AEM Accepted Manuscript Posted Online 5 June 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.00835-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 1

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Phylogenetic and VNTR analysis Identified Non-pathogenic Lineages within Xanthomonas

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arboricola Lacking the canonical Type Three Secretion System

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Salwa Essakhia *, Sophie Cesbrona, Marion Fischer-Le Sauxa, Sophie Bonneaua, Marie-Agnès

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Jacquesa, Charles Manceaua, b#

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a

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b

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Angers, France

INRA, UMR 1345, Institut de Recherche en Horticulture et Semences, Beaucouzé, France ; Anses, Laboratoire de la Santé des Végétaux, Unité Expertise – Risques Biologiques,

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Running Head: Non-pathogenic X. arboricola strains lacking T3SS

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Journal: Applied and Environmental Microbiology

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#

Address correspondence to Charles Manceau, [email protected]

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*Present address: Clermont Université, VetAgro Sup, INRA UMR 1095 GDEC, BP 10448,

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F-63000, 18 Clermont-Ferrand, France.

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 2

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ABSTRACT

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Xanthomonas arboricola is conventionally known as a taxon of plant-pathogenic bacteria,

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including seven pathovars. This study showed that X. arboricola encompasses also non-

30

pathogenic bacteria causing no apparent disease symptoms on their hosts. The aim of this

31

study was to assess the X. arboricola population structure associated to walnut including non-

32

pathogenic strains, in order to gain a better understanding of the role of non-pathogenic

33

xanthomonads in walnut microbiota. A multi-locus sequence analysis (MLSA) was performed

34

on a collection of 100 X. arboricola strains including 27 non-pathogenic strains isolated from

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walnut. Non-pathogenic strains grouped outside clusters defined by pathovars and formed

36

separate genetic lineages. A multi-locus variable-number tandem repeat analysis (MLVA)

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conducted on a collection of X. arboricola strains isolated from walnut, showed that non-

38

pathogenic strains clustered separately from clonal complexes containing Xanthomonas

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aroboricola pv. juglandis strains. Some non-pathogenic strains of X. arboricola did not

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contain the canonical type III secretion system (T3SS) and harboured only one to three type

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III effectors (T3E) genes. In non-pathogenic strains CFBP 7640 and CFBP 7653, neither

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T3SS genes, nor any of the analysed T3E genes were detected. This finding raises the

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question about the origin of non-pathogenic strains and evolution of plant pathogenicity in X.

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arboricola. T3E genes that were not detected in any non-pathogenic isolates studied represent

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excellent candidates to be those responsible for pathogenicity in X. arboricola.

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 3

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INTRODUCTION

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Eubacteria constitute a major component of the commensal microbiota and their interaction

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with plants is still unknown (1). Xanthomonas strains living in close association with plants

54

but causing no apparent disease symptoms on their host have been reported (2, 3). Based on

55

amplified fragment length polymorphism (AFLP) analysis, Gonzalez et al. (2) showed that

56

non-pathogenic Xanthomonas strains colonizing cassava were clearly distinguished from

57

Xanthomonas axonopodis pv. manihotis strains that cause cassava bacterial blight. Although

58

some of these non-pathogenic strains have been characterized genetically and phenotypically,

59

little is known about their epidemiological or ecological importance.

60

In previous studies, the genetic diversity and population structure of Xanthomonas have been

61

performed using DNA-DNA hybridization (4, 5); repetitive-sequence PCR (rep-PCR) (4–7);

62

AFLP (4, 8), and fluorescent AFLP (9, 10). As an alternative to these methods, the

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comparative sequence analysis of protein-encoding genes has also been widely explored. For

64

example, Parkinson et al. (11) used the gyrB gene, which encodes the subunit B protein of

65

DNA gyrase, for establishing a phylogenetic relationship among 203 Xanthomonas pathotype

66

strains. Young et al. (12, 13) used a multi-locus sequence analysis (MLSA) based on four

67

genes (dnaK coding the chaperone protein, fyuA coding one tonB-dependent transporter, gyrB

68

and rpoD coding the RNA polymerase sigma factor) to study the phylogenetic and taxonomic

69

relationships within the genus Xanthomonas. MLSA is a powerful technique for inferring

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phylogenetic relationships at the interspecific and intraspecific levels, as well as for

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evolutionary studies and systematics and can be useful in bacterial taxonomy as a

72

complementary tool for defining species and for identification of new strains (14, 15). MLSA

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provides a robust method for the differentiation of most Xanthomonas spp. One of its most

74

important contributions, applied to Xanthomonas, is that it allows strains to be allocated to

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known species or be indicated as members of new species more easily. Moreover, MLSA

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 4

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generally mimics grouping generated by DNA-DNA hybridization within Xanthomonas,

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AFLP and rep-PCR and may therefore offer a refined method for differentiation of species

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(13). In some cases, MLSA is insufficient for discriminating closely related isolates and

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studying intra-species genetic diversity. Thus, highly discriminative typing methods are

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needed for surveillance and outbreak studies. Multi-locus variable-number tandem repeat

81

(VNTR) analysis (MLVA) has been successfully developed for many bacterial species

82

(16–21). It is a bacterial typing method involving amplification and fragment size analysis of

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polymorphic regions of DNA containing variable numbers of tandem repeat sequences.

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VNTRs can be rapidly characterized by PCRs with specific primers based on the flanking

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regions of the tandem repeats. MLVA based on a few highly variable VNTRs usually displays

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a high level of discriminatory power in distinguishing closely related isolates for the

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investigation of disease outbreaks and epidemiological studies (16–21).

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Vauterin et al. (3) suggested the investigation of hrp (hypersensitive reaction and

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pathogenicity) genes to distinguish the non-pathogenic Xanthomonas strains from the

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pathogenic ones. hrp genes are known to be involved in induction of hypersensitive response

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(HR) in resistant host and non host plants and pathogenicity in susceptible host plants

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(22, 23); hrc (hrp conserved) genes, are considered to be critical for pathogenicity and

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initiation of disease and encode the type III secretion system (T3SS), a highly conserved

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protein secretion system (22, 24). Previous studies reported that the distribution of Type III

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effectors (T3Es) within Xanthomonas strains may suggest a basic role in host specificity (25).

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T3Es are candidate determinants of host specificity of pathogenic bacteria since it has been

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shown that many T3Es can act as molecular double agents that betray the pathogen to plant

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defences in some interactions and suppress host defences in others (26). More recently, Hajri

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et al. (27) investigated the variability of T3E repertoires in the species X. arboricola, and their

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potential role in structuring its populations according to host range and confirmed that T3SS

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 5

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is an essential virulence mechanism in X. arboricola.

