TITLE: USDA LTAR Common Experiment measurement: Aboveground biomass
AUTHORS: Brook Wilke, Lori J. Abendroth, Stacey VanderWulp

[DESCRIPTION]
The most important ecosystem service from agriculture is the provision of food, fiber, feed, and fuel. These outputs from the system are almost always a function of the amount of biomass accumulated above the soil surface (except with root crops). Measuring the aboveground biomass is necessary for determining the amount of plant material allocated to vegetative and reproductive organs. The amount of biomass allocated to these organs will vary to some degree each year due to interactions among the genetics, environment, and stressors. Measuring the vegetative-to-reproductive biomass ratio across treatments, landscapes, and years is an important metric and this ratio is termed the “harvest index.” The harvest index provides insight into improving management and cost efficiency in response to positive and negative impacts incurred from local (e.g., soil quality, pest and disease pressure, and weather), technological (e.g., genomic enhancement, precision inputs, and remote sensing/AI), or systematic (e.g., market, policy, and climate) sources. The amount of biomass remaining in the field is additionally important for determining the amount of C, N, P, K, S, and other nutrients returned to the soil. These data, paired with nutrient export and off-site losses, enable the construction of nutrient balances critical in evaluating tradeoffs among ecosystem services provided by agricultural landscapes.

This protocol describes the methods used to collect aboveground biomass which includes everything that:
Stays in the field: such as stem, stalk, branches, leaves, grain enclosures (such as pod shells and husk leaves), and grain support structures (such as cob and ear shank), and
Leaves the field: such as grain, hay, fiber, and plant material for bioenergy production.

[STEPS]
SECTION: Overview
1. This protocol describes methods for collecting whole plant samples taken by hand from random or predetermined sampling areas in plots and fields. These samples constitute the collection of all plant material (including non-crop species). Following the collection process, the individual will then separate the biomass into two categories: staying versus leaving the field. These samples provide the data and material for biomass, plant diversity, and nutrient analyses. 

There are four protocols that are dependent on the physical aboveground biomass samples collected in this protocol; these are the first three bulleted items. The fourth protocol in the bulleted list is as an additional protocol to carry out in tandem with this protocol; see Step 13 below.  

USDA LTAR Common Experiment measurement: Concentration of carbon and nitrogen in aboveground biomass (Cavigelli and Strickland, 2024)
USDA LTAR Common Experiment measurement: Concentration of phosphorus, potassium, and sulfur in aboveground biomass (Kovar and Fortuna, 2024)
USDA LTAR Common Experiment measurement: Obtaining quality metrics in forage aboveground biomass (Cassida and Collins, 2024)
USDA LTAR Common Experiment measurement: Cropland plant diversity (Wilke, 2024)
SECTION: Type of plant
2. There are important considerations to account for based on the type and timing of the plant material (crop) being sampled.  
SECTION: Type of plant
3. Annual grains

These grains include maize, soybean, wheat, rice, and grain sorghum. Collect biomass during maximum vegetative biomass and maximum reproductive biomass. The crop may not reach maximum vegetative and reproductive at the same time or may not maintain the vegetative structures to totality throughout the reproductive developmental stages. Therefore, it may be necessary to sample not just once but twice a season. Determine whether your crop of interest has "leaf drop" before maximum reproductive development, such as with indeterminate soybean. If yes, additional documentation may be needed on your method. Two approaches are typically used when collecting aboveground biomass from crops with leaf drop:
SECTION: Type of plant
3.1. Collect samples right before leaf drop occurs and remove the grain by threshing. Use all non-grain components for the maximum vegetative biomass component. The grain can be discarded because only the vegetative biomass is needed. The grain will be collected when sampling occurs again at maximum reproductive biomass (typically referred to as physiological maturity). 
SECTION: Type of plant
3.2. Collect whole-plant samples slightly before or during harvest and collect leaves that fell on the ground within the sampling area. It is likely that some leaves are from neighboring plants with this approach. Do not collect leaves from crops that were grown prior to the current crop of interest. This combined sample of standing biomass plus leaves on the ground make up the entirety of the aboveground biomass sample. 
SECTION: Type of plant
4. Root crops

These crops include potatoes and sugar beet. Although this protocol is for aboveground biomass, a similar approach should be utilized for belowground biomass when it is the marketable, economic component. Collect biomass during maximum vegetative biomass and maximum reproductive biomass. 
SECTION: Type of plant
5. Weeds 

