Journal of Plant Pathology (2021) 103:539–547

https://doi.org/10.1007/s42161-021-00787-4

ORIGINAL ARTICLE

Association of Pantoea stewartii subsp. stewartii with ChrysSA genus

flea beetles in jackfruit crops
Alejandro Hernández‑Morales1 · Ruth Elena Soria‑Guerra2 · Néstor Isiordia‑Aquino3 · Juan Campos‑Guillén4 ·
Juan Ramiro Pacheco‑Aguilar4 · Abril Bernardette Martínez‑Rizo5 · Jackeline Lizzeta Arvizu‑Gómez6 

Received: 1 December 2020 / Accepted: 12 February 2021 / Published online: 1 April 2021
© Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2021

Abstract
Pantoea stewartii subsp. stewartii is the causal agent of the emerging disease in jackfruit crops (Artocarpus heterophyllus
L.), known as "Fruit Bronzing". This disease affects the quality of fresh jackfruits, resulting in economic production losses.
Thus far, the biology of the P. stewartii subsp. stewartii bacterium during the stages that make up the fruit bronzing disease´s
cycle is poorly understood. Therefore, to gain further insight into this emerging disease, we evaluated the association
between P. stewartii subsp. stewartii and the jackfruit flea beetle, which could be involved in the dissemination of this
bacterium in this crop. Five bacterial isolates were obtained from two of the six groups of field-collected flea beetles (Group
I and Group VI). Morphological characteristics and PCR amplification employing taxon-specific primers both indicated
the isolates were P. stewartii subsp. stewartii. AG085-I, AG086-I, AG087-I, and AG088-I were isolated from Group I
flea beetles, and AG024-VI was isolated from Group VI flea beetles. Molecular analysis results showed that Group I flea
beetles correspond to Chaetocnema minuta, while that Group VI flea beetles correspond to the ChrysSA genus. Finally,
the molecular identification of bacterial isolates by analysis of the 16S rDNA and cpsD regions showed that the AG085-I,
AG086-I, AG087-I, AG088-I isolates correspond to P. ananatis and only the AG024-VI isolate obtained from the Group VI
flea beetles ChrysSA corresponds to P. stewartii subsp. stewartii. The AG024-VI isolate showed pathogenic potential against
jackfruit. These results demonstrate the presence of CrhysSA sp. flea beetles associated with P. stewartii subsp. stewartii in
jackfruit crops.

Keywords  Fruit bronzing · Jackfruit · Insect · P. stewartii subsp. stewartii

Introduction

“Fruit Bronzing” is the term used to refer to the emerging

* Jackeline Lizzeta Arvizu‑Gómez disease of the jackfruit crop (Artocarpus heterophyllus
lizzeta28@gmail.com
L.) caused by the Pantoea stewartii subsp. stewartii
1
Facultad de Estudios Profesionales Zona Huasteca, bacterium. It is considered an important disease since it
Universidad Autónoma de San Luis Potosí. Ciudad Valles, generates economic production losses. Typical symptoms
San Luis Potosí, México of the disease are the presence of yellowish-orange to
2
Facultad de Ciencias Químicas, Universidad Autónoma de reddish discoloration of the affected pulps and rags without
San Luis Potosí, San Luis Potosí, México external symptoms on the fruit surface (Gapasin et al. 2014;
3
Unidad Académica de Agricultura, Universidad Autónoma Abidin et al. 2018). Jackfruit bronzing disease had only
de Nayarit, Tepic, México been reported in the Philippines and subsequently also in
4
Universidad Autónoma de Querétaro, Santiago de Querétaro, Malaysia (Gapasin et al. 2014; Zulperi et al. 2017). Recently,
México P. stewartii subsp. stewartii has also been identified as the
5
Unidad Académica de Medicina, Universidad Autónoma de cause of this disease in Mexico (Hernández-Morales et al.
Nayarit, Tepic, México 2017).
6
Secretaría de Investigación y Posgrado. Centro Nayarita P. stewartii subsp. stewartii is widely known as the
de Innovación y Transferencia de Tecnología, Universidad causal agent of Stewart’s vascular wilt and leaf blight
Autónoma de Nayarit, Nayarit, México

13
Vol.:(0123456789)
540
of maize (Zea mays), the most serious bacterial disease
of sweet corn and maize in the USA and Europe, where
this bacterium is considered a quarantine pathogen. The
dissemination of P. stewartii subsp. stewartii to corn
plants can be carried out mechanically or through seed,
although this occurs with a low frequency of transmission.
The spread of P. stewartii subsp. stewartii to corn
plants is mainly through the flea beetle Chaetocnema
pulicaria Melsheimer (Coleoptera: Chrysomelidae).
This insect is the main known vector of the bacterium.
Corn flea beetles C. pulicaria acquire the P. stewartii
subsp. stewartii bacterium from infected plants and the
bacterium overwinters in the gut of the flea beetle (Correa
et  al. 2008). The introduction of the bacteria into the
plant is via scratching wounds created by the flea beetle
as it feeds on the corn plant. P. stewartii subsp. stewartii
colonizes both the intercellular spaces of the leaf tissue
and the xylem, which leads to systemic spread (Roper
2011). Other known vectors of the P. stewartii subsp.
stewartii bacterium include Diabrotica undecempunctata
(both adult and larva), Chaetocnema denticulata, larvae
of Delia platura, Agiotes mancus, Phyllophaga sp., and
Diabrotica longicornis (EPPO 2016; Jeger et al. 2018).
