Genetic diversity of non-pathogenic Clavibacter strains isolated from tomato seeds

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Abstract

Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-transmitted, quarantine pathogen which causes bacterial wilt and canker of tomato. Despite efforts to prevent seed contamination, new introductions are regularly detected, associated with new regions of tomato seed production. It seems as if the expanding diversity of Cmm also challenges the limited host range.

Clavibacter-like isolates from tomato seed are phenotypically similar to Cmm in the common diagnostic semi-selective media and are identified as Cmm in the customary tests but are not pathogenic to tomato. In our first study four representatives formed a separate cluster in gyrB sequence analysis and in MALDI-TOF MS. Their presence on seed prevents clear judgment on the health status of tomato seeds. As their nature and function are unclear we aimed to investigate and compare them to Cmm.

Twenty strains described as Clavibacter-like isolated from tomato seed and not pathogenic to tomato plantlets were selected. Leaf spots, wilting or cankers were not induced after local or systemic inoculation. Tomato stems were not colonized nor was there evidence of survival in tomato stems. Total DNA–DNA hybridization and sequence analysis of gyrB and dnaA proved that they belong to the Cm species but can be unambiguously separated from Cmm. Some of the genes encoding virulence determinants in Cmm strains were also detected in some of the non-pathogenic isolates. Moreover, Cmm strains formed a coherent group, while non-pathogenic Cm strains were heterogenic. The latter was confirmed by BOX-PCR. We speculate that tomato seeds likely represent a larger reservoir of unexplored Clavibacter diversity.

Introduction

Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne Gram-positive phytopathogen causing bacterial wilt and canker of tomato. The disease can become very destructive in commercial tomato production worldwide [13] and therefore international plant health regulations are implemented on seeds and transplants to prevent its introduction and spread [3]. Seed contamination allows the pathogen to disseminate over long distances and frequently new populations are introduced into Europe and elsewhere from South American or Asian countries where tomato seeds were produced [12]. Weakly virulent and avirulent Cmm are also frequently isolated from tomato seeds [2]. The challenge to seed health lies in accurately differentiating Cmm from seed contaminants and to determine the significance of seed-borne Cmm with varying levels of virulence, i.e. hypovirulent (only causing canker at the inoculation point) or avirulent. Furthermore, Cmm coexists with numerous other bacteria on tomato seed, some of which share similar colony morphologies on the selective media used in the seed assay. Some Clavibacter-like strains were isolated from tomato seed lots imported into Europe but for others the country of origin is not known. They cross react with commercially available antisera for Cmm, are positively identified as Cmm by the commonly used PCR tests but fail in the pathogenicity test [26]. A set of Clavibacter-like strains, isolated from tomato seeds, was recently investigated by gyrB sequence analysis and MALDI-TOF MS (matrix-assisted laser desorption/ionization mass spectrometry) [51]. The strains clustered in a separate C. michiganensis clade outside Cmm and were most closely related to C. michiganensis subsp. tessellarius (Cmt).

Isolates of Clavibacter, specifically related to C. michiganensis, were reported on rice seed [10], [11] and were recovered from healthy aerial tissues of prairie plants. The latter were found to colonize the plant species from which they were isolated as endophytes without causing disease symptoms. The endophytic nature in tomato plants of the Clavibacter-like strains from tomato seed has not yet been investigated. Colonization of tomato plants by Cmm and its resultant disease is the consequence of several virulence factors. These are located on two circular plasmids, i.e. pCM1 (∼27.5 kb) and pCM2 (∼70. kb) [15] and each plasmid carries a single virulence factor. The large plasmid harbors the pat-1 gene which encodes a serine protease [14]. The small plasmid contains the celA gene which encodes an endo-β-1,4-glucanase [34]. These are sufficient for induction of disease. Plasmid content differs in Cmm isolates with most of the strains carrying two plasmids [29]. Loss of one plasmid delays symptom development in tomato plants [34]. A Cmm strain which contains neither of the plasmids is non-virulent but bacterial titers in planta are as high as for the wild type strain [34]. The strain is thus converted into an endophyte and this suggests that the genes required for host recognition, invasion, suppression of host defense and colonization are encoded on the Cmm chromosome [16]. The most prominent in the Cmm chromosome is the chp/tomA region which may be a pathogenicity island. The chp genes encode a family of serine proteases and they are homologs to the plasmid-borne virulence factor pat-1 [45]. Homolog genes of the pat-1 gene, named phpA/B, are also present on the pCM2 plasmid but it was demonstrated that these genes are not involved in pathogenesis [8].

