Biology and management of

bacterial spot in tomato and pepper

Jeff Jones
University of Florida
 Bacterial spot of tomato and pepper

Photo credit: Vallad

• Xanthomonas euvesicatoria – pepper and tomato

• Xanthomonas hortorum pv. gardneri - pepper and tomato

•Xanthomonas perforans – tomato and recently pepper

•Xanthomonas vesicatoria - pepper and tomato

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-3059.2001.00644.x/epdf
Original isolation of Xanthomonas spp. associated
with tomato

Xanthomonas vesicatoria ca. 1920

Xanthomonas gardneri. 1957

Xanthomonas perforans 1991

Xanthomonas euvesicatoria ca. 1920

Distribution of A (Xanthomonas euvesicatoria)
and B (X. vesicatoria)

Xanthomonas vesicatoria

Xanthomonas euvesicatoria (85-10)

Distribution of Xanthomonas gardneri

X. gardneri was in recent years determined to be

the most prevalent on tomato in Canada
 Distribution of Xanthomonas perforans

§First identified in Florida on tomato in 1991

§Two novel avr genes (avrXv3 and avrXv4)

§Only infects tomato. (avrXv3 elicits HR in pepper and tomato (Xv3)

§X. perforans produces 3 bacteriocins inhibitory to X. euvesicatoria.

Recent spread of X. perforans and X. hortorum pv. gardneri

Xanthomonas vesicatoria
Xanthomonas euvesicatoria
Xanthomonas perforans
Xanthomonas gardneri
Xanthomonas sp.

Figure 1. Current worldwide distribution of xanthomonads associated with pepper and tomato
Potnis et al. 2015 Mol. Plant Path
 Evolution of of bacterial spot of tomato
pathogen in Florida
X. euvesicatoria
1950 1990 2000 2010 2015
First showed First showed copper
antibiotic resistance resistance in plant
in the field (Stall and pathogenic
Thayer, 1962; Thayer resistance (Marco
and Stall, 1962) and Stall, 1983)
 Bacterial spot of tomato in Florida

X. perforans
T3
1950 1990 2000 2010 2015
X. euvesicatoria

X. perforans produces 3
bacteriocins against X.
euvesicatoria
 X. perforans dominated Florida fields after 1991 and
completely replaced X. euvesicatoria on tomato by 2006
Species wipe-out on tomato in Florida
Water treated

Bcn mutants

Zone of inhibition of X. WT T3 treated

euvesicatoria due to
diffusion of bacteriocin
produced by X. perforans
strain.
Growth of X. euvesicatoria T1 strain in
tomato leaf over time following inoculaton in
leaflet previously inoculated with wild-type
X. perforans and mutants.

Bacteriocin production by X. perforans suppresses growth of X. euvesicatoira

Hert et al. 2005 Appl Env Microbiol

Hert et al. AEM 2005

 Xanthomonas perforans

§First identified in Florida on tomato in 1991

§Two novel avr genes (avrXv3 and avrXv4)

§Produces 3 bacteriocins inhibitory to X. euvesicatoria.

§Only infects tomato (avrXv3 elicits HR in pepper and tomato (Xv3)

 Bacterial spot Xanthomonas races continue to evolve

T1 was only know race in Florida before 1991.

T3 became prevalent after 1991.

In 2006 survey, approx 77% of the strains

collected were T4 type.

In 2012 100% were X. perforans race T4

Race shift in Florida population

X. perforans X. perforans X. perforans

(T3) (T3,T4) (T4)
1950 1990 2000 2010 2015
X. euvesicatoria (T1)

Stall et al. 2009, Ann. Rev. Phytopathol.

Horvath et al, 2012, Plos One
Schwartz, 2015, Frontiers in Microbiology
 Bacterial spot of tomato in Florida

Tomato strains in
Florida designated All strains
X. perforans
tomato race 1 (T1) copper-
T3
in late 1980s tolerant
1950 1990 2000 2010 2015
X. euvesicatoria X. perforans
T4

–AvrXv3

T4 strains vary in ability to produce bacteriocins but are insensitive to T3-

produced bacteriocins
 Host range on pepper

In 2010, our lab received infected pepper leaf samples with characteristic leaf perforation
symptoms suggestive of X. perforans from South Florida.

Host-range shift/expansion in Florida population

 Host range expansion to pepper by Group 2 strain

Evidence for recombination between X. perforans and X. euvesicatoria

Strains lacF lepA gyrB fusA gapA gltA

Xp Type Xp1 Xp1 Xp1 Xp1 Xp1 Xp1
Xe Xe1 Xe1 Xe1 Xe1 Xe1 Xe1
Xp Group 2 Xp1 Xp1 Xe1 Xp1 Xe1 Xp1

The 2010 X. perforans strain from

pepper is Group 2 strain.