Collect weed biomass (if present) during aboveground biomass sampling. Use the same sampling area used for collecting crop biomass but collect weed biomass in a separate bag for weight and analysis. Weed biomass is valuable for aboveground net primary productivity (ANPP) calculations and cropland plant diversity, as described in other protocols. 
SECTION: Type of plant
6. Forages

If the crop has multiple cuttings, site personnel need to collect biomass before each cutting, process, analyze, and then sum the values for a total per calendar year. 
SECTION: Type of plant
7. Biofuel/energy crops

Same as forages except only one cutting typically occurs in the fall. 
SECTION: Type of plant
8. Fiber crops

This category includes cotton and hemp. It is likely that separation of the fiber components (such as cotton bolls) will be needed from the rest of the plant. 
SECTION: Type of plant
9. Non-cash crops, such as cover crops

Collect biomass at 1 to 5 days before termination in the spring. If the cover crop is not terminated but simply has the cash crop planted into the green stand, wait until the maximum biomass of the cover crop is reached before sampling. If the crop typically experiences “winter kill,” sampling should occur in the fall before the first hard freeze.
SECTION: Type of plant
10. Prairie and conservation plantings

Collect biomass once at physiological maturity of the dominant plant community, typically in late summer or early fall. If possible, collect twice per year, aligning with maximum growth for cool-season plants in the spring and warm-season plants in the fall. 
SECTION: Type of plant
11. Intercropping

Collect biomass at maximum vegetative and reproductive biomass based on the crops grown which will require multiple samplings throughout the season. If sampling crops together, plant and seed separation will be necessary in-field or during processing.  
SECTION: Sample collection and processing
12. The suggested area (referred to as “quadrat”) for sampling is 1 m2, but the dimensions of the sampling quadrat may vary with treatment and plot size. For crops planted in 76 cm (30”) rows, the ideal quadrat size is 1.5 m × 0.65 m (4’ 11” × 2’ 2”). For crops planted in narrow rows, drilled, or broadcast, quadrats equal to 2.0 m × 0.5 m (6’ 7” × 1’ 8”) work well. Record the quadrat dimensions for subsequent area-based calculations. Quadrats are oriented with the long side parallel to the direction of crop planting and allow for the inclusion of row and inter-row plant communities in the sampling area. 
SECTION: Sample collection and processing
13. Determine the location for a quadrat based on random or preselected stations in each treatment replicate. The number of quadrats will depend on the experiment based on the scale of the plot or field and how best to account for spatial variability. After placing the quadrat, count and record the number of individual plants or reproductive stems within that area. All plants rooted within the boundary of a sampling quadrat are hand-clipped at ground level (or just above the crown of perennial grasses). Remove all standing plant material from within the quadrat. If any plant material from the intended crop is dead and lying on the soil surface (termed “surface litter”), collect it and add it to the bagged sample. However, if the surface litter is heavily soiled or compromised do not add it to the sample due to the presence of minerals that will impact nutrient analyses later conducted on the sample. Also, do not collect surface litter if it is from past seasons and unassociated with the current crop.

 

SECTION: Sample collection and processing
14. Separate plant samples into vegetative and reproductive components (or "fractions") before the processing steps below. In mature crops, grain removal is fairly easy, which allows your tissue and grain samples to be distinct before drying. However, in non-mature crops (such as before leaf drop), it may be easier to separate the grain following drying. 
SECTION: Sample collection and processing
15. Depending on oven space, it may be necessary to dry a subsample instead of whole plants. 
SECTION: Sample collection and processing
15.1. If subsampling, chop the vegetative components and reproductive components separately. Chop each sample into pieces a few inches in size and mix fully in a large pail/can (Figure 7). Collect a series of handfuls randomly from the pail/can to fill a one (1) gallon container or paper bag. This specimen is your subsample. Put it in the dryer. Collect the fresh weight of the whole sample and the subsample, as these weights are needed later for Calculation 1a (see below). 
SECTION: Sample collection and processing
15.2. If oven space is not a limitation, chopping is unnecessary. If grain threshing occurs after drying, briefly re-dry tissue and grain to remove ambie moisture gained during the threshing process.  
SECTION: Sample collection and processing
15.3. If considerable weed seed is in the sample or two crops were grown together in an intercropping scenario, separate the seed for each plant type. 
SECTION: Sample collection and processing
16. Dry the plant and grain samples at 60°C or lower in a forced air oven (Figure 11). Temperatures higher than 60°C will degrade sample quality, which could negatively impact nutrient analyses performed later. Depending on the size of the samples and drying temperature, drying times should range from 2-4 days. Remove the sample from the dryer when the sample weight has reached equilibrium. If unsure, it is best to leave it in the dryer for an extra day so that all samples are dried consistently. Aboveground biomass weight data are to be reported to the LTAR centralized data portal as oven-dry weight. 
SECTION: Sample collection and processing
17. Collect the dry weight (g) of the samples. Ensure the bag weight is not recorded in the final biomass weight.
SECTION: Sample collection and processing
18. Calculate total dry weight (g) and the total dry weight per area (kg/ha) using the equations (1a, 1b, 2) below. 
SECTION: Concurrently sampled covariate metrics
22. Plant population. Count the number of plants within the quadrat for the area calculation below.
Management data. Provide planting and harvest dates, tillage, fertilizer/manure applications, pesticide applications, and other field operations as appropriate in the farm management entry interface.
Collect grain density (test weight) and moisture content at the time of harvest of the grain sample. This information could also come from the combine when mechanically harvesting.
Elemental concentration of nitrogen, phosphorus, potassium, sulfur, and carbon from the ground tissue samples.
SECTION: Calculations
20. Calculation 1a. Weight basis (g) for when a subsample is dried: 