In relation to the P. stewartii subsp. stewartii-jackfruit
pathosystem, the knowledge existing about the cycle of
the jackfruit bronzing disease is very scarce. Thus far,
how the bacterium spreads to jackfruit crops is completely
unknown. Likewise, there are not reports that establish
the existence of an association of the bacterium with any
insect present in jackfruit crops, which is probably related
with the dispersion of the bacteria in these crops just as
it happens in corn. Furthermore, it has not been reported
whether flea beetles, such as C. pulicaria inhabit jackfruit
crops, being also able to be the insect vector of P. stewartii
subsp. stewartii in this crop. Though corn is the primary
host of the corn flea beetle, the insect has been recorded
to feed on a large variety of secondary host plants such as
orchard grass, Kentucky bluegrass, yellow foxtail, giant
foxtail, fall panicum, and several other kinds of grasses.
Wheat, barley, oats, and Timothy have also been identified
as food plants for the flea beetle (Cook and Weinzieri
2004). In this manner, so far, the only known fact about
jackfruit bronzing disease´s cycle is that P. stewartii subsp.
stewartii is the causal agent of this disease. Based on the
above, this study was undertaken with the objective to
unravel the bacterium biology and possible strategies used
by the P. stewartii subsp. stewartii bacterium during its
spread or transmission to jackfruit crops. In a particular
manner, this study was focused on determining the
existence of an association of P. stewartii subsp. stewartii
pathogenic bacteria with insects present in jackfruits crops,
particularly flea beetle.
13
Journal of Plant Pathology (2021) 103:539–547
Materials and methods
Collection and identification of flea beetles
Flea beetles were collected in regions of the Nayarit
state, Mexico, in the major jackfruit growing areas of the
municipality of San Blas, Nayarit; “El Llano” (21° 25′
06.7′’N, 105° 11′ 18.1′’ W; 21°25′ 17.3′’N, 105°11′ 03.9′’
W; 21°24′ 38.5′’N, 105° 10′ 24.6′’ W) and “La Palma”
(21°30′ 55.7′’ N; 105°11′ 01.1′’ W), in winter 2019.
The jackfruit crops sampled do not have crops or maize
plantations in their vicinity. The monthly collection of
insects was performed by sweep netting and knockdown
using insecticides containing pyrethrin, as previously
reported (Cambero et al. 2010). Subsamples of collected
beetles were used for confirming species identity by
the taxonomic keys. Furthermore, the species identity
was confirmed by amplification and sequencing of the
mitochondrial cytochrome C oxidase subunit 1 gene
(cox1) using primer pairs LCO 1490: GGT​CAA​CAA​
ATC​ATA​A AG​ATA​T TG​G and HCO 2198: TAA​ACT​
TCA​G GG​TGA​C CA​A AA​A AT​CA. DNA extraction was
performed from full insects previously disinfected with
ethanol 70% for 3 min, and rinsed with sterilized water.
The Dneasy Blood & Tissue kit (QIAGEN) was used for
DNA extraction following the manufacturer´s instructions.
The PCR program consisted of 3 min at 94 °C for 1 cycle,
45 s at 94 °C, 45 s at 51.2 °C, and 1 min at 72 °C for
35 cycles, with one step of final extension at 72 °C for
7 min. PCR products were sequenced at the Biotechnology
Institute-UNAM. The insects were identified by sequence
comparison against the BOLD (http://bolds​ystem​s .org/
index​.php/datab​ases) and GenBank databases using the
BLASTn search algorithm (http://blast​.ncbi.nlm.nih.gov).
Isolation of P. stewartii from field‑collected flea
beetles
Isolates of P. stewartii from field-collected flea beetles
were obtained by crushing and culture technique using
protocols previously described, with some modifications
(Correa et  al. 2012). The insects were disinfected with
ethanol 70% for 3 min, before being rinsed with sterilized
water. Three disinfected insects pool were ground in 300
μL of 0.85% NaCl solution. One-hundred μL of serial
dilutions of the homogenates were plated on Luria medium
and incubated at 30  °C for 48  h. Single colonies with
morphological characteristics of P. stewartti such as round
colonies, yellow-pigmented, smooth, translucent, with
bacillar cell form and Gram-negative (Roper 2011), were
Journal of Plant Pathology (2021) 103:539–547
recuperated in Luria medium. Initially, the identities of the
isolates as P. stewartii were evaluated by colony-PCR using
the set of primers CPSL1 (CCT​GTC​AGT​CTC​GAACC) and
CPSR2c (ATC​TCG​AAC​CGG​TAACC) for the cpsDE gene
region and ES16 (GCG​AAC​TTG​GCA​GAGAT) and ESIG2c
(GCG ​ C TT ​ G CG ​ T GT​ -TAT ​ G AG ​ ) for 16S-23S rRNA/
ITS region. Both primers sets reported as specifics for P.
stewartii subsp. stewartii (Coplin et al. 2002). The PCR
program was performed for 5 min at 94 °C for 1 cycle; 35 s
at 94 °C, 40 s at 55 °C, and 1.5 min at 72 °C for 35 cycles;
and 7 min at 72 °C for 1 cycle. The P. stewartii subsp.
stewartii JM5´ Mexican isolate was used as a positive
control for these assays (Hernández-Morales et al. 2017).
