Plant Health Progress 䉬 2022 䉬 0:1–7
1094/PHP-08-21-0109-DG
Diagnostic
Practical and Comprehensive Diagnostic Guide for Black Rot
Q:1 of Brassicas

niel-Go
Mario Da
mez,1 Ella Reeves,2 and Inga M. Meadows2,†
1
Microbiological Research Center (CIMIC), Department of Biological Sciences, Universidad de Los Andes, Bogot
a, 111711, Colombia
2
Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695, U.S.A.
Accepted for publication 19 April 2022.
Q:2 Keywords: bacteriology, diagnostics, ornamentals, plant pathology, vegetables
Q:3 Hosts The genus Xanthomonas includes several economically
important plant pathogenic bacteria that cause disease on various
crops (Hayward 1993; Mansfield et al. 2012; Tang et al. 2021). In
this guide we focus on the vascular pathogen X. campestris pv.
campestris (Pammel) Dowson. This bacterium has a relatively nar-
row host range, causing black rot in Brassica spp. (cabbage, calabr-
ese, canola, cauliflower, broccoli, rapeseed, etc.). However, the
pathogen can also affect crops such as radish (Raphanus spp.), and
ornamental brassicas (Brassica spp., Erysimum cheiri, and
Matthiola incana) (da Silva et al. 2019; Krauthausen et al. 2018;
Tang et al. 2021; Vicente et al. 2001; Williams 1980). A list of sev-
eral high value crops susceptible to X. campestris pv. campestris and
the most common symptoms they display can be found in Table 1.
Pathogen Xanthomonas campestris pv. campestris is a mesophilic,
gram-negative, aerobic, non-spore forming, motile, rod-shaped bacte-
rium responsible for causing black rot in brassica plants, which
results in vascular decay that has the potential to devastate crop yield
and quality (Hayward 1993; Thind 2020). Black rot was first
described by Garman in 1894 (Vicente and Holub 2013) and the
causal bacterium has undergone several name changes over time
(Arlat et al. 1991; Pammel 1895; Rimmer et al. 2007; Thind 2020).
Currently, the preferred scientific name is Xanthomonas campestris
pv. campestris (Pammel 1895) described by Dowson in 1939
(Dowson 1939). The pathogen has been encountered on every conti-
nent where brassica crops are grown (Vicente et al. 2001).
Other bacterial diseases that affect brassicas include bacterial
leaf spot (Pseudomonas syringae. pv. maculicola), bacterial soft rot
Q:4 (Pseudomonas marginalis pv. marginalis), and Xanthomonas leaf
spot (X. campestris pv. armoraciae/X. campestris pv. raphani)
(Rimmer et al. 2007).
Taxonomy
Domain Bacteria; phylum Proteobacteria; class Gammaproteo-
bacteria; order Xanthomonadales; family Xanthomonadaceae;
genus Xanthomonas; species Xanthomonas campestris; pathovar
Xanthomonas campestris pv. campestris. Like all members of the
Xanthomonas genus, X. campestris pv. campestris is a member of
†
Corresponding author: I. M. Meadows; inga_meadows@ncsu.edu
The author(s) declare no conflict of interest.
© 2022 The American Phytopathological Society
PLANT HEALTH PROGRESS 䉬 2022, Vol. 0, No. 0 䉬 Page 1
https://doi.org/10.
the rRNA homology group I (Fialho et al. 1990). The taxonomy
data presented are based on data found in the Taxonomy Browser
Tool on the National Center for Biotechnology Information website
(https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi).
Much debate has surrounded the classification of X. campestris
pv. campestris, as this pathovar, as well as X. campestris pv.
raphani (causal agent of Xanthomonas leaf spot on crucifers and
some solanaceous plants) and X. campestris pv. incanae (causal
agent of bacterial blight on ornamental Brassica spp.), also cause
disease in Brassicaceae crops, ornamentals, and weeds (Tang et al.
2021). Nevertheless, pathogenicity assays have mostly restricted
the campestris pathovar to strains infecting vascular tissue
(though incidents of the bacterium causing bacterial leaf spot in
brassicas have been reported; Wechter et al. 2008). Recent studies
have identified 11 races within the X. campestris pv. campestris
pathovar capable of causing black rot on differential brassica
plants (Cruz et al. 2017; Fargier and Manceau 2007).
Symptoms and Signs
The disease can appear at any point during the growth of the
plant, but seedling infection is of significant importance as early
detection may aid growers in terminating the crop promptly and
thus minimize the misuse of time and resources. If left in the crop,
the disease slows the formation of the stem, which stunts growth,
and the bacterium clogs veins, which impedes transpiration. This
may lead to the appearance of too many symptoms on the market-
able part, rendering the crop not worth harvesting and bringing on
monetary loss.
Bacterial proliferation then causes blackening along the margin of
cotyledons, which later wither and fall off. Infected seedlings may
deteriorate, wilt, and die. Furthermore, seedlings can be systemi-
cally infected from infested seed. In this case, leaves may become
generally yellowish without discrete lesions and veins become
blackened. Symptoms on seedlings are similar to those observed on
mature plants but may be observed depending on environmental
conditions during germination and early growth.
In mature plants, the symptoms of infection (Fig. 1) initially appear Q:5
as yellowing along the margin of affected leaves. Infection pro-
gresses inward from the entry points (stomata, hydathodes, or
wounds; Thind 2020) on the leaf margins toward the base of the leaf.
Lesions appear V-shaped, and the center turns brown. This change in
leaf coloration is followed by a blackening of the vascular tissue,
which is a characteristic symptom of the disease and is a result of the
proliferation of the pathogen and the production of extracellular pol-
ysaccharides, namely xanthan (Katzen et al. 1998). Because the
bacterium can be systemic, new lesions and more blackening of the
vascular tissue may occur throughout the plant (Vicente and Holub
2013). In time, this proliferation in the vascular system can cause
stunted growth, chlorosis, wilting, and infection of the seeds within a
seed crop, which further propagates the disease (Rimmer et al. 2007;
Thind 2020).
In infested fields, bacteria survive between crops on or in seeds
and in debris left in the field (Schultz 1986). Long distance spread
is achieved primarily through the movement of infested seeds.
Transplanting infected seedlings can introduce the pathogen to a
field where black rot had previously not occurred. Secondary
spread is primarily achieved through water splash, with infection
facilitated by the fact that species of Xanthomonas possess a fla-
gellum and are thus motile (Hayward 1993; Thind 2020); how-
ever, human activity in the crop that entails hands or equipment
touching leaves, especially when they are wet, such as harvesting,

