield. After completing my measurements, I mowed down the entire field and then used an organic insecticide on subplots and again erected cages. Aspiration of PLH into designated cages occurred 14 days after harvest. In the lab, a subsample of the harvest sample was dried at 60 Degrees Celsius for 2 days. I ground subsamples using a Cryomill (Retsch Mill) available at the University of Maryland Entomology Department. After I ground and weighed subsamples, I determined natural nitrogen isotope ratios (15-N/14-N). To do this, I took ground subsamples to the University of Maryland Geology Department for natural nitrogen isotope analysis using a Micromass/Elementar Isoprime mass spectrometer. I completed multiple harvests during the 2018 growing season, and I repeated the described procedure for each harvest. From these data, I used ANOVA to compare yield, nitrogen yields, and relative proportion of 15-N/14-N across all treatment combinations for each harvest. I analyzed each harvest individually due to the repeated measures from each experimental unit. Overall, I anticipated any negative effects induced by PLH feeding would compound over time, resulting in alfalfa with continually reduced nitrogen fixation throughout the growing season.

Objective 2: Compare the response of resistant and susceptible alfalfa cultivars to PLH injury with regard to nitrogen fixation and soil nitrogen uptake.

To learn if resistant alfalfa is a viable management tool in combating nitrogen losses from PLH feeding, I conducted a greenhouse experiment to compare nitrogen uptake in resistant and susceptible alfalfa. Seeds of a leafhopper-susceptible variety and a leafhopper-resistant variety were grown in standard potting mixture and allowed to germinate and develop for two weeks. At this early stage of development, nodules did not form and any nitrogen present in the potting mixture did not confound the study. After two weeks, seedlings were removed from the potting mixture. Seedling roots were washed in a Sinorhizobium (nitrogen fixing bacteria) inoculum and then three seedlings were transplanted into 10 cm ceramic pots filled with sand. At the conclusion of the experiment, sand facilitated separating root tissue from growth media. Pots were watered daily through a hydroponics set up, pausing watering once a week to add nitrogen-free Hoagland’s solution. Using this solution forces the plants to use the atmosphere instead of the soil as their source of nitrogen. After nine weeks of growth, the plants were cut back to 2.5 cm of stubble, simulating harvest in the field, and initiating experimental manipulation.

All treatments were replicated twelve times and arranged in a randomized complete block design along two greenhouse benches. Treatments represent all possible combinations of three factors: 1) susceptible or resistant variety, 2) sand amended with 15-N or not, and 3) PLH present or absent on plants. For PLH treatment, pots were covered with plastic cylindrical cages at 21-28 days after cutting. Half of the cages received 10 PLH and the other half did not. This time period represents when nitrogen fixation is most vulnerable to PLH injury. Additionally, I amended pots with 15-N (16 mg of 15-N labeled potassium nitrate diluted in 50 mL of water). For pots that did not receive 15-N amendments, I added 16 mg of potassium chloride diluted in 50 mL of water to balance the amount of potassium received by all plants (chloride is not a required nutrient and would not impact alfalfa growth). 35 days after cutting, plants will be separated from sand and divided into shoot, crown, and root portions. Tissues were dried in an oven at 60°C for two days, ground, and subject to nitrogen isotope analysis as previously described. ANOVA was used to compare the response of nitrogen fixation in resistant and susceptible alfalfa under pest pressure.