Analysis of Tomato Post-Harvest

Properties: Fruit Color, Shelf Life, and

Fungal Susceptibility
Vera Thole,1 Philippe Vain,1 Ray-Yu Yang,2 Juliana Almeida Barros da Silva,3
Eugenia M. A. Enfissi,3 Marilise Nogueira,3 Elliott J. Price,3 Saleh Alseekh,4
Alisdair R. Fernie,4 Paul D. Fraser,3 Peter Hanson,2 and Cathie Martin1,5
1
Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich,
United Kingdom
2
AVRDC–The World Vegetable Center, Shanhua, Tainan, Taiwan
3
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey,
United Kingdom
4
Max Planck Institute for Molecular Plant Physiology, Potsdam-Golm, Germany
5
Corresponding author: cathie.matrin@jic.ac.uk

A wide variety of fresh market and processing tomatoes (Solanum lycoper-

sicum) is grown and consumed worldwide. Post-harvest losses are a major con-
tributing factor to losses in crop productivity and can account for up to 50% of
the harvest. To select and breed elite tomato varieties, it is important to char-
acterize fruit quality and evaluate the post-harvest properties of tomato fruits.
This includes the analysis of shelf life (the period during which a fruit remains
suitable for consumption without qualitative deterioration), color, and pathogen
susceptibility. Tomato shelf life depends upon the rate of fruit softening which
accompanies fruit ripening and exacerbates damage during transport and han-
dling. Furthermore, the susceptibility of tomatoes to fruit pathogens is also of-
ten linked to fruit ripening, especially for necrotrophic fungi such as Botrytis
cinerea, also known as gray mold. The methods described here are critical for
determining fruit quality and fungal susceptibility during storage. © 2020 The
Authors.
Basic Protocol 1: Fruit color as a determinant of fruit quality
Basic Protocol 2: Shelf life test of tomato fruits
Basic Protocol 3: Botrytis cinerea pathogen test of tomato fruits
Support Protocol: Preparation of Botrytis spore inoculum
Keywords: Botrytis r fruit color r fruit quality r fruit storage r fungal suscep-
tibility r post-harvest analysis r shelf life

How to cite this article:

Thole, V., Vain, P., Yang, R.-Y., Almeida Barros da Silva, J., Enfissi,
E. M. A., Nogueira, M., Price, E. J., Alseekh, S., Fernie, A. R.,
Fraser, P. D., Hanson, P., & Martin, C. (2020). Analysis of tomato
post-harvest properties: Fruit color, shelf life, and fungal
susceptibility. Current Protocols in Plant Biology, 5, e20108.
doi: 10.1002/cppb.20108

INTRODUCTION
Tomato (Solanum lycopersicum L.) is one of the most important horticultural crop plants
Thole et al.
and a well-established model system for fleshy fruits, fruit development, and ripening.
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Current Protocols in Plant Biology e20108, Volume 5
Published in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cppb.20108
© 2020 The Authors. This is an open access article under the terms
of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original
work is properly cited.
 Tomatoes are a great source of phytonutrients, including vitamins, carotenoids, and
polyphenols, and are consumed fresh as well as in many cooked and processed prod-
ucts.

In a climacteric fruit such as tomato, ethylene triggers the onset of the ripening process,
and ethylene is also required for normal, full fruit ripening (Alexander & Grierson, 2002).
During fruit ripening, tomatoes undergo dramatic changes in color, texture, flavor, and
aroma. The change in fruit color is accompanied by the degradation of chlorophyll that
unmasks pre-existing pigments, as well as the synthesis and accumulation of different
types of compounds such as carotenoids.

Commercially, tomatoes destined for the fresh market are picked at different ripening
stages, depending on the proximity between production and market place, and vary from
green and firm to almost ripe for local markets. Long-distance transportation of tomatoes
involves storage at low temperatures to limit post-harvest losses and to extend shelf life.
Fruits often also undergo ethylene treatment to induce color and ripening before reaching
the supermarket shelf. Artificial ripening and cold storage have, however, detrimental ef-
fects on tomato flavor, aroma, and texture (Baldwin, Plotto, Narciso, & Bai, 2011; Zhang
et al., 2016).

Tomatoes, like other fleshy fruits, are particularly prone to post-harvest losses. The shelf
life of food is defined as the period during which a stored product remains suitable for
consumption and is normally determined by the degree of softening, shriveling, and rot-
ting of fruit. Post-harvest shelf life is one of the most important traits for commercially
grown tomatoes and can be shortened as a result of rapid over-ripening induced by var-
ious factors such as changes in temperature and pathogen exposure. Fruit softening and
over-ripening leading to cell breakdown favors pathogen takeover and increases suscep-
tibility to pathogens such as the necrotroph Botrytis cinerea, commonly known as gray
mold, the second-most-important fungal plant pathogen (Cantu et al., 2008, 2009; Dean
et al., 2012).

Important challenges for tomato production and commercialization include post-harvest

losses and reduced quality due to fruit senescence and pathogen infection. Here, we de-
scribe three effective and robust protocols that represent standard operating procedures
for assessing color, shelf life, and susceptibility to gray mold (B. cinerea) to define tomato
fruit quality parameters important for consumers. The protocols can be applied to all
types of tomato genotypes from wild tomato species to commercially grown tomato va-
rieties.

BASIC FRUIT COLOR AS A DETERMINANT OF FRUIT QUALITY

PROTOCOL 1
Tomato fruit color and uniformity play a pivotal role in consumer perception of fruit
quality. Tomato fruits display various colors depending on the fruit ripening stage, the
dominant carotenoids, and other phytochemicals produced in the fruits, as well as envi-
ronmental factors. It is an esthetic parameter whereby the consumer associates color with
a better tasting, more nutritious, and fresher product. The color value is also an impor-
tant fruit quality indicator for breeders and industry for the selection of varieties and the
monitoring of quality.