Molecular identification of bacterial isolates
obtained from field‑collected flea beetles
The cpsDE region, involved in the synthesis of capsular
polysaccharide stewartan reported as exclusive of P.
stewartii subsp. stewartii (Coplin et al. 2002), and the 16S
rDNA region were used for the molecular identification
of the bacterial isolates. Genomic DNA was isolated, as
previously described (Chen and Kuo 1993). The primers
CPSL1 and CPSR2c, mentioned above, were used for
amplification of the cpsDE region. The 16S rDNA region
was amplified with the bacterial universal primers FD1 and
RD1 (Weisburg et al. 1991). The products were purified
using the Gen Elute PCR Clean-up kit (SIGMA, USA) and
sequenced at the Biotechnology Institute-UNAM. Bacterial
isolates were identified by sequence comparison against the
GenBank databases using the BLASTn search algorithm.
Phylogenetic analyses
Phylogeny reconstruction analysis was performed in MEGA
7 software package, sequences were aligned by ClustalW
algorithm and the phylogenetic tree was constructed using
the statistical method of Maximum parsimony and the
phylogeny test Bootstrap method with 1000 replications.
Pathogenicity test
The pathogenic potential of the P. stewartii subsp. stewartii
AG024-VI isolate obtained from flea beetles was evaluated
by Koch´s postulates as previously reported (Hernández-
Morales et al. 2017). A bacterial suspension (10 ml 0.85%
NaCl, ­108 cfu ml−1) of the AG024-VI isolate was inoculated
by syringe into detached jackfruits. Fruits inoculated
with sterile saline solution (0.85%) were evaluated
simultaneously and used as control. Furthermore, the P.
541
stewartii subsp. stewartii JM5´ Mexican isolate was used as
a positive control for these assays. Three replicates for each
treatment were performed. The presence of the characteristic
symptoms of the fruit bronzing disease was evaluated after
fourteen days post-inoculation. The bacteria re-isolation
from inoculated jackfruits was performed as previously
described (Hernández-Morales et al. 2017). Morphological
and molecular analyses (cpsDE gene) were used for the
identification of the re-isolated bacteria.
Accession numbers
The sequences of the 16S rDNA and cpsDE regions of the
bacterial isolates obtained from field-collected flea beetles
were deposited in GenBank with accession numbers:
AG085-I (MW092177, MW115979), AG086-I (MW092178,
MW115980), AG087-I (MW092179, MW115981), AG088-I
(MW092180, MW115982), and AG024-VI (MW092176,
MW115978). Likewise, the sequences of the mitochondrial
cytochrome C oxidase subunit 1 gene (cox1) from field-
collected flea beetles Group I and Group VI were deposited
in GenBank with accession numbers MW136280 and
MW13628, respectively.
Results
Presence of flea beetle associated with Pantoea
stewartii subsp. stewartii in jackfruit crops
To evaluate the existence of an association of P. stewartii
subsp. stewartii with insects present in the jackfruit crops,
particularly flea beetles, we started this study performing
collection of insects in the major jackfruit growing areas of
the Nayarit state, Mexico, in winter 2019. The collection of
flea beetles was performed by sweep netting and knockdown
techniques. The specimens collected were grouped based on
similar morphological macroscopic characteristics. A total
of six groups (I-VI) were formed, each one with a variable
number of specimens. The presence of field-collected flea
beetle associated with P. stewartii subsp. stewartii was
evaluated initially by using the crushing and culture on agar
plates technique. A total of 1250 colonies with morphological
characteristics particular to P. stewartii subsp. stewartii
were obtained. The identity of these colonies obtained from
flea beetle was initially evaluated by PCR using the set of
primers CPSL1 and CPSR2c (cpsDE gene region) and ES16
and ESIG2c (16S-23S rRNA/ITS region). Both primers sets
reported as specifics for P. stewartii subsp. stewartii (Coplin
et al. 2002). The PCR assays with the CPSL1 and CPSR2c
primers pair showed the presence of an amplicon of the
expected size (1.1. kb) in only five of the obtained bacterial
13
542
Fig. 1  PCR analyses of the bacterial isolates obtained from field-
collected flea beetles. a) PCR assays using the CPSL1 and CPSR2c
primers (cpsDE gene region). b) PCR assays using the ES16 and
ESIG2c primers (16S-23S rRNA/ITS region). The images show the
presence of amplicon in the sizes expected, corresponding to 1.1 kb
and 0.92  kb for the cpsDE and ITS region, respectively. Lane 1,
isolates (Fig. 1a). The AG085-I, AG086-I, AG087-I, and
AG088-I isolates obtained from flea beetles belonging to
Group I, and the AG024-VI isolate obtained from the Group
VI flea beetles. Likewise, the PCR analyses with the ES16
and ESIG2c primers showed the presence of amplicon
(0.92  kb) in only these five bacterial isolates (Fig.  1b).
These results demonstrate the presence of P. stewartii subsp.
stewartii in flea beetle inhabitants in jackfruit crops.
Identification of field‑collected flea beetle
associated with Pantoea stewartii subsp.
stewartii
After the presence of flea beetles associated with P. stewartii
subsp. stewartii in jackfruit crops has been determined
by PCR assays, we performed the identification of these
insects. The morphological identification analysis of sub-
samples of the insects of the I and VI groups showed the
presence of particular characteristics of the Chaetocnema
genera (Coleoptera, Crysomelidae, Alticinae), such as:
apical segment of posterior tarsi not swollen; apical spur
of posterior tibiae terminating in a single point; antennae
11-segmented; tarsal claws simple or appendiculate;
pronotum lacking distinct impressions; elytra regularly
punctate-striae; middle and posterior tibiae with corbel
(McDaniel et al. 1992).
