Sampling methods for: Plant Tillers | Seeds | Urine | Grain | Hay or Forage
HOW TO SAMPLE PLANT TILLERS
When to sample: The endophyte does not necessarily grow when the plant is growing. It grows best from late spring until late fall. Therefore, sampling is best from mid-April until January in the Deep South; the first of May until December in the mid-South, mid-West, and West coast; the first of July until the first of October in the Intermountain West and Northeast.
Method 1: A sample consists of tillers (stems) of plants that have been cut with a razor blade or sharp knife at the soil surface (Note: it is very important to cut the tiller at the soil surface!). Avoid taking stems that have seed heads on them, but do not take small or immature tillers either. Tillers with stems having a thickness of 1/8 inch or larger work best. Take about 10-20 more tillers than is necessary for the test you desire to make sure a good working sample can be obtained at the laboratory. IT IS IMPORTANT to make sure your take your tiller samples while walking back and forth across your field so you get roughly the same number of tillers from all sections of the field. Measure up about 4" from the base of the stem and cut the remaining plant tissue distant from the stem base. Save the stem bases but discard the tissue containing the leaves. Place the stem pieces into a plastic zip lock bag. Put a damp (not wet) paper towel in the bag to prevent drying of the tissues. Mark your name, address, telephone number, e-mail address, and field identification on the zip lock bag with a permanent marker (make sure to note the field identification for your records and put it in a safe place). Place the plastic bag containing the sample in a cooler with a little ice while sampling other fields.
Sample your fields on a Sunday through Tuesday. Ship your samples the same day or day after you remove them from the fields. Pack you samples in a box with a block of blue ice and ship the box overnight express to Agrinostics Ltd. Co. (address will be provided when order is placed). Keep your box refrigerated until it is posted. This assures that your samples will not sit in a hot post office room or truck where they will begin to decay.
The endophyte does not live indefinitely within the seed. Research has demonstrated time and again the endophyte lives longest when the seed is stored under cool dry conditions. Leaving seed in the back of pick-up trucks or in cars will accelerate the rate at which the endophyte dies. Seed stored under barn conditions is also ill-advised since heat and humidity are often a problem. Therefore, good seed storage is the first step in getting a good sample of seed otherwise the seed sample will not be indicative of what the endophyte level was in the seed following harvest, at the time of sale, or at planting. Seed grow-out analysis is the only way to determine viable endophyte status of your seed. However, if you want to confirm seed to be endophyte-free, storage conditions are not as critical.
Method 2: If you desire to obtain endophyte data for a commercial seed lot, it is advisable to obtain a certified seed sample from your State Crop Inspection Service or Certified Seed Program to submit to Agrinostics. Place the seed into a box with a blue-ice block and ship overnight express to Agrinostics Ltd. Co. (express mail address will be provided when order is placed). Seed being verified for endophyte free does not have to be shipped overnight.
If you wish to obtain information concerning the status of the seed you purchased or planted, the sampling procedure is rather simple. For seed you purchased, use a seed probe and take a sample from the middle of each bag. Bulk the seed you sampled and place it into a zip-lock sandwich bag. Identify the bag with your name and address (email address if you have one), variety, seed company and seed lot identification number from the seed label. Keep seed samples from bags with different company names and seed lot numbers separate. Do not bulk seed samples from different sources, or seeds that have been stored differently into the same seed sample. Place the seed into a box with a block of blue ice and ship overnight express to Agrinostics Ltd. Co. (express mail address will provided when order is placed). Seed being verified for endophyte free does not have to be shipped overnight.
If you wish to obtain endophyte information about the seed you are planting, sample seed from each bag as you dump it into your planting equipment, bulk the sample, label it and send it to Agrinostics Ltd. Co. as stated in the previous paragraph.
Sampling urine from a large animal can be dangerous and tricky. DO NOT attempt to collect urine unless you are a seasoned animal handler and are familiar with the animal from which you are collecting the sample.
