Yield response and ozone injury on Phaseolus vulgaris

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Yield response and ozone injury on

Phaseolus vulgaris

Revised and updated from previous versions


Experimental Protocol
ICP Vegetation, 2010

Essential Requirements

  • Pot size must be 12-15 litre volume

  • Use slow release fertiliser

  • Exposure site away from main roads and buildings

  • Record date of first flowering

  • Exposure until 6 weeks after flowering

  • Visible injury assessments

  • Harvest of pods 6 weeks after flowering

  • Ozone and meteorological data from site or nearby

  • Send data by the end of November

Experimental aim

  • To identify the extent of occurrence of visible injury on ozone-sensitive and ozone-resistant Phaseolus vulgaris in the differing ozone and physical climates of Europe.

  • To quantify the reduction in pod yield of the ozone-sensitive Phaseolus vulgaris across Europe.

  • To establish a stomatal conductance database for the ozone-sensitive and ozone-resistant Phaseolus vulgaris for the development of a flux-effect relationship.

This study will use the ozone-sensitive (S156) and ozone-resistant (R123) genotypes of Phaseolus vulgaris (Bush bean, French Dwarf bean) that have been selected at the USDA-ARS Plant Science Unit field site near Raleigh, North Carolina, USA. The bean lines were developed from a genetic cross reported by Dick Reinert (described in Reinert and Eason (2000)). Individual sensitive (S) and tolerant (R) lines were identified, the S156 and R123 lines were selected, and then tested in a bioindicator experiment reported in Burkey et al. (2005). A trial of this system occurred in central and southern parts of Europe during the summers of 2008 and 2009. Kent Burkey of USDA-ARS kindly provided seeds for the trials.

Experimental Requirements
Experimental Plot: The site should be situated in an open field, at least 200m from main roads and 50m from buildings. The plot should be fenced to prevent birds, rabbits and small mammals from eating the bean plants and should be surrounded by grass or an artificial weed reducing material to prevent excessive dust formation, mud-splashes or overshadowing by weeds. Slugs like bean plants. Slug pellets or biological control should be used if necessary.
Replication: Use a minimum of 8 pots for each genotype of bean plants.
Pots: 12 - 15 litre volume with a surface diameter of approximately 25cm. Each pot should be kept well-watered using a method suitable for the climate. For hot climates, some modifications may be necessary to try to minimise heat uptake by the pots, evaporative losses from the soil surface and wilting when the beans have a lot of foliage. Suggestions are:

  • Regular, possibly daily watering

  • white pots and/or reflective material covering pots

  • Stones or other material placed on the soil surface

  • Shading net over the plot

Soil mixture and fertilizer: Use whichever soil mixture/compost is appropriate for the area. Please add slow-release fertilizer according to the manufacturer’s instructions.
Seeds: Seeds of ozone-sensitive (strain S156) and ozone-resistant (strain R123) lines of Phaseolus vulgaris will be supplied by the Coordination Centre. These seeds have been provided by Kent Burkey of USDA-ARS .
Monitoring equipment: Participants should have access to pollution and climate data monitored at or close to the experimental site. Hourly mean data for ozone, temperature, humidity and solar radiation are the most useful and are required to enable ozone fluxes to be calculated. If these data are not available at the experimental site, please send data from the nearest appropriate monitoring station
Data submission: Please note that only data submitted by the end of November, 2010 will be processed in time for the Task Force Meeting (see later for information on data submission).

Setting up the experiment
The experiment will last for approximately 60 days. The seeds should be sown at an appropriate time, depending on climate. As a guide, sowing in late May/early June would be ideal for most central and southern areas of Europe.
At the experimental site, fill the self-watering pots with compost and water well to ensure that the soil is fully wetted. For eight pots per genotype either:
a) Sow two seeds 5 cm apart near to the centre of each pot at a depth of 3 cm. Ensure that the pots are protected from birds, animals, slugs and snails. When the first true leaves have emerged, thin to one plant per pot. Day 0 of the experiment is the day that the plants are thinned to one per pot.

b) Sow one seed per pot into degradable fibre pots (approx 6 cm diameter) at a depth of 3 cm. When the first true leaves have emerged, place one fibre pot containing a bean plant into each large experimental pot. Do not remove the plant from the fibre pot, the roots can grow through. Day 0 of the experiment is the day that the plants are transplanted into the large pots.

Note: bean seedlings can be susceptible to root pathogens (root rot). There are commercial chemical treatments (e.g. Ridomil Gold, a fungicide containing the active ingredients, mefenoxam and chlorothalonil) that can be added as a soil drench if this becomes a problem.
Note: the first true leaves are very susceptible to a variety of environmental and biotic stresses. The plants will usually grow through this. Any injury symptoms on these leaves that look like ozone injury are usually non-specific and should not to be included in assessments.

