Bulletin OEPP/EPPO Bulletin (2016) 46 (2), 202–225 ISSN 0250-8052. DOI: 10.1111/epp.12302 European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/42 (3) Diagnostics Diagnostic PM 7/42 (3) Clavibacter michiganensis subsp. michiganensis Specific scope Specific approval and amendment This Standard describes a diagnostic protocol for Approved in 2004-09. Clavibacter michiganensis subsp. michiganensis.1,2 Revision adopted in 2012-09. Second revision adopted in 2016-04. The diagnostic procedure for symptomatic plants (Fig. 1) 1. Introduction comprises isolation from infected tissue on non-selective Clavibacter michiganensis subsp. michiganensis was origi- and/or semi-selective media, followed by identification of nally described in 1910 as the cause of bacterial canker of presumptive isolates including determination of pathogenic- tomato in North America. The pathogen is now present in ity. This procedure includes tests which have been validated all main areas of production of tomato and is quite widely (for which available validation data is presented with the distributed in the EPPO region (EPPO/CABI, 1998). Occur- description of the relevant test) and tests which are currently rence is usually erratic; epidemics can follow years of in use in some laboratories, but for which full validation data absence or limited appearance. is not yet available. Two different procedures for testing Tomato is the most important host, but in some cases tomato seed are presented (Fig. 2). In addition, a detection natural infections have also been recorded on Capsicum, protocol for screening for symptomless, latently infected aubergine (Solanum dulcamara) and several Solanum tomato plantlets is presented in Appendix 1, although this weeds (e.g. Solanum nigrum, Solanum douglasii, method has yet to be fully validated. Solanum trifolium). Other solanaceous plants are suscepti- ble upon artificial inoculation (Thyr et al., 1975). Several solanaceous and non-solanaceous plants, e.g. Datura 2. Identity stramonium, Chenopodium album and Amaranthus Name: Clavibacter michiganensis subsp. michiganensis retroflexus, have been identified as reservoirs for epi- (Smith, 1910; Davis et al., 1984). phytic survival and spread (Chang et al., 1992). The sig- Synonyms: Corynebacterium michiganense subsp. nificance of these epiphytic populations is not fully michiganense (Smith, 1910; Carlson & Vidaver, 1982), understood, although they seem to contribute to infections Corynebacterium michiganense pv. michiganense (Smith, through pruning wounds (Carlton et al., 1994). 1910; Dye & Kemp, 1977), Corynebacterium michiganense The pathogen is frequently seed transmitted, both inter- (Smith, 1910; Jensen, 1934). nally in seed and on the seed surface. The quantity of bacteria Taxonomic position: Procaryotae Kingdom, Division II 2 4 in infected seed usually averages between 10 and 10 cfu Firmicutes Gibbons & Murray 1978, Class I Firmibacte- per seed. Latently infected young plants are also considered ria. The genus Clavibacter was designed to accommodate important for spread (De Leon et al., 2011). Further informa- the plant pathogenic coryneform bacteria, the cell wall tion can be found in the EPPO data sheet on peptidoglycan of which contains 2,4-diaminobutyric acid C. michiganensis subsp. michiganensis (EPPO/CABI, 1997). as a dibasic amino acid (Davis et al., 1984). Strictly aer- obic; Gram-positive rods which do not produce endo- spores. V, Y and palisade arrangements of cells are 1 This Standard should be used in conjunction with PM 7/76 Use of usually present. EPPO diagnostic protocols. EPPO code: CORBMI. 2The use of names of chemicals or equipment in these EPPO Standards implies no approval of them to the exclusion of others that may also be Phytosanitary categorization: EPPO A2 List no. 50, EU suitable. Annex designation II/A2. 202 ª 2016 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 46, 202–225 PM 7/42 (3) Clavibacter michiganensis subsp. michiganensis 203 Sample Extraction Dilution plating on media Suspected colonies yes no Identification tests on pure culture (1) At least two tests positive no yes Sample with suspected presence of Clavibacter michiganensis subsp. michiganensis Pathogenicity test (critical cases ) (2) Sample with suspected presence of Clavibacter symptoms no michiganensis subsp. michiganensis yes Sample negative for Sample positive for Clavibacter Clavibacter michiganensis subsp. michiganensis subsp. michiganensis michiganensis Fig. 1 Flow diagram for the detection and identification of C. michiganensis subsp. michiganensis in symptomatic samples. (1) At least two tests based on different characteristics of the pathogen (e.g. combinations of biochemical, serological or molecular tests) or two molecular tests based on different