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Walnut blight (WB), caused by Xanthomonas arboricola pv. juglandis (5) is a major disease

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of walnut in France and the most important one in all walnut-growing areas (28). Common

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symptoms include stem, fruit and leaf spots; catkin necrosis as well as fruit drop. Previous

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studies showed that X. a. pv. juglandis has been also isolated from tissues affected by brown

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apical necrosis (29–31). Hajri et al. (10) reported the association of X. a. pv. juglandis with

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vertical oozing canker (VOC) and clarified the taxonomic position of VOC strains as

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belonging to a singular lineage within X. a. pv juglandis. During surveys in the two main

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production areas of walnut in France (Grenoble in the southeast and Périgord in the

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southwest), we noticed that non-pathogenic strains of X. arboricola were isolated from

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healthy and diseased walnuts. These strains were characterized by pathogenicity tests on

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walnut seedlings and a range of other plants. The main aim of the present study was to assess

113

the X. arboricola populations structure associated to walnut including non-pathogenic strains,

114

in order to gain a better understanding of the role of non-pathogenic xanthomonads in walnut

115

microflora. Knowledge pertaining to the population structure of X. arboricola isolated from

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walnut, should shed light on the epidemiology of diseases associated with X. a. pv. juglandis,

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with the final aim of helping in development of reliable identification and specific detection

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tools that will facilitate ecological and epidemiological studies. Hence, the genetic structure of

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non-pathogenic strains was investigated using MLSA and MLVA approaches and the type of

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interaction that this group of bacteria develops with host and non-host plants was

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characterized. In this context, we investigated the distribution of T3Es and T3SS coding genes

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in non-pathogenic strains.

123 124 125

MATERIAL AND METHODS

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 6

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Bacterial strains. The bacterial strains used in this study are listed in Table 1. Strains of X.

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arboricola were obtained from the International Center for Microbial Resources, French

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Collection

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(http://www.angers.inra.fr/cfbp/) or isolated from buds of healthy walnuts in the two main

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walnut-growing areas in France (Rhône-Alpes region in the south-east and Périgord in the

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south-west). Bacterial strains were routinely cultured at 27°C on YPGA medium (7 g L-1

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yeast extract; 7 g L-1 peptone; 7 g L-1 glucose; 15 g L-1 agar) for 24 to 48 h.

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Plant material. Seedlings of walnut (Juglans regia cv. Fernor and cv. Franquette), peach

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(Prunus persica cv. Dixired), radish (Rhaphanus sativus var. Kocto), tomato (Lycopersicon

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esculentum cv. marmande) and pepper (Capsicum annum cv. ECW) were used for

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pathogenicity tests. Hypersensitivity reaction was induced on leaves of Nicotiana

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benthamiana. Plants were grown in a greenhouse under 18°C at night and 24°C during day

138

with a 12-h photoperiod. For negative controls, plants were inoculated with sterile distilled

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water. For positive controls, plants were inoculated with X. a. pv. juglandis strains CFBP

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2528PT and CFBP 7179 for walnut, X. a. pv. pruni CFBP 3894PT for peach, Xanthomonas

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vesicatoria CFBP 1941 for tomato, Xanthomonas euvesicatoria CFBP 5618 for pepper, X.

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campestris pv. campestris CFBP 5241 for tests on R. sativus and N. benthamiana plants.

143

Pathogenicity tests.

144

Walnut seedlings were grown in a greenhouse until four to six young leaves. Bacterial

145

suspensions (1 x 108 CFU ml-1) were sprayed onto the foliage and plants were maintained for

146

two days under plastic bags and incubated in growth chambers. The plastic bags were then

147

removed and the plants were maintained in the growth chamber under the same climatic

148

conditions. Plants were checked for symptoms weekly for up to 30 days after inoculation.

149

Two-year-old peach seedlings were planted in 30 cm diameter pots. Young leaves (third to

150

sixth leaves from shoot tip) were detached from peach seedlings, collected and inoculated

for

Plant

associated

Bacteria,

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(CIRM-CFBP),

INRA,

Angers,

France

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 7

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using a detached leaf assays as decribed by Randhawa and Civerolo (32). Detached leaves

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were disinfected for 40–60 s with 70% ethanol and then rinsed in sterile water. These leaves

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were then immersed in a bacterial suspension (1 x 107 CFU ml-1) and a 0.1 bar vacuum was

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applied for 2 min. Inoculated leaves were placed in a sterile tube with the leaf upright, and the

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petiole immersed in 6% water agar. Symptom development was recorded daily for 3 weeks

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after inoculation. For a positive result, after 6–9 days all inoculated sites should exhibit

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confluent water soaking, becoming dark brown and brittle necrotic spots often surrounded by

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a greyish white or purple margin.

159 160

On radish, two leaves per plant at the stage of four full-expanded leaves were inoculated by

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the leaf-clipping method. The last completely expanded leaf was cut with scissors dipped in

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bacterial suspensions (1 x 108 CFU ml-1). Ten leaves were inoculated for each strain. For a

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positive result, V-shaped lesions appeared at the leaf margin after approximately two weeks

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of inoculation.

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Leaves of tomato and pepper were punctured at four locations with a sterile needle and 1 ml

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of bacterial suspension (1 x 106 CFU ml-1) was infiltrated through wounds. For a positive

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reaction, inoculated leaves of tomato should exhibit dark brown irregular shaped splotches

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with chlorosis surrounding lesions, while inoculated leaves of pepper should hold small,

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yellow-green lesions that became deformed and twisted.

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Leaves of N. benthamiana were punctured at four locations with a sterile needle and 1 ml of

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bacterial suspension (1 x 106 CFU ml-1) was infiltrated through wounds. Necrosis of the

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infiltrated area after 24 – 48 hr was considered as hypersensitive reaction (HR).

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The disease occurrence was monitored by quantification of symptoms and bacterial

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populations at 2, 7, 14 and 21 days after inoculation in plant tissues. Inoculated plants were

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 8

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maintained in growth chambers under 18°C at night and 20°C during day with a 15-h

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photoperiod (light intensity of 85 μE m−2 s−1) and a high relative humidity.

177

Multi-locus sequence analysis (MLSA). Gene fragments of atpD, dnaK, efP, fyuA, glnA,

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gyrB and rpoD were amplified from genomic DNA of the 27 non-pathogenic strains using

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primers described by Fargier et al. (14). A list of genes and primer sequences used for PCR

180

amplification and sequencing is provided inTable 2. PCR amplifications were carried out in a

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total volume of 25 µL containing 1 x GoTaq buffer (Promega, Fitchburg, WI, USA), 200 µM

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(each) deoxynucleoside triphosphates (dNTPs), 0.5 µM (each) primers, 0.4 U of GoTaq

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polymerase (final concentrations), and 5 µl of boiled bacterial cells (3 x 108 CFU ml-1). The

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PCR cycling conditions consisted of an initial denaturation step at 94°C for 5 min, followed

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by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for all loci (except 62°C for

186

efP) for 60 s, extension at 72°C for 30 s, and a final extension step at 72°C for 7 min.

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PCR amplicons sequencing was performed by the Biogenouest platform (Nantes, France).

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Nucleotide sequences were corrected using Geneious v. 4.8.4 (33) and edited using BioEdit

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(34). These sequences were aligned together with sequences of 73 representative strains of

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Xanthomonas

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(http://www.angers.inra.fr/cfbp/) using Clustal W (35). A neighbor-joining tree was generated

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with MEGA v. 5.0.3 (36) using the Kimura two-parameter model (37) and 1,000 bootstrap

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replicates. X. campestris pv. campestris strain CFBP 5241 was used as an outgroup.

