Protocol on estimating leaf herbivore damage and pathogen infection

This is a detailed protocol on how to assess herbivore and pathogen damage in your BugNet Experimental plots. We ask you to assess damage in year three of your experiment (third year of pesticide application). However, if you have the time, you can assess damage at a more regular basis (see Ideas for student projects). At the bottom of the page there are more detailed tips and tricks on how to assess damage and a picture gallery of the most common damage types.

Where to assess damage?

If your system is very productive and will be mown regularly, damage and disease will be measured in the small plot dedicated for the plant cover (small plot i). If your system is very unproductive and removing individuals for damage assessment would have strong influences on the vegetation, then measure the herbivore damage and fungal infection in one of the other small plots dedicated for destructive sampling (ii, iii, iv), or alternatively, assess damage in the cover small plot without removing individuals.

When to assess damage?

Assess herbivore and pathogen damage at peak biomass, when you also collect your biomass samples and assess the cover of plant community.

On which species and individuals to assess damage?

Selection of species: We aim to measure the community weighted mean damage and disease per plot. Start with the species having the highest cover per plot, followed by the species with the second highest cover, and so on, until the cover-sum of the species per plot reaches 80% (relative cover excluding bare ground, rocks). However, do not assess damage on more than five plant species per plot. E.g. if Species A has a relative cover of 50%, Species B a relative cover of 20%, Species C a relative cover of 10% and Species D a relative cover of 8% assess damage on A, B and C. If Species A has already a relative cover of 90%, then only assess damage on species A. If Species A, B, C, D, and E have each a relative cover of 15% and species F, G, H, I… a cover of 5% then only assess damage on the five most abundant species (A,B,C,D and E) although the sum of their cover does not add up to 80%.

Selection of individuals per species: If your species has less than 10 individuals, select all individuals. If your species has between 10 and 20 individuals per plot, mark all individuals in the 1m² with grill sticks numbered from 1-20. Use a random number generator (or quickly select 10 numbers in your head) to select 10 individuals (it is important that the individuals are selected randomly, and that there is no bias towards particularly damaged or undamaged ones). If your species has more than 20 individuals, divide your 1m² plot into four quadrants, and estimate the proportion of individuals in each of the four quadrants. Randomly select individuals per quadrant in proportion to their numbers of individuals, e.g., if quadrant 1 contains 80% of all individuals, and quadrant 3 20%, then randomly select eight individuals of quadrant 1 and two of quadrant 3. Particularly, if the distribution of your species is very patchy (e.g. one large patch with many individuals, and 3 isolated individuals) this methods prevents that you will select isolated individuals with a higher likelihood (see Fig. 1).

**Fig. 1:** Selection of individuals per plant species. For each selected plant species, select 10 individuals on which you will assess the presence/absence of damage signs of the different damage categories (incidence). Of these 10 individuals you will select five individuals which you will cut at ground level (respectively a subset of them), bag, and bring to the lab for the assessment of % leaf damage and the measurements of plant traits.

For more guidance – this youtube video gives a tutorial on how to select species and individuals for damage incidence and damage % measurements.

How to measure?

Measurements: On each of the selected individuals per species (max. of 10), record the presence or absence (0,1) of damage signs by chewing, mining, galling and sucking/rasping herbivores, and pathogen disease symptoms of the categories downy mildews, powdery mildews, rusts and leaf spots (see damage gallery at the bottom of this page). This will give us an estimation of damage incidence.

In addition, randomly pick five of the ten individuals (select them using the grill sticks with the first five random numbers from your random-number list), and measure the maximal height (stretch out if necessary). Then, cut them at ground level. If your individual is very large, or builds large tussocks as is often the case for grasses, then take a subsample from the middle of the individual which contains at least five leaves. If you selected only one plant species per plot because this species had a relative cover of 80% or more, then cut all 10 individuals that were used to assess incidence. On these individuals you will estimate the leaf area (%) that has been damaged. You can either do this directly in the field, or you can bag the plants in labelled plastic bags, place them in a cooler, bring them to the lab, and do the % damage assessment there. In any case, visually survey five random, mature, and non-senescing leaves (or leaflets if your leaves are very small) per individual for damage and disease symptoms. For easier inspection you can use hand lenses to better assign damage types or use a stereomicroscope. On each of the five leaves estimate the leaf area (%) that has been removed by chewing herbivores, mining, galling and sucking/rasping herbivores, and the leaf area that is covered by pathogenic disease symptoms of the categories downy mildews, powdery mildews, rusts and leaf spots (see damage gallery below). Some plant individuals will have fewer than 5 leaves, and for these all leaves should be surveyed (but leave out senescent leaves). Note that in some cases, damage is present on only the underside of leaves, so remember to check both sides of the leaf for damage. Below you will find more tips and tricks on how to assess damage and also check our photo gallery of the most common damage types. We also highly recommend that you train your damage assessment with the ZAX Herbivory Trainer app by Zoe Xirocostas and Angela Moles!