In general, fruit color can be measured by visual analysis or different instrumental meth-
ods such as colorimetry, spectrophotometry, or a computer vision system (for reviews see
Pathare, Opara, & Al-Said, 2013; Wu & Sun, 2013). Most color measuring instruments
use mathematically defined color spaces standardized by the International Commission
on Illumination (Commission Internationale de l’Éclairage; CIE). The two-color space
Thole et al. system named CIELAB (also known as CIE L*a*b*, L*a*b*, or Lab) is commonly used
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Figure 1 Tomato fruit color measurement using the CIELAB color space system. (A) Diagram representing
the CIELAB color scale. The L* axis indicates the degree of lightness or luminosity from black to white (L* = 0 to
100). The a* axis represents the redness or greenness on a scale from red (positive number) to green (negative
number). The b* axis approximates yellowness and blueness on a scale from yellow (positive number) to blue
(negative number). The center is achromatic (neutral or gray). The a* and b* values can be used to predict
chroma (C*) and hue (hab ). Chroma indicates the intensity or saturation level of a particular hue/vividness or
dullness of a color. Hue indicates the perception of a color of an object (e.g., red, green, yellow, according to the
color wheel). (B) NE 4000 colorimeter used to measure L*, a*, and b* values of a fresh tomato puree sample
(indicated by black arrow).

commercially for food color measurement as it closely approximates the color percep-
tion by humans. CIELAB, which is related to the alternative Lab color space Hunter Lab,
mathematically describes all perceivable colors in three dimensions and measures three
values: the achromatic component L* (light vs. dark) and two color descriptors, the a*
(red vs. green) and b* (yellow vs. blue) values (Fig. 1A).

This protocol is intended for researchers to rapidly become familiar with the method
routinely used by industry and breeders to determine tomato fruit color.
Materials
6 to 12 mature tomato fruits per genotype or tomato products (e.g., tomato puree or
juice)
Cloth, to clean tomatoes
Knife or razor blades, to cut tomatoes
Blender (e.g., Panasonic MX-188) or homogenizer
Gauze fabric, 100% cotton, 20 S, non-sterile, unbleached without fluorescent
chemicals (e.g., Charry Co. Ltd.)
50-ml centrifuge tubes or glassware, to store tomato puree
Benchtop colorimeter and accessories (e.g., Nippon Denshoku Industries Color
Meter NE 4000 or ZE6000) or portable handheld colorimeter (e.g., Hunter Lab
MiniScan EZ 4500)
Computer and software for color data analysis and management (e.g., ColorMate 5)
Sample preparation
1. Harvest tomato fruits at mature ripening stage, and transfer to the laboratory.
For tomatoes, the degree of ripening is often estimated by color charts in an industrial
setting (e.g., visual aid TM-L-1; United States Department of Agriculture, 2017). It can
also be specified by days post anthesis or post breaker stage (see Basic Protocols 2
and 3).
An undesirable yellowish fruit color can be observed in unripe or ripe fruits as a result
of abiotic and/or biotic stress, such as high temperature, or can be caused by diseases Thole et al.

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 a
Table 1 Colorimeter Setup for Calibration and Sample Reading

Setup Zero calibration Standard calibration Sample measurement

Upper ZERO box Standard White Plate ZERO box
30- glass plate 30- glass plate 30- sample cell
30-mm sample stage for 30-mm sample stage for 30-mm sample stage
standard calibration standard calibration
Lower 30- aperture 30- aperture 30- aperture
a As described in Basic Protocol 1 steps 11 to 13.

during fruit ripening. Fruit color is also influenced by growing conditions such as light
intensity, use of fertilizers (type and amount), and watering regimen.

2. Select 6 to 12 fruits per tomato variety, depending on fruit size and sampling proce-
dure of the study design.
3. Remove fruit calyx and remaining stem parts. Clean fruits with a cloth to remove
dirt.
4. Cut each fruit into two to four pieces, and avoid juice loss as much as possible.
Alternatively, a whole fruit can be used for color measurements.