The species identity of the insects was confirmed
by amplification and sequencing of the mitochondrial
cytochrome C oxidase subunit 1 gene (cox1) from sub-
samples specimens of these insects. Insects were identified
13
Journal of Plant Pathology (2021) 103:539–547
AG024-VI isolate; lane 2, AG085-I isolate; lane 3, AG086-I isolate;
lane 4, AG087-I isolate and lane 5, AG088-I isolate. M: GeneRuler™
1  kb DNA ladder (Thermo Scientific). C-: negative control (PCR
reaction without DNA). C + : P. stewartii subsp. stewartii JM5´ Mexi-
can isolate (positive control)
by sequence comparison against the BOLD and/or GenBank
databases. On the one hand, the cox1 sequence obtained
from Group I flea beetles (Fig. 2a) showed 91% similarity
with Chaetocnema minuta (BOLD: ABA9953, GenBank
accession No. KR483900). On the other hand, the analyses
of the cox1 sequence obtained from Group VI flea beetles
(Fig. 2b) showed 99.82% similarity with ChrysSA sp.47
genus insects (BOLD:AAN6169), using BOLD database.
The comparison of this sequence against GenBank
database showed only 85% similarity with Melandryidae
sp. (KM849706.1). This genus does not match at the
morphology level with the specimens belonging to Group
VI. It should be noted that no sequence for ChrysSA genus
insects has been deposited in the GenBank database.
Phylogenetic analyses were conducted by the Maximum
Parsimony method. These analyses showed cox1 sequence
from Group I flea beetles is clustered together in the clade
of C. minuta. with a high bootstrap value of 99%. Also, the
cox1 sequence obtained from Group VI flea beetles was
grouped with cox1 sequences from ChrysSA sp. 47 genus
insects (Fig. 3). These results reveal the presence of ChrysSA
genus flea beetles and C. minuta in jackfruit crops.
Molecular identification of bacterial isolates
obtained from flea beetles collected
in jackfruit crops
Although the obtaining of amplicon in the expected size by
using oligonucleotides such as CPSL1- CPSR2c and ES16-
ESIG2c, reported as specifics for P. stewartii subsp. stewarti,
Journal of Plant Pathology (2021) 103:539–547
Fig. 2  Jackfruit crops collected-
flea beetles. The image shows
field collected flea beetles from
which were obtained bacterial
isolates with morphological
characteristics and amplification
by PCR with specifics primer of
P. stewartii subsp. stewartii. a)
Group I flea beetles. b) Group
VI flea beetles
it has been considered in diverse literature as a strategy of
confirmation and identification of this bacterium, including
phytosanitary tests (EPPO 2006,2016; Palacio-Bielsa et al.
2009). We decide to confirm the identity of the AG085-I,
AG086-I, AG087-I, AG088-I, and AG024-VI bacterial
isolates obtained from flea beetles by amplification and
sequencing of the 16S rDNA and cpsDE regions. Bacterial
isolates were identified by sequence comparison against the
GenBank databases using the BLASTn search algorithm. The
Fig. 3  Molecular phylogenetic
analysis of field-collected flea
beetles based on coxI gene
sequences. The evolutionary
history was inferred using the
Maximum Parsimony method.
The percentage of replicate
trees in which the associated
taxa clustered together in the
bootstrap test (1000 replicates) 69
are shown next to the branches.
Evolutionary analyses were
conducted in MEGA7 (Kumar
et al. 2016). The sequence of
Anuraphis catoni was included
as an outgroup
20
543
determined sequences for the cpsDE region from the AG085-I,
AG086-I, AG087-I, AG088-I bacterial isolates, obtained from
Group I flea beetles corresponding to C. minuta, showed
99% similarity with those from reference strains of Pantoea
ananatis (CP028033.1). Besides, the sequence analysis of the
cpsDE region from the AG024-VI bacterial isolate obtained
from Group VI flea beetle corresponding to ChrysSA sp. 47
genus, was 99% identical to reference strains of P. stewartii
subsp. stewartii (MH752487.1).
MH118683.1 Chaetocnema minuta voucher MPQCIQCHR1106-01
64
KR483900.1 Galerucinae sp. BOLD-2016 voucher BIOUG05614-H04
99
SMTPB10823-13.COI-5P Chaetocnema minuta
99
RRSSC5060-15.COI-5P Chaetocnema minuta BOLD:ABA9953
99
HQ984056.1:23-655 Chaetocnema sp. BOLD:AAN5988
75
Group I flea beetle
45 MK253723.1:34-681 Chaetocnema sp. W.Moore lab DNA4753
KU913225.1 Chaetocnema hortensis voucher ZFMK-TIS-15667
HQ989937.1 Chaetocnema sahlbergii voucher BIOUGCAN :10PROBE-20872
21
45 KF654598.1:18-655 Chaetocnema obesa voucher BMNH:850800
31 KJ207904.1 Chaetocnema pulicaria voucher BIOUG03566-E06
KX943408.1:2575-3217 Chaetocnema depressa
15 JICCB1220-16.COI-5P ChrysSA sp. 47 BOLD:AAN6169
100 Group VI flea beetle
KM849706.1Melandryidae sp. BOLD:ACG3777 voucher BIOUG05861-G05
KT878790.1 Anuraphis catonii isolate 1 voucher SO3173
13
544
The sequence analysis of the 16S rDNA region confirmed
these results. The determined sequences for the 16S rDNA
from the AG085-I, AG086-I, AG087-I, AG088-I bacterial
isolates showed 99% similarity with reference strains of
Pantoea ananatis (MT212809.1, MT367850.1, MT212809.1,
ID: MT212809.1, respectively) while the 16S rDNA sequence
from the AG024-VI isolate showed 99% similarity with
reference strains of P. stewartii subsp. stewartii (MN894069.1).