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The nucleotide sequences obtained in this work were deposited in GenBank under accession

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numbers KF904342 to KF904442 for atpD, KF904443 to KF904543 for dnaK, KF904544 to

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KF904644 for efP, KF904645 to KF904745 for fyuA, KF904746 to KF904846 for glnA,

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KF904847 to KF904947 for gyrB, KF904948 to KF905048 for rpoD.

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MultiLocus VNTR Analysis (MLVA). Seventeen loci of 6 to 15 bp tandem repeats (TR)

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units previously developed (38) were used in the MLVA scheme (Table 3). PCR and capillary

arboricola

available

from

-8-

the

CIRM-CFBP

sequence

data

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 9

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electrophoresis were conducted as in (38). Output data from capillary electrophoreses were

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managed with BioNumerics v.6.5 (Applied-Maths, St-Martens-Latem, Belgium) and

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chromatograms were also checked with Peakscanner™ Software v. 1.0 (Life Technologies).

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The allele scores based on the fragment sizes were converted into repeat numbers and used as

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character data for cluster analysis. A minimum spanning tree (MST) was generated using

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BioNumerics v. 6.5 (Applied-Maths, St-Martens-Latem, Belgium) using the categorical

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coefficient and the maximum number of single-locus variants (SLVs) as a priority rule (39).

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Amplification of T3E and T3SS genes. Non-pathogenic strains used in this study were

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tested for the presence/absence of hrp genes by PCR amplification of genomic DNA using

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primers described by Hajri et al. (27) (Tables 4 and 5). For detection of T3E genes, PCRs

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were carried out in a total volume of 20 µl containing 1 x GoTaq buffer (Promega), 200 µM

211

(each) dNTPs, 0.5 µM (each) primers, 0.4 U GoTaq polymerase (final concentrations), and 5

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µl of boiled bacterial cells (3 x 108 CFU ml-1). All PCRs were performed with the following

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cycling conditions: initial denaturation step at 94°C for 2 min, 30 cycles of denaturation at

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94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min, and a final

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extension step at 72°C for 10 min.

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RESULTS

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Identification of non-pathogenic strains of X. arboricola on walnut. Xanthomonas-like

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strains isolated from walnut buds were characterized by pathogenicity tests on walnut

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seedlings and on a range of other plants. Strains were identified as X. arboricola based upon

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phenotypic characteristics and biochemical tests as described by Schaad. (40) and

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amplification of a specific PCR fragment using the XarbQ-F and XarbQ-R PCR test (41). A

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set of 27 strains formed convex, yellow-pigmented colonies, which were characterized as

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Gram-negative rods able to perform oxidative metabolism of glucose, galactose, mannose,

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cellobiose, trehalose and arabinose; hydrolysis of gelatin, esculin, starch and Tween 20

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 10

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(except for strains CFBP 7645 and CFBP 7636 that did not hydrolyze Tween 20). A single

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amplicon at the correct size of 432 bp was obtained for each of these 27 strains using the PCR

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test (41). None of the 27 strains induced symptoms on walnut (Juglans regia), the plant from

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which they were isolated, after inoculation on walnut seedlings. Dynamics of the bacterial

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population sizes revealed that these strains were not able to reach bacterial population sizes

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higher than 1 x 106 CFU per g leaf tissue, the typical level observed for pathogenic

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interactions on walnut, whereas X. a. pv. juglandis CFBP 7179 induced typical necrotic leaf

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spots (Fig. 1) and reach about 1 x 107 CFU per g seven days after inoculation (see Fig. S1 in

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the supplemental material).

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The pathogenicity of five strains representing each clonal group identified by MLSA and

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MLVA (CFBP 7634, CFBP 7645, CFBP 7651 and CFBP 7652 and CFBP 7653) was

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evaluated on plants known to be hosts of other X. arboricola pathovars: P. persica, R. sativus,

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L. esculentum and C. annum. While positive control strains developed characteristics

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symptoms on their respective hosts, none of the five X. arboricola strains isolated from

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walnut and non-pathogenic on walnut (CFBP 7634, CFBP 7645, CFBP 7651 and CFBP 7652

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and CFBP 7653) induced any disease symptom on these plants (Fig. 2). Cell death in

241

inoculated area was observed on tomato and pepper plants following inoculation of two

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strains: CFBP 7651 and CFBP 7652 (Fig. 3). The dynamics of bacterial population sizes

243

showed that the five strains were not able to reach population sizes equal to 1 x 108 CFU per

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g, the typical level of pathogenic interactions on the tested plants (see Fig. S1 in the

245

supplemental material).

246

The ability of these strains to induce cell death was also tested on N. benthamiana. CFBP

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7651 and CFBP 7652 cause cell death on N. benthamiana within 48-72 h after inoculation;

248

while CFBP 7634, CFBP 7653 and CFBP 7645 did not elicit any plant reaction on

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incoculated N. benthamiana leaves (Fig. 4).

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 11

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MLSA confirmed that non-pathogenic strains isolated from walnut belong to X.

251

arboricola. Partial sequences of atpD, dnaK, efp, fyuA, glnA, gyrB and rpoD genes were used

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in the present study to investigate the phylogenetic relationships between pathogenic and non-

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pathogenic strains of X. arboricola isolated from walnut. A phylogenetic tree was constructed

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based on the Neighbour-joining method using the concatenated nucleotide sequences of the

255

seven gene fragments of 27 non-pathogenic strains and 73 representative strains of X.

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arboricola. The sizes of the seven gene fragments were 750 bp (atpD), 759 bp (dnaK), 339 bp

257

(efp), 753 bp (fyuA), 675 bp (glnA), 735 bp (gyrB), and 609 bp (rpoD), repectively, leading to

258

a total of 4,620 bp for the concatenated dataset. Except for the strain CFBP 7653, all strains of

259

X. arboricola used in the MLSA scheme clustered in a large clade separately from X. c. pv.

260

campestris, used as outgroup in the phylogenetic analysis (Fig. 5). Phylogenetic analyses

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distinguished several groups with high bootstrap values corresponding to different pathovars

262

of X. arboricola. This clear correspondence between phylogenetic clustering and pathovar

263

classification was not supported by phylogenetic trees based on individual loci (see Fig. S2 in

264

the supplemental material). These observed incongruences might be explained by

265

recombination events that shuffles the phylogenetic signal and by the fact that each individual

266

locus does not harbor enough phylogenetic information. Thus, the addition of non-pathogenic

267

strains in the phylogenetic tree does not modify the phylogenetic relationships between

268

pathovars of X. arboricola (pruni, corylina, fragariae, populi, celebensis, and juglandis).

269

Based on the phylogenetic position of non-pathogenic strains in the NJ tree, we confirmed

270

that non-pathogenic strains definitely belonged to X. arboricola, grouped outside clusters

271

defined by pathovars and formed separate genetic lineages. Non-pathogenic strains isolated

272

from walnut were polymorphic as they were distributed into three separated clusters within X.

273

arboricola (Fig. 5). Thus, three main groups, termed NP1, NP2 and NP3 and four single

274

branches were revealed by MLSA. The high bootstrap values of non-pathogenic strains

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 12

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depicted the robustness of these lineages; NP2 and NP3 with a bootstrap value equal to 100%,

276

and NP1 with a bootstrap value equal to 98%. For NP3, CFBP 7640 present differences in

277

nucleotide sequences of the seven genes used in the phylogenetic analysis, whereas CFBP

278

7636 and CFBP 7645 are identical. Each remaining strains, CFBP 7630, CFBP 1022 and

279

CFBP 7652 did not cluster with either NP1 or NP2 with high bootstrap support.

