IPM and Biological Control of Meloidogyne chitwoodi and the Colorado Potato Beetle

2.1 Field Experiments
Field plots in Prosser, WA in 2006 and 2007 were set up with 5 replications in a randomized complete block design to control for the uneven distribution of M. chitwoodi in the field. Plots with soil type sandy loam were 8 ft x 20 ft with 12 inch inter-row spacing and three rows per plot, the middle row being buffered by two border rows. Lengthwise, plots were separated by Xft of planted potatoes without treatment between plots. Mustard seed meal of Brassica carinata was applied at 9.75 pounds per plot or 1 Ton/acre on May 30, 2006 and May 1st, 2007 followed by 2 inches of irrigation.  Potatoes were planted fifteen days post application on June 15th, 2006 and May 15th, 2007. On the same day of potato planting, entomopathogenic nematodes Steinernema feltiae strain 75 and S. riobrave strain 355 (BeckerUnderwood) were applied at 3 billion IJ/acre with 2.3 L of water per plot using a backpack sprayer (brand?). EPN were sprayed after 5pm to avoid UV and heat damage to the EPN. The experiment was a 2 x 3 multifactorial design examining main effects of EPN, mustard meal, and interaction between EPN and mustard meal biocontrol treatments. Treatments were mustard meal, S. feltiae, S. riobrave, mustard meal + S. feltiae, mustard meal + S. riobrave, chemical Mocap control, and no treatment control.

2.1a EPN Monitoring
The presence of EPN from the first treatment application and prior to the second application was evaluated for residual EPN presence in the field using standard Galleria mellonella wax worms (Rainbow mealworms) on August 4th, 2006.  For each plot, one 118 ml perforated bucket (perforated to allow for water drainage and ventilation) were filled with soil from each plot and 10 waxworms were placed in each bucket, capped and left in the field for 48 hours to ascertain residual presence of EPN. Waxworms were collected August 7th, 2006 and placed into a petri dish and observed for discoloration and dissected for EPN infectivity 4-5 days later. Due to the negative results from 2006, EPN monitoring was not considered useful for 2007 and therefore was not conducted a second time.
2.1b Field Soil Sampling for Plant Parasitic Nematodes
Prior to the first treatment applications, soil samples were taken with a field core sampler (brand) on May 30th, 2006 and May 1st, 2007. Three samples to the depth of 12 inches were taken from each plot and combined and put into cold storage (4ºC).  Within 1-2 weeks, total nematodes were extracted from 250 cc of the mixed soil using the sugar centrifugation technique (Bird and Bird et al date) and analyzed for number and identity of plant parasitic nematodes. Prior to the second mid-season EPN applications, on August 8th, 2006 and July 6th, 2007 soil samples were taken again in the same method to assess mid-season plant parasitic nematode populations. Lastly, soil samples were taken at harvest on October 30, 2006 and October 15, 2007 to assess end of season plant parasitic nematode populations and to assess the change in nematode populations after the second mid-season EPN application.

2.1d Colorado potato beetle field infection
For each plot, two 118 ml perforated buckets (perforated on the bottom and top to allow for water drainage and ventilation) were filled with soil from each plot and EPN were sprayed in the field at 2 billion ij/acre using a backpack sprayer with 2.3L water per plot. After spraying the soil including the soil filled buckets, 10 CPB 4th instar larvae were placed in each bucket and capped and left in the field for 48 hours. Buckets were taken from the field and CPB larvae were put inside individual Petri dishes to observe for death and EPN infection. The larvae were dissected under a dissecting microscope and recorded as infected if EPN were visibly present.

2.1d Potato Infection Ratings
Potato plots were harvested on October 30, 2006 and October 15, 2007. Middle rows of each plot were dug with a potato harvester and bagged into burlap sacks and put into cold storage until processed. Potato plots were weighed and sorted for culls and #1 and #2 spuds. Twenty potato tubers from each plot were randomly chosen from each plot and assessed for percent total M. chitwoodi infection of tubers and infection index of tubers. To assess percent infection, the numbers of tubers with visible knot symptoms on the outer tuber skin were counted as infected. To assess infection index, the twenty potato tubers were peeled and inspected under magnifying lense with light for presence of females in the potato cortex. The number of females were counted and assigned an infection rating using the infection index scale of 0 = 0 females, 1 = 1-3, 2 = 4-5, 3 = 6-9, 4 = 10-50, 5 = 100+, 6 = 200+.

2.2 Greenhouse Experiments
Greenhouse trials were conducted in Pullman, WA in 2006 and 2007 in a completely randomized design with ten replications and repeated three times. Four inch square pots with 500 grams of fumigated 2:1 sand: soil mixture was used in the experiment. Greenhouse was kept at 16:8 (dark:light) photoperiod and an average temperature of 26.6ºC.  M. chitwoodi eggs were inoculated into each pot at a rate of 2 eggs/g soil at the same time as the treatments. Mustard seed meal was applied by mixing 500 grams of soil with 1 Ton/acre or 2.61 grams per pot of Brassica carinata ‘Biofence’ mustard seed meal. The seed meal was thoroughly mixed with the soil in a plastic bag prior to putting soil into the pots. S. feltiae and S. riobrave were applied at 3 billion ij/acre or 7652.28 IJ/pot by creating a small hole in the soil and pipetting in the EPN. Treatment combinations of mustard meal and EPN were done by first mixing the seed meal with the soil, potting the soil then adding in M. chitwoodi and the respective EPN species, S. feltiae or S. riobrave. Ten days after treatment, five week old tomato seedlings (Lycopersicum esculentum variety Rutgers? were transplanted into treated pots. CPB larvae infection was tested in pots during the second EPN application (2 billion IJ/acre or 5101.53 IJ/pot) by placing 5 larvae in mesh bags and burying them in the soil prior to EPN application. EPN were applied in the same manner as the first application using a pipette to inoculate into a small indentation in the soil. CPB larvae were observed for infection or mortality and dissected for EPN reproduction, the percent of CPB larvae infected by S. feltiae or S. riobrave were recorded. Roots of tomato seedlings were acid-fuchsin stained and number of M. chitwoodi females in roots was recorded after two months.