Phylogenetic analyses of sequences were conducted in the
MEGA 7 software by the Maximum Parsimony method with
a Bootstrap test including 1000 replicates (Kumar et al. 2016).
Analysis based on cpsD and 16S rDNA sequences indicated
that AG085-I, AG086-I, AG087-I, and AG088-I isolates
clustered together in the clade of P. ananatis. Moreover, the
cpsD and 16S rDNA sequences obtained from the AG024-VI
bacterial isolate grouped respectively with cpsD and 16S rDNA
genes from available genomes of P. stewartii subsp. stewartii
(Fig. 4) (Suplementary file). These results demonstrate and
confirm the presence of P. stewartii subsp. stewartii in ChrysSA
genus flea beetle inhabitants in jackfruit crops. Moreover,
these results suggest that ChrysSA genus flea beetles could
participate in the spread of the P. stewartii subsp. stewartii
bacterium into jackfruit crops.
63
100 MH752487.1 Pantoea stewartii subsp. stewartii strain NWm6 CpsD
78
100 CP034148.1 Pantoea agglomerans strain L15
61
98
50
Fig. 4  Molecular phylogenetic analysis of bacterial isolates based on
cpsD gene sequences. The evolutionary history was inferred using the
Maximum Parsimony method. Boostrap values after 1000 replicates
are expressed as percentages. The percentage of trees in which the
associated taxa clustered together is shown next to the branches. Evo-
lutionary analyses were conducted in MEGA7 (Kumar et  al.  2016).
13
Journal of Plant Pathology (2021) 103:539–547
P. stewartii subsp. stewartii obtained
from ChrysSA genus flea beetles collected
in jackfruits crops, shows pathogenic
potential
To determine the potential of the P. stewartii subsp. stewartii
AG024-VI bacterial isolate in the development of the fruit
bronzing disease, the Koch´s postulates were evaluated.
Pathogenicity tests were performed by the inoculation of
a bacterial suspension (10 ml, 1­ 08 cfu ml−1) of the selected
strain (AG024-VI) injected into jackfruits. The search for
characteristic symptoms of the fruit bronzing disease was
performed 14 days post-inoculation. The results showed the
presence of reddish-brown spots or yellowish discoloration
which extends from the point of inoculation toward the pulp
and rags in those jackfruits inoculated with the P. stewartii
subsp. stewartii AG024-VI isolate. Similar symptoms were
observed in jackfruits inoculated with the control strain P.
stewartii subsp. stewartii JM5´ (positive control) (Fig. 5).
These symptoms correspond to the characteristics of the
fruit bronzing disease. No symptom related to the disease
was observed in the control jackfruits inoculated with saline
KY965964.1 Pantoea stewartii First Mexican Isolate
AG024-VI isolate
CP049115.1 Pantoea stewartii strain ZJ-FGZX1
AB894429.1 Pantoea stewartii subsp. stewartii
EU215384.1 Pantoea stewartii subsp. stewartii isolate DOAB 022
99
AB894428.1 Pantoea stewartii subsp. stewartii
CP028033.1 Pantoea ananatis strain SGAir0210
AG086-I isolate
100
AG087-I isolate
72 AG085-I isolate
AG088-I isolate
CP034469.1 Pantoea agglomerans strain CFSAN047153
CP014129.2 Pantoea vagans strain FDAARGOS 160
CP028349.1 Pantoea vagans strain PV989
AJ300463.1 Erwinia pyrifoliae
The AG085-I, AG086-I, AG087-I, AG088-I bacterial isolates,
obtained from Group I flea beetles C. minuta, are clustered with Pan-
toea ananatis.The AG024-VI bacterial isolate obtained from Group
VI flea beetle ChrysSA sp. 47 genus, corresponding to P. stewartii
subsp. stewartii. The sequence of Erwinia pyrifoliae was included as
an outgroup
Journal of Plant Pathology (2021) 103:539–547
Fig. 5  Pathogenicity tests.
The image shows symptoms
of the fruit bronzing disease
showing reddish-brown spots
affecting the pulp and rags
(arrows). a) jackfruits inocu-
lated with P. stewartii subsp.
stewartii JM5´mexican isolate.
b) jackfruits inoculated with
the P. stewartii subsp. stewartii
AG024-VI isolate
solution. The macroscopic, microscopic, and molecular
analysis of the bacterial isolates obtained from artificially
inoculated jackfruits with presence of symptoms, showed
that these isolates have identical characteristics to those
of the initial strain (Data not shown). Thus, these results
demonstrate that isolates of P. stewartii subsp. stewartii,
present in ChrysSA genus flea beetles have pathogenic
potential against jackfruit.