5. Put cut tomatoes in a blender, and blend for 1 to 2 min until pureed.
6. Filter puree through a two-layered gauze cloth into 50-ml centrifuge tubes to remove
seeds and skin debris.
7. Stir tomato puree before color measurement (step 13).
Color measurement
The specific procedure depends on the type and model of the instrument used. Steps 8
to 13 relate to the use of a desktop colorimeter (e.g., Color Meter NE 4000 or ZE 6000;
Fig. 1B) and the measurement of L*, a*, and b* values for tomato fruit puree. For other
colorimeter models follow the manufacture’s guidelines for setting up the instrument,
standardization, and reading samples.
8. Preparing for measurements: Power on and set operating functions using the display
menu. Set color space system to L*a*b* (six other color space options are available;
e.g., Lab, XYZ, and RGB). Select light source mode/viewing angle for the CIE Stan-
dard Daylight Illuminant D65/10° (alternatives are CIE illuminant/observer angle
C/2° or D65/2°), and set measurement mode to reflectance (the other selectable op-
tion is transmittance).
9. Select sample type (i.e., powder/paste if using tomato puree or solid sample if using
whole tomato fruits) from the drop-down menu.
10. Select measuring area (6, 10, or 30 mm) and aperture (6, 10, or 30 ). Choose
appropriate sample stage (holding/supporting the sample cell) corresponding to the
measuring area (6, 10, or 30 mm) and sample type (solids, powder, or paste). For
analyzing color of tomato puree, choose a wide aperture of 30 , a 30-mm sample
stage, and a round cell (i.e., sample cell/cuvette for powder and paste samples).
11. Zero calibration: Place 30- aperture, corresponding sample stage (30 mm) for
standard calibration, and 30- glass plate into the instrument. Cover with “ZERO
box” (black box) to prevent light exposure, and press the “zero-calibration (0-CAL)”
key (Table 1).
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12. Standard calibration: Remove “ZERO box,” and replace with the “Standard White
Plate.” Press the “standard-calibration (S-CAL)” key (Table 1).
13. Sample measurement: Remove “Standard White Plate,” 30- glass plate, and
30-mm sample stage used for calibration, and replace with 30-mm sample stage for
sample measurement. Fill sample cell with tomato puree (from step 7) to the top,
and place sample cell on top of the sample stage. Cover sample cell with “ZERO
box” (Table 1), and read L*, a*, and b* values.
Readings can be saved and analyzed (see step 14).
The L* value indicates the level of lightness from black (L* = 0) to white (L* = 100); a
low number indicates dark (L* = 0 to 50), while a high number indicates light (L* = 51
to 100). The a* value expresses greenness (negative number) or redness (positive num-
ber). The b* value indicates blueness (negative number) or yellowness (positive number).
Neutral gray values are represented at a* = 0 or b* = 0 (Fig. 1A).
The CIELAB L*, a*, and b* values measured in this protocol can be converted to color
values measured by other color space systems such as CIE XYZ and Hunter Lab using
manual calculation or colorimeter software programs.
Data presentation
14. Present color value as L*, a*, b*, and a*/b*.
The value of a*/b* is the most widely used reference parameter for color quality in com-
mercial practice and scientific publications on tomato fruits and products.
A true red reference color point has the coordinates L* = 50, a* = +60, and b* = 0; a
green reference value has the coordinates L* = 50, a* = –60, and b* = 0 (López Camelo
& Gómez, 2004). For example, immature green tomatoes have an a*/b* ratio <0, which
increases to 0 and above as the tomatoes ripen and turn redder (Hanson et al., 2004); L*
values decrease with darkening of the red fruit color (López Camelo & Gómez, 2004).
Color parameters such as tomato color index, chroma difference, total color difference,
and hue angle can be determined using L*, a*, and b* values to express color changes
and evaluate fruit quality (López Camelo & Gómez, 2004; Yildiz & Baysal, 2007). The
fruit lycopene content can also be calculated based on the L*, a*, and b* color readings
(Arias, Lee, Logendra, & Janes, 2000).
Fruit color can also be measured by visual analysis using a standard color chart under
controlled illumination conditions.

SHELF LIFE TEST OF TOMATO FRUITS BASIC

PROTOCOL 2
Here we provide a detailed description of the analysis of shelf life properties of tomato
fruits, including fruit texture (firmness/softness), weight, and rotting. We describe the
scoring of fruit quality before and during storage, tomato fruit harvest, preparation of
fruits for storage, and storage conditions, as adapted from Zhang et al. (2013).
Materials
10 to 15 tomato fruits per tomato genotype
Sterile water
10% (v/v) bleach (e.g., Parozone Thick Bleach Original)
70% (v/v) ethanol (e.g., VWR, cat. no. 20821.330)

Labels (e.g., colored paper clips, small paper pricing tags)

Clipboard, paper, and writing instrument
Computer running spreadsheet analysis software
15-cell and 40-cell plant potting trays, ∼6.5 cm × 6.5 cm per cell and ∼3.5 cm ×
3.5 cm per cell, respectively (e.g., Desch Plantpak)
Plastic plant propagator trays (e.g., Desch Plantpak) Thole et al.

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 Masking tape
Scale (e.g., Sartorius Practum 612-1S)
Lint-free paper towel sheets (e.g., Kimberly-Clark WypALL or Kimwipes)
500-ml containers (e.g., Phytatray II; Sigma-Aldrich, cat. no. P5929)
50-ml centrifuge tubes, disposable, sterile (e.g., Corning, cat. no. 430290)
Zip-lock polythene bag, ∼33 cm × 45 cm (e.g., Allpack Packaging Supplies, cat.
no. SS16)
18°C, 70% humidity growth cabinet with 24-hr dark cycle (e.g., Sanyo, Fitotron)
1. Monitor development and ripening of tomato fruits daily or every other day. When
fruits have reached breaker stage (i.e., when skin color changes from green to pink,
red, or yellow on ∼10% of the fruit surface), label and record breaker date. Label
individual fruits with a paper tag annotated with the breaker date. For large-scale
experiments (∼100 tomatoes per day), use colored paper clips or unique tags to rep-
resent a specific date. Log breaker date of each fruit in a spreadsheet to facilitate
subsequent scheduling of fruit harvest for experiments and to monitor the number of
fruits already available for a given genotype.
It is recommended to use at least 10 tomatoes per genotype for shelf life tests considering
background fruit-to-fruit variability. Tag at least 12 to 15 fruits per genotype to account
for possible fruit damage or loss.
In large-scale experiments, color or shape coding of labels is necessary to identify specific
fruits among hundreds without the need to read each fruit label individually.

2. Harvest tomato fruits 2 weeks after breaker stage into labeled, clean containers, such
as disposable 15-cell plant potting trays inserted into a plastic plant propagator tray;
label individual cells using strips of masking tape. Collect only fruits that are in perfect
condition. Avoid using wrinkled or damaged tomatoes, fruits with a dry calyx, or fruits
grown on damaged or diseased branches or plants.
For large-scale tests (≥100 fruits per day), it is helpful to have a tick list of fruits to be
harvested on the day before collection.
In large-scale experiments, fruits aged between 13 and 16 days after breaker can be used
without significant deviation of performance compared with fruits aged 14 days after
breaker. However, it is not recommended to use much younger or older fruits.

3. Remove any remaining calyx, measure individual fruit weight, and record color. As-
sess fruit color according to Basic Protocol 1 or using visual annotation (e.g., purple,
dark red, red, yellow-red). Score fruit firmness using a scale of 1 to 5 (i.e., 1 = very
firm, 2 = firm, 3 = medium, 4 = soft, 5 = very soft). Score fruit firmness by holding
the fruit with both hands and moving the fingers around the whole equator of each
fruit.
Very firm tomatoes (1) have a totally rigid surface, while very soft fruits (5) have a "water
balloon–like" feel all around. Tomatoes scored as (3) are in between. This method enables
the quick and nondestructive assessment of fruit firmness as a whole, which is particularly
important as tomato fruits can be heterogeneous in their maturation (i.e., some sectors or
areas are more mature than others).