Timilsina et al. 2015 AEM

 Insights into population structure and host range shift of
Xanthomonas perforans using whole genome sequencing

South Georgia

During the field surveys in 2006, 2012 and 2013,

bacterial spot strains were isolated from
Infected tomato leaves.

All 2012 and 2013 strains were T4.

Schwartz et al. 2015, Frontiers in Microbiology

Multiple genetic groups of X. perforans in Florida
Core genome phylogeny

Group 3

Group 1

Group 2

Timilsina, et al.,2019. Frontiers in Microbiol.

Multiple genetic groups of X. perforans in Florida
Core genome MLST fingerprints indicate recombination

Group 3 142 genes with specific allele

45% from X. euvesicatoria
5 alleles from X. a. citrumelo

Group 1 Varying numbers of

X. euvesicatoria alleles

Group 2 96 genes with specific allele

81% from X. euvesicatoria

Timilsina, et al.,2019. Frontiers in Microbiol.

Multiple genetic groups of X. perforans in Florida
Core genome is correlated with expansion to pepper Xp 91-118
Xp5-6 Xp17-12 Xp2010 TB9
avrXv3 mutant

Group 3

Xp 91-118
avrXv3 mutant Xp5-6 Xp17-12 X
9
Figure 2. Symptoms produced on pepper b
8
Group 1 that do not have AvrBsT. The first two pan
strains, Xp17-12 is a unique strain, and the
7

Log CFU/cm2
Group 2 strains. Xp2010 was isolated from
6
others were isolated from tomato.
5
Figure 3. In planta growth of mutant and w
4 genetic background (indicated in white tex
3 on day 8 post-inoculation. AvrBsT knock-o
2 1A 1A 2 2 1A 1A
9 2 1A 1B 2 background (blue and purple bars) have m
2
magnitude higher growth than knock-outs Figu
Xp 91-118

GEV872 avrBsT
GEV909 avrBsT
GEV1001 avrBsT
GEV872 avrBsT

TB15
+ cAvrBsT
GEV1001
GEV839
GEV872
GEV909

GEV839 avrBsT

Xp91-118 avrXv3
8 that
avrXv3 mutant Xp5-6 Xp17-12 Xp2010 TB9
background TB15 The Grou
(green and orange).
7 the same level of growth in pepper as strain
TB1

Log CFU/cm2
Group 2 6
knock-outs show much greater growth tha
Grou
Group 2 strain that naturally lacks AvrBsT
other
5
mutated in the race 3 strain Xp91-118Figu (ligh
4 lacks AvrBsT. gene
3 on da
2 1A 1A 2 2 1A 1A 2 1A 1B 2 back
2
magn

GEV872 avrBsT
GEV909 avrBsT
GEV1001 avrBsT
GEV872 avrBsT

TB15
+ cAvrBsT
GEV1001
GEV839
GEV872
GEV909

GEV839 avrBsT

Xp91-118 avrXv3
9
back
Figure 2. Symptoms produced on pepper by X. perforans strainsthe s
8 that do not have AvrBsT. The first two panels are Group 1B Grou
7 strains, Xp17-12 is a unique strain, and the last three panels are knoc
Log CFU/cm2

Group 2 strains. Xp2010 was isolated from pepper, while the muta
6
others were isolated from tomato. lacks
5
Figure 3. In planta growth of mutant and wildtype strains by
4 genetic background (indicated in white text on bars) in pepper
3 on day 8 post-inoculation. AvrBsT knock-outs in the Group 2
2 1A 1A 2 2 1A 1A 2 1A 1B 2
2 Neha Potnis, Auburn U.
background (blue and purple bars) have more than an order of
magnitude higher growth than knock-outs in the Group 1
872 avrBsT
909 avrBsT
1001 avrBsT
872 avrBsT

TB15
+ cAvrBsT
GEV1001
GEV839
GEV872
GEV909

839 avrBsT

-118 avrXv3
background (green and orange). The Group 2 knock-outs show
the same level of growth in pepper as TB15 (dark grey), a
Group 2 strain that naturally lacks AvrBsT. The Group 1A
knock-outs show much greater growth than when avrXv3 is
 Xanthomonas perforans in Florida over time
Prior to 1991, X. euvesicatoria was the only species found to cause bacterial spot of tomato in Florida. X. perforans
quickly took over. X. perforans success thought to be due to its production of bacteriocins against X. euvesicatoria.

100% T4 strains;
X. perforans tomato race phylogenetic groups 1 and 2;
4 (T4) strain first appears. 32% resistant to
streptomycin.

1991 2006 2017

1998 2012
X. perforans first appears as
a tomato race 3 (T3) strain;
70% T4 / 30% T3;
phylogenetic groups 1 and 2 ?
phylogenetic group 1; dominant, one phylogroup 3 strain;
sensitive to streptomycin. 5% resistant to streptomycin.