Discussion
The fruit bronzing disease is a newly emerging disease of
jackfruit crops whose causal agent is the P. stewartii subsp.
stewartii bacterium (Gapasin et  al. 2014). Thus far, the
biology of this bacterium during the stages that make up
the cycle of the fruit bronzing disease is poorly understood.
In the present work, we initiated the study of insects that
carry P. stewartii subsp. stewarti in jackfruits crops,
as approaching to the strategies used by this bacterium
during its spreads in this crop. In this study, we report the
association of flea beetles of the ChrysSA genus with P.
stewartii subsp. stewartii bacteria, which have pathogenic
potential against jackfruits. From the results of this work, we
hypothesize a role of these insects during the spread stage
of the bacterium into the cycle of the fruit bronzing disease.
Microbiological and molecular analysis performed using
oligonucleotides widely reported as specifics for P. stewartii
subsp. stewartii, allowed us to demonstrate the presence
of flea beetles infested with P. stewartii subsp. Stewartii
in jackfruits crops. Only five bacterial isolates of the total
evaluated, AG085-I, AG086-I, AG087-I, AG088-I, and
AG024-VI, obtained from insects belonging to Group I and
Group VI, showed the presence of amplicon in expected
sizes. AG085-I, AG086-I, AG087-I, AG088-I isolated from
Group I flea beetles, and AG024-VI isolated from Group
VI flea beetles. Bioinformatic analyses based on the cox1
sequence showed that the insect specimens belonging to
Group I correspond to C. minuta. While the identity of
the flea beetles belonging to Group VI was determined as
545
ChrysSA genus. To our knowledge, this is the first report
of these genus/species of flea beetles in jackfruit crops.
Even more, this is the first report of flea beetles present
in jackfruit crops. In general, most species of flea beetle
attack only one plant group or closely related groups.
Common agricultural and garden hosts include members of
Brassicaceae (mustard, broccoli, cabbage, etc.), solanaceous
families (potatoes, tomatoes, eggplant, peppers, etc.), and
cereals (including maize). Other hosts of flea beetle include
alder, currant, evening primrose, sedum, skunkbrush, sumac,
willow, and a variety of weeds and grasses (Bunn et al. 2015;
Cranshaw 2014; Gikonyo et al. 2019).
Finally, the identity of the bacterial isolates obtained
AG085-I, AG086-I, AG087-I, AG088-I, and AG024-VI, was
validated by analysis of the cpsDE and 16S rDNA region
sequences. Surprisingly, the bioinformatic analysis showed
the AG085-I, AG086-I, AG087-I, AG088-I isolates obtained
from C. minuta (Group I) correspond to P. ananatis and not
to P. stewartii subsp. stewartii as might be expected after
obtaining amplicons of the expected size in these isolates
using oligonucleotides specifics for this bacterial subspecies.
In addition, the analysis of the cpsDE and 16S rDNA regions
of the AG024-VI isolate obtained from the ChrysSA genus
insects (GroupVI) showed that this isolate corresponds to P.
stewartii subsp. stewartii. Thus, the results demonstrate the
existence of an association with P. stewartii subsp. stewartii-
ChrysSA genus flea beetles in jackfruit crops. Furthermore,
these results could suggest a role of the ChrysSA genus
flea beetles in the spread of the P. stewartii subsp. stewartii
bacterium into jackfruit crops.
The fact that isolates of bacterial species different from
P. stewartii, such as P. ananatis, showed the presence of
amplicons of identical size to that of P. stewartii subsp.
stewartii by using oligonucleotides reported as specific for
this subspecies (CPSL1- CPSR2c and ES16-ESIG2c), result
of importance. Some literature, including phytosanitary
guides, have reported the use of these set of primer pairs as
part of the strategies of identification or confirmation of P.
stewartii subsp. stewartii (Gapasin et al. 2014; EPPO 2006,
2016; Palacio-Bielsa et al. 2009). Previously, the obtention
13
546
of amplicons in bacterial species different from P. stewartii,
particularly in P. ananatis (formerly P. ananas), by using the
primers ES16-ESIG2c had been reported (Coplin et al. 2002;
Coutinho and Venter 2009). Likewise, some P. agglomerans
isolates showed the presence of amplicon after their
analysis with the primers CPSL1-CPSR2c. However, these
amplifications obtained from bacterial species different from
P. stewartii occurred in very low proportion or frequency
besides appearing as faint bands or even of variable size
(Coplin et  al. 2002). Furthermore, P. ananatis isolates
had already been reported, which showed the presence of
amplicons of identical sizes to those of P. stewartii subsp.
stewartii, after they were analyzed by PCR with both pairs
of primers CPSL1-CPSR2c and ES16-ESIG2c (Pérez et al.
2012). These findings together with those obtained in this
work, still highlight the need to develop more specific
molecular techniques for the identification of this species-
subspecies, and further emphasize the fact that current
diagnostic strategies for P. stewartii subsp. stewartii should
be based on the development of diverse analyses.
On the other hand, the fact that a higher proportion of
P. ananatis isolates were obtained from field-collected
flea beetles versus the P. stewartii subsp. stewartii isolates
could be related to the particular biology of P. ananatis.