Core activities
1: Assessment of ozone injury
Ozone injury on Phaseolus vulgaris consists of bronze-coloured lesions that gradually join together to cover large parts of the leaf surface (Figure 1). Weekly assessments of injury are encouraged wherever practical. If this is not possible, as a minimum, there will be two assessment times:

  1. The onset of flowering (when 50% or more of the plants are flowering)

  2. Two weeks after the onset of flowering

Please take photographs of any injury that develops.

(a) (b)

(c) (d)

Figure 1: Ozone injury on a trifoliate leaf of Phaseolus vulgaris (a) scored as 5-25% of leaf injured, see below, (b) and (c) severely damaged leaf (scored as >25% injury) and (d) senesced leaf
At each visual assessment, please ignore any symptoms on the primary leaves as this is often non-specific, and record the following for each plant:

  • Plant genotype

  • Plant number

  • Health of plant (key described below)

  • Total number of trifoliate leaves1 (excluding primary leaves and any cotyledons still present)

  • No of trifoliate leaves1 with 1 – 5% injury

  • No. of trifoliate leaves1 with 5 – 25% injury

  • No. of trifoliate leaves1 with >25% injury

  • No. of dead/senesced trifoliate leaves that remain on the plant

1 One trifoliate leaf comprises one central and two side leaflets
Key for the health of each plant
Grade each plant as either healthy (H) or abnormal, with the cause of the abnormality graded as 1 (slight), 2 (moderate), or 3 (severe) using the following key:

S Stunted

D Diseased

I Insect damage

Sl Slug damage

A Animal (rabbits, deer, birds etc.)

V Virus
Please record the date of flowering (when 50% or more plants have flowers)

2: Final Destructive harvest
Pods should be harvested 6 weeks after the onset of flowering (it does not matter whether or not the pods are still green). This is because it is important to harvest both genotypes at the same time, but with high levels of ozone stress, all of the S156 leaves may have abscised and all pods will have become brown before the R123 genotype reaches the 50% brown pods stage.
By harvest, a large proportion of the leaves will have already fallen from the plant and thus we only require information about the biomass of the pods. Do not harvest or count pods < 2cm. Dry the pods (> 2cm) to constant weight. Separate pods into two size classes, <4cm and >4cm. For each size class, count and record the dry weight of (1) pods that have seeds in and (2) pods that have failed (i.e. have no seeds in).

Optional additional studies
3. Stomatal conductance: Collection of stomatal conductance data for flux-effect modelling

The aim is to establish a comprehensive stomatal conductance database for the sensitive (S156) and resistant (R123) genotype of snap/French bean (Phaseolus vulgaris L.) to enable the parameterisation of the DO3SE1 flux model for bean.

We would like to encourage colleagues to carry out as many stomatal conductance measurements as possible on both bean genotypes grown at their experimental site(s). The final dataset is expected to indicate the dependence of stomatal conductance on meteorological parameters such as Photosynthetically Active Radiation (PAR, μmol m-2 s-1), air temperature (°C) and Vapour Pressure Deficit (VPD, kPa), and will help to identify the maximum stomatal conductance (gmax) of these two genotypes. The collated data will be analysed using the boundary line approach which will then enable the parameterisation of the DO3SE flux model.

Single-sided porometers as well as steady-state gas exchange systems (IRGA) can be used for stomatal conductance measurements, however, when using the former make sure to take measurements on both sides of the leaf since Phaseolus vulgaris has amphistomatous leaves (i.e. stomata on both sides of the leaf). Once you have taken approximately 50 alternate measurements with the single-sided porometer (i.e. lower-upper-lower-etc.) on each genotype, subsequent measurements can then be made just for one leaf surface (that with the highest conductance). The ratio of the two surfaces calculated from the 50 alternate measurements can then be used to convert single-sided conductance to conductance for the entire leaf (total gs). However, it is recommended that measurements continue to be taken on both sides of the leaf if possible.

If the measuring device measures conductance in cm s-1 (velocity units), air pressure and temperature data are required to convert the values into mol units.

Stomatal conductance should be measured according to the following criteria:

  • Use a range of climatic conditions, including sunny and overcast days. Please make sure that for the derivation of gmax enough measurements (200 +) are made under “ideal” meteorological conditions for the gas exchange of the plants (i.e. sunny, warm and humid days);

  • Measure at various developmental stages of the plants (e.g. before flowering, at flowering, during pod formation)

  • Measure at various times over the course of the day, ideally at frequent intervals between sunrise and sunset;

  • Use the youngest fully developed and healthy leaf, e.g. the second or third from the top of the plant;

The measurements should be recorded in an Excel table (see Appendix 1), indicating:

Leaf conductance

Meteorological data (with measuring height)


Leaf number (count from the top of the plant)

% injury of the measured leaf

Phenological stage (before flowering, flowering, during pod formation)

Leaf surface used (when using a single-sided porometer, i.e. upper or lower surface)

Surface reference area

Measuring device

Gas measured (e.g. H2O, CO2)

Measuring unit (e.g. mmol H2O m-2 s-1, cm s-1)

Time and date of the measurement.