No effective resistance has been deployed commercially to select against particular strain types.
Timilsina et al. (2016, 2019)
 2017-18 Florida collection
• 585 Xanthomonas strains, from field-grown
tomatoes planted Fall 2017, representing:
o 70 fields
o 22 farms
o 15 grower operations Transplant facility
o 8 transplant facilities Farm
o 8 counties
o 23 cultivars
o 8 seed producers

• Overall, this collection represents relative Counties (by field)

proportions of tomato production by county.
4%2%
7%
• This is the largest bacterial spot collection 7% 36%
within a single season, and we are 7%
characterizing all strains for certain
7%
phenotypic and genotypic characteristics.
30%

Manatee Collier
Desoto Decatur (GA)

Klein-Gordon et al.
ISME 2021
Phytopathology. 2020
Multiple genetic groups of X. perforans in Florida
2017-2018 statewide survey

Six Xp core gene clusters in

the population, whereas
only three clusters were
identified in previous
Florida-based genomic
studies

Fig. 1: Phylogenetic tree of Xanthomonas perforans based on genetic

distance of core gene SNPs, corrected for recombination.
Klein-Gordon et al., 2021 ISME
Worldwide collection of X. perforans

270 strains of X. perforans from seed and large-scale tomato producers of the world.

Strain breakdown:
USA – 180
Canada – 4
Mexcio – 5
Brazil – 11
Nigeria – 10
South Africa – 4
Ethiopia – 5
Italy – 5
Iran – 5
China – 9
Thailand – 18
Australia – 9
South East Asia – 4

Timilsina et al. (In preparation)

 Core genome phylogeny

• Based on 887 genes.

• The tree does not include the 9
genomes from Nigeria and 2 from
Thailand that skewed the tree due
to the variation these strains carry.
• Relative geographic clusters with
genomic signals representing strain
migration between the two
countries is evident. (Further shown
based on genomic clustering in the
NMDS plots)

Timilsina et al. (In preparation)

 0 40 100
% ID

Global snapshot of X. perforans

core genome variation GEV2410
GEV1911
GEV1912
GEV2109
GEV2420
GEV2123
GEV2072
GEV1916
GEV1917
GEV1913
GEV2089
GEV1919
GEV2384
GEV2389
GEV1918

GEV2048
GEV2118
GEV2400
GEV2407
North America
GEV2047
GEV2050
GEV2052
GEV2059
GEV1920
Africa
GEV2393
GEV2403
GEV2113
GEV2067
GEV2088
South America
Australia
GEV2060
GEV1993
GEV1992
GEV2058
GEV2114

Asia
GEV2133
GEV2087
GEV2011
GEV1914
GEV1915

Europe
NC-47
NC-373
GEV2055
GEV2013
GEV2391
GEV1991
GEV2119
Mexico-LT3
NC-252

NC, FL
NC-289
NC-242
MRS-30P-011
NC-67
16-1165A1
GEV2111
GEV2126
GEV2408
GEV2399
GEV2388
GEV2110
GEV2117
GEV2135
GEV1063
GEV2132
GEV1001
Xp10-13
GEV2015
GEV2049
GEV1989
Xp8-16
GEV1921
GEV2129
Xp2010
GEV2004
GEV2120
GEV2116
GEV2115
GEV2009
GEV2098
GEV1044
GEV1054
GEV839D
TB6
GEV2063
TB15
TB9
Xp9-5
Xp7-12
Xp3-15
Xp4-20
Xp18-15
Xp15-11
Xp11-2
Xp4B
Xp5-6
GEV968D
GEV940D
GEV993D
GEV909D
GEV936D
GEV915D
GEV872
GEV893D
GEV917D
GEV904D
GEV1026
Bzl13
Bzl8
Bzl5
Bzl10

Brazil
Bzl11
BZL-21
Bzl-16
Xp3-8
Xp5-14
Bzl14
Xp5-9
Xp1-6
X10-B85
X59-BD1351
X2-B14
Scott-1
Xp1805
Xp3-16
Xp3-12
Xp1-5
SM-1806
SM-1830
SM-1813
SM-1811
SM-1814
SM-1828
SM-1815
NC-204
16-1181-2
16-1402A

USA and Mexico

SM-1829
SM-1807
SM-1812
GEV485
SM-1809
SM-1810

(FL, IN, NC, OH)

SM-1808
SM-1831
16-1205A
16-974C
Xp1920
NC-112
Bzl7
Mexico1
Xp1550
Xp1856
Xp1912
Xp1797
Xp1564
Mexico-LT1
Xp1275
Xp1183
Xp1144
Xp1118
91-118
Aus3
Aus15