TABLE 1
Common symptoms on crops affected by Xanthomonas campestris pv. campestris
Symptomsa
V-shaped
lesions Vascular Defoliation/ Stunted Head
Host (common name) Chlorosis on leaf margins blackening leaf drop growth discoloration
Brassica oleracea var. capitata (cabbage) + + + + + n/a
Brassica oleracea var. botrytis + + + + + +
(cauliflower)
Brassica oleracea var. italica (broccoli) + + + + + +
Brassica oleracea var. gemmifera + + + + + n/a
(Brussels sprouts)
Brassica oleracea var. acephala (kale) + + + + + n/a
Brassica napus subsp. napus (rapeseed) + + + + + n/a
Eruca vesicaria (arugula) + + + – – n/a
Erysimum cheiri (wallflower) + + + n.d. n.d. n/a
Matthiola incana (Brompton stock) + + + n.d. n.d. n/a
a
Not all symptoms may be observed simultaneously and may vary from plant to plant. + indicates that the symptom is present in the host.
– indicates that the symptom is not present in the host. n.d. indicates that the symptom has not been described but its presence cannot be ruled
out. n/a indicates that the symptom is not applicable to the host.

FIGURE 1
Distinctive symptoms and signs of Xanthomonas campestris pv. campestris on representative hosts: A, characteristic V-shaped lesions in the margins of
cabbage in the field; B, bacterial streaming from cut leaf (yellow arrow) (note that light reflection [black arrow] can be confused with bacterial streaming);
C, vascular tissue blackening in cabbage indicating systemic infection; D, lesions on young cabbage leaves; E, marginal chlorosis and vein blackening in
leaves of field-grown Brussels sprouts; and F, chlorosis of the lower leaves, and marginal lesions of ornamental cabbage. Images courtesy G. Holmes (C),
T. Creswell (D) via Bugwood.org, and M. McGrath (E and F).