280

MLVA distinguished several clonal complexes within X. arboricola strains isolated from

281

walnut. Ninety three X.arboricola strains isolated from walnut, including 27 non-pathogenic

282

strains were typed using Multi-Locus Variable number tandem repeat Analysis. A minimum

283

spanning tree (MST) was constructed using the highest number of single locus variants

284

(SLVs) as the priority. The minimum spanning tree is an undirected network in which all the

285

samples within the population studied are linked together with the smallest possible linkages

286

between nearest neighbours. MLVA types were distinguished to define clonal complexes that

287

grouped strains that differ from each others by at most three locus variants (Fig. 6). Strains

288

causing VOC grouped separately from WB strains and from non-pathogenic strains isolated

289

from walnut. VOC causing strains were grouped into one clonal complex and two singletons,

290

whereas, WB strains were more heterogeneous; they were separated into two clonal

291

complexes and 16 singletons. Non-pathogenic strains were grouped separately from clonal

292

complexes containing the WB and VOC causing strains and confirmed to be heterogeneous as

293

they were divided into six clonal complexes and four singletons. Non-pathogenic strains

294

belonging to NP1 clustered in two clonal complexes and one singleton (CFBP 7638), while

295

NP2 strains clustered into three clonal complexes and two singletons, NP3 strains were

296

divided into one clonal complex and one singleton (Fig. 6).

297

In order to better understand the correlation between the population structure of non-

298

pathogenic strains and their geographical origin, we performed an MST on the 27 non-

299

pathogenic strains. Some clonal complexes grouped only strains belonging to the same

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 13

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geographic origin, i.e. isolated from the same field such as the P10 orchard located at Saint-

301

Romains (CFBP 7644, CFBP 7646, CFBP 7649, CFBP 7650) or P8 orchard located at Laval

302

(CFBP 7629, CFBP 7632, CFBP 7633, CFBP 7634). However, other clonal complexes

303

grouped strains isolated in different geographic locations such as clonal complexes grouping

304

CFBP 7631, CFBP 7637, CFBP 7648 and CFBP 7656 (Fig. 7). These clonal complexes

305

grouped strains belonging to different geographic locations from both eastern and

306

southwestern area and other non-pathogenic strains belonging to different clonal complexes

307

populations were recovered in the same orchards. Thus, the non-pathogenic strains were not

308

structured according to their geographical origins based on this MLVA.

309

Several non-pathogenic strains lack the hrp-hrc cluster coding for the Type III Secretion

310

System. Most non-pathogenic strains isolated from walnut were unable to elicit an HR on N.

311

benthamiana and to cause disease on walnut and on other plant species tested. These

312

observations suggest that these strains may lack the T3SS. We monitored the distribution of 9

313

genes coding the highly conserved genes of this secretion apparatus (27). Based on PCR

314

results, no T3SS was detected in NP1 strains, NP3 strains, and CFBP 7653 since the 9 hrp-hrc

315

gene primer pairs failed to amplify any DNA fragment for these strains. However, NP2

316

strains, CFBP 1022, CFBP 7630, and CFBP 7652 harboured genes of a typical T3SS of the

317

Hrp2 family based on our PCR results (Fig. 8).

318

The composition of T3E repertories differs between non-pathogenic strains. In this study,

319

we investigated the distribution of 18 T3Es present in X. a. pv. juglandis (27). Many

320

differences between non-pathogenic strains in the size and composition of their T3E

321

repertoires were noticed. Strains that belonged to NP2 and contain T3SS have more effector

322

encoding genes (seven in total) in comparison with strains of NP1 that do not contain T3SS

323

and harbour only three effectors (xopR, avrBs2 and avrXccA1). In addition, strains belonging

324

to NP2 and CFBP 7630, CFBP 1022 and CFBP 7652 showed a homogeneous pattern of T3Es

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 14

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genes. All these strains harboured seven T3Es encoding genes; six of them being orthologues

326

of avrBs2, xopF1, xopA, hrpW, hpaA and xopR (Fig. 8). These genes are considered to be the

327

ubiquitous set of T3E genes for X. arboricola strains (27). XopB encoding gene, which was

328

detected previously only in VOC strains and XopAH encoding gene which is present only in

329

WB isolates of X. a. pv. juglandis were not detected in any of the non-pathogenic strains

330

tested. Two strains from NP3 (CFBP 7636 and CFBP 7645) have only the XopR encoding

331

gene, while two strains (CFBP 7640 from NP3 and the singleton CFBP 7653) do not have any

332

of the 18 T3Es encoding genes tested. A total of 11 out of the 18 T3E genes studied were not

333

detected in any non-pathogenic isolates studied.

334

DISCUSSION

335

X. arboricola is conventionally known as a taxon of plant-pathogenic bacteria, including

336

seven pathovars. However, this study showed that X. arboricola encompasses also non-

337

pathogenic bacteria that do not cause any disease neither on plants from which they were

338

isolated nor on a panel of plants representative of various plant species usually used for plant

339

bacteria interaction studies. Knowledge pertaining to the population structure of X. arboricola

340

isolated from walnut should shed light on the epidemiology of diseases associated with

341

a. pv. juglandis, the evolutionary mechanism of this pathogen, the taxonomy and ecology of

342

non-pathogenic xanthomonads and to increase the effectiveness of detection and management

343

of plant diseases associated with Xanthomonas taxa. During epidemiological surveys

344

conducted in the two main production areas of walnut in France (Grenoble in the southeast

345

and Périgord in the southwest), 27 bacterial strains isolated from asymptomatic buds were

346

identified as non-pathogenic based on phenotypic and genotypic characteristics and

347

pathogenicity tests on walnut seedlings. Phylogenetic analyses, performed by MLSA,

348

distinguished several groups with high bootstrap values corresponding to different pathovars

349

of X. arboricola (pvs. pruni, corylina, fragariae, populi, celebensis, poinsetticola and

- 14 -

X.

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 15

350

juglandis). Non-pathogenic strains definitely belong to X. arboricola and grouped outside

351

clusters defined by pathovars that form separate genetic lineages. Non-pathogenic strains

352

were polymorphic as they were distributed into three separated clusters within X. arboricola

353

termed NP1, NP2 and NP3 and four single branches. Clustering of phylogenetic position of

354

non-pathogenic strains is not correlated to their geographical origins indicating the absence of

355

genotype-geographic structure. NP2 showed a high percentage of similarity and clustered

356

strains collected in both eastern and southwestern regions in France. It is remarkable that all

357

non pathogenic xanthomonad strains isolated from walnut belong to the same species X.

358

arboricola that is the only pathogenic bacterium known on walnut so far. It suggests that a

359

link would exist between these lineages.

360

MLVA distinguished several clonal complexes within X. arboricola strains isolated from

361

walnut. Strains causing VOC grouped separately from WB strains. VOC strains are divided

362

into one clonal complex and two singletons, whereas, WB strains are more heterogeneous and

363

divided into two clonal complexes and 16 singletons. Thus, strains causing VOC might be the

364

result of a natural selection of some WB strains that gain additional features necessary to

365

cause canker in woody parts. One VOC strain (12714) was not grouped with other VOC

366

strains and was already found to belong to a separate lineage by MLSA studies (10). We

367

could hypothesize that the gain of features could occur several times on the WB populations.