Australia
Aus10
Aus5
Aus1
Aus11
Aus16
Aus7
SEA-23
Bzl3
Bzl6
ETH25
ETH21
ETH5
Xp894
Xp909
XV0938
Xp1268
Xp1241
16-1184A
Mexico-LT5
16-990C
ETH33
ETH11
GEV2121
GEV2125
GEV2130
Xp17-12
GEV2097
GEV2112
GEV2122
GEV2127
GEV2134

FL & NC (group 3),

GEV2124
GEV2010
GEV2397
GEV2392
GEV2396
GEV2390
NC-350

Australia, China
NC-101
Aus14
GEV2128
GEV2065
NC-14
GEV2099
GEV2108
CHI-5
CHI-7
CHI-10
TOM816
F215
F210

Iran
K41
TOM801
CHI-6
CHI-15
CHI-12

China
CHI-3
CHI-18
SEA-21
SEA-3

Italy
SEA-5
2P4S1
1P6S1
2P4S1D
2P6S1
1P4S1D
THA-54
THA-72
THA-81A

XopAG
XopT
XOO4824
AvrXccA1
AvrBs1
XopAX
XopH
XopAJ
THA-45
THA-14
THA-40
THA-8
THA-119

Thailand
THA-112
THA-128
THA-100
THA-132
THA-116
THA-126
THA-135
THA-157A
14-463-1A
16-990A
16-1182A
Mexico3
Bzl2
Bzl1
CHI-8
NC-282
Xp1861
KS5
14A
4D

Canada
4A
12A

4.0E-5

S. Timilsina

Timilsina et al. (In preparation)

 Summary of worldwide strains

Ø US had highest diversity based on both core genome and the

effector profiles (strains were collected from different times in
the US). Nigeria, Thailand and Mexico also have shown
diversity.

Ø Strains are moving all over the world. We see a distinct cluster
of Florida group 3 strains with strains in Australia and seed
production area of China.

Ø Strains from Southeast Asia and Thailand were also found in

Italy.
 Disease Control

qChemical

qCultural

qBiological

6/30/23
Chemical Control
Bactericides Common bactericides
Copper products:
Inorganic Compounds copper sulfate
copper oxychloride
basic copper sulfate
copper hydroxide
cupric oxide
cupric carbonate

Organic compounds: copper resinate

copper oleate
copper oxinate
copper linoleate

http://www.apsnet.org/publications/apsnetfeatures/Pages/AntibioticsForPlants.aspx
Organic compounds

Carbamates: Mancozeb
(Thiram, Ziram,
ferbam, Maneb,
Zineb)

Antibiotics Streptomycin(*) (*) 25% of apple

Gentamicin acreage; 35-40% of
Tetracycline (i.e: pear acreage; and 8%
mycoplasma of peach and
yellowing in palm) nectarine acreage
Oxytetracycline utilize Str for control
(Terramycin) of
fire blight.

http://www.apsnet.org/publications/apsnetfeatures/Pages/AntibioticsForPlants.aspx
 Evolution of of bacterial spot of tomato
pathogen in Florida
X. euvesicatoria
1950 1990 2000 2010 2015
First showed First showed copper
antibiotic resistance resistance in plant
in the field (Stall and pathogenic
Thayer, 1962; Thayer resistance (Marco
and Stall, 1962) and Stall, 1983)
 Copper bactericides have been key component of
management of many bacterial diseases

Discovery of Cu resistance in plant pathogenic bacteria:

• 1963, Smith and Jones: enteric bacteria in pigs. J. Appl. Bacteriol.

• 1983, Marcó and Stall: X. euvesicatoria (tomato). 1st report for
plant pathogenic bacteria.
 Mechanism of Copper resistance

• Copper resistance in Xanthomonas and Pseudomonas is mediated

by different plasmids.
Copper Resistance (Plasmid
mediated - pXvCu)

Self transmissible Not self transmissible

Result of selection pressure From Pseudomonas

Homology sectors presume a common origin

Nanoparticles as alternative to copper bactericides

Ø Silver-based

Ø Copper-based

Ø Magnesium-based

Ø Hybrid nanoparticles
 Ag Nanoparticles (<100 nm) and Plant Pathology
• Inorganic Ag compounds have germicidal
properties and have been used in the field of
medicine (bactericides against E.coli, P.
aeruginosa, and S. aureus).
• AgNPs can be deposited on graphene oxide
(GO) in order to prevent AgNPs from
aggregating and to enhance the antibacterial
effect.
• AgNPs generate ROS and interact with proteins
and enzymes on bacterial cell membranes
which lead to structural deformation of the cell
membrane.
• The uptake of free Ag ions which can
cause a disruption in ATP production and
DNA replication.

