Preferred name: Clavibacter insidiosus
Authority: (McCulloch) Li et al.
Taxonomic position: Bacteria: Actinobacteria: Micrococcales: Microbacteriaceae
Other scientific names: Aplanobacter insidiosum McCulloch, Clavibacter michiganensis subsp. insidiosus (McCulloch) Davis et al., Corynebacterium insidiosum (McCulloch) Jensen, Corynebacterium michiganense pv. insidiosum (McCulloch) Dye & Kemp
Common names in English: bacterial blight of lucerne, bacterial root rot of lucerne, bacterial wilt of lucerne, vascular wilt of lucerne
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Notes on taxonomy and nomenclature EPPO Categorization: A2 list
EU Categorization: RNQP (Annex IV)
view more categorizations online...
EPPO Code: CORBIN

The main host of C. insidiosus is Medicago sativa (lucerne or alfalfa). Other Medicago species such as M. falcata and M. truncatula have been reported as natural hosts of this bacterium as well. C. insidiosus has also been found on Melilotus albus, Onobrychis viciifolia, Lotus corniculatus and Trifolium sp. (Bradbury, 1986; Lu et al., 2015). On inoculation, C. insidiosus can induce symptoms in several other Medicago species. A report of C. insidiosus on Zea mays is considered doubtful (Bradbury, 1986).

Host list: Lotus corniculatus, Medicago falcata, Medicago sativa, Medicago truncatula, Melilotus albus, Onobrychis viciifolia, Trifolium sp.

Lucerne plants with symptoms caused by C. insidiosus were first reported in Illinois and Wisconsin in 1924 rapidly followed by reports from other states throughout the USA, indicating that the pathogen was already widespread before this first report (Jones, 1925; Jones and McCulloch, 1926). During the remainder of the 20^th century, the pathogen was found in the most important lucerne production areas in the USA and Canada, and was also detected in other continents. In several countries with historical findings of C. insidiosus, the disease no longer occurs (e.g. in Brazil, Canada, South Africa). In the EPPO region, findings were mainly sporadic and since the 1980s, the disease is either no longer found or no significant outbreaks have been reported.

EPPO Region: Kazakhstan, Lithuania, Poland, Russia (Central Russia, Southern Russia), Tunisia, United Kingdom
Africa: Tunisia
Asia: Iran, Kazakhstan, Saudi Arabia, Turkmenistan
North America: Mexico, United States of America (Arkansas, California, Iowa, Kansas, Kentucky, Maryland, Minnesota, Montana, Nebraska, Nevada, New York, North Dakota, Ohio, Oklahoma, Oregon, South Dakota, Texas, Utah, Washington, Wisconsin)
Oceania: Australia (New South Wales, South Australia, Tasmania, Victoria), New Zealand

The bacterium can enter its host through wounds, such as mowing wounds, wounds caused by freezing and thawing, or feeding wounds caused by nematodes or insects. C. insidiosus usually spreads from plant to plant in water. Following entry into the plant, the bacterium is able to spread rapidly through the vascular system. C. insidiosus is thought to be primarily a disease of the perennial plant parts and as such it colonizes the taproot and crown of lucerne plants. From there it can spread to newly formed stems (EFSA, 2014; Hunt, 1971; Jones and McCulloch, 1926; Koehler and Jones, 1932).

In inoculation experiments, symptoms can be observed after three weeks (Hale, 1972). In the field, the disease is rarely observed in one year old plants. In the second year after sowing, symptoms may become visible on the above ground parts but it usually takes three years for a field to become severely affected (Koehler and Jones, 1932). In the laboratory and on solid growing media, C. insidiosus grows best at 23 °C, but in the field disease incidence has been shown to be greater at 16 °C compared to 24°C and 28°C. Abundant soil moisture aids the progress of the disease (EFSA, 2014; Jones and McCulloch, 1926; Koehler and Jones, 1932).

Overwintering generally occurs in the roots and crowns of diseased plants. Bacteria remain viable for 10 years in lucerne stems stored at 20-25°C, but they survive only poorly in non-sterile soils (Carroll and Lukezic, 1971; Cormack, 1961; Koehler and Jones, 1932).