368

This hypothesis of gaining features is supported by the ability of VOC strains to cause

369

cankers on trunks and necrotic spots on leaves and fruits as well whereas WB cause only

370

necrotic symptoms on leaves and fruits. Non-pathogenic strains were grouped separately from

371

clonal complexes grouping WB and VOC strains and revealed to be genetically

372

heterogeneous as they were divided into six clonal complexes and four singletons. There is a

373

concordance between MLSA and MLVA results. All genetic lineages identified by MLSA

374

were distinct from each other in the MLVA scheme as well. However, MLVA confirmed to

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Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 16

375

be a more discriminative typing method in comparison to MLSA, given that some genetic

376

lineages defined by MLSA were divided into several clonal complexes and singletons in the

377

population structure defined by MLVA. Strains that appear identical by MLSA may present

378

different VNTR profiles, such as strains belonging to NP2 that formed a single genetic

379

lineage with a 100% internal similarity in the MLSA scheme and are divided into three clonal

380

complexes and two singletons in the MLVA. In addition, population structure of clonal

381

complexes defined by VNTR analysis is not correlated to their geographical origins given that

382

some clonal complexes grouped strains belonging to different geographic locations from both

383

Rhône-Alpes and Périgord regions. The occurrence of the same genotype in different

384

geographic areas supports the fact that non-pathogenic strains have been spread all over

385

walnut growing areas in France. It would be useful to check the occurrence of these bacterial

386

lineages on other cultivated plants and weeds in both areas to gain a better understanding of

387

the ecology of these bacteria and to highlight the role of walnut in the ecology of X.

388

arboricola strains. VNTR markers proved to be relatively easy and rapid to use and provide

389

informative data for subtyping bacterial strains. VNTR analysis will gain more attention in the

390

future because of the availability of more Xanthomonas genomes sequenced since VNTRs can

391

rapidly be characterized by PCRs with specific primers based on the flanking regions of the

392

tandem repeats.

393

Based on the population structure of X. arboricola described in this study, we can presume

394

that pathovars result from a selection of host-specialized strains that have been further

395

developed as a single clonal lineage. Non-pathogenic X. arboricola strains were more

396

polymorphic than pathovars and were spread in different geographic locations, suggesting that

397

the plant-bacteria interaction of these non-pathogenic strains occurs differently from plant-

398

pathogen interactions. In this context, the plant bacteria interaction of representative strains of

399

non-pathogenic X. arboricola defined by MLSA and MLVA (CFBP 7634, CFBP 7645, CFBP

- 16 -

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 17

400

7651, CFBP 7652 and CFBP 7653) were assessed on a range of plants including P. persica,

401

R. sativus, L. esculentum, C. annum and N. benthamiana. None of the xanthomonad strains

402

tested induced disease symptoms when inoculated into Prunus and Raphanus

403

However, L. esculentum, C. annum and N. benthamiana were resistant to strains CFBP 7651

404

and CFBP 7652 as local cell death was noticed at the point of inoculation, while CFBP 7634,

405

CFBP 7653 and CFBP 7645 did not induce any disease symptoms on these plants. According

406

to the results obtained from pathogenicity and HR assays, the following questions arise: i)

407

Why some non-pathogenic strains induced cell death when inoculated into tomato, pepper and

408

tobacco leaves whereas others do not? ii) What are the molecular pathogenicity determinants

409

that differ between pathogenic and non-pathogenic strains and induce the inability of non-

410

pathogenic strains to cause disease on plant species tested and especially on walnut?

411

Previous studies reported that the repertoires of T3Es within Xanthomonas strains play a basic

412

role in aggressiveness and host specificity (25) and more recently, Hajri et al. (27)

413

investigated the variability of T3E repertoires in the species X. arboricola, showed that T3SS

414

is an essential virulence mechanism in X. arboricola and suggested the use of non-pathogenic

415

strains to test whether a modification in T3E repertoire would lead to changes in the

416

pathogenic behavior of the bacterium. Thus, the distribution of 18 T3Es, which are the core

417

sets of T3Es present in X. a. pv. juglandis (27) was investigated together with the

418

presence/absence of genes coding for the highly conserved T3SS of the Hrp2 family. We

419

noticed congruence between the composition of T3E repertoires and phylogenetic structure of

420

the non-pathogenic strains in three major groups. Interestingly, no T3SS was detected in NP1,

421

NP3 and CFBP 7653 although these strains clearly belong to X. arboricola. The groups of

422

strains defined by MLSA, i.e. NP1, NP3 and the strain CFBP 7653, lack a hrp-hrc cluster,

423

whereas strains CFBP 1022, 7630, 7652 and NP2 encoded a typical hrp-type T3SS. The

424

major common attribute of strains belonging to NP1 and NP3 is the lack of T3SS genes and

- 17 -

leaves.

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 18

425

the inability to elicit any HR or cause disease symptoms in any of the plants tested. Hence, we

426

can hypothesize that non-pathogenic strains lacking T3SS and containing T3Es xopR,

427

avrXccA1 and avrBs2, are unable to translocate effectors into plant cells, which may explain

428

their inability to cause disease on plant species tested and to elicit an HR.

429

Given that hrpF gene functions as a translocon of effector proteins into the host cell (42, 43),

430

we can assume that T3Es present in NP1 strains are not translocated into plants inoculated

431

due also to the absence of hrpF, which might explain their inability to elicit an HR on non

432

host plants. In fact, previous studies showed that mutation of the hrpF locus of X. oryzae pv.

433

oryzicola strain resulted in the loss of pathogenicity in rice and the ability to induce HR in

434

non-host tobacco (44). Similarly, mutations in hrpF of X. c. pv. vesicatoria strain or X.

435

axonopodis pv. glycines strain resulted in strains that were nonpathogenic in host plants and

436

unable to elicit race-specific HRs (44, 45).When corresponding R and avr genes are present in

437

both host and pathogen respectively, the result is disease resistance (46). AvrBs2 is a

438

functional protein reporter for avrBs2-dependent HR activity in plant cells (47). Transient

439

expression of AvrBs2 in BS2 pepper leaves induced a strong HR response. The AvrBs2/Bs2

440

reporter system has been previously used as a tool to identify translocated effectors in

441

bacterial pathogens that infect other naturally occurring or transgenic BS2 plant lines (48). In

442

addition, we showed that within the species X. arboricola, two isolates, CFBP 7640 and

443

CFBP 7653, do not contain either an hrp-hrc cluster coding for a T3SS or known

444

Xanthomonas T3E genes. Given that the successful establishment of a disease relies on the

445

presence of a T3SS and on the translocation of T3Es (26, 49, 50), it appears that these strains

446

may depend on an entirely non-pathogenic lifestyle. Previous work reported that non-

447

pathogenic Pseudomonas isolates lacking a T3SS are common leaf colonizers of healthy

448

plants and grow as well as or better than other P. syringae strains on nonhost species without

449

causing disease (51). Clarke et al. (50) showed later that these strains contain an unusual

- 18 -

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 19

450

hrp/hrc cluster that is only distantly related to the canonical P. syringae hrp/hrc cluster.