     Total biomassDW = Total biomassFW × (Subsample biomassDW / Subsample biomassFW) 

     DW = dry weight, FW = fresh weight

Calculation 1b. Weight basis (g) for when the whole sample is dried:

      No calculations are needed. Simply collect the dry weight after removing it from the dryer. 
SECTION: Calculations
21. Calculation 2. Area basis (kg/ha):

     Total biomass (kg/ha) = Total biomassDW × (## m2 sampling area) × 10

     ## = the area used to collect the sample, likely quadrat dimensions multiplied
SECTION: Quality assessment and quality control
23. We recommend a “calibration” for all members of sampling teams before sample collection so that everyone follows the same approach to sample collection. This uniformity is important when personnel vary in tenure and perhaps experience. Address quadrat area selection, quadrat orientation, counting techniques, safe clipping practices (across electric, gas, and manual tools), sorting criteria, labeling samples, and transport to chopping or drying facilities. 
SECTION: Archiving
24. Collect a minimum of a 300 gram representative subsample of each plant component (grain, stems/stalk/leaves, weeds, cover crops, etc.) harvested from each plot. Coarsely chop and grind samples through a 1 mm screen, using a mill such as in Figure 12. If samples have not recently been dried to 60°C, place into a dryer for a day to remove any ambient moisture prior to long-term storage. Store 150 grams of ground sample in a labeled plastic container (Figure 8) for utilization in elemental (2 grams) and/or forage NIR (100 grams) analysis. Utilize the remaining ground sample to fill a labeled 4 ounce glass container with a screw-top metal lid, which will be archived at room temperature for future analyses. Archived samples can also be stored as whole seed, rather than as a ground sample, if preferred.

 

 

SECTION: Illustrative information
25. Equipment and supplies needed will vary among sites, but the items shown here are often used by researchers when collecting, transporting, chopping, drying, weighing, and storing biomass samples. 
SECTION: Illustrative information
27. Mesh and paper bags for sample collections. It is not always possible to process samples immediately so keeping them aerated is important. Large mesh bags like this are often termed "onion bags" or "cabbage bags" because of their use in the food industry for transporting these products.  

 

SECTION: Illustrative information
28. Woodchipper

 

SECTION: Illustrative information
30. Large tubs, garbage cans, tins, and/or bags for processing samples

 

SECTION: Illustrative information
31. Airtight vials for shipping and archiving. Consider plastic vials for shipping and glass vials for archiving. 

 
 
SECTION: Illustrative information
32. Scales for large and small samples

 

 

SECTION: Illustrative information
33. Forced air oven

 

SECTION: Illustrative information
34. Mill for grinding

 
 
SECTION: Recommendations for data collection
35. Table 1. Summary of recommendations for the measurement of aboveground biomass staying in the field and leaving the field. 

 

SECTION: Sample collection and processing
19. Once weight data has been collected, this marks the completion of this protocol for collecting data on aboveground biomass. The next steps are to prepare the sample for archiving and the likely analysis of nutrient content on it. This is explained under the Archiving Section below. 
SECTION: Illustrative information
29. Thresher

 

SECTION: Illustrative information
26. Tools for cutting plants; this can include electric, gas, and manual tools such as a hand pruner, tree shearer, cordless trimmer, or cutter bar.