Summary
All published semiselective media for Clavibacter michiganensis subsp. michiganensis (Cmm) proved to be not satisfactory for a sensitive detection of Cmm in infected tomato plants and seeds. Therefore new selective media for Cmm were developed in three steps: 1) Selection of a basic medium allowing good growth of Cmm but excluding or slowing down several other bacterial species; 2) screening a wide range of antibiotics and other inhibitors for selective inhibition of often accompanying bacterial or fungal species; 3) optimizing the composition of inhibitors and nutrient components.
Initial tests for selection of antibiotics which did not inhibit Cmm were conducted with 30 strains of accompanying pathogenic and non-pathogenic bacterial species isolated from tomato seeds and plants that were obtained from different locations. For these experiments, tomato plants were cultivated in the field and artificially inoculated with very low concentrations of a rifampicin and streptomycin resistant strain of Cmm. These tomato plants did not develop disease symptoms but were latently infected with the pathogen. On the other hand, homogenates from leaves, stems or tomato fruits were heavily contaminated with various microorganisms (bacteria and fungi). The exact concentration of Cmm cells contained in the homogenates was determined by dilution plating on NGY agar medium supplemented with rifampicin, streptomycin and a fungicide. Parallely, dilution plating assays from the same homogenates were conducted on many newly designed compositions for a potential semiselective medium. The best suited new media were then tested for isolation of Cmm from naturally infected plants obtained from different locations in Germany, Syria and Austria, in order to enlarge the diversity of naturally occurring microorganisms on or in tomato plants.
Compared with all previously recommended semiselective media for Cmm, the new media (BCT and BCT-2) proved to be well suited for sufficient and fast growth of a wide range of Cmm strains.On the other hand, the new media inhibited growth of naturally occurring microorganisms to an extent of 98 to 100%. By testing tomato seeds and plants which were
Chapter 1 Conclusions
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latently infected with Cmm and highly contaminated with different saprophytic bacteria, the Cmm population was always detected on the new media, whereas all published semiselective media revealed false negative results under these conditions.
Additional tests revealed that the new selective media were also well suited for isolation and identification of the Clavibacter michiganensis subspecies nebraskensis, insidiosus and tessellarius, but neither for C. m. ssp. sepedonicus nor for Curtobacterium flaccumfaciens pv.
flaccumfaciens.
Conclusions
The new media BCT and BCT-2 are superior to all published semiselective media for Cmm and are denoted as selective media because:
the mean plating efficiency amounted up to 89%, all the 30 Cmm strains from a wide range of different origins grew on the new media (one exception for BCT-2),
high selectivity, accompanying bacterial species occurring on tomato plants and seeds or obtained from culture collections were inhibited to an extent of 98 to 100%, and
remarkable detection sensitivity. Thus, very low Cmm populations occurring in plant and seed material in the presence of high concentrations (thousand-fold more) of non-target accompanying bacteria were detected on the new media but never on the published semiselective media.
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Introduction
Clavibacter michiganensis subsp. michiganensis (Cmm) (Smith, 1910) Davis et al., 1984 can cause a very destructive wilt disease of tomato plants, especially in greenhouses. Therefore, the pathogen has been classified as an A2 quarantine organism by the European Plant Protection Organization (OEPP/EPPO, 2005; Council Directive 2000/29/EC). The disease may result in serious losses, and very strict hygienic measures must be applied once it appears (Strider, 1969).
Infested seeds and transplants are responsible for disease transmission into new areas (Chang et al., 1991; Strider, 1969; Werner et al., 2002), whereas transmission by soil appears to be of minor importance (Ftayeh, 2004; Ftayeh et al., 2004; Strider, 1969). Thus, indexing of tomato seed for the canker pathogen is a key for disease control (Biggerstaff et al., 2000).