The P. ananatis phytopathogenic bacterium, which shows
morphological characteristics similar to those of P.
stewartii subsp. stewartii, has been found associated with
diverse insects (Coutinho and Venter 2009; Walterson
and Stavrinides 2015). In fact, P. anantis is recognized as
a common inhabitant of the gut microflora of invertebrate
hosts including fleas, thrips, ticks, and mulberry pyralids
(Murrell et al. 2003; Watanabe and Sato 1999; Walterson
and Stavrinides 2015; Wells et al. 2002). In contrast, P.
stewartii subsp. stewartii has been reported to be associated
with only some insects and whose association, in particular
with its main Chaetocnema pulicaria vector insect has
been reported as variable showing temporal changes
within the seasons (Esker and Nutter 2003; Walterson and
Satvrinides 2015). Thus, a previous analysis about the P.
stewartii- infested corn flea beetle populations estimated that
only 10 to 20% of C. pulicaria populations were infested
with P. stewartii subsp. stewartii. Furthermore, the analysis
of the temporal dynamics of P. stewartii-infested corn flea
beetle populations showed proportions of P. stewartii-
infested beetles ranged from 0.04 to 0.86 within the seasons
(Esker and Nutter 2003). This in a way makes sense with
what was obtained in this work where only the AG024-VI
isolate of P. stewartii subsp. stewartii was obtained from
field-collected ChrysSA genus flea beetles during the
winter season in which, in the case of corn crops, showed
the lowest proportion of P. stewartii-infested C. pullicaria
beetles (0.04–0.11) in the field (Esker and Nutter 2003). In
this study, a total of thirty individuals of ChrysSA genus
13
Journal of Plant Pathology (2021) 103:539–547
flea beetles were collected, of which a proportion of 0.10
(3/30) showed association with P. stewartii subsp. stewartii.
The low number of ChrysSA genus individuals collected
during the sampling could be related to the existence of
temporary fluctuations of populations of these insects in a
similar way to that reported for C. pullicaria in corn, where
it has been observed that the populations of C. pullicaria
are variable throughout the seasons, dates and locations
of sampling. Even more, the existence of C. pullicaria
beetle-free periods in corn has been reported (Esker and
Nutter 2003). Therefore, analysis of the temporal dynamics
of ChrysSA flea beetles and P. stewartii-infested flea beetle
populations in jackfruit crops is necessary. Finally, in order
to evaluate the pathogenic potential of the P. stewartii subsp.
stewartii AG024-VI isolate against jackfruit, pathogenicity
assays were performed. These analyses demonstrated that
P. stewartii subsp. stewartii isolates obtained from ChrysSA
genus flea beetles can infect the jackfruit crop causing the
fruit bronzing disease. We hypothesize that ChrysSA genus
flea beetle is the vector insect of P. stewartii subsp. stewartii
in jackfruit crops and could be involved in the inoculation
stage during the cycle of the fruit bronzing disease. More
experimental evidence is necessary to corroborate this
hypothesis. Currently, the information in relation to ChrysSA
genus flea beetles is very scarce and there are reports only
on the repository of specimens of these insects.
In general, the results of this work demonstrate the exist-
ence of an association of P. stewartii subsp. stewartii patho-
genic bacteria with ChrysSA genus flea beetles in Jackfruit.
Supplementary Information  The online version contains supplemen-
tary material available at https:​ //doi.org/10.1007/s42161​ -021-00787-​ 4.
Funding  No funding was received for conducting this study.
Declarations 
Conflicts of interest  The authors declare that they have no conflict of
interest.
References
Abidin N, Zulperi D, Ismail S, Termizi Yusof M, Ismail-Suhaimy N,
Singh Karam D, Hakiman M (2018) Diagnostic Approach and
Genetic Diversity of Jackfruit Bronzing Bacterium in Malaysia.
Asian J Plant Pathol 12:46–55. https​://doi.org/10.3923/ajppa​j.
2018.46.5
Bunn B, Alston D, Murray M (2015) Flea Beetles on Vegetables
(Coleoptera: Chrysomelidae) Utha State Univeristy Extension
ENT-174–15. https​://utahp​ests.usu.edu/uppdl​/files​-ou/facts​heet/
flea-beetl​es.p
Cambero OJ, Johansen R, Retana A, García O, Cantú M, Carvajal C
(2010) Thrips (Thysanoptera) del aguacate (Persea americana) en
Nayarit, México. Rev Colomb Entomol 36:47–51
Journal of Plant Pathology (2021) 103:539–547
Chen W, Kuo T (1993) A simple and rapid method for the preparation
of gram negative bacterial genomic DNA. Nucleic Acids Res
21:226
Coplin DL, Majerczak DR, Zhang Y, Kim W, Jock S, Geider K (2002)
Identification of Pantoea stewartii subsp. stewartii by PCR and
Strain Differentiation by PFGE. Plant Dis 86:304–311
Correa VR, Majerczak DR, Ammar E, Merighi M, Coplin DL, Pratt
RC, Redinbaugh MG, Hogenhout SA (2008) Characterization of
a Pantoea stewartii TTSS gene required for persistence in its flea
beetle vector. Phytopathology 98:S41
Correa VR, Majerczak DR, Ammar E, Merighi M, Pratt RC,
Hogenhout SA, Coplin DL, Redinbaugh MG (2012) The
bacterium Pantoea stewartii uses two different type III secretion
systems to colonize its plant host and insect vector. Appl Environ
Microbiol 78:6327–6336
Cook KA, Weinzieri R (2004) Corn Flea Beetle (Chaetocnema
pulicaria Melsheimer).University of Illinois inetgrated pest
managment. Insect fact sheet. https​://ipm.illin​ois.edu/field​crops​/
insec​ts/corn_flea_beetl​e.pdf
Coutinho TA, Venter SN (2009) Pantoea ananatis: an unconventional
plant pathogen. Mol Plant Pathol 10:325–335. https​://doi.org/10.