The genetic diversity of Cmm in disease outbreaks is being investigated only fairly recently [4], [12], [36]. The studies are performed with genomic typing methods, i.e. BOX-PCR, AFLP [12] and ISSR [4]. The genomic variability is used to track strains in epidemiological studies [4] and to associate outbreaks with origin of primary contamination (farm-borne or new introduction). Knowledge of the extent and nature of the genetic diversity of pathogens in their natural habitats is essential for epidemiological studies but also for understanding the ecological and evolutionary forces that govern their distribution and population dynamics.

The host specialization of Cmm is known to be very strict, with natural infections occurring on tomato although other solanaceous and non-solanaceous plant species were reported as potential hosts after artificial inoculation [17], [48]. Just recently, Cmm was diagnosed on pepper (Capsicum annum) in Korea [50] displaying a distinct orange colony morphology which was noticeably different from the common Cmm phenotype from tomato. However, ELISA, FAME, 16S rRNA gene sequencing and ITS-PCR confirmed their high similarity to Cmm. The Cmm strains from pepper were highly virulent on its host of origin but only weakly virulent in tomato plants. Furthermore, no amplicons were produced for the plasmid-borne virulence factors, nor for the virulence related genes located on the chromosomal pathogenicity island. Atypical Cmm strains lacking pat-1 gene and inducing weaker symptoms on tomato were found on symptomatic plants in greenhouses in Italy [5]. Not pathogenic to tomato Clavibacter strains which cross reacted with identification methods for Cmm were investigated by Jacques et al. [26].

So, it is evident that various ‘atypical’ strains of Cmm exist with clear differences in phenotype, genotype and virulence. This paper reports on the investigation of a novel group of Clavibacter-like isolates from tomato seeds. In our initial work we observed a stable separate grouping of these strains within the C. michiganensis clade using gyrB amplicon sequences and MALDI-TOF MS [51]. These strains also appeared to be not pathogenic to tomato plants. Their apparent lack of virulence on tomato and the risk of erroneous identification as Cmm make them an extremely interesting study object. In the present study the genetic diversity of Clavibacter-like strains was investigated by gyrB and dnaA gene sequence analysis and by BOX-PCR. These new isolates from tomato seeds were then compared with Cmm. Subsequently, we confirmed their separate taxonomic position within the genus Clavibacter and tested their pathogenicity on tomato, both in planta and by investigation of the known pathogenicity factors of Cmm.

Section snippets

Bacterial strains and growth conditions

The strains used in this study were obtained from the BCCM/LMG Bacteria Collection (Ghent, Belgium), the PD collection (Wageningen, The Netherlands) and the GBBC (ILVO Plant Clinic, Merelbeke, Belgium). This Clavibacter strain subset consisted of five type strains of the species C. michiganensis subsp. michiganensis (Cmm) LMG7333T, C. michiganensis subsp. nebraskensis (Cmn) LMG5627T, C. michiganensis subsp. sepedonicus (Cms) LMG2889T, C. michiganensis subsp. insidiosus (Cmi) LMG3663T, C.

Pathogenicity, colonization tests and bacterial colony morphology

After analysis of genetic factors, pathogenicity tests on tomato plants of Cmm, Clavibacter-like strains and members of the family Microbacteriaceae were performed (Table 1). After 21 days of incubation the plants were checked for specific symptoms. None of the Clavibacter-like strains induced disease symptoms, whereas Cmm strains produced wilting or/and canker (Fig. 1; Table 1). The Cmm group contained very aggressive strains (e.g., PD 1664) which led to fast disease development and complete

Conclusions

From an economical perspective, it is most imperative to confirm that seeds contaminated only with non-pathogenic strains are safe and thus can be released onto the market. However, our results clearly demonstrated that, although they are not pathogenic, Clavibacter-like strains seem to contain some pathogenicity genes, both on plasmids and on the chromosome. Moreover, not all virulence factors were investigated and we cannot exclude their eventual latent state in tomato plants. A change in the

Acknowledgements

We thank the PD, GBBC and BCCM/LMG collections for providing necessary strains. The authors wish to acknowledge Harrie Koenraad (Naktuinbouw) and Bert Wundt (Syngenta) for helpful discussions and for providing a collection of non-pathogenic strains. This work was performed in the Seventh Framework Programme of project KBBE-2008-1-4-01 (QBOL) nr 226482 funded by the European Commission. The Belgian NPPO (FAVV) is acknowledged for partially financing ILVO-research and help with in planta test.

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