451

This study reports on the occurrence of non-pathogenic isolates within the species X.

452

arboricola, that do not contain an hrp-hrc cluster coding for a T3SS and of strains that harbor

453

some T3Es but no T3SS-encoding genes. The present finding raises the question about the

454

origin of these non-pathogenic strains and evolution of plant pathogenicity in X. arboricola.

455

Mohr et al. (51) suggest that loss of the T3SS in one pathogenic strain has been the initial

456

event in the evolution of T3SS lacking isolates. They assumed that non-pathogenic

457

Pseudomonas strains most likely evolved from a pathogenic strain ancestor through the loss

458

of its T3SS. The hrp-hrc cluster and most of the effector genes were deleted during the

459

evolution of non-pathogenic strains.

460

In contrast, pathogenic strains might have evolved from their non-pathogenic ancestors after

461

i) acquisition of pathogenesis-associated gene clusters (In this context, Lu et al. (52)

462

suggested that acquisition of the hrp clusters were critical steps in the evolution of plant

463

pathogenicity in Xanthomonas) ii), mutations in genes related to virulence or avirulence

464

function, and iii) horizontal gene transfer. Non-pathogenic strains were present together with

465

pathogenic ones. They grow or at least survive, epiphytically on plants without causing

466

disease. Consequently, these strains have been largely overlooked because of their lower

467

economic importance.

468

The identified non-pathogenic strains provide excellent tools to elucidate the difference in HR

469

and pathogenicity reactions between these strains and pathogenic ones in relation to their T3E

470

repertories. T3E genes that were not detected in any non-pathogenic isolates studied represent

471

an excellent candidate to be those responsible for pathogenicity in X. arboricola.

472

Furthermore, these strains could be used to study pathogenicity factors, molecular

473

determinants, particularly effectors and to better understand the basis of host range in

474

Xanthomonas and to gain insight into molecular determinants of plant resistance and how

- 19 -

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 20

475

bacterial pathogenicity works. In fact, it would be interesting to engineer these isolates back

476

into pathogens with different host ranges by adding a functional T3SS or different assortment

477

of effector genes to see whether a modification of the interaction between these strains and

478

plants tested would be observed.

479

Because of the practical obstacle of extensive pathogenicity tests, the possibility cannot be

480

excluded that some of these non-pathogenic isolates could be pathogenic Xanthomonas strains

481

with unknown host plant(s), particularly strains belonging to NP2, CFBP 7630, CFBP 1022

482

and CFBP 7652 that harboured genes of a typical T3SS of the Hrp2 family and seven T3Es

483

encoding genes. Therefore, the use of the term non-pathogenic for these strains might not

484

always be correct. In this context, Mohr et al. (51) assumed that non-pathogenic P. syringae

485

strains can live without causing disease on plants for extended periods of time, but can cause

486

disease when they find themselves on a susceptible plant under favourable environmental

487

conditions.

488

Further work is underway to conduct a complete genomic comparison of pathogenic and non-

489

pathogenic strains sequenced, in order to find bacterial functions involved in the epiphytic

490

colonisation of plants and to determine common and differential genes within these strains

491

(and other xanthomonads) that could be linked to pathogenicity. Further analysis of

492

sequenced genomes would aid in understanding the function of the missing components of a

493

T3SS in non-pathogenic strains and to provide novel insights into other pathogenicity

494

determinants that may play a role in the plant-bacteria interaction. Among them, a particular

495

attention should be payed to genes involved in adhesion, biofilm formation, flagellum

496

synthesis, motility, lipopolysacharide synthesis, quorum sensing, and finally type IV or VI

497

secretion system to answer the question regarding the possibility of involvement of other

498

pathogenicity determinants like T4SS, T6SS in NP1 strains that carry T3Es but do not

499

harbour any gene of T3SS. Understanding acquisition and evolution of type III effectors in X.

- 20 -

Non-pathogenic X. arboricola strains lacking T3SS, Essakhi et al. Page 21

500

arboricola may help us to deduce the roles of these proteins in pathogenicity and will help in

501

understanding functions of most T3Es identified in X. arboricola.

502

ACKNOWLEDGEMENTS

503

This project has been financed by Direction Générale de l’Armement (REI project # 2010

504

34007). We thank the Collection Française de Bactéries associées aux Plantes (CIRM-

505

CFBP), INRA, Angers, France, for providing X. arboricola strains and their corresponding

506

sequences of the seven investigated genes and Annie Micoud, Anses, Lyon for providing X.

507

arboricola isolates. We thank Jacky Guillaumès and Mathilde Mullard for their contribution

508

in biochemical and pathogenicity tests.

509 510

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1 2

TABLE 1. Bacterial strains used in this study

Taxon Xanthomonas arboricola pv. celebensis X. arboricola pv. celebensis Xanthomonas arboricola pv. corylina X. arboricola pv. corylina X. arboricola pv. corylina X. arboricola pv. corylina X. arboricola pv. corylina X. arboricola pv. corylina X. arboricola pv. corylina Xanthomonas arboricola pv. fragariae X. arboricola pv. fragariae X. arboricola pv. fragariae X. arboricola pv. fragariae X. arboricola pv. fragariae X. arboricola pv. fragariae X. arboricola pv. fragariae

Strain no. CFBP 3523PT CFBP 7150 CFBP 1159PT CFBP1846 CFBP1847 CFBP1848 CFBP 2565 CFBP 5956 CFBP 6101 CFBP 6771PT CFBP 6762 CFBP 6763 CFBP 6770 CFBP 6772 CFBP 3548 CFBP 3549

Xanthomonas arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni

CFBP 3894PT CFBP 3893 CFBP 3898 CFBP 3900 CFBP 3901 CFBP 3921

Other collection accession numbers & strain no. Host plant

Geographic origin

Yr. of isolatio n

LMG 677, NCPPB 1832, ATCC 19045 LMG 676, NCPPB 1630, ICMP 1484

Musa acuminata Musa acuminata

New Zealand New Zealand

1960 1960

LMG 689, NCPPB 935, ATCC 19313

Corylus maxima Corylus avellana C. avellana C. avellana C. avellana C. avellana C. avellana

United States France Algeria United Kingdom France France France

1939 1975 1977 1977 1985 1979 1979

Fragaria × ananassa Fragaria × ananassa Fragaria × ananassa Fragaria × ananassa Fragaria × ananassa Fragaria sp. Fragaria sp.

Italy Italy Italy Italy Italy France France

1993 NA NA 1994 NA 1986 1986

Prunus salicina Prunus persica Prunus domestica P. persica Prunus armeniaca P. persica

New Zealand Italy United States United States United States Italy

1953 1989 1989 1987 1987 1996

ICMP 11956

LMG 19145, PD 2780 PD 2694 PD 2697 LMG 19144, PD 2696 PD 2803 LMG 19146, PD 3164 PD 3160 NCPPB 416, ATCC 19316, ICMP 51, CFBP 2535.