As few as 0.01-0.05% contaminated seeds or transplants can cause an epidemic in suitable conditions (Chang et al., 1991). New outbreaks of canker diseases of tomato (Solanum lycopersicum L) caused by Cmm were recently reported in several locations in Europe, including Austria, Belgium, Czech Republic, France, Germany, Netherlands, Serbia, Slovakia, Slovenia and Spain (CABI/ EPPO, 2009), as well as in Syria (Chapetr 4; Ftayeh et al., 2008b), and several countries worldwide. The disease occurred in some locations for the first time, although infected plants were originally obtained from tomato seeds and transplants that were certified as pathogen free. Since health certification documents had been issued according to international standard detection and testing methods, many questions arose on the reliability of the presently used diagnostic and detection protocols for Cmm. Due to obvious insufficiencies of these protocols, the here presented research project was started at the University of Göttingen in 2006. At the end of 2008, an external evaluation by a European collaborative study was organised between research institutions as well as seed companies in several European countries to determine the weaknesses of diagnostic methods and “to open perspectives for the development of alternative methods” (Olivier et al., 2009).
Protocols for detection of Cmm in tomato seeds and symptomless plant tissues, recommended by EPPO, the European Plant Protection Organization (OEPP/EPPO, 2005) and by ISHI, the International Seed Health Initiative (ISHI, 2008) are based on isolation by dilution plating of seed extracts and tissue homogenates on semiselective media, confirmed by identification tests of pure bacterial cultures by a pathogenicity test. According to the EPPO protocol, the identity of the pathogen must be also confirmed by at least one other test, such as biochemical characteristics, SA-agglutination test, IF test, ELISA, PCR, genomic fingerprinting or
SDS-Chapter 1 Introduction
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Semiselective media are valuable and essential tools in phytobacteriology for disease diagnosis, indexing and epidemiological studies (Roy and Sasser, 1990). Direct isolations and plating assays onto semiselective media remain the most widely used detection methods and have several advantages for detecting bacterial diseases. Plating onto semiselective media is easier to do, less expensive and results in recovery of viable bacterial cultures that can be used to determine pathogenicity (Schaad, 1982; Schaad et al., 1997).
Semiselective media are based on knowledge of the nutritional requirements and physiological tolerances of the target bacterium. This includes choosing suitable carbon and nitrogen sources that allow growth of the target organism but that are not readily used by other bacteria, minimizing the growth of non-target organisms. After optimizing carbon and nitrogen concentrations, inhibitors such as antibiotics and dyes can be incorporated to enhance selectivity (Gitaitis and Walcott, 2007). Other methods which could increase selectivity of semiselective media include pH levels (Burbage et al., 1982), osmotic concentrations imposed by extremely high concentration of sucrose (Crosse and Goodman, 1973) and incubation temperatures (Gitaitis et al., 1997) that allow growth of the target bacterium but inhibit growth of the background microflora.
Development of semiselective media for coryneforms is difficult because of their fastidious nature and inherent susceptibility to antibiotics and inhibitors (De la Cruz et al., 1992).
Semiselective media developed for Cmm differ in basal components and in inhibitors added.
Inhibitors contailned in previously used semiselective media for Cmm include cycloheximide, polymyxin B sulfate, nalidixic acid, nicotinic acid, nystatin, lithium chloride, boric acid, potassium tellurite and sodium azide. Inhibitors may differ in mode of action and in their interactions with components of the basic media, thus effecting selectivity, plating efficiency and growth speed of the target bacterium and as a result sensitivity and reliability for detection of Cmm. However, the protocols recently recommended by EPPO and ISHI (OEPP/EPPO, 2005; ISHI, 2008) for detection of Cmm in tomato seeds and plants are not sensitive enough, because the suggested semiselective media proved to be not satisfactory.
Therefore, the aim was to develop a new selective and highly sensitive medium that can be used for routine seed testing and for a reliable isolation and detection of Clavibacter michiganensis subsp. michiganensis in infested seeds and latently infected plants.
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