4. Surface clean tomato fruits by rinsing twice with sterile water directly in the 15-cell
potting tray previously used for harvest (step 2). To avoid handling fruits, pour wa-
ter over fruits in the tray, and then remove carefully by tilting the tray. After rinsing,
wipe dry the surface of each fruit with a lint-free towel sheet, and place fruits back
into the tray on top of a clean paper towel pushed into the bottom of the cell. From
this point onward, handle fruits with gloves to maintain semi-sterile conditions. Next,
dip each fruit for 10 to 15 s into ∼400 ml of 10% (v/v) bleach, and briefly rinse in 400
Thole et al. ml sterile water placed in 500-ml containers. Replace bleach and water after ∼10 to
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Figure 2 Shelf life test. (A) Storage of tomato fruits in a tray placed in a sealed bag. (B) Recording
of individual fruit position. Shown is an example for storage of 10 fruits (A) and the corresponding
log sheet (B).

12 dips. Following rinsing, surface dry fruits with a clean paper towel. Finally, care-
fully wipe each fruit using a paper towel sheet sprayed with 70% ethanol.
The cleaning step with bleach can be omitted; however, some fruits may develop unwanted
fungal growth, especially in cases of prolonged storage. This might produce an undesirable
“batch” effect for the other fruits stored within the same container.

5. Surface clean (e.g., spray) a 40-cell plant potting tray or any equivalent container with
70% ethanol. Divide tray into two parts that will be placed on top of each other to
increase rigidity. Label each fruit with a permanent marker near the calyx attachment
zone (optional), and place, top up, in a cell. Adjust the number of tomatoes per tray
according to size (up to 12 tomatoes per tray). When using small fruits (like cherry
tomatoes), add plastic caps from 50-ml centrifuge tubes (or equivalent support) on the
bottom of the cell. Place each tray in a new, clean zip-lock polythene bag to prevent
extensive water loss during fruit storage. Add one or two 50-ml centrifuge tubes into
empty cell(s) as a “spacer” between the tomatoes and the bag to avoid direct contact
between them (Fig. 2A). Record the position of individual tomatoes in each tray (Fig.
2B).
Tubes can also be used for labeling (e.g., tray identifier, experiment date).

6. Incubate tomatoes at 18°C in the dark with 70% humidity in a controlled environment
room or cabinet (Fig. 3) until the end of the storage test (step 7).
7. Each week, evaluate fruit weight and quality by scoring fruit softness on a scale of
1 (very firm) to 5 (very soft; see step 3) and by rating fruit surface integrity from 1
to 4 (D1 = little, D2 = medium, D3 = severe fruit shriveling or skin damage, D4
= split fruits or tomatoes presenting major necrosis or wounds). Wear clean gloves
when handling or weighting fruits. Compile all data (i.e., genotype name, breaker
date, date of fruit harvest [number of days after breaker]) and weekly fruit weight,
softness, and surface integrity into a spreadsheet to enable data analysis. Collect data
for at least 10 biological replicates (scattered in different trays according to their date
of maturity), and compare to well-characterized, control genotypes.
Very soft fruits (5) or tomatoes with severe surface damage or rotting (D3 or D4) are
considered unsuitable for commercialization or consumption and are removed from each
Thole et al.
tray weekly. It can take several weeks (sometime months) until the last tomato is discarded.
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 Figure 3 Shelf life test. Storage of tomato fruits in a controlled environment cabinet.

The total weight loss of tomato fruits is the difference between the initial and the final
weight at the time of its removal from the tray.
As an order of magnitude, 100% of fruits from S. lycopersicum cv. M82 are, on average,
discarded after 4 weeks of storage at 18°C.

BASIC BOTRYTIS CINEREA PATHOGEN TEST OF TOMATO FRUITS

PROTOCOL 3
Tomato fruit quality and shelf life properties are also greatly influenced by the susceptibil-
ity to diseases, especially fungal infections. B. cinerea is one of the most important post-
harvest pathogens in tomato, although it can also cause significant damage and losses
during plant growth and development. Here, we describe the analysis of fruit pathogen
susceptibility with respect to B. cinerea, from harvest to wound inoculation of tomato
fruits, which was adapted from Zhang et al. (2013). We also present a method for isola-
tion of Botrytis spores used as an inoculum for wound inoculation (see Support Protocol).
Materials
10 to 15 tomato fruits per tomato genotype
Sterile water
70% (v/v) ethanol (e.g., VWR, cat. no. 20821.330)
1 × 105 spores/ml Botrytis macrospore inoculum (see Support Protocol)
10% (v/v) bleach (e.g., Parozone Thick Bleach Original)
Labels (e.g., colored paper clips, small paper pricing tags)
Clipboard, paper, and writing instrument
Computer running spreadsheet analysis software
15-cell and 40-cell plant potting trays, ∼6.5 cm × 6.5 cm per cell and ∼3.5 cm ×
3.5 cm per cell, respectively (e.g., Desch Plantpak)
Plastic plant propagator trays and transparent lids (e.g., Desch Plantpak)
Masking tape
Thole et al.
Scale (e.g., Sartorius Practum 612-1S)
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Figure 4 Fungal susceptibility test. (A) Experimental setup for fruit wound inoculation. (B) Log sheet. Shown
is an example of an inoculation tray for 16 fruits (A) and the corresponding log sheet (B).

