Analysis of virulence factors of Chilean

Pseudomonas syringae pv. actinidiae isolates: causal agent

of bacterial canker of Kiwifruit
Ms. Camila Prince1, Dr. Oriana Flores1, Mr. Mauricio Núñez1 , Dr. Ximena Besoain2, Dr. Carolina Yañez1, Dr. Roberto Bastías1.
1. Laboratory of Microbiology, Institute of Biology, Faculty of Sciences, Pontifical Catholic University of Valparaíso, Chile.
2. Laboratory of Phytopatology, Faculty of agronomy, Pontifical Catholic University of Valparaíso, Chile.

Abstract RESULTS
Background: Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes a severe economic losses in
kiwifruit production of worldwide, including Chile. Phylogenetic analysis by MLST and Rep-PCR suggest Chilean Psa
isolates are genetically homogenous. In this work, 18 Chilean Psa isolates obtained from VII and VIII regions, were
Floatting-biofilm formation and motility of Chilean Psa isolates
characterized according to virulence factors such as motility, biofilm formation, indole production, and presence of B
effectors genes. Methods: Swimming and swarming motility were evaluated using LB medium with different agar
compositions at 72 h post-inoculation. Biofilm production in surface adhered were determined by microtiter dish assay
after 7 days of incubation, while air-liquid interface in static conditions were observed at 96 h post-
inoculation. Tryptophan-dependent indole production was evaluated using the Salkowski assay. Presence indole Figure 3. Floatting-biofilm
production-pathway and effectors genes were analyzed by PCR reactions with specific primers. Results: All Chilean Psa formation and motility of
isolates had swimming motility and only for Psa 889 isolate the movement were reduced. Swarming phenotype were Chilean Psa isolates. A. Assays
reduced for Chilean Psa isolates and only Psa 510 had a slightly greater displacement. Psa isolates had a lower ability to for biofilm pellicle type
produce a surface-biofilm, however, a thin layer of cell aggregates air-liquid interface were observed. Indole production formation in air-liquid interface
by tryptophan-dependent pathway were detected in Psa isolates, which are similar to indoleacetic acid (IAA) produced were performed in top of culture
by plant growth-promoting rhizobacteria. No amplicons were obtained for indole-3-acetamide (IAM) pathway genes, a of Chilean Psa isolates. Evolution
common mechanism for IAA production in P. syringae pathovars, however, all isolates harbor iaaL gene that encoded of biofilm were registred by
for enzyme that produce indole-3 acetyl-3-L-lysine (IAA-Lys), which would have a rol in fitness and virulence in others photographed each 24 h for 96 h
phytopathogens. Presence of 16 effector genes belongs to Biovar 3 cluster, the hyper-virulent pandemic form of post-inoculation. B,C. Abilities in
Psa, were confirmed in all Chilean Psa isolates. Conclusion: In this work, we contrast the high genetic homogeneity swimming or swarming
between Chilean Psa isolates with phenotypic variation found in virulence factors as biofilm production and bacterial movement were determined
motility. Our results are the first report about the ability of Psa Biovar 3 to produce indole by tryptophan -dependent using LB medium plates with
pathway, however, further studies are needed to determined importance of IAA-Lys in Psa virulence and kiwifruit 0.3% and 0.5% agar, respectively.
infections. Bacterial suspensions were
inoculated onto the center on
plate and movement of bacteria
were observed by halo
Introduction formation arround. Plates were
photographed and the diameter
Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes the bacterial canker of kiwi, a disease that has
of each halo was measured at 72
caused great economic losses worldwide. The most characteristic symptoms of this disease are leaf spots, and cankers with
h post-inoculation. Names of
reddish exudations on the trunk and branches, which finally produce the wilting of the plant until it causes its death (Figure
isolates are shown.
1). At present, 5 biovars or different biotypes of Psa are known according to their genetic, phenotypic and virulence
characteristics. Psa was detected for the first time in Chile in 2010 and since then it has expanded rapidly in several regions of
the country, causing large losses at the national level. Phylogenetic analysis by MLST suggest Chilean Psa isolates belong to
Psa Biovar 3 cluster (Figure 2)