PLANT HEALTH PROGRESS 䉬 2022, Vol. 0, No. 0 䉬 Page 2

transplanting, and even planting, can contribute to pathogen spread.
Cultivated Brassica crops that have become established as weeds in
and around crop production fields may serve as important pathogen
reservoirs in warmer climates (Schaad and Dianese 1981), but not
in colder climates (Lange et al. 2022). It has also been reported that
insect activities such as pollination may contribute to disease devel-
opment, as insects can serve as mechanical vectors and may create
an entry point for infection by feeding on leaves (Thind 2020; Van
Der Wolf and Van Der Zouwen 2010).
In the presence of free moisture (dew, rain, irrigation, etc.), the
bacterium can multiply at temperatures as low as 16 C (60 F), but
the optimum temperature for the development of the disease is
between 25 C and 28 C (77 F and 82 F). Correspondingly, free
moisture (dew, rain, irrigation, etc.) is needed for initiation and
progression of the disease. Under these circumstances, symptoms
may begin to develop 10 to 14 days postinfection (Thind 2020).
This disease may look similar to other diseases such as Verticil-
lium wilt and Fusarium wilt. Symptoms of Verticillium wilt
(Verticillium spp.) include leaf yellowing, plant wilt, and drying
out of lower leaves. In contrast, leaves affected with black rot do
not turn entirely yellow or dry out. Additionally, mature plants
affected by black rot are not known to wilt (Rimmer et al. 2007).
Fusarium wilt of cabbage or Fusarium yellows (Fusarium oxyspo-
rum f. sp. conglutinans) can cause yellow leaves, plant stunting,
and, in advanced stages, discoloration of the vascular tissue
(Rimmer et al. 2007; Sherf 1979). However, with Fusarium yel-
lows, symptoms typically appear more severe on one side of the leaf
or plant, which is not observed with black rot. Finally, symptoms
similar to black rot can be caused by environmental conditions such
as droughts and floods, as well as by poor cultural practices such as
overwatering or excessive use of fertilizer (Dicklow et al. 2013).
Host Range
The host range of X. campestris pv. campestris comprises many,
if not all, species in the Brassica genus, as most known races have
been isolated from plant species within it (Cruz et al. 2017;
Hayward 1993; Vicente and Holub 2013). including the model
organism Arabidopsis thaliana (Simpson 1990).
Geographic Distribution
X. campestris pv. campestris is distributed worldwide and is pre-
sent wherever Brassica crops are cultivated (Thind 2020). Races
1 and 4 are considered to be prevalent worldwide, with other races
having more modest and localized distributions (Vicente and Holub
2013). The predilection of X. campestris pv. campestris for warm
and humid climates make it more prevalent in tropical areas with
high rainfall, but it is an economically important pathogen in tem-
perate areas and is one of the most common diseases of these crops
in areas such as the northeastern U.S. (Thind 2020; Vicente and
Holub 2013). Though it can survive for short periods of time in the
soil, its survival and distribution are mostly dependent on the pres-
ence of infested seeds and plant debris (Silva J
unior et al. 2020).
Pathogen Isolation
X. campestris pv. campestris can be isolated from crop debris
and in some cases soil (Silva J
unior et al. 2020), though the most
common isolation is done from symptomatic tissue and/or seeds.
It is important to use aseptic technique throughout the procedure
and adequate biosafety measures, if pertinent. It is important to
note that the procedure can be negatively affected by the presence
of contaminant microbiota in samples (Silva J
unior et al. 2020).
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Isolation from Seeds
Isolation from seeds is especially important for detection and
crop protection, as a very low level of seed infestation can result
in high disease incidence in the field (Roberts et al. 1999). How-
ever, it can be difficult to monitor seed infestation by X. campest-
ris pv. campestris because the pathogen may be present on the
seed at levels too low for detection, but adequate to start an epi-
demic (ISTA 2015). A more comprehensive protocol for the
detection of X. campestris pv. campestris has been published else-
where (ISTA 2015), and we are limiting the scope of this diagnos-
tic guide to isolation from plant tissue.
Isolation from Plant Tissue
To confirm bacterial presence in symptomatic leaf tissue, a small
piece of the sample (approximately 0.5 cm × 0.5 cm) spanning the
margin of the lesion can be placed in sterile saline solution
(0.85% NaCl) (Bila et al. 2013) or sterile distilled water on a glass
slide and observed for the presence or absence of bacterial ooze
streaming out of tissue with the aid of a stereoscope or compound
light microscope. If ooze is observed, the bacterial suspension can
then be serially diluted and streaked onto nutrient-rich media
(nutrient agar [NA], King’s B, or yeast dextrose carbonate agar
[YDC]) (Schaad et al. 2000) or selective media such as
mCS20ABN, FS agar, or NSAR/NSARF (nutrient agar, 5 g/liter
of sucrose, and 100 lg/ml of rifampicin [NSARF: NSAR supple-
mented with chlorothalonil and 0.01 g/liter of thiophanate-