DNA sequence targets in the genome. (2) Information on critical cases is available in PM 7/76 Use of EPPO diagnostic protocols. ª 2016 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 46, 202–225 204 Diagnostics Seed sample Procedure A Procedure B Extraction Extraction (refrigerated soaking and mechanical crushing) (soaking at ambient temperature) Dilution plating on semi IF test selective media Suspected Typical cell colonies morphology yes yes Dilution plating on non- no selective media PCR (3) no Suspected colonies PCR positive no yes yes Identification tests on pure culture (1) & (2) no Bioassay no At least two tests positive Isolation yes Sample with suspected presence of Clavibacter michiganensis subsp. michiganensis Pathogenicity test (critical cases) (4) Sample with suspected presence of Clavibacter symptoms no michiganensis subsp. michiganensis yes Sample negative for Sample positive for Sample negative for Clavibacter Clavibacter Clavibacter michiganensis subsp. michiganensis subsp. michiganensis subsp. michiganensis michiganensis michiganensis Fig. 2 Flow diagram for the detection and identification of C. michiganensis subsp. michiganensis in seed samples. (1) At least two tests based on different characteristics of the pathogen (e.g. combinations of biochemical, serological or molecular tests) or two molecular tests based on different DNA sequence targets in the genome. (2) Validation data provided in this protocol for Procedure A is based on a combination of dilution plating and two PCR tests. (3) Validation data provided in this protocol for Procedure B is based on the PCR test described in Appendix 5. (4) Information on critical cases is available in PM 7/76 Use of EPPO diagnostic protocols. ª 2016 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 46, 202–225 PM 7/42 (3) Clavibacter michiganensis subsp. michiganensis 205 3. Detection 3.1 Disease symptoms Generally, C. michiganensis subsp. michiganensis causes systemic infection of tomato plants. The pathogen can also cause spots on leaves, petioles, peduncles and fruits as a result of a local infection, usually under overhead irrigation. There are a wide range of symptoms which vary with place of production (glasshouse or field), age of the plant at the time of infection, cultural practices, cultivar, etc. Symptoms can be divided into two types, depending on whether infec- tion is systemic within the vascular tissue or superficial. Additional pictures of symptoms are available on the EPPO Global Database (https://gd.eppo.int/). 3.1.1 Symptoms in systemic infections In systemic infections, the disease can often be recognized at an early stage by dull green, oily areas between the leaf veins which rapidly desiccate. Subsequently, pale brown necrotic areas appear which are frequently marginal, giving the plant a scorched appearance. Small affected areas may coalesce and produce larger necrotic zones (Fig. 3). Down- ward turning of one or a few of the lower leaves occurs as the systemic infection progresses and often the leaflets Fig. 4 Flaccidity and wilting of leaflets. along one side of a leaf become flaccid (Fig. 4), at least during periods of enhanced evapotranspiration. Under within a few days (Fig. 5). Finally, the whole plant wilts favourable conditions for symptom development (25–30°C and desiccates. Under less favourable conditions, irre- and evapotranspiration stress), entire leaves wilt and shrivel versible wilting will be delayed and the plant may not show Fig. 3 White desiccated leaf areas. Fig. 5 Wilting and shrivelling of tomato plants. ª 2016 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 46, 202–225 206 Diagnostics any wilting when defoliation is done for crop management. Fruits of systemically infected plants may fail to develop, ripen unevenly or fall. They can appear marbled with longi- tudinal chlorotic streaks and internal bleaching of vascular and surrounding tissues. Typical leaf or fruit spot symp- toms, arising from external infection, are less frequently found in glasshouse crops than in open field crops. Following systemic infections, the vascular tissues of transversely cut stems of wilted plants usually appear dark yellow to brown, in particular at the nodes (Fig. 6). The vascular parenchyma in particular has a mealy appearance and is soft, resulting from bacterial degradation and ooze production (Fig. 7). The pith may collapse completely. 3.1.2 Symptoms in superficial infections Superficial infections occur when bacteria multiply on the Fig. 7 Infected vascular stem tissues with mealy appearance of plant surface or within surface wounds and stomata. vascular parenchyma. Leaves, stems and calyces may show a mealy appearance, may expand to a few millimetres with brown, roughened as if dusted by coarse