Lint-free paper towel sheets (e.g., Kimberly-Clark WypALL or Kimwipes)

50-ml centrifuge tubes, disposable, sterile (e.g., Corning, cat. no. 430290)
200-μl pipette tips, for wounding fruit (e.g., StarLab, cat. no. S1113-1810-C)
Parafilm, 50 mm × 76 mm (e.g., R&L Slaughter, cat. no. 291-1214)
20°C, 70% humidity growth cabinet with 12-hr light/12-hr dark cycle, 70% light
intensity (e.g., Sanyo, Fitotron)
Caliper (e.g., Draper Dual Reading Digital Vernier Caliper)
CAUTION: Perform all steps involving Botrytis macrospore inoculum in a laminar flow
hood, dedicated incubator, or laboratory area suitable for working with biosafety level
(BSL) 2 pathogens.
Production and preparation of tomato fruits
1. Monitor development and ripening of tomato fruits daily or every other day. When
fruits have reached breaker stage (i.e., when skin color changes from green to pink,
red, or yellow on ∼10% of the fruit surface), label and record breaker date. Label
individual fruits with a paper tag annotated with the breaker date. For large-scale
experiments (∼100 tomatoes per day), use colored paper clips or unique tags to
represent a specific date. Log breaker date of each fruit in a spreadsheet to facilitate
the subsequent scheduling of fruit harvest for experiments and to monitor the number
of fruits already available for a given genotype.
It is recommended to use at least 10 tomatoes per genotype for pathogen tests consider-
ing background fruit-to-fruit and inoculation variability. Tag at least 12 to 15 fruits per
genotype to account for possible fruit damage or loss.

2. Harvest tomato fruits 2 weeks after breaker stage into labeled, clean containers, such
as 15-cell plant potting trays inserted into a plastic plant propagator tray; label in-
dividual cells using strips of masking tape. Collect only fruits that are in perfect
condition. Avoid using wrinkled or damaged tomatoes, fruits with a dry calyx, or
fruits grown on damaged or diseased branches or plants.
For large-scale tests (≥100 fruits per day), it is helpful to have a tick list of fruits to be
harvested on the day before collection (e.g., Fig. 4).
In large-scale experiments, fruits aged between 15 and 17 days after breaker can be used
without significant deviation of performance compared with fruits aged 14 days after
breaker. However, it is not recommended to use much younger or older fruits.

3. Remove any remaining calyx, measure individual fruit weight, and record color.
Assess fruit color as described in Basic Protocol 1 or Basic Protocol 2 (step 3; rapid
method). Score fruit firmness using a scale of 1 to 5 (see Basic Protocol 2 step 3). Thole et al.

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 4. Surface clean tomato fruits by rinsing twice with sterile water directly in the
15-cell potting tray previously used for harvest (step 2). To avoid handling fruits,
pour water over fruits in the tray, and then remove carefully by tilting the tray. Af-
ter rinsing, wipe dry the surface of each fruit with a lint-free towel sheet, and place
fruits back into the tray on top of a clean paper towel pushed into the bottom of
the cell. From this point onward, handle fruits with gloves to maintain semi-sterile
conditions. Finally, carefully wipe each fruit using a paper towel sheet sprayed with
70% ethanol.
An additional cleaning step with 10% (v/v) bleach (see Basic Protocol 2 step 4) can also
be performed; however, due to the short incubation time of the pathogen test (3 days), it is
not essential. Omitting this step reduces the workload and saves time during large-scale
experiments.

5. Surface clean (e.g., spray) with 70% ethanol 40-cell plant potting trays, propagator
trays, and lids or any equivalent containers in which inoculation and incubation will
be subsequently undertaken. To maintain a highly humid environment during incu-
bation (step 8), add two towel sheets soaked with 100 to 150 ml sterile water into
the propagator tray, underneath the 40-cell tray inlay. Label each fruit with a perma-
nent marker near the calyx attachment zone, and place, top up, in a cell. When using
small fruits (like cherry tomatoes), add plastic caps from 50-ml centrifuge tubes (or
equivalent support) on the bottom of the cell. Label each tray, and record the position
of individual tomatoes on the tray (Fig. 4B).
Tomatoes should be well spaced in the tray (16 to 20 fruits per 40-cell tray, depend-
ing on fruit size) to allow easy wounding and inoculation without touching other fruits
(Fig. 4A). It is also important to make sure that the tomatoes are stably positioned and
cannot move to ensure that the inoculum droplets remain on the fruit surface (step 7).
Rolled masking tape or double-sided tape can be placed at the base or on the side of
each fruit to secure fruits into position.
Include control fruits in each experiment and/or tray. It may be useful to include geno-
types with a known level of fungal susceptibility. Including standard genotypes such
as S. lycopersicum cv. M82 or cv. MoneyMaker will enable accounting for potential
experiment-to-experiment and year-to-year variations.

Pathogen test
6. Wounding surface-sterilized tomatoes: Adjust the number of wounds according to
tomato fruit size, and space wounds evenly around the upper half of the fruit. Wound
large tomatoes (e.g., S. lycopersicum cv. M82 fruits; ∼40 to 50 cm high × 40 to
50 cm wide × 50 to 60 cm deep) four times at 0°, 90°, 180°, and 360° positions.
Wound medium-sized tomato fruits (∼30 to 40 cm × 30 to 40 cm × 40 to 50 cm)
three times, and wound small tomatoes (e.g., S. pennellii or S. lycopersicum cv.
MicroTom fruits; ∼20 to 30 cm × 20 to 30 cm × 30 to 40 cm) only once or twice.
Use a 200-μl pipette tip to pierce and wound (1 to 2 mm deep) fruit surface (Fig.
5A). Change tip between genotypes to avoid sap cross contamination.
Adjustments to the number of wounds are made to prevent lesions overlapping with each
other and to ensure consistent levels of infection. As a rule of thumb, wounds should be
separated by ∼20 mm. It is also important to wound fruits consistently.
After wounding, fruit sap may come out of the wounds (especially for fruits that are very
soft 2 weeks after breaker) and should be carefully dried off with a clean paper towel to
prevent subsequent dilution or spillage of inoculum. Usually, tomatoes are wounded in
batches of 16 to 32 tomatoes (i.e., one to two experimental trays of tomato fruits) before
proceeding with wound inoculation (step 7).