methyl]) medium (ISTA 2015). If nonselective media are used, be
aware that other contaminating bacteria may be present. It is
important to subculture suspect colonies to obtain a pure culture.
Isolation from leaf samples can also be done through cutting
small pieces (0.25 cm2 to 1 cm2) from symptomatic tissue span-
ning the margin of the lesion. These pieces can then be suspended
in 10 to 15 ml of sterile water and vortexed for 2 to 3 min. The
bacterial suspension can then be diluted into serial tenfold dilu-
tions and streaked onto nutrient-rich and/or selective media (FS,
mCS20ABN), ensuring the adequate distribution of the liquid
with a sterile bent rod or loop. Plates should be incubated at 28 C
for 2 to 4 days before checking for growth of X. campestris pv.
campestris. Again, if tissue is not surface sterilized and nonselec-
tive media is used, be aware that other contaminating bacteria
may be present.
Pathogen Identification
The pathogen can be identified by testing infected plant material
or an isolated pure culture (Fig. 2). If testing plant material, the
Agdia ImmunoStrip (Agdia, Elkhart, IN, USA) or the Agdia
ImmunoBlot Kit (Agdia, Elkhart, IN, USA) are relatively afford-
able and reliable to detect Xanthomonas spp. or X. campestris,
respectively. A species-specific PCR assay developed by Park
et al. (2004) can be used with plant or seed tissue to detect
X. campestris. However, none of these tests will allow the user to
identify the pathovar (Fig. 3).
The most reliable methods to detect X. campestris (or X. campestris
pv. campestris) begin with a pure culture. When grown on nutrient-
rich media, X. campestris pv. campestris colonies are yellow, round,
translucent, raised, and mucoid (Fig. 2), but this is not a reliable mor-
phological characteristic for identification to species. After initial iso-
lation, X. campestris pv. campestris can be identified through the use
of several biochemical and molecular techniques listed in Table 2
with their advantages and disadvantages. A classic biochemical assay
such as the analytical profile index (API) can provide an inexpensive
identification but may be subject to false positive and inaccurate
results and is not capable of species or pathovar differentiation
(Schaad et al. 2000; Thind 2020), so additional analyses are recom-
mended. The following assays have been used in recent years to suc-
cessfully identify X. campestris from pure culture: traditional
enzyme-linked immunosorbent assay (ELISA) (Alvarez and Lou
1985; Franken et al. 1992), Biolog identification system (Massomo
et al. 2003), polymerase chain reaction (PCR) with species-specific
primers (Berg et al. 2005; Inoue et al. 2021; Park et al. 2004), PCR
followed by sequencing (Cruz et al. 2017), species-specific quantita-
tive PCR (qPCR) (Eichmeier et al. 2019), and pathogenicity tests
(Fargier and Manceau 2007) (Fig. 3). ELISA, Biolog, biochemical
assays, and pathogenicity tests may be performed with a pure culture,
but only pathogenicity testing will provide identification to the path-
ovar level. PCR followed by sequencing (Berg et al. 2005) and
species-specific PCR (Park et al. 2004) each can identify X. campest-
ris but cannot distinguish pathovar campestris from other pathovars.
Pathovar identification can be completed by using the pathovar-
specific assay by Rubel et al. (2019), which can detect X. campestris
pv. campestris and does not cross-react with other pathovars, or by
pathogenicity tests (Bila et al. 2013; Eichmeier et al. 2019). Repeti-
tive PCR-based fingerprinting (rep-PCR) (Lema et al. 2012) can be
used to identify X. campestris pv. campestris races 1, 3, 4, and 6
(Afrin et al. 2018, 2020; Rubel et al. 2017), but other races cannot
yet be identified using molecular tools. Therefore, the most reliable
option for race identification is the use of differential cultivars and
subsequent observation of the presence or absence of a hypersensi-
tive response (Table 3). Resistant lines exhibit cell death or necrosis
around the inoculation point, a hypersensitive response, while sus- Q:6
ceptible lines do not (Cruz et al. 2017; Fargier and Manceau 2007;
Vicente et al. 2001).
Pathogen Storage
Prior to preservation, pure cultures of X. campestris pv. campest-
ris can be grown on nutrient-rich media and incubated at 28 C for
24 to 48 h (Liao and Shollenberger 2003). Strains can be stored for
a prolonged period of time (6 to 10 years) in sterile distilled water
or phosphate-buffered saline (PBS) (pH 7.2; KH2PO4, 15.44 lm;
NaCl, 1.55 mm; Na2HPO4, 27.09 lm; Life Technologies, Rock-
ville, MD, USA) at room temperature (20 C), but moderately low
temperatures (£10 C) under these conditions can reduce the viabil-
ity of the bacteria (Ruissen et al. 1993). The preferred method for
long term storage and preservation of strains is in screw-capped
vials containing nutrient broth supplemented with 30% glycerol
(v/v) and frozen at −70 to −80 C (Silva J
unior et al. 2020; Vicente Q:7
et al. 2001). The bacteria can be recovered on nutrient-rich media
such as NA, brain heart infusion agar (BHIA), Pseudomonas agar F
(PAF), or yeast-malt (YM) agar (Liao and Shollenberger 2003).