Collate and process all data, and send to Felicity Hayes at the Coordination Centre (fhay@ceh.ac.uk) as raw data and ideally as plots of gs versus measured environmental parameters (such as temperature, VPD, PAR).

4. Other effects
a: Pod Quality. This could include counts of the number of beans per pod, mean weight per bean and chemical analysis of pod quality.
b: Photosynthesis measurements and ACi curves.

Data collection
We request that all data is returned to Felicity Hayes (fhay@ceh.ac.uk) at the Coordination Centre by the end of November to ensure that it can be processed before the next Task Force Meeting, early in the following year.
Pollutant and climate data: should be sent by email to Felicity Hayes as a spreadsheet preferably in Excel format. The file should have a separate row for each hour of data i.e. the spreadsheet should have parameters such as ozone concentration and temperature along the top, and day and hour in separate columns down the left-hand side. It is important that gap-filled data is readily distinguishable from ‘real’ data. Template spreadsheets and instructions for ‘gap filling’ for missing data are available from Felicity Hayes and are also available on the ICP Vegetation web-page. Preferably, please send data for the period from May (or earlier if your experiment starts earlier) though to October. The template spreadsheet will automatically calculate parameters such as AOT40, 12h mean (7am – 7pm, local time) and mean daily maximum ozone concentration.
Please note: due to time constraints, we are no longer able to process files that are not in the above format and/or are sent as multiple files that need joining together and gap filling.
Plant data: should also be sent to Felicity Hayes by email. Example spreadsheets are available on request and are also available on the ICP Vegetation web-page.
References cited
Burkey, K.O., J.E. Miller and E.L. Fiscus (2005). Assessment of ambient ozone effects on vegetation using snap bean as a bioindicator species. J. Environmental Quality 34:1081-1086
Reinert.A. and G. Eason (2000). Genetic control of O3 sensitivity in a cross between two cultivars of snap bean. J. American Society of Horticultural Science 125(2):222-227.

Appendix 1: Example table for recording stomatal conductance



Measuring device:

Surface reference area:

Gas and unit of measurements:

Height of meteorological sensor(s):

Additional information:



Leaf number (from top)


% injury of measured leaf

Phenological stage

Leaf surface (upper/lower)

PAR/PPFD (e.g. μmol m-2 s-1)


temperature (°C)

VPD (kPa)

RH (%)

Air pressure (e.g. hPa)

gs (e.g. mmol H2O m-2 s-1)







































































Appendix 2: Self-watering pots
Here are some ideas (we have not examined or tested these products):
Adjustable Reservoirs Create Self-Watering Planters

These Adjustable Water Reservoirs convert your favorite pots into self-watering planters, keeping your plants from drying out and reducing the time you spend watering. Just place this self-contained reservoir in the bottom of your pot, adjust the refill tube and cover with soil. The Large Reservoir holds 4 quarts and fits pots between 16" and 20" in diameter. The Small Reservoir holds 1 quart and fits pots up to 10-14" diameter. Simply fill the reservoir with water through the convenient Fill Tube. The soil will be moistened through evaporative action from the reservoir below.

 A vacation watering solution!

 Includes a water level indicator

 May be used with a liquid or a water-soluble fertilizer


Riviera Vision Planter - 13" Grey

The hidden reservoir catches and stores water inside the planter, while a unique fabric wicking system delivers steady, measured hydration to thirsty roots. The handy water gauge completely eliminates guesswork - you know exactly when its time to refill. Caring for plants has never been this easy! Attractive, contemporary design in durable polypropylene never cracks, chips, or peels. Available in Speckled Beige and Speckled Gray. Outer Diameter: 13.7" Inner Diameter: 12"

Appendix 3: Fibre pots
Here are some ideas (we have not examined or tested these products):

Top of Form

Top of Form


Value Pack of 96
Colour: Natural

Ideal pots for seeds, seedlings, transplanting and cuttings

Easy to plant as you plant the whole pot the roots will grow uninterrupted through the pot and they are not damaged
No pot to discard

Now with reduced peat (maximum 25%)

Comprise mostly of wood pulp from renewable sources
Better for the environment
Better for plants as wood pulp is easier to re-wet



1 DO3SE: Deposition of Ozone and Stomatal Exchange model

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