Oscoy et al., 2013; Strayer et al., 2016

 Silver nanoparticles
Ø Silver-based nanocomposite, Ag-dsDNA-GO, and its
potential as an alternative to copper

Ø The combination of dsDNA and graphene oxide (GO) were

important in minimizing aggregation, maintaining small
particle size and increasing interaction with bacterial
membranes

(Occoy et al., 20130) Adv. Mater.

(Occoy et al., 20130) ACS Nono
 In vitro activity of various concentrations of Ag-dsDNA-GO and
copper (Kocide 3000) on Xanthomonas perforans survival

Ag-dsDNA-GO kills copper tolerant

within 15 min at all concentrations
Copper-tolerant
Copper has slight effect

Ag-dsDNA-GO kills copper sensitive

Copper-sensitive within 15 min at all concentrations

Copper has delayed effect

Strayer et al. 2016 Plant Dis.

 Greenhouse assay

Chemical Xp
treament Inoculation
Effect of Ag-dsDNA-GO concentrations and copper-mancozeb on control of bacterial
spot of tomato under greenhouse conditions. AUDPC = area under the disease
progress curve.

Ag-dsDNA-GO concentrations tested

significantly reduced bacterial spot
severity relative to copper-mancozeb

Strayer et al. 2016 Plant Dis.

 Summary and Comments – Silver nanoparticles

Ø Ag-dsDNA-GO toxic to copper tolerant X. perforans at

concentrations as low as 10 μg/m
Ø Ag-dsDNA-GO concentrations tested significantly reduced bacterial
spot severity relative to copper-mancozeb
Ø Ag-dsDNA-GO concentrations tested significantly reduced bacterial
spot severity relative to copper-mancozeb
Ø Phytotoxicity at higher concentrations
Ø Ag nanocomposite contain graphene oxide which reduced
aggregation of AgNPs
Ø Long-term effects in the environment and on plant heath need to
be evaluated
 Copper Nanomaterials?
o Several studies have demonstrated that antibacterial
activity of metallic compounds is size dependent:
o Smaller particles with larger surface to volume
ratios
o Interact more closely with microbial membranes
o Releases more metal ions in solution
o Thus, we evaluated three advanced copper
nanomaterials, Core-shell Copper (Cs-Cu), Multivalent
copper (MV-Cu), and Fixed Quat Copper (FQ-Cu)
against copper-tolerant X. perforans (in vitro) and
bacterial spot of tomato in 3 greenhouse experiments
and 3 field trials
Fig. Scanning electron microscopy (SEM) and high-resolution transmission electron
microscopy (TEM) images of three copper composites: core shell silica copper (CS-
Cu), multivalent copper (MV-Cu), and fixed quaternary ammonium copper (FQ-Cu).

CS-Cu MV-Cu FQ-Cu

Strayer et al. 2018 Phytopathology

 In vitro activity of three copper composites on Xanthomonas perforans
survival

Copper-sensitive All copper composite concentrations and

Kocide 3000 completely inhibited copper
sensitive strain within 1 h of exposure.

Only copper composites inhibited copper

tolerant strain within 1 h of exposure.
Copper-tolerant

Strayer et al. 2018 Phytopathology

 Greenhouse assay

Chemical Xp
treament Inoculation

Disease severity
SNK
300.000
c
c
250.000 bc bc
200.000
b b
AUDPC

b
150.000 b b

100.000

50.000 a

0.000
1000 500 200 100 1000 500 200 100
MgO Kocide 3000 Cu-EBDC UT
Treatments (µg/ml)
LIAO, Y.-Y., et al. Phytopathology, 2019
 Effect of various metallic copper composites on
bacterial spot of tomato under greenhouse conditions

Strayer et al. 2018 Phytopathology

 TABLE. Effect of copper (Cu) composites and standard copper
bactericides on bacterial spot disease severity in the field
AUDPC
Treatment Experiment 1 Experiment 2
CS-Cu, 100 µg/ml 599±74 ab 837±46 a
CS-Cu, 200 µg/ml 588±94 ab 838±48 a
MV-Cu, 100 µg/ml 480±31 a 887±16 a
MV-Cu, 200 µg/ml 538±63 a 832±87 a
FQ-Cu, 100 µg/ml 590±74 ab 869±7 a
FQ-Cu, 200 µg/ml 662±69 ab 938±87 a
Kocide, 540 µg/ml 671±99 ab 1,135±60 ab
Copper-mancozeb, 540 µg/ml 596±185 ab 1,188±144 ab
HO
2 774±140 b 1,402±112 b
Strayer et al. 2018 Phytopathology
 Summary and Comments - Copper composite nanoparticles
Ø CS-Cu and FQ-Cu at 100 µg/ml completely inhibited bacterial growth
of a copper-tolerant strain of X. perforans within 1 h, whereas micron-
sized metallic copper at 1,000 µg/ml had no effect after 48 h

Ø Greenhouse and field experiments revealed that all three copper

composites were either more effective or statistically equivalent at
reducing bacterial spot disease compared with micron-sized metallic
copper

Ø Advantage of the copper composites is significantly reduced disease

using 50 to 90% less metallic copper than micron-sized metallic
copper
Strayer et al. 2018 Phytopathology
 Magnesium oxide(MgO)
• Micron-sized and nano-sized MgO particles have antimicrobial
activity against several mammalian pathogens.
• MgO is not on the list of the EPA’s Toxic Release Inventory (TRI)
Program.
• The FDA generally recognized MgO as safe (GRAS) compound
(Clydesdale 1997).