FIGURE 2
Growth of Xanthomonas campestris pv. campestris on: A, nutrient agar (NA); B, NSAR medium; C, King’s B (KB) medium; and D, yeast dextrose carbonate
(YDC) agar. Image courtesy F. Rotondo, The Ohio State University (D). Images A to C were taken 5 days after inoculation and image D was taken 2 days after
inoculation.

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 Plant sample (plant ssue)

Rapid diagnosis Isolaon

Typical colony Non-typical colony

morphology morphology

Agdia Agdia ELISA Race

DNA Extracon DNA Extracon
Xan Immunoblot idenficaon
Immunostrip® Kit for Xc
Biolog
RT-PCR* idenficaon
(Eichmeier et al. 2019) system

Xanthomonas sp. Species-specific PCR* Biochemical

(Berg et al. 2005, tests (API)
Eichmeier et al. 2019,
Xanthomonas campestris Inoue et al. 2021)

PCR,
Xanthomonas campestris pv. campestris sequencing

*To date, no primers used in real-me polymerase chain reacon (RT-PCR) or PCR exist that can separate pathovars of X. campestris, although they can detect pv.
campestris

FIGURE 3
Diagnostic workflow outlining assays used to identify Xanthomonas campestris pv. campestris, the causal agent of black rot of brassicas.

TABLE 2
Advantages and disadvantages of assays used for the detection and identification of Xanthomonas campestris pv. campestris
Assay Advantage Disadvantage
Agdia ImmunoStrip  Result within minutes  Identification to genus only
 Low cost  Low sensitivity
 Transportable
 No technical skills required
 Detection in plant material
Agdia ImmunoBlot  Result within hours  Low sensitivity
 Low cost
 Minimal technical skills required
 Detection in plant material
 Identification to species
Enzyme-linked immunosorbent  Result within hours  Identification to genus only
assay (ELISA)  Low cost  Pure culture required
 Minimal technical skills required
Biolog  Identification to species  High initial cost
 Minimal technical skills required  Result in days
 Pure culture required
Polymerase chain reaction  Result within hours  High initial cost
(PCR) (species specific)  Identification to species (or pathovar)  Some technical training required
 Detection in plant material
Polymerase chain reaction  Identification to species  Some technical training required
(PCR) followed by  High initial cost
sequencing  Result in 2 to 3 days
 Pure culture required
Quantitative polymerase chain  Identification to species  Some technical training required
reaction (qPCR)  Result within hours  High initial cost
 Detection in plant material
Analytical profile index (API)  Identification to species  Prone to inaccurate results
 Low cost  Result in days