7. Inoculating wounded tomatoes: Following wounding of fruits, carefully inoculate

Thole et al. 5 μl of 1 × 105 spores/ml pregerminated spore suspension on top of each wound
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Current Protocols in Plant Biology
Figure 5 Fungal susceptibility test. (A) Wounding of tomato fruit surface with a 200-μl pipette tip.
(B) Inoculation of surface wound with a 5-μl spore inoculum droplet. (C) Medium-sized tomato fruit
with one inoculation droplet (indicated by a white arrow) on each of the four wound sites.

Figure 6 Fungal susceptibility test. (A) Storage of inoculated tomato fruits in a controlled environ-
ment cabinet in sealed containers. (B) Optional adjustment of light intensity using sheets of white
paper.

(Fig. 5B). Deposit inoculum onto wound surface; do not inject inside fruit. Shake
spore suspension frequently (after every third or fourth fruit inoculation) to ensure
that spores do not sediment. After all fruits have been inoculated, seal lid tightly
onto the propagator tray using Parafilm (Fig. 6).
Inoculum usually remains visible as a droplet on top of the wounds up to 1 day post
inoculation (DPI; Fig. 5C).
Sealing trays is important for biosafety reasons and to maintain high levels of humidity
within the tray.
Handle containers with inoculated tomatoes very carefully to ensure that spore suspen-
sion droplets remain on top of each wound. The less handling the better.
8. Incubate trays with tomatoes for 3 days at 20°C and 70% humidity with a 12-hr
light/12-hr dark cycle at 70% light intensity (∼270 μmol) in a controlled environ-
ment room or cabinet. Cover lid of each propagator tray with a sheet of white paper
to reduce light intensity (Fig. 6B) if the lighting level of the incubator cannot be
adjusted to ∼270 μmol.
9. Measure and record lesion size (width and height) at 3 DPI using a caliper
(Fig. 7A). Score and record lesion aspect (e.g., presence or absence of mycelium
growth on fruit surface; Fig. 7B,C). Combine data with information previously col-
lected on fruit color, texture and softness, age (e.g., 14 to 17 days after breaker), and
weight (see step 3). Analyze data and combine for at least 10 biological replicates.
Thole et al.
Compare to well-characterized, control genotypes.
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Current Protocols in Plant Biology
 Figure 7 Fungal susceptibility test. (A) Digital caliper used to measure fungal lesions. (B) Lesions
on tomato fruits 3 days after wound inoculation. The width and height (white dotted arrows) of each
lesion (white dotted circle) are measured. (C) Examples of lesions on different types of tomato
fruits. Mycelium developing on certain wounds is indicated by a white arrow, and some lesions are
highlighted with a dotted circle.

Lesions are usually circular or ovoid soft tissue areas with a noticeable color change
around the wounding point.
Fruit volume (height × width × depth) can be used as an alternative to weight, but de-
termining fruit volume is more labor intensive.
As an order of magnitude, lesions of S. lycopersicum cv. M82 have a diameter of
∼15 mm at 3 DPI. Expect limited to no fungal growth for fruits from tolerant or resistant
genotypes; expect lesions exceeding 20 mm for (very) susceptible genotypes.
10. Dispose of tomato fruits inoculated with Botrytis and other contaminated consum-
ables (i.e., 40-cell plant potting tray) according to best laboratory practice (i.e., dou-
ble bagged, treated as BSL 2 waste, and autoclaved). Treat propagator trays and lids
that have not been in direct contact with tomatoes using bleach, and rinse with water.
Experiments with the pathogen B. cinerea can be carried out only in designated labora-
tories holding a license approving work with BSL 2 pathogens.

SUPPORT PREPARATION OF BOTRYTIS SPORE INOCULUM

PROTOCOL
Here we report a protocol for the isolation of Botrytis spores used as an inoculum for
fruit wound inoculations based on the methods described in Schoonbeek, van Nistelrooy,
and de Waard (2003); Stefanato et al. (2009); and Zhang et al. (2013).
Materials
B. cinerea strain B05.10 in 15% (v/v) glycerol, stored at −80°C (see Schoonbeek
et al., 2003; Stefanato et al., 2009; Zhang et al., 2013)
Potato dextrose agar (PDA) solid medium (see recipe)
Malt extract yeast agar (MEYA) solid medium (see recipe)
Tween 80 (e.g., Sigma-Aldrich, cat. no. P4780)
Sterile distilled water
Potato dextrose broth (PDB) liquid medium (see recipe)
20°C, 70% humidity incubator with 12-hr light/12-hr dark cycle, 70% light
Thole et al.
intensity
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Current Protocols in Plant Biology
 Scalpel with no. 11 blade (e.g., R&L Slaughter, cat. nos. 233-5454 and 233-5334)
Fine forceps, no. 5 (e.g., TAAB Laboratories Equipment, cat. no. T083)
Micropore tape (e.g., 3M, cat. no. 1530-0)
L-shaped spreader, sterile (e.g., StarLab, cat. no. E1412-1005)
5-ml pipette tip (e.g., StarLab, cat no. E1070-2596) plugged with glass wool (e.g.,
Sigma-Aldrich, cat. no. 18421)
15-ml centrifuge tube, disposable, sterile (e.g., Corning, cat. no. 430790)
Benchtop centrifuge (e.g., Eppendorf Centrifuge 5810)
1.5-ml microcentrifuge tube
Hemocytometer (e.g., Thoma cell counting chamber; 0.0025-mm2 mini squares,
0.1 mm-depth)
CAUTION: Perform all steps involving Botrytis macrospore inoculum in a laminar flow
hood suitable for working with BSL 2 pathogens.
1. Culture a 15% glycerol stock aliquot of fungal pathogen B. cinerea strain B05.10
on PDA solid medium at 20°C with a 12-hr light/12-hr dark cycle. After 1 week,
cut three small agar blocks (about 0.02 cm3 ) with B. cinerea mycelium from the
PDA solid medium, and using fine forceps place blocks upside-down (i.e., mycelium
against medium) onto MEYA solid medium. Incubate mycelium for about 10 to 14
days at 20°C with a 12-hr light/12-hr dark cycle until sporulation is observed. Plates
are sealed with micropore tape.
To prepare a 15% glycerol stock of B. cinerea strain B05.10, use freshly isolated spores
resuspended in water (see step 2), and add sterile glycerol to a final concentration of 15%
(v/v). For example, add 150 μl sterile 100% glycerol to 850 μl spores suspended in water.
Make several aliquots of the glycerol stock, and store at –80°C until use. Avoid freeze–thaw
cycles.
To inoculate PDA plates, the glycerol stock aliquot needs to be completely thawed as spores
sediment to the bottom of the tube.
2. Isolate spores (macroconidia):
a. Flood B. cinerea mycelium developed on a MEYA plate (step 1) with 10 to 15 ml
water containing 0.05% (v/v) Tween 80.
b. Carefully scrape surface of mycelium with a sterile L-shaped spreader to detach
spores.
At this point, a color change from light to dark gray is observed.
c. Pipette out crude spore suspension, and filter through a 5-ml pipette tip plugged
with glass wool placed into a sterile 15-ml centrifuge tube.
d. Centrifuge filtered suspension 5 min at 129 × g, 20°C, and then carefully discard
supernatant.
e. Resuspend pelleted spores in 10 ml sterile water, and count spores using a 10-fold
diluted aliquot in a hemocytometer.
f. Centrifuge spores 5 min at 129 × g, 20°C, and resuspend in sterile water to adjust
the concentration to 2.5 × 107 spores/ml (final stock solution) in 1.5-ml microcen-
trifuge tubes.
Spore stocks can be kept in water at 4°C for about 2 weeks. Minimize exposure to room
temperature to prevent spore germination in a non-nutrient solution.