X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni X. arboricola pv. pruni Xanthomonas arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi

CFBP 411 CFBP 5229 CFBP 5529 CFBP 5530 CFBP 5580 CFBP 5722 CFBP 5723 CFBP 5724 CFBP 6653 CFBP 7098 CFBP 7099 CFBP 7100 CFBP 3123PT

ATCC 10016, ICMP 12475 NCPPB 1607

CFBP 2113 CFBP 2666 CFBP 2669 CFBP 2983 CFBP 2985

X. arboricola pv. populi CFBP 2986 X. arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi

CFBP 3004 CFBP 3121 CFBP 3122 CFBP 3124 CFBP 3338 CFBP 3342

ICMP 9140 LMG 9713, ICMP 9367

P. persica Prunus sp. P. persica Prunus persica Prunus japonica P. persica Prunus sp. Prunus amygdalus P. persica P. domestica P.domestica Prunus dulcis

United States Argentina Australia Italy France Brasil Uruguay United States France Spain Spain Spain

1963 1996 1964 1989 2000 1991 NA NA 2000 2002 2003 2006

Populus × canadensis Populus × interamericana Populus × interamericana Populus × canadensis Populus × canadensis Populus × interamericana Populus × interamericana Populus × interamericana Salix alba Salix alba Populus × generosa Populus × interamericana Salix sp.

Netherlands

1979

Netherlands

1980

France France Italy

1983 1987 1989

Belgium

1989

Belgium

1989

France Netherlands Netherlands New Zealand

1989 1980 1980 1986

France New Zealand

1991 1988

X. arboricola pv. populi X. arboricola pv. populi X. arboricola pv. populi Xanthomonas arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis

CFBP 3343 CFBP 3344 CFBP 3839 CFBP 2528T CFBP 176 CFBP 878 CFBP 2564 CFBP 2568 CFBP 2632 CFBP 6557 CFBP 7071 CFBP 7072 CFBP 7244 12572 12573 12575 12576 12577 12579 12581 12586 12707 12710 12680 12783 12763* 12574* 12578* 12580* 12582*

LMG 747, NCPPB 411, ATCC 49083, ICMP 35

ICMP 11955 ICMP 11963

CFBP 7179

Populus sp. Salix sp. Populus deltoides

New Zealand New Zealand Belgium

1988 1988 1984

Juglans regia J. regia J. regia J. regia J. regia J. regia J. regia Juglans sp. Juglans sp. J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia cv. Fernor J. regia cv. Franquette J. regia J. regia J. regia cv. Fernor J. regia J. regia J. regia J. regia

New Zealand France France Italy Italy Spain Italie Spain Spain France France France France France France France France France France France France France France France France France France

1956 1961 1966 1985 1985 1984 1999 1993 1993 1978 2001 2001 2001 2001 2001 2001 2001 2001 2002 2002 2002 2003 2002 2001 2001 2001 2001

X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis

12583* 12584* 12585* 12587* 12588* 12589* 12591* 12592* 12681* 12708* 12709* 12711* 12712* 12713* 12714* 12715* 12762* 12765* 12766* 12768* 12769* 12770* 12772* 12774* 12775* 12776* 12777* 12778* 12779* 12780* 12781*

J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia cv. Fernor J. regia cv. Fernor J. regia cv. Fernor J. regia cv. lara J. regia cv. Fernor J. regia cv. Fernor J. regia cv. Fernor J. regia cv. Fernor J. regia cv. Fernor J. regia J. regia J. regia J. regia cv. Fernor J. regia cv. Fernor J. regia J. regia J. regia J. regia J. regia J. regia J. regia cv. Fernor J. regia

France France France France France France France France France France France France France France France France France France France France France France France France France France France France France France France

2001 2001 2001 2001 2001 2001 2001 2001 2002 2002 2002 2002 2002 2002 2002 2002 2002 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003

X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis X. arboricola pv. juglandis Xanthomonas arboricola sp. X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola X. arboricola

12782* 12784* 12785* CFBP 7643 CFBP 1022 CFBP 7654 CFBP 7653 CFBP 7651 CFBP 7652 CFBP 7647 CFBP 7641 CFBP 7635 CFBP 7637 CFBP 7638 CFBP 7645 CFBP 7640 CFBP 7636 CFBP 7633 CFBP 7634 CFBP 7639 CFBP 7632 CFBP 7629 CFBP 7630 CFBP 7656 CFBP 7646 CFBP 7644 CFBP 7650 CFBP 7649 CFBP 7648 CFBP 7631 CFBP 7655

CB1 CS2 CS5F SPS1 P2-4 P2-7 P2-21 P3-6 P3-24 P7-4 P7-18 P7-27 P8-6 P8-10 P8-14 P8-15 P8-20 P9-12 P9-21 P10-8 P10-14 P10-19 P10-25 P11-12 P11-21 P16-11

J. regia J. regia cv. Vina J. regia cv. Franquette J. regia J. regia J. regia J. regia J. regia J. regia J. regia cv. Franquette J. regia cv. Franquette J. regia cv. Franquette J. regia cv. Franquette J. regia cv. Franquette J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia J. regia

France France France France France Périgord, France Périgord, France Isère, France Périgord, France Isère, France Isère, France Isère, France Isère, France Isère, France Loire, France Loire, France Loire, France Rhône, France Rhône, France Rhône, France Rhône, France Rhône, France Isère, France Isère, France Rhône, France Rhône, France Rhône, France Rhône, France Isère, France Isère, France Isère, France

2003 2003 2003 2009 1967 2008 2008 2008 2008 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009

Xanthomonas campestris pv. campestris

CFBP 5241

Brassica oleracea

United Kingdom

1957

3 4

CIRM/CFBP, Collection Française de Bactéries associées aux Plantes, INRA, Angers, France; ICMP, International Collection of Microorganisms from Plants,

5

Auckland, New Zealand; LMG, BCCM/LMG Bacteria Collection, University of Ghent, Ghent, Belgium; NCPPB, National Collection of Plant Pathogenic

6

Bacteria, York, United Kingdom; ATCC, American Type Culture Collection, Manassas, VA; PD, Culture Collection of Plant Pathogenic Bacteria, Plant

7

Protection Service, Wageningen, Netherlands. Superscript T and PT indicate type strain of a species and pathotype strain of a pathovar respectively.

8

*Xanthomonas arboricola pv. juglandis strains isolated from vertical oozing canker symptoms on trunks and branches (10). NA, not available.