Hypothesis: Mg nano-materials will provide

better antibacterial properties and disease
control compared to commercial Cu
bactericide.
 New nanomaterials are being formulated to
prevent aggregates

• Mg nanomaterials
1. SgMc (~10nm)
2. SgMg #3
3. SgMg #2.5
4. Mg-Cu
SgMc, SEM (Huang, unpublished)
5. Mg double coated

50
 Mg nanomaterials: Mg-Cu, Mg double coated
SNK, p=0.05
Change in Bacterial Population (Xp GEV485) Overtime
5 e d c e d
Bacterial Popultaion (log CFU/ml)

c d d c
4 c b
3 b
b
2
1 1h
a a a a a a a a
0 4h
1000 100 1000 100 1000 100 24 h
Mg-Cu MgO double coated Kocide 3000 UT
formulated product Cu bactericide water
Treatments (µg/ml)
Green: live cells
Red: dead cells
Water Kocide 100ppm MgCu 100 ppm Mg double coated 100 ppm
 Viability assay: Xp treated with Nano-MgO (20 nm)
TEM A1 A2 A3 A4

observation
Untreated
control Alive
Dead
B1 B2 B3 B4 D2
LIVE/DEAD
BacLight
100µg/ml Bacterial
Nano- Viability kit
MgO (L7007,
treated
Molecular
Probes,
Invitrogen)
C1 C2 C3 C4 D3

100µg/ml
Kocide
3000
treated

Nano-MgO is bactericidal.

LIAO, Y.-Y., et al. Phytopathology, 2019

 Mg nanomateirals: SgMc, SgMg #3, SgMg#2.5
Green: live cells
Red: dead cells
UT Heat treated SgMc 100µg/ml Kocide 3000 100µg/ml

Greenhouse study
400 e e e e
de de
350 c-e
b-e
300 c-e
a-d a-d
250 a-c
AUDPC

a-c
200 ab
ab ab
150 a
100
50
0
500 200 100 500 200 100 500 200 100 500 200 100 500 200 100 - -
NP SgMc SgMg #3 SgMg #2.5 Kocide K+M UT
cr ude MgO nanoparticle formulated Mg-based nano-materials Cu bacter icide grower's inoculated
standard control
Treatments (µg/ml) SNK stats
 Field experiments
o Completely randomized block design. o Treatment list:
o 4 replications per treatment. • MgO 1000, 200 µg/ml
• 1 chemical application pre-inoculation (20nm, 0.3µm, 0.6µm)
• Cu: Kocide 3000
• Inoculation with a suspension of 10 CFU/ml
8
• Grower’s standard:
Cu-tolerant Xp GEV485.
Cu-EBDC
(Kocide Penncozeb®75DF)
• Water
• 7 weeks of follow-up chemical application and rating.
 Field study: Efficacy of Nano-MgO against
bacterial spot disease of tomato.
Treatment Rate (µg/ml) Quincy, FL Wimauma, FL Quincy, FL
2015 Fall 2016 Spring 2016 Spring
Nano-MgO 1,000 805.0 az 866.4 ab 913.5 a
Nano-MgO 200 836.9 a 580.1 a 853.6 a
Kocide 3000 2,100 1,196.4 ab 972.1 ab 1,135.4 ab
Cu-EBDC 1,092.9 ab 773.4 ab 1,188.0 ab
Water (Untreated) 1,330.9 b 1,136.8 b 1,402.1 b

SNK analysis p=0.05; zNumber with different character in the same column has significant difference.

• No phytotoxicity.
• No significant yield reduction.
LIAO, Y.-Y., et al. Phytopathology, 2019
Field study: using Mg-Cu and Mg double coated against
bacterial spot of tomato
(2019 Fall Quincy, FL)
Treatment Rate (µg/ml) AUDPC
Mg-Cu 100 2603.9 cd
Mg-Cu 500 1574.9 a
Mg double coated 100 2055.6 ab
Mg double coated 500 1729.1 a
Nano-MgO 100 2048.0 ab
Nano-MgO 1,000 1730.6 a
Kocide 2,100 2636.1 cd
Cu-EBDC 1974.8 ab
water 2188.2 bc

Incomplete plot design, LSD, p=0.05

 Take home message
• Nano-MgO significantly reduced disease
severity in the field.
• No excessive metal accumulation in the fruit.
• No phytotoxicity.
• No significant yield reduction.
• Formulated Mg nano-materials have potential
to be an alternative to Cu bactericide.