3. For fruit inoculum, dilute 2.5 × 107 spores/ml spore stock solution in 0.25× PDB
liquid medium to 1 × 105 spores/ml (e.g., 40 μl spore stock suspension in 10 ml of
0.25× PDB).
The spore suspension concentration can be adapted to fruit size; that is, small fruits such
as S. lycopersicum cv. MicroTom fruits can be inoculated with a half-strength suspension of Thole et al.

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Current Protocols in Plant Biology
 5 × 104 spores/ml (Zhang et al., 2013). However, when a range of genotypes is compared,
it is recommended to use the same spore concentration across the entire experiment.

4. Preincubate B. cinerea spores in 0.25× PDB liquid medium for 1.5 to 2 hr at room
temperature with occasional shaking (i.e., tilting inoculum tube every 30 min) to stim-
ulate and synchronize spore germination.
Following pregermination, the spore suspension can be used to inoculate surface-cleaned
and wounded tomato fruits (see Basic Protocol 3 step 7).
For small-scale experiments, there is generally enough time to clean and prepare fruits
(Basic Protocol 3 steps 4 to 6) while the spores are pregerminating. For large-scale exper-
iments, several batches of spore pregermination need to be timed to coincide with batches
of fruit inoculation.

REAGENTS AND SOLUTIONS

MEYA solid medium
50 g/L malt extract agar (e.g., Thermo Fisher Scientific, cat. no. CM0059)
2 g/L yeast extract (e.g., Merck, cat. no. 103753)
5 g/L agar (e.g., Neogen, cat. no. NCM0236)
Pour into 9-cm petri dishes (e.g., R&L Slaughter, cat. no. 101R2O)
Store sterile medium in 200-ml aliquots at room temperature for up to 6 months
Store solid medium poured in plates, sealed with plastic wrap, at 4°C for up to
3 months
Malt extract agar contains 30 g/L malt extract, 5 g/L mycological peptone, and 15 g/L agar.

PDA solid medium

20 g/L glucose
4 g/L potato extract
17 g/L agar
Pour into 9-cm petri dishes (e.g., R&L Slaughter, cat. no. 101R2O)
Store sterile medium in 200-ml aliquots at room temperature for up to 6 months
Store solid medium poured in plates, sealed with plastic wrap, at 4°C for up to
3 months
PDA medium can also be purchased from commercial sources (e.g., ForMedium, cat. no.
PDA0101).

PDB liquid medium

20 g/L glucose
4 g/L potato extract
Store sterile medium in 200-ml aliquots at room temperature for up to 6 months
PDB medium can also be purchased from commercial sources (e.g., ForMedium, cat. no.
PDB0101).