Method 3. It only required several milliliters (one fluid ounce) to get a sufficient quantity for alkaloid and creatinine determinations. Having good animal handling facilities is important because the animal must be immobilized in many cases. For cattle, we have found bunching them into a crowding alley forces them to focus on the individual immediately in front, and in back of them. When crowded they do not pay as much attention to animal handlers, thus, providing you with an opportunity to gather a sample with relative ease. Cattle often urinate after being moved about. So, gathering the animals up and putting them into a crowding alley usually is sufficient to elicit urine flow. Affix a cup or other capturing device on the end of a stick and hold it to capture the urine. DO NOT stick your hand or arm under or behind the animal to collect the urine. If the animal does not urinate immediately, simply wait until they do. Place the samples on ice as they are gathered and freeze the samples prior to shipping. Ship the samples frozen (with freezer blocks) in an insulated shipping container (Styrofoam is best) via overnight express to Agrinostics Ltd. Co.
Method 4. Obtaining an accurate analysis for Fusarium in grain is dependent upon whether you obtain a representative sample of the grain. The process is relatively easy for small lots of grain (small plots, stored samples, plant breeding populations). Mix the grain in a rotary mixer, by hand, or by inverting the grain numerous times in a large bag. Retrieve 10-15 grams of seed and keep in a separate vessel (disposable centrifuge tubes works well).
If you are sampling from a larger amount of grain (load on a truck,
grain elevator, malting room, etc.) simply take samples from multiple
locations using a sampling probe. Be sure to sample deep into the grain
as well as on the surface. Combine the samples into a clean 5 gallon
plastic bucket and mix thoroughly. Retrieve 10-15 grams of seed and
keep it in a separate vessel (disposable centrifuge tubes or screw
cap specimen cups work well). Place all samples into a sturdy box (one
that will not break during shipment) and send through regular surface
mail or parcel service. IT IS NOT NECESSARY to ship grain samples via
Method 5. Sampling hay or fresh forage for alkaloid analysis is more about getting a representative sample than it is about getting volume.
HAY can be sampled using a hay coring device which is inserted into bales of a hay lot. Select several bales from which to sample. Get approximately 20 core samples and composite them. Place the cores onto a sheet of newspaper and let it air dry in a cool dry environment. NOTE: Hay must be ground through a 1-mm screen in a Udy or Cyclone type mill prior to submission for analysis. This permits the maximum amount of alkaloids to be extracted from the plant tissue. Put ground hay into a plastic vial or bag, seal and label the bag so you will be able to identify the sample, and submit the sample to Agrinostics. The alkaloids are stable in dry tissue, so it is not necessary to ship the samples overnight.
FRESH FORAGE preparation depends upon how the forage is harvested. If the forage is harvested using plot equipment, sample the forage by mixing the harvested tissue, take a 200-300 g subsample and put it into a cloth or paper bag, freeze the tissue, and dry the tissue using a freeze-drier. NOTE: Oven-drying the tissue will destroy between 50-75% of the alkaloids. Do not prepare samples for alkaloid analysis by drying them in an oven. Dry fresh forage must be ground through a 1-mm screen in a Udy or Cyclone type mill prior to submission for analysis. This permits the maximum amount of alkaloids to be extracted from the plant tissue. Put ground tissue into a plastic vial or bag, seal and label the bag so you will be able to identify the sample, and submit the sample to Agrinostics. The alkaloids are stable in dry tissue, so it is not necessary to ship the samples overnight.
FRESH FORAGE can also be sampled using the hand-grab method. Hand-grabbing tends to produce a truer measure of livestock nutrient intake than clipping because the hand-grab method more closely mimics the apprehensal, or gathering, abilities of grazing livestock. To perform the hand-grab method on standing forage, randomly select a handful of forage and wrap fingers tightly around the upper third portion. Make a swift pull and place the entire sample into the container. Move to another location within the pasture and repeat. Make separate samples where obvious differences in soil and forage structure occur within each pasture. Samples should be evenly distributed across the pasture or area. As time passes and/or forage is removed, forage quality will change. Mix the harvested tissue, take a 200-300 g subsample and put it into a cloth or paper bag, freeze the tissue, and dry the tissue using a freeze-dryer. NOTE: Oven-drying the tissue will destroy between 50-75% of the alkaloids. Do not prepare samples for alkaloids analysis by drying them in an oven. Dry fresh forage must be ground through a 1-mm screen in a Udy or Cyclone type mill prior to submission for analysis. This permits the maximum amount of alkaloids to be extracted from the plant tissue. Put ground tissue into a plastic vial or bag, seal and label the bag so you will be able to identify the sample, and submit the sample to Agrinostics. The alkaloids are stable in dry tissue, so it is not necessary to ship the samples overnight.