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The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis : C. m. subsp. michiganensis , C. m. subsp. sepedonicus , C. m. subsp. insidiosus C. m. subsp. nebraskensis , and C. m. subsp. tessellarius . Specific primers designed for amplification of the housekeeping genes recA , rpoB , and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C . michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA , rpoB , and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease Fnu DII.

Clavibacter michiganensis subsp. insidiosus (Cmi) is a seed-borne pathogen that causes bacterial wilt of lucerne (alfalfa, Medicago sativa). The pathogen has a limited distribution worldwide and is included in the A2 pest list of the European and Mediterranean Plant Protection Organisation (OEPP/EPPO). In South Africa, Cmi was first reported in 1967 in a few regions (Western Cape province and in the Eastern Cape), with the last doubtful listing in 1983 also reporting it from the Transvaal region (currently known as Gauteng, Limpopo, North West and Mpumalanga provinces). Subsequently, lucerne plants exhibiting bacterial wilt symptoms have not been observed in any region of South Africa. Nevertheless, for South Africa, Cmi is listed as \"present, restricted occurrence\" by the OEPP/EPPO. Therefore, the status of Cmi in South Africa was re-investigated through field inspections and seed testing. Field inspections of lucerne seed crops conducted in all production regions from 2006 to 2014 did not reveal any symptomatic plant. Seed testing of 67 representative commercial seed lots showed that Cmi was absent from all of the seed lots. Prior to testing the commercial seed lots, artificially inoculated seed lots were used to show that the serial dilution plate method was sensitive (0.1% detection limit). It was also shown that a published Cmi semi-selective glucose-yeast-carbonate agar medium containing kanamycin and cycloheximide was able to more readily reveal the presence of presumptive Cmi colonies than the media of the EPPO diagnostic protocol, and the International Seed Health Initiative ring test.

The European Commission requested EFSA's Panel on Plant Health to perform the pest categorisation for Clavibacter michiganensis subsp. insidiosus. The identity of the bacterium responsible for the bacterial wilt of lucerne is clearly defined. C. michiganensis subsp. insidiosus is present in only a few MSs in the EU and it is listed in the Annex IIAII of the Directive 2000/29/CE. Only sporadic disease outbreaks occur, and not in countries where lucerne production is of importance. The pathogen causes yield and quality loss only if susceptible cultivars are grown and conditions are favourable for disease expression. The pathogen is not reported in the main lucerne‐producing MSs. There are no indications that in last decade the pathogen has a high impact on lucerne production in the EU, possibly because of the use of bacterial wilt‐resistant varieties. C. michiganensis subsp. insidiosus is seed‐borne and probably seed‐transmitted, although with some uncertainty. The main pathway for long‐distance dispersal of this pathogen is very likely via seeds, while machines and contaminated hay may also potentially play some role in the dissemination of the pathogen. The pathogen can be easily detected and identified on the basis of various microbiological and molecular tests and disease symptoms, including leaf mottling, reduction in plant height, and “witches’ broom” syndrome. Effective management strategies are available and include the use of resistant cultivars and, probably, the use of pathogen‐free seeds. Finally, the Panel concluded that severe consequences, in terms of yield and quality losses, are expected for lucerne only if bacterial wilt‐susceptible varieties are grown and if weather conditions are conducive to the disease.

A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34-41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%-16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life = 7.3 h) than that from SBL11 (half-life = 6.4 h at 50 °C), while both showed the same temperature optimum of 37 °C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.Key words: biodegradation, monocrotophos, phosphotriesterase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11.