1111/J.1364-3703.2009.00542​.X
Cranshaw WS (2014) Flea Beetles.Colorado State University Ext Fact
Sheet No.5.592. http://www.ext.colos​tate.edu/pubs/insec​t/05592​
EPPO (European and Mediterranean Plant Protection Organization),
(2006) PM 7/60 (1) Pantoea stewartii subsp. stewartii.
EPPO Bulletin 36:111–115. https​: //doi.org/10.1111/j.1365-
2338.2006.00920​.x
EPPO (European and Mediterranean Plant Protection Organization),
(2016) PM 7/60 (2) Pantoea stewartii subsp. stewartii. EPPO
Bulletin 46:226–236. https​://doi.org/10.1111/epp.12303​
Esker PD, Nutter FW (2003) Temporal Dynamics of corn flea beetle
populations infested with Pantoea stewartii, causal agent of
Stewart´s disease of corn. Phytopathology 93:210–218. https​://
doi.org/10.1094/PHYTO​.2003.93.2.210
Gapasin RM, Garcia RP, Advincula CT, De la Cruz CS, Borines
LMFruit Bronzing: a New Disease Affecting Jackfruit Caused
by Pantoea stewartii (Smith) Mergaert, et al (2014) Annals of
Tropical Research 36:17–31
Gikonyo MW, Biondi M, Beran F (2019) Adaptation of flea beetles to
Brassicaceae: host plant associations and geographic distribution
of Psylliodes Latreille and Phyllotreta Chevrolat (Coleoptera,
Chrysomelidae). ZooKeys 856:51–73. https​://doi.org/10.3897/
zooke​ys.856.3372
Hernández-Morales A, Pérez-Casillas JM, Soria-Guerra RE,
Velázquez-Fernández JB, Arvizu-Gómez JL (2017). First report
of Pantoea stewartii subsp. stewartii causing jackfruit bronzing
disease in Mexico. J Plant Pathol 99:807
Jeger M, Bragard C, Candresse T, Chatzivassiliou E, Dehnen-Schmutz
K, Gilioli G, Grégoire JC, Jaques Miret JA, MacLeod A, Navajas
547
M, Niere B, Parnell S, Potting R, Rafoss T, Rossi V, Urek G,
Van Bruggen A, Van der Werf W, West J, Winter S, Manceau
C, Pautasso M, Caffier D (2018) Pest categorisation of Pantoea
stewartii subsp. stewartii. EFSA Journal 16:5356. https​://doi.
org/10.2903/j.efsa.2018.5356
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular
Evolutionary Genetics Analysis version 7.0 for bigger datasets.
Mol Biol Evol 33:1870–1784
McDaniel B, Clay SH, Scholes C (1992) Morphology of three imported
Aphthona flea beetles used as Biological Control Agents of Leafy
Spurge. Technical Bulletins 98. South Dakota State University. 56
p. Available at: https​://openp​rairi​e.sdsta​te.edu/agexp​erime​ntsta​_
tb/10/. https​://doi.org/10.13140​/RG.2.2.34491​.00807​
Murrell A, Dobson SJ, Yang X, Lacey E, Barker SC (2003) A survey of
bacterial diversity in ticks, lice and fleas from Australia. Parasitol
Res 89:326–334
Palacio-Bielsa A, Cambra MA, López MM (2009) PCR detection and
identification of plant-pathogenic bacteria: updated review of
protocols (1989–2007). J Plant Pathol 91:249–297
Pérez Y, Terrón R, Carmona JC, Cebada JA, Munive JA (2012)
Bacterial pathogenicity in corn. CIBA Revista Iberoamericana
de las Ciencias Biológicas y Agropecuarias 1:24–39
Roper MC (2011) Pantoea stewartii subsp. stewartii: lessons learned
from a xylem-dwelling pathogen of sweet corn. Mol Plant Pathol
12:628–637
Walterson AM, Satvrinides J (2015) Pantoea: insights into a highly
versatile and diverse genus within the Enterobacteriaceae. FEMS
Microbiol Rev 39:968–984. https:​ //doi.org/10.1093/femsr​e/fuv02​7
Watanabe K, Sato M (1999) Gut colonization of an ice nucleation
active bacterium, Erwinia (Pantoea) ananas, reduces the cold
hardiness of mulberry pyralid larvae. Cryobiology 38:281–289.
https​://doi.org/10.1006/cryo.1999.2169
Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S
ribosomal DNA amplification for phylogenetic study. J Bacteriol
173:697–703
Wells ML, Gitaitis RD, Sanders FH (2002) Association of
tobacco thrips, Frankliniella fusca (Thysanoptera: thripidae),
with two species of bacteria of the genus Pantoea. Ann
Entomol Soc Am 95:719–723. https​://doi.org/10.1603/0013-
8746(2002)095[0719:AOTTF​F]2.0.CO;2
Zulperi D, Manaf N, Ismail S, Karam Singh DS, Yusof M (2017)
First report of Pantoea stewartii subspecies stewartii causing
Fruit Bronzing of Jackfruit (Artocarpus heterophyllus), a
New Emerging Disease in Peninsular Malaysia. Plant Dis
101:831. https​://doi.org/10.1094/PDIS-11-16-1689-PDN
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
13