Symptoms

Within lucerne crops, symptomatic plants may be scattered throughout the field or found in patches. Lower wetter parts of the field are often affected first. Symptoms are often inconspicuous in the first year and affect the lucerne crop rather uniformly, hence the 'insidious' species name. Mild symptoms include leaf-mottling and cupping or upward curling of leaf margins, with some reduction in plant height. Moderate infection additionally leads to a proliferation of the stems, giving a witches' broom effect. In severe infections, young plants or newly formed stems are only a few centimeters high, have thin and spindly stems, leaflets are small and thicker, often distorted and show marginal or entire bleaching, and ultimately plants usually die. In the field, severely affected plants can be easily spotted due to their yellow green color and stunted growth. When drought stress occurs, infected plants only show enhanced wilting, compared to healthy plants. In the absence of, or in addition to aerial symptoms, there is a yellow to pale-brown discoloration of the young woody root tissue at the junction of the cortex and vascular cylinder. This is visible when peeling off the cortex or cutting the tap-root (Close and Mulcock, 1972; Jones, 1925; Jones and McCulloch, 1926; Koehler and Jones, 1932).

Morphology

C. insidiosus is an aerobic, Gram-positive, capsulated, non-motile rod, 0.4-0.5 x 0.7-1.0 µm, which does not produce chains. Agar colonies are generally pale-yellow, round or amorphous, smooth, glistening, flat or slightly raised. Characteristic blue pigment granules occur irregularly in cultures on media high in available sugars after 6-8 days at 21°C. DNA G+C content of the type strain (LMG 3663^T) is 72.7% (Close and Mulcock, 1972; Li et al., 2018; Mcculloch, 1925).

Detection and inspection methods

The EPPO Standard on diagnostics PM 7/99 (2) (EPPO, 2022) provides comprehensive information on the available methods for detecting C. insidiosus in both symptomatic plant material and seeds. It also provides information on isolation and identification methods. Laboratory analysis should be performed on cut sections of the main root or on seeds. For detection of C. insidiosus with immunofluorescence, two antisera are suggested and some performance characteristics are provided. For detection with PCR, two conventional PCR tests (Samac et al., 2017; Ward et al., 2008) and two real-time PCR tests (Samac et al., 2017; Ward et al., 2008) are suggested, with the conventional PCR by Ward et al. (2008) being suggested only for plant material and not for seed testing. In addition to these immunofluorescence and PCR tests, DNA barcoding (Zaluga et al., 2011), BOX-PCR (Louws et al., 1998) and MALDI-TOF MS (Zaluga et al., 2011) can be used for identification.

Short distance spread of C. insidiosus mainly occurs through the movement of water (or water droplets) containing the pathogen. The spread by contaminated machinery and especially mowers appears to be important. Mowing lucerne crops when the foliage is wet creates ideal conditions for bacterial dissemination since both infected and non-infected plants are wounded and the mower spreads infected plant material and contaminated water droplets over the field (Jones and McCulloch, 1926; Koehler and Jones, 1932; University of Illinois Department of Crop Sciences, 1988). There have also been reports of local spread by insects and nematodes, and the presence of nematodes in the field favors infection (Hawn, 1963; Hunt, 1971; Kůdela et al., 1984).

Long distance spread can occur through contaminated seed lots. C. insidiosus can be present on particles present in the seed lot (e.g. plant debris), on the surface of the seeds or inside the seeds. The exact role of these seed contaminations in new outbreaks is uncertain as data on the transmission of the bacterium from seed to seedling is generally missing. In experiments, the percentage of symptomatic plants that transmitted the bacterium to its seeds was 7%, which is relatively low (EFSA, 2014; Samac et al., 1998).

Economic impact

In the past, C. insidiosus caused serious damage with major economic impact on lucerne crops in countries including the USA, Canada and New Zealand (Hale and Close, 1974; Koehler and Jones, 1932; Peake and Cormack, 1955). However, at the moment in most parts of the world the presence of the pathogen is limited and its impact is strongly reduced. Canada is now free of the pathogen and in the USA major impact is no longer observed. In the European Union the impact of C. insidiosus is also low. In Australia growers have reported that even infected fields remain highly productive (Ophel-Keller, 2005). However, in some parts of the world, e.g. Iran, the pathogen still caused damage in lucerne fields (Heidari and Khodakaramian, 2012).