COMMENTARY
Background Information
The methods to assess post-harvest prop-
erties reported here could also be used by
breeders and researchers to produce new or
improved tomato varieties that show a pro-
longed shelf life and reduced susceptibility
to B. cinerea by means of exploiting natu-
ral variation, plant breeding, or transgenic ap-
proaches. In the past, genetically engineered
Thole et al.
purple and indigo tomato fruits containing
14 of 17
high levels of delphinidin-type anthocyanins
have been shown to exhibit an enhanced shelf
life and were less susceptible to B. cinerea in-
fections (Zhang et al., 2013).
In our protocol for the analysis of shelf
life, we adapted the storage tests commonly
used by the tomato-producing industry for
laboratory-based analytical studies. Mechani-
cal tests can also be carried out using a texture
analyzer to assess tomato firmness by pressure
Current Protocols in Plant Biology
load or shear press tests. These mechanical
analyses can be destructive and lead to punc-
turing of the fleshy fruits. Consequently, these
fruits cannot be used any longer in shelf life as-
says, and this in turn increases the number of
fruits that need to be analyzed for each geno-
type in a given experiment to account not only
for fruit-to-fruit variability but also for loss of
fruit. What is gained in precision using these
numerical methods can be lost due to sampling
errors as tomato fruits do not always mature
uniformly, with some parts of the fruits being
more mature than others.
Critical Parameters
Tomatoes should be harvested from healthy
plants. To allow efficient growth of tomato
plants and to enable good fruit set, yield, and
quality, plants that are grown in glasshouses
should be planted into large pots (minimum
10-L pots) with occasional use of fertilizers
if required. When fungal susceptibility tests
are performed, standard preventative spraying
should be restricted as fungicides, particularly
systemic fungicides, will affect fungal suscep-
tibility tests.
Tomato fruits should be harvested at the
same ripening stage (e.g., 2 weeks after
breaker) to have comparable and reliable re-
sults across experiments. Fruit ripeness is gen-
erally optimal at 2 weeks (and up to 2.5 weeks)
after breaker for post-harvest studies. To cor-
rectly interpret fungal susceptibility levels, it
is critical to perform the pathogen test con-
sistently at the optimal ripening stage as un-
ripe fruits are less susceptible to fungal infec-
tions, especially when necrotrophic fungi are
used. Fruit harvest for shelf life assays has also
been reported at an earlier developmental or
ripening stage (e.g., 7 to 12 days after breaker),
which is an alternative way to analyze storage
properties if performed consistently through-
out an experiment.
During fungal susceptibility tests, it is crit-
ical to ensure that the inoculation droplets
remain on the wounding sites. This can be
achieved by careful handling and minimal
transportation of wound-inoculated tomatoes,
as well as by removing any fruit sap drip-
ping out through the wound sites before in-
oculations. It is also important that the spore
suspension is mixed frequently to maintain
a homogenous inoculum. To promote fungal
development, a high humidity level must be
maintained during the incubation period (e.g.,
by placing water in the bottom of the incuba-
tion container). It is also important to measure
fungal lesion width and length as lesions are
Current Protocols in Plant Biology
not always regularly shaped; a single measure-
ment is a source of spurious variability that
may require a larger number of repetitions.
The Botrytis susceptibility test should be eval-
uated at 3 DPI, as at later stages the fungal
lesions generally overgrow and develop into
each other; this would hinder accurate scor-
ing of lesion size. Mycelium that develops on
the inoculation sites can also start to sporulate
around 4 DPI, and for biosafety reasons the
experiments should be evaluated before any
sporulation.
For shelf life analyses, fruits should be
stored in single layers, not stacked, without
fruit-to-fruit contact or pressure, and prefer-
ably in sealed clean containers. Judging fruit
texture and firmness by using hands enables
the successive scoring of the same fruit dur-
ing the entire duration of storage; this is in
contrast to mechanical texture analysis which
is destructive and requires large quantities of
fruits for weekly measurements.
For assessment of color, shelf life, and
fungal susceptibility, cleaning of fruits is
critical to remove surface contaminants. Each
batch of fruits to be analyzed should include
control fruits, enabling batch-to-batch and
year-to-year comparisons, especially when
large-scale experiments are performed. In gen-
eral, the use of a minimum of 10 biological
replicates per genotype is essential in post-
harvest studies to enable meaningful statistical
analysis.
Troubleshooting
No or poor fungal growth on wound sites
including on control fruits could indicate
that spore stock solutions were not stored or
handled properly (e.g., prolonged exposure
to temperatures above 4°C while resus-
pended in water) or that there was insufficient
pregermination of spores due to a shortened
pregermination period or the use of an in-
correct inoculation buffer. Poor growth could
also be caused by non-homogeneous, poorly
mixed fungal spore suspension, unfavorable
incubation conditions such as low tempera-
tures (below 18°C to 20°C) and low humidity
levels (below 70%), or not harvesting fruits at
the optimal ripening stage.
Unexpected, early rotting of fruits or de-
terioration of fruit quality could indicate that
fruits were picked from diseased plants or that
fruits were not surface cleaned before experi-
ments.
If experiencing unexpected year-to-
year variation of performance for some
Thole et al.
control genotypes, check seed stocks for
15 of 17
 contamination or off-type deviation. Use only
well-characterized seed stocks.
Understanding Results
In our hands, the protocols described here
enabled the reliable and accurate determina-
tion of fruit color, shelf life, and fungal sus-
ceptibility levels of a collection of more than
40 different tomato accessions for researchers
and breeders as part of the TomGEM project
that was designed to evaluate the impact of cli-
mate change on tomato yield and quality in the
context of the European Union’s Horizon 2020
Programme.
Time Considerations
At ∼2 weeks after tomato seed germi-
nation (8 to 10 seeds per genotype), prick
seedlings into 15-cell potting trays or 7-cm2
pots when seedlings are about 5 cm tall with
well-developed cotyledons and when the first
real leaves are emerging.
Transplant 4- to 4.5-week-old tomato
plantlets (3 to 4 plants per genotype) about
1 to 1.5 months after sowing into 11-cm2
pots, when plants reach ∼15 to 20 cm
in height. Subsequently, about 6-week-old
tomato plantlets are potted an additional time
into 4- to 20-L pots.
Depending on tomato genotype and culti-
vation conditions, about 1.5 to 2 months after
sowing, flowering will start. Fruit set will be
observable about 2 to 2.5 months after sow-
ing, and fruits will start reaching breaker stage
about 4 to 6 weeks later (about 3 to 4 months
after sowing).
Score tomato fruit ripening every day to
every other day. Label breaker stage, and ∼2
weeks after breaker perform post-harvest as-
says.
For storage tests, analyze fruit firmness and
weight weekly until fruits are overripe and
can be discarded. This is highly dependent
on genotypes and can last from zero to many
weeks (i.e., months).
For fungal susceptibility tests, score fungal
lesion size 3 days after wound inoculation of
fruits.
Acknowledgments
The authors received funding from the
Horizon 2020 Research and Innovation Pro-
gramme of the European Union through
the TomGEM project under grant agree-
ment no. 679796. The authors thank Dr.
Henk-Jan Schoonbeek (John Innes Centre,
Norwich, UK) for his help and stimulating
Thole et al. discussions.
16 of 17
Conflicts of Interest
The authors declare they have no conflicts
interests.
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