With this method you assess damage on a minimum of 10 individuals (if one species had a cover of 80% or more) or on up to 25 individuals (if you selected 5 plant species with each 5 individuals) per plot.

Shrublands: If you work in shrublands, you may not find 10 individuals of a species in your 1m² plot but very likely will have only one or two individuals. You may also have spreading shrubs where it can be difficult to distinguish individuals. In this case, randomly pick 50 leaves from 10 different branches per shrub species throughout the plot (5 leaves per branch). The five leaves per branch should always originate from the same leaf position, i.e. from the tip of each branch, count the first ten leaves and start sampling from there (leaf 11 to 15). Make sure that the 10 branches are from either as many shrub individuals as possible, or else take branches from different positions and orientations within the shrub patch. Assess how many of those 50 leaves are damaged. This allows us to assess the proportion of leaves that are damaged by the different damage categories per shrub individual (incidence). In addition, assess the % leaf area damaged on at least 25 randomly chosen leaves per shrub species.

If your shrub species is leafless and instead has photosynthetic stems (e.g. Retama ssp.), instead of picking leaves, randomly cut 5 cm pieces from 10 different branches throughout the plot. On these, assess how many of those pieces show damage signs of the different damage categories (incidence). In addition, on five of those branches, assess the % photosynthetic area that is damaged. You can assess % damage either in the field or collect the leaves to assess damage in the lab.

More tips and tricks on how to assess damage

1. How can I distinguish the different damage types?

We will differentiate between the following damage types, see also our damage type gallery:

Herbivores

Chewing damage: any missing area of a leaf blade, such as holes within the leaf, or cuttings at the edges, that go all the way through the leaf, including tissue removal at leaf margins. Exceptions are holes surrounded by dead, brown tissues, often fading into yellow. These are the symptoms of leaf spot disease, caused by bacterial or fungal infections which results in necrosis where dry dead tissue can break off leaving holes.
Sap-sucking or rasping damage: this is a bit more difficult to identify. Sucking herbivore damage is often visible as regularly shaped, circular punctures, surrounded by lighter colored tissue. Against the light it looks like yellow, discolored spots. Irregularly shaped discolored spots or areas instead are likely to be caused by pathogens. Rasping damage leaves at least the epidermis on top or bottom intact, and causes no hole (often caused by molluscs).
Leaf-mining damage: transparent traces or feeding corridors on a leaf (due to missing mesophyll tissue), where the two epidermal layers are still intact. You can often see the excrement of the mining insects in the mine. Please also count any brownish area next to the mines, as this damaged area is attributed to the mining. Mostly caused by moth (Lepidoptera), sawflies (Symphyta), flies (Diptera), sometimes beatles (Coleoptera).
Galling: swelling growth on the external tissues of plants. They can be caused by various organisms, from viruses, fungi, insects and mites.

Pathogens

Rust fungi (Pucciniomycotina): They are easily recognized with the yellow, red, or brown urediniospores released from little “volcanos” or “pillows”, where most spores can be brushed away by your finger except some which look black and not easily brushed away. Rusts can be found on both sides of the leaves, so don’t forget to check both sides. Often the presence of a yellow spot on the top of a leaf may indicate the presence of a rust underneath.
Downy mildews (Oomycetes): obligate parasites of plants.Initial symptoms are large, angular, pale green to yellow areas which are visible on the upper surface of leaves, and later turn brown. On the undersides, these areas are covered with white to grayish, cotton-like (downy) structures, which appears watersoaked.
Powdery mildews (Erysiphales):white-greyish powderycircles (mycelium) on the upper leaf surface which can easily be brushed off. Powdery mildew can also be on the backside of the leaf. When ripe, little dots can be seen at the underside of the leaf. Powerdery mildews are obligate biotroph and relies on living substrate to survive.
Leaf spots: Leaf spot symptoms are discolored, sometimes reddish to brown circles of necrotic plant tissue, which often have a center of necrosis or cell death, and sometimes several rings around the center. They are caused by a different fungal pathogens, bacteria or viruses.