57
 Systemic Acquired Resistance

• SAR triggered by exposing plant to biotic and

abiotic factors; dependent on salicylic acid
and accumulation of Pathogensis-related
(PR) proteins etc.

• ISR by plant growth-promoting

rhizobacteria. Relies on pathways regulated
by jasmonate and ethylene.

• SAR and ISR different on the basis of nature

of elicitor and regulatory pathways
involved

• Production of Salicylic acid also shown by

PGPRs but no induction of PR proteins.

Use of SAR in controlling BLS of tomato

• Acibenzolar-S-Methyl (Actigard)
o Analog of Salicylic Acid
o Up-regulation of Pathogenesis-Related
proteins.
Effect of application rates and frequencies of acibenzolar-S-methyl (ASM) on
bacterial spot and yield. ASM reduced disease comparable to standard (copper-
mancozeb). Yield significantly lower.

Huang et al. 2012. Effect of application frequency and reduced rates of acibenzolar-S-methyl on the
field efficacy of induced resistance against bacterial spot on tomato. Plant Dis. 96:221-227.
 Summary

•Chemicals used to control plant diseases are in general

"protectants" and must be applied before infection to protect
the plant from the pathogen.

•Both the correct recommendation of a control compound

and the time of application are very important decisions in
crop management.

•The use of the same compound for several seasons favors

the development of resistance.

•When resistance appears, attempting to eliminate this new

characteristic of the pathogen is almost impossible, so it is
necessary use alternatives for control.
 Biological control

•PGPRs

•Bacteriophages

•Bacteriocins

•SARs

•Niche competition

6/30/23
 PGPRs (Plant growth promoting rhizobacteria)
Plant growth promoting rhizobacteria (PGPRs)
Various non-pathogenic Pseudomonas rhizobacteria have the ability to induce a state of
systemic resistance in plants, which provides protection against a broad spectrum of
phytopathogenic organisms including fungi, bacteria and viruses. ISR acts through a
different signaling pathwayto that regulating systemic acquired resistance (SAR), the ISR
pathway is induced when the plant is challenged by pathogenic organisms.
Bloemberg and Lugtenberg. 2001. Molecular basis of plant growth promotion
and biocontrol by rhizobacteria 2001.CURRENT OPINION IN PLANT
BIOLOGY, 2001

They are sold commercially. Do they work?

6/30/23
 Obradovic et al. 2005. Control of bacterial spot of tomato with PGPRs.

PGPRs

Do they work here? It depends on who you ask.

PGPRs were ineffective in disease control

6/30/23
 Bacteriophages

Used previously to control bacterial plant

pathogens with limited success

6/30/23
 Phage therapy for control of bacterial spot of tomato
(Xanthomonas campestris pv. vesicatoria)

6/30/23
 Disease control with phages

Important Considerations
n Apply before pathogen ingress - ineffective once enters plant.
n Apply at high concentrations: ~108 PFU/mL is desirable
n Phage resistance can develop
n Apply in mixture – strain variation
n Wide host-range phages
n Host-range mutant phages
n Monitor bacteria and adjust phage mix if needed

n Goal - maintain a high phage population on the plant

foliage. This is difficult to achieve since phages are
extremely sensitive to sunlight.
 Why have phages been ineffective?

ØUse of only one phage (likelihood for build-up of

phage-resistant strains

ØVariation within bacterial strains necessitate use of

multiple phages

ØEnvironmental factors affect bacteriophage

survival or maintenance on leaf surfaces (UV,
desiccation, rain (leaching))

6/30/23
 Approach

Use mixture of lytic phages (kills bacterial cells) to reduce likelihood

for development of resistant strains

6/30/23
 Phage attachment to bacterial cell

6/30/23
 Table. Effect of field treatment on yield
Extra large
Treatment 1997 1998
Flaherty, J.E., Jones, J.B., Harbaugh, B.K., Somodi, G.C. and Jackson, L.E. (2000) Control of
bacterial spot on tomato in the greenhouse and field with H‐mutant
Phage 143 A 154 A bacteriophages. HortScience, 35, 882– 884.

Copper 119 B 121 B

Control 126 B 123 B

Formulated phage works better

a
b
160 c bc
cd
140 d Formulating the phage with various
120 Copper-Mancozeb compounds such as skim milk
PCF
100
improves efficacy.
AUDPC

Casecrete
80
Skim milk
60 Non-formulated
40 Untreated control
20
0 Balogh, B., Jones, J., Momol, M., Olson, S., Obradovic, A., King, P. and Jackson, L.E. et al.
(2003) Improved efficacy of newly formulated bacteriophages for management of bacterial
spot on tomato. Plant Dis. 87, 949– 954.