Control

The most important strategy to control C. insidiosus is the use of resistant lucerne cultivars. In the USA and Canada such cultivars have already been available for decades and their use has probably led to the strong reduction of damage caused by C. insidiosus. In Europe, lucerne cultivars with reduced susceptibility have also been developed (EFSA, 2014; Kozová et al., 2003; Víchová and Kozová, 2004). Other practices that can help to reduce the risk of damage due to C. insidiosus are: avoiding poorly drained soils, harvesting young stands before old ones, harvesting fields with symptoms last, steam cleaning equipment with steam between fields, mowing only when foliage is dry, growing other crops for two or three years before reseeding lucerne, reducing injuries to crowns, and using pathogen-free seed from regions where the bacterium is absent (EFSA, 2014; University of Illinois Department of Crop Sciences, 1988).

Phytosanitary risk

C. insidiosus has not been reported in most EPPO countries where lucerne is grown, even though there are suitable climate and soil conditions in these areas. The use of resistant cultivars and strict phytosanitary measure have likely strongly reduced the risk of spread and establishment of this pathogen (EFSA, 2014).

Seeds should come from areas known to be free of C. insidiosus. Seeds of lucerne from areas where C. insidiosus occurs should come from a field which, along with adjacent fields, was found free from C. insidiosus during the last growing season, and where no lucerne was grown during the 3 years prior to the sowing of this crop. Alternatively, seeds should come from cultivars which are considered highly resistant to C. insidiosus (EPPO, 2019).

Bradbury JF (1986) Guide to Plant Pathogenic Bacteria. Farnham Royal, Slough, UK, CAB International, 332 pp.

Carroll RB & Lukezic FL (1971) Preservation of Corynebacterium insidiosum in a sterile soil mix without loss of virulence. Phytopathology 61, 688-690.

Close R & Mulcock AP (1972) Bacterial wilt Corynebacterium insidiosum (McCulloch, 1925) Jensen, 1934 of lucerne in New Zealand. New Zealand Journal of Agricultural Research 15, 141-148.

Cormack MW (1961) Longevity of the bacterial wilt organism in alfalfa hay, pod debris, and seed. Phytopathology 51, 260-261.

Davis MJ, Gillaspie AG, Vidaver AK, Harris RW (1984) Clavibacter: a new genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. cynodontis subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and bermudagrass stunting disease. International Journal of Systematic Bacteriology 34(2), 107-117.

EFSA (2014) Scientific Opinion on the pest categorisation of Clavibacter michiganensis subsp. insidiosus (McCulloch) Davis et al. EFSA Journal 12(12), 3910. https://doi.org/10.2903/j.efsa.2014.3910

EPPO (2019) Evaluation of the Regulated Non-Quarantine Pest (RNQP) status for Clavibacter michiganensis subsp. insidiosus. Available at https://pra.eppo.int/pra/8a8b3bad-4c8d-4f07-9884-a91562a340fd

EPPO (2022) EPPO Standards. Diagnostics. PM 7/99 (2) Clavibacter insidiosus. EPPO Bulletin 52, 67-86.

Hale CN (1972) Rapid identification methods for Corynebacterium insidiosum (McCulloch, 1925) Jensen, 1934. New Zealand Journal of Agricultural Research 15, 149-154.

Hale CN & Close RC (1974) A survey of the occurrence of bacterial wilt of lucerne in New Zealand. New Zealand Journal of Experimental Agriculture 2, 75-77.

Hawn EJ (1963) Transmission of bacterial wilt of alfalfa by Ditylenchus dipsaci (Kühn). Nematologica. 9, 65-68.

Heidari A & Khodakaramian G (2012) Comparison of pathogenicity of Clavibacter michiganensis subsp. insidiosus and Pseudomonas viridiflava strains on alfalfa. Archives of Phytopathology and Plant Protection 45, 922-931.

Hunt OJ (1971) The effects of root knot nematodes on bacterial wilt in alfalfa. Phytopathology 61(3), 256-259.

Jones FR (1925) A new bacterial disease of alfalfa. Phytopathology 15, 243-244.

Jones FR & McCulloch L (1926) A bacterial wilt and root rot of alfalfa caused by Aplanobacter insidiosum. Journal of Agricultural Research 33, 493-521.

Koehler B & Jones FR (1932) Alfalfa wilt as influenced by soil temperature and soil moisture. University of Illinois Agricultural Experiment Station Bulletin 378, 58 pp.

Kozová Z, Víchová J, Babinec J & Vaverka S (2003) The resistance of alfalfa varieties to Clavibacter michiganensis subsp. insidiosus (McCulloch) Davis et al. Czech Journal of Genetics and Plant Breeding. 39, 272-274.