See also our picture gallery of plant damage at the end of the document!

2. How to estimate damage?

We largely follow the protocols for damage estimation from “The Herbivory Variability Network“ (HerbVar, herbvar.org), who kindly allowed us to use their description here – so most of the text has been written by members of herbvar. Thus, we recommend using visual estimation because digital methods are slower and more laborious. Also, careful visual estimations have been shown to do a surprisingly good job, especially after some practice. To increase precision in visual estimation of percentage damage we recommend that you train your damage assessment with theZAX Herbivory Trainer app by Zoe Xirocostas and Angela Moles!

How does visual estimation work?

Visual estimation is very simple. Look at a leaf and guess what percent was removed or damaged or shows pathogen symptoms. Try to estimate at a high resolution of at least 2.5 % (see Table 1). Even if your estimate has an error, a high resolution estimates will still be closer to the true value than estimates that are reported in coarse categories.
When you first look at a leaf, do a quick mental calibration before estimating damage. For this, imagine to cut the leaf into a range of proportion. For example, think about what half the leaf would look like (50%), then imagine a quarter (25%) of the leaf. Do the same for a tenth of the leaf (10%) by mentally cutting the leaf into 10 equally-sized pieces. Then mentally cut one tenth in half to get an idea of 5% leaf area. Half of that unit would then be 2.5 %.
When it is time to do the actual herbivory estimate, one strategy that works well for contiguous blocks of damage is to use fractional thinking, starting with larger fractions and gradually working your way down to smaller fractions – honing from a coarse estimate to a precise estimate. For example,
- If ~12.5% of a leaf were damaged, then…
Mentally cut the leaf into quarters
See that less than a quarter (25%) is damaged
Mentally cut the quarter with damage in half, yielding eighths (12.5%)
See that the area damaged is equal to an eighth and record 12.5%
If ~30% of a leaf were missing, then…
- Mentally cut the leaf in half
- See that less than half is damaged
- Mentally cut the leaf into quarters
- See that more than a quarter (25%) is damaged
- Take mental note of the 25% damaged, and then focus on estimating how much more than that 25% is damaged
- Mentally halve the quarter of the leaf with the excess damage above 25%, yielding eighths (12.5%)
- See that the damage above 25% is a little less than half of one of those eighths, which means it’s a little less than a sixteenth or 6.25%
- 25% plus a little less than 6.25% comes close to 30%, record it!
If your leaf has more than one area of damage, try mentally consolidating each area of damage into one area and then estimate the size of that using the method above. Alternatively, if mental consolidation isn’t working well, you can mentally divide the leaf into fractions that are as small as the smallest patch of herbivore damage. Then simply mentally tally the number of patches of that size that would be damaged.
For complexly pinnate leaves (e.g., Apiaceae), it is probably best to divide the leaf into leaflets or pairs of leaflets, then follow the methods above.
If damage by herbivores is very high and very little leaf tissue remains, take a large and small leaf and compare the leaf base width, petiole and midrib size to compare. Use these comparisons to visually reconstruct the leaf, and deduce % damage from there
Through all of this, make sure you are correctly identifying what is herbivore damage versus disease versus physical damage. Please have a look at our Picture Gallery of plant damage. We are trying to avoid damage caused by abiotic stress. E.g. wind damage can manifest as browning.
Mines should be included in percent damage.
Galls should only be counted, and not included in percent damage because galls are actually extra tissue. The removed tissue is internal and can’t be seen.

**Tab. 1:** *Recommended resolution for recording percent damage. Adopted from herbvar.org.*

We thank again the Herbivore Variability Network (herbvar.org) for using parts of their protocols! They also created an illustrated guide to percent leaf damage, which might be helpful for training.