Indole production in Psa isolates

Figure 2. Phylogenetic tree of chilean
Psa isolates derived from Multi Locus
Sequencing Analysis (MLST). Sequences
of four genes housekeeping (gapA, gltA,
gyrB and rpoB) were concatenated and
alignment. The phylogenetic tree was
inferred using the Neighbor-Joining
method. Tip labels show country and
year of isolation. Biovar clade is
indicated. P. syringae pv. tomato (Pst
DC3000) genes were included in
analysis.
Figure 1. Symptoms of canker bacterial
of kiwifuit orchards by Psa
infection in 2011 (SAG)
Objetive
Characterize chilean Pseudomonas syringae pv. actinidiae isolates according to virulence factors such as motility, biofilm
formation, indole production, and presence of effectors genes.
Metodology
Chilean Psa isolates were obtained from the Agricultural and Livestock Service (SAG) from orchards of kiwifruit plants located
in the regions of Maule and Bío Bío, which presented the characteristic symptoms of Psa infection. In this work, virulence
factors described in Psa Biovar 3 (Biofilm formation, Motility, Indole formation, and effectors genes presence) were
determined in Chilean Psa isolates and phenotypic diversity between them were analyzed.
Table 1. Chilean Psa isolates
Floatting biofilm ✓ Air-liquid interphase
formation
Chilean Psa
✓ Swimming
isolates Motility plate assay
✓ Swarming
✓ Salkowsky assay
Indoles production ✓ iaaL/matE genes
identification
Presence of effectors ✓ Identification
genes in bacterial genome
✓ PCR detection
Figura 3. Indole production and iaaL/matE genes
identification in Chilean Psa isolates. A. Bacterial
indole production were analyzed by colorimetric
technique using Salkowski reagent and, the color
obtained was measured using the
spectrophotometer and the values were compared
with a standard curve. The indole production
obtained for each isolate as shown. A. brasiliensis
(Ab) and S. bongori (Sb) were used for positive and
negative control, respectively. B. Amplicons from
PCR reactions by iaaL (top) and matE (below) genes
identification vizualized in agarose electrophoresis.
Genomic DNA extracted from Chilean isolates, A.
brasiliensis (Ab) and S. bongori (Sb) were used in PCR
with specific primers for iaaL (⁓ 1,2 kb) and matE (⁓
1,5 kb) genes detection. 1Kb: Molecular weight
marker. Molecular weight markers: 1Kb and 100 bp.
Control (-): Reaction without DNA. Bacterial names
are indicated
Presence of effector genes
Table 2. Identification and detection of effectors genes in
Chilean Psa isolates. Psa isolates of this work were clustering in
Psa biovar 3, which have been considered as “Hyper-Virulent”
group and repertory of effector gene could be important bacterial
in virulence. According to previously works, we analyzed the
presence of putatives effector genes has been identified in Psa
biovars (Presence gene: pink; Absence gene: white; Variable
presence: variable; Incomplete sequence: Imcomplete). For
detection of type III effector genes, specific primers were
designed considering the sequences present in Biovar 3 and in
contigs of Chilean Psa genomes, ICMP 19439 (ANJM00000000.1)
and ICMP 19455 (ANJK00000000.1) availables in Genbank (NCBI).
Seventeen Type III effector genes (Bolded), were identified all
Chilean Psa isolates by PCR reactions including genes encoded by
genome and conjugative DNA plasmid (hopAV1 and hopAU1,).
Plasmid presence not have been reported in Psa Chilean isolates
and further studies are necessary for demonstrate its existence
and importance in bacterial virulence.

Conclusions
In this work, we contrast the high genetic homogeneity between Chilean Psa isolates with phenotypic
variation found in virulence factors as biofilm production and bacterial motility. Our results are the first
report about the ability of Psa Biovar 3 to produce indole by tryptophan -dependent pathway, however,
Supported By: Acknowledgment further studies are needed to determined importance of IAA-Lys in Psa virulence and kiwifruit infections
s
Acknowledgments References
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plant-pathogen interactions: a key molecule for in planta bacterial virulence and fitness. Res. Microbiol. 167,774-787
FONDECYT Postdoctorado 2017 N°3170567
2. HC. McCann et al. 2017. Origin and Evolution of Kiwifruit Canker Pandemic. Genome Biol Evol. 9:932-944
3. SAG web page: http://www.sag.cl/ambitos-de-accion/bacteriosis-del-kiwi-psa