6/30/23
Management of tomato bacterial spot in the field by foliar
applications of bacteriophages and SAR inducers

Obradovic et al., 2004. Plant Dis. 88, 736– 740.

 Fall 2001 contrast comparison of total marketable fruit yield: Cu-Mz vs Phage
and Actigard vs No Actigard

Yield response: Phage Yield response: Actigard

treated > No Phage treatment did not
enhance yield

Obradovic et al., 2004. Plant Dis. 88, 736– 740.

6/30/23
Resistance to
bacterial spot
pathogens
Pepper and
tomato races
 Non-hypersensitive resistance in Capsicum annuum
In preliminary greenhouse tests ECW12356 was shown to have high level of resistance to all
known Xcv races. The resistance associated with the ECW12356 is recessive and has been
designated as bs5, and bs6

ECW12346 bs6

Disease reactions (105 CFU/ml)

with race 6 strain

bs5 ECW123
Bs1, Bs2, Bs3
Population dynamics of pepper race 3 and pepper race 6 strain on ECW12356
5= bs5 and 6= bs6

10
Log10 CFU/cm2

8
6
4
2
0 2 4 6 8 10
Days after inoculation
P3-ECW123 P6-ECW123 P6-ECW12356

bs5 and bs6 suppress P6 populations in pepper leaves

Sharma et al., 2023 Front Plant Sci

Vallejos et al., 2010. Theor. Appl. Genet.
Szabó et al., 2023. Theor. Appl. Genet.
 Is this resistance durable?
ØChemical mutagenesis (nitrosoguanidine) resulted in no mutant
strains.
ØTransposon mutagenesis has also been ineffective in identifying
mutant strains that are virulent on this resistance.
ØThis recessive resistance appears to be durable.

Seed companies are using bs5 resistance in their breeding programs

in combination with Bs1, Bs2 and Bs3. There are commercially
available varieties with bs5 resistance available.
 Is this resistance durable?
X. gardneri is virulent on bs5 and bs6

XV444
XV444Δavrhah1

X gardneri has been found in many countries.

Genotypes with bs5, bs6 are tolerant to exotic strains
 Would bs5 and bs6 be durable in Mexico?

• Xanthomonas gardneri is cool temperature

pathogen. X. euvesicatoria is a warm weather
pathogen.

• It is very likely that resistance derived from bs5 and

bs6 would be durable in Mexico given that X.
gardneri is unlikely to become established. However,
Bs2 and Bs3 should be introgressed with recessive
resistance to minimize likelihood of mutant strains
developming that overcomes bs5 resistance.
 Is there resistance to X. gardneri?
X. gardneri is virulent on bs5 and bs6

XV444
XV444Δavrhah1

X gardneri has been found in many countries.

Genotypes with bs5, bs6 are susceptible to X. gardneri
Identification and Mapping of bs8, a Novel Locus Conferring Resistance
to Bacterial Spot Caused by Xanthomonas gardneri

Fig. 1 Resistance against Xanthomonas gardneri.

Fig. 1. Scoring scale for phenotyping the F population for resistance against Xanthomonas gardneri.
2

Published in: Anuj Sharma; Gerald V. Minsavage; Upinder S. Gill; Samuel F. Hutton; Jeffrey B. Jones;
Phytopathology® 2022, 112, 1640-1650.
Copyright © 2022 The American Phytopathological Society • DOI: 10.1094/PHYTO-08-21-0339-R
Bs8 in pepper has resistance to Xanthomonas gardneri

Fig. 2. Resistance phenotype of parents and F as seen from abaxial leaf

3
surface when inoculated with Xanthomonas gardneri
2 strain Xg51 at 10
CFU/ml. For Early Calwonder (ECW) at 18 days. Top, intact leaf; and
bottom, defoliated leaf.

Published in: Anuj Sharma; Gerald V. Minsavage; Upinder S. Gill; Samuel F. Hutton; Jeffrey B. Jones; Phytopathology® 2022, 112, 1640-1650.
Copyright © 2022 The American Phytopathological Society • DOI: 10.1094/PHYTO-08-21-0339-R
 ACKNOWLEDGEMENTS

University of Florida University of Central Florida

Erica Goss Swadesh Santra
Fanny Iriartemb
Jeannie Klein-Gordon
Gerald V. Minsavage
Mathews Paret
Anuj Sharma
Sujan Timilsina
Gary Vallad

Funding sources:
USDA-SCRI, Vallad, Goss, Jones et al.
FDACS Specialty Crop, Vallad and Jones
Florida Tomato Committee Grants Program