9

Table 2. Primers used in MLSA for amplification and sequencing Primer sequences Gene

PCR fragment size (bp)

Forward

Reverse

atpD

GGGCAAGATCGTTCAGAT

GCTCTTGGTCGAGGTGAT

750

dnaK

GGTATTGACCTCGGCACCAC

ACCTTCGGCATACGGGTCT

759

efP

TCATCACCGAGACCGAATA

TCCTGGTTGACGAACAGC

339

fyuA

ACCATCGACATGGACTGGACC

GTCGCCGAACAGGTTCACC

753

glnA

ATCAAGGACAACAAGGTCG

GCGGTGAAGGTCAGGTAG

675

gyrB

ACGAGTACAACCCGGACAA

CCCATCARGGTGCTGAAGAT

735

rpoD

ATGGCCAACGAACGTCCTGC

AACTTGTAACCGCGACGGTATTCG

609

Table 3. Primers used for amplification of VNTR loci and their corresponding position on the genome of X.a.pv. pruni CFBP 5530 Primer sequences* Multiplexe A

B

C

D

E

F

G

VNTR locus

Forward

Reverse

Position start

end

TR50I

V-cgtgcatcagacgcttgcgt

gttgcgagatcgggcgcttc

3413992

3414042

TR33I

P-ctcgcaaaacccttgccatc

cgagtggatgttatggcgtgg

2022442

2022490

TR68I

F-aaatcatcggcgcctgaaac

cttgcggtactggctgttca

4140738

4140827

TR19I

N-gattgacggcacccacacag

ccaggacgttgtgccgtggt

1523902

1523949

TR36I

P-cgatcgcatctgtgtgggttag

gcaggagaaggaaagcgccag

2284172

2284214

TR58I

V-accaacaccgagcttgcctc

atctgttgctggccgagagc

3538491

3538528

TR3I

N-ggttgcttggtcgttgatcg

gacattcgccgggagtgcag

150355

150401

TR40I

P-tggaatgtggaggctgttcg

tatcaggcagcgcaccagct

2426642

2426699

TR15I

N-tcgagcggttcctgcggttgt

gccatgtcgccgggaaacga

1310624

1310662

TR37I

P-ccaacagaaccccgcaccca

atggaggatgcggttgcggct

2348164

2348197

TR05II

F-cagatgctgtcccgattcccg

gtcgacgggttcgcggaaggt

340287

340353

TR39II

P-ggtacggaaggtggtggtctgc

cccgcatactgatgcagttcg

1940044

1940160

TR06II

F-gtgcagcaccagccaaaggca

tcataggctggggattgggga

340223

340303

TR21II

V-acacggacgtacttgcggcgt

ggagcgtattgcttgaacggga

1114440

1114597

TR58II

P-tctgatcggtgctgagcgtct

ggaagagtacccggcaattct

3391120

3391216

TR38II

N-cccgtagctgtatcagtgcct

tctcggtatcgatgtgggtgc

1930537

1930595

TR67II

P-agctcgcaactgcttttcccga

gatacaaggcgaacgcgatga

3641116

3641204

* Forwards primer pairs were marked with one of the fluorescent dyes: F, FAM; V, VIC; N, NED; P, PET

Table 4. PCR primers used to amplify T3Es genes T3E gene avrBs2 xopF1 xopA hrpW hpaA xopR xopN xopX xopZ xopQ xopK xopV xopL xopAI avrXccA1 avrXccA2 xopAH xopB

Forward primer ACCGCGCTGGCCACACCG TGCCGGTGTTGATGCACGA TGAAACTCACCAGCAATATCG AGGCCATCGACCCCAAGATCC ATGGACTCATCTATCGGAAACTT TGCAGACGATGGGCATCG ATGCAACGCATGCTCAGCGACAT AAGGTCGTCACCGCGC ATGATCCGGCGCATTTCGCCAG CGCTGGATGGCATGGACGACG ATGCGCCTGAGTCAGTTGTTT CGTGCGGCCCTGATCGC ATGAAGTCATCCGCATCCGTCGAT GTCATGACCCAGGGCGC ATGGAGATCAAGAAACAGCAAACCGC GTGGAAAACAACCTGGG GCACTTGCGGATACTAATGCGG TTCGGCCGCGGCTCGGC GTGCCCGCAGGCGCTCATGCAA ACCCCGACGATGT GACGCCCTTGCTTCAGCGAAC CTCGGCATCCAGGGC ATGAAAGTCTCCGCAACCCTT ACACGCCTGTTCGTCTC ATGCGACGCGTCGATCAACCG CCACCGACCGTGGGCGCTTCATCATTA ATGACTTCGGTAAGCCAGCGCGAATC AGAGCAGACCACGCCCTCTACG GTGGTTCGCTGCGATGGC GATGGGCGGCACCG GCCGATGGCTGCCGCCGGCGCTA ACGGCCCGTTCTTTCCGCAAAGCC ATGAAGAACACGTCTGTCCCT ATTGTGGTATGGGCCTAGGC ATGAAGGCAGAGCTCACACGA AGCATTTGGCCCAAGCGCTTT

Reverse primer TCACTCGCCCGGCTCGATC TCGGTCAGCAGGCTTTC CTAGCGAAGCGCCTCGCTC GTTCTTGGCCTTGAGCGCATTCC GCCGGTGATGCTCGACAG CTGCATCAGCTGCATCACGATC GTCTTCAGGTTCGCCAGCTTCAC GTCCTGCACGACCTTGTCT TCATGCACGAATCTCCTGAGCGGC CGTCTGAGCGTCTGGTCGGCGGC GTAGCCGTTGTCGATTGCCTCTT GTAGCCCTGCATCATGCGTT CTCGATCGGTTCGGGCTACTCG GGTGATGGCGGTGTGCTG GGCGACAGGCTTTGCACATATCTGG CCCCAGTTCATCGCC GTCGACGAAGTCCTGCAATTGG GCACGGCATGGCGCGCTCC CCTTGGCGTGAACAGCATGCC TTGTTGTAGGCGCG TTCGGTGGCCAGCAACGTGCC GACAAAGCCCTTGTTCCA TCAGGTTGCGAAAGGTGAGG GCGATGTTCCATTTGTA CTACTGATGGCCTGAGGGTTCCG ACATCTGCACTGCCTTGGCCAGC TCGATCTGGCTTTGATAAATCCTCAGAC GAATATTCTTCGGGAAGCGAGTGC TCACCCAGCCAGCGGG ATCGCCACGCACCTG TTGGTGTTCCAGTTCCGATCCAGG CAACGGGCGCTCCGGCGACG CTACTTCTGCGTGGGAGGC TGCTTGGCGTACTCGTAGAAT TCAGGCGCGGGTTGGTGCGAAGTA CGCTTCGGTTGTCGTCATATTGG

PCR fragment Size (bp) 2118 850 1996 779 381 239 905 399 816 292 1230 303 2092 864 1865 827 2868 1012 1224 484 2454 357 1023 236 1863 1324 950 507 813 163 1442 371 1002 220 1835 574

Table 5. PCR primers used to amplify T3SS genes Gene

Forward primer

Reverse primer

PCR fragment Size (bp)

hrcR

GCTGGTGGTCATCATGCTGG

GTGTTTGAGGAGGAATTGC

292

hrcN

ATGTCAACGTGATCGTGC

CTGGCTCATCACCCGGCTC

524

hrcT

GTCGTTCTACGCGCTGG

GTTGGCGGCATCGTGCAA

376

hrcC

ACCGAAGTGCAGGTGTTTC

ATCTCGATGATGGTGGCATCGAT

575

hrcV

GCGCCATGAAATTCGTCAAGG

GCCAGCAGCAGGAACAGC

367

hrcU

GGCGTGGTGCTGTGG

GGTTGACCACCATCACCTTG

340

hrcJ

CTCGGCGAGATGTTCAAG

GCCACCAATACAGCGC

436

hrpB1

CTGATCACGGTCGG

TCGGCATCGGCGTC

287

hrpF

ACGCTGGACACCATC

TTCTTGTAGCCGGTGAT

188