Kůdela V, Havlíčková H & Vacke J (1984) Sitona lineatus as a vector of Corynebacterium michiganense pv. insidiosum. Sborník ÚVTIZ, Ochrana Rostlin 20, 267-271.

Li X, Tambong J, Yuan K, Chen W, Xu H, Lévesque CA & De Boer SH (2018) Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses. International Journal of Systematic and Evolutionary Microbiology 68, 234-240.

Louws FJ, Bell J, Medina-Mora CM, Smart CD, Opgenorth D, Ishimaru CA, Hausbeck MK, de Bruijn FJ & Fulbright DW (1998) rep-PCR-mediated genomic fingerprinting: a rapid and effective method to identify Clavibacter michiganensis. Phytopathology 88, 862-868.

Lu Y, Samac DA, Glazebrook J & Ishimaru CA (2015) Complete genome sequence of Clavibacter michiganensis subsp. insidiosus R1-1 using PacBio single-molecule real-time technology. Genome Announcements 3(3), e00396-15. https://doi.org/10.1128/genomeA.00396-15

Mcculloch L (1925) Aplanobcater insidiosum n. sp. the cause of an alfalfa disease. Phytopathology 15, 496-497.

Ophel-Keller K (2005) Bacterial wilt of lucerne: A market access issue for lucerne seed growers. Report for the Rural Industries Research and Development Corporation. Publication No. 05/10116 pp.

Peake RW & Cormack MW (1955) Effect of bacterial wilt on hay yield of irrigated alfalfa. Canadian Journal of Agricultural Science 35, 202-210.

Samac DA, Nix RJ & Oleson AE (1998) Transmission frequency of Clavibacter michiganensis subsp. insidiosus to alfalfa seed and identification of the bacterium by PCR. Plant Disease 82, 1362-1367.

Samac DA, Ophel-Keller K & Caffier D (2017) Detection of plant-pathogenic bacteria in seed and other planting material. Second Edition: 16, Chapter 16: Detection of Clavibacter michiganensis subsp. insidiosus in alfalfa seeds by M. Fatmi, R. R. Walcott and N. W. Schaad. American Phytopathological Society, 103-107.

University of Illinois Department of Crop Sciences (1988) Bacterial wilt of alfalfa. Report on plant disease 300, 1-3.

Víchová J & Kozová Z (2004) The virulence of Clavibacter michiganensis subsp. insidiosus strains and tests of alfalfa varieties for resistance to the wilt pathogen. Journal of Plant Protection Research 44, 147-154.

Ward LJ, Gourley J & De Boer SH (2008) Molecular detection of Clavibacter michiganenis ssp. insidiosus in alfalfa seed. Canadian Journal of Plant Pathology 30, 492-497.

Zaluga J, Heylen K, Van Hoorde K, Hoste B, Van Vaerenbergh J, Maes M & De Vos P (2011) GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter. Systematic and Applied Microbiology 34, 400-407.

Zgurskaya HI, Evtushenko LI, Akimov VN & Kalakoutskii LV (1993) Rathayibacter gen. nov., including the species Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., Rathayibacter iranicus comb. nov., and six strains from annual grasses. International Journal of Systematic and Evolutionary Microbiology 43(1), 143-149.

This datasheet was extensively revised in 2024 by Robert A.M. Vreeburg and Michiel J.C. Pel (NIVIP, Netherlands Institute for Vectors, Invasive plants and Plant health). Their valuable contribution is gratefully acknowledged.

EPPO (2024) Clavibacter insidiosus. EPPO datasheets on pests recommended for regulation. https://gd.eppo.int (accessed 2024-12-22)

This datasheet was first published in the EPPO Bulletin in 1982 and revised in the two editions of 'Quarantine Pests for Europe' in 1992 and 1997, as well as in 2024. It is now maintained in an electronic format in the EPPO Global Database. The sections on 'Identity', ‘Hosts’, and 'Geographical distribution' are automatically updated from the database. For other sections, the date of last revision is indicated on the right.

CABI/EPPO (1992/1997) Quarantine Pests for Europe (1^st and 2^nd edition). CABI, Wallingford (GB).

EPPO (1982) Data sheets on quarantine organisms, Corynebacterium insidiosum. EPPO Bulletin 12(1), 5-9. https://doi.org/10.1111/j.1365-2338.1982.tb01949.x