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 TABLE 3
Differentially susceptible cultivars and their race-specific responses to the different Xanthomonas campestris pv. campestris
races currently known (adapted from Cruz et al. 2017; Fargier and Manceau 2007)
X. campestris pv. campestris race pathogenicity
Differential
cultivars 1 2 3 4
Wirosa F1 (B. oleracea) +a + + + +
Just Right Hybrid Turnip + + + –b
(B. rapa)
Seven Top Turnip (B. rapa) + – + –
PI 199947 (B. carinata) – + – –/(+)c
Florida Broad Leaf Mustard – + – –
(B. juncea)
Miracle F1 (B. oleracea) + – – +
a
+ indicates compatible interaction (susceptibility).
b
– indicates incompatible interaction (resistance).
c
(+) indicates weak pathogenicity.
Pathogenicity Tests
Pathogenicity tests should be performed when X. campestris pv.
campestris has been isolated from a plant previously not reported
as a host, when attempting to determine pathovar, and for identify-
ing weed hosts and susceptibility. Protocols for pathogenicity tests
involving X. campestris pv. campestris have been described by
Romero et al. (2008), Rosenthal et al. (2018), and others. To per-
form pathogenicity tests with X. campestris pv. campestris, fresh
inoculum of the bacterium is needed. Frozen or recently purified
cultures should be grown in nutrient broth or plated on NA and
incubated at 28 C between 24 and 48 h; if in liquid culture, the
containers should be maintained on a shaker to ensure thorough
colonization. After incubation, the bacterial culture should be
resuspended in distilled water and the absorbance measured in a
spectrophotometer at a wavelength of 600 nm while using dis-
tilled water as a blank. The OD600 of the inoculum should be
between 0.08 and 0.1, equivalent to a bacterial concentration of
approximately 108 cfu/ml. However, a standard curve should be
generated by dilution plating of known X. campestris pv. cam-
pestris strains to confirm the concentration of bacteria at this
wavelength. Pathogenicity assays should be performed using
susceptible cultivars such as those listed in Table 3. Plants
should be a minimum of 3 weeks old before inoculation. Follow-
ing the protocol employed by Rosenthal et al. (2018), the foliage
of the plants is sprayed with the bacterial inoculum until runoff
occurs, then the plants are enclosed in plastic bags to maintain
high humidity for infection. After 48 h, bags are removed, and
plants are maintained in a greenhouse (20 to 22 C) for 14 to 16
days. At this time, symptoms such as chlorotic lesions or vascu-
lar blackening should be apparent in the inoculated plants. Inoc-
ulated plants should be compared with mock-inoculated negative
control plants that were sprayed with the same liquid medium
but lacking the inoculum and with a known host inoculated with
the pathogen to serve as a positive control. The bacterial patho-
gen should then be reisolated from the infected tissue and com-
pared with the previously isolated bacteria, thereby fulfilling
Koch’s postulates (Rosenthal et al. 2018).
Acknowledgments
The authors thank Dr. Lina Quesada-Ocampo and the WolfPack
Colombia Internship program at NC State University in the College of
Agriculture and Life Sciences for providing this avenue for collabora-
tion. The authors are grateful for the in-depth and thoughtful comments
and suggestions provided by the reviewers that have helped to improve
the completeness of this guide.
PLANT HEALTH PROGRESS 䉬 2022, Vol. 0, No. 0 䉬 Page 6
5 6 7 8 9 10 11
+ + + + + +
+ + + + – + +
+ + + – – – –
(+) + + – – + +
+ + – – – – +
+ + + – – + +
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