Taxonomy is the science of grouping organisms according to shared properties; it consists of classification, nomenclature, and identification. Bacteria are classified using both phenotypic and phylogenetic approaches. This chapter discusses techniques such as DNA sequencing of rRNA genes, DNA–DNA reassociation, multilocus sequence typing (MLST), and average nucleotide identity (ANI). Once a species, subspecies, or pathovar can be accurately and unambiguously identified, the organism must be named using standardized nomenclature. Nomenclature rules in microbiology are regulated by the International Code of Nomenclature of Bacteria.

Our highly concentrated monoculture makes crops vulnerable to pests and diseases. An increase in emerging non-indigenous bacterial diseases poses a real threat to US agriculture. The United States has 100,000 miles of shoreline and 6,000 miles of border, making possible easy introduction of crop pests and diseases. Most threatening to crops are the cross-domain enteric bacteria. In contrast to animals, crops have hundreds of major diseases and development of molecular-based detection protocols for each pathogen is impossible with current technology. Rathayibacter toxicus, a neurotoxin-producing bacterium transmitted by a seed gall nematode, is an example of a high-risk Select Agent. The bacterium infects seeds of grasses without showing any symptoms, often resulting in the death of grazing cattle. A prerequisite for the control of any disease is sensitive detection and proper identification of the causal organism. Detecting bacteria in samples of plants showing symptoms is relatively simple, whereas detection in asymptomatic tissues is difficult due to the extremely low numbers of the target pathogen present. Rapid serological assays work well with symptomatic tissues but not from asymptomatic tissue when bacteria levels are below sensitivity limits. Classical agar-plating assays are 1,000 fold more sensitive then serology or PCR. However, agar plating assays take from 3 to 5 days and require pathogenicity tests to confirm the identity. PCR-based assays allow for rapid, accurate identification but are insensitive due to use of 1 microL sample in comparison to 100 microL used for agar plating. To overcome this disadvantage, an enrichment technique termed BIO-PCR can be used in combination with agar plating for detection with asymptomatic tissues. The key to developing a successful BIO-PCR protocol is to determine the time required for development of pin point-size colonies to appear. For most plant pathogens 15 to 24 hours is sufficient time, whereas for the cross-domain bacteria only 1 to 2 hours is needed. For greater sensitivity, BIO-PCR can be combined with 96-well microliter plates with membranes to detect a single viable cell per 10 mL of an aqueous sample.

Xanthomonas citri (synonym = Xanthomonas axonopodis pv. citri) (3) has been reported in several countries in Africa (1) but not Somalia. During 2006 and 2007, hyperplasia-type lesions, often surrounded by a water-soaked margin and yellow halo, typical of citrus canker caused by X. citri were found on 8- to 10-year-old lime (Citrus limetta) and grapefruit (Citrus × paradisi Macfed.) trees in northern and southern Somalia, respectively. Ten leaf samples diagnosed presumptively as citrus canker by Xac ImmunoStrip test kits (Agdia, Elkhart, IN) were mailed to the USDA Foreign Disease-Weed Science Research Unit at Ft. Detrick, MD. To confirm the identification of X. citri, isolations were made from several lesions from each sample onto yeast-dextrose-CaCO3 (YDC) agar (2). Yellow, xanthomonad-like mucoid, convex colonies were purified and stored on YDC slants. Phenotypic tests were done as described (2), and real-time PCR assays were done using primers XCit8F and XCit5R with probe XCitP2 (N. W. Schaad, unpublished). For pathogenicity tests, cultures were grown overnight in liquid nutrient broth-yeast (4) medium adjusted to contain 1 × 105 CFU/ml and inoculated into leaves of lime seedlings with the blunt end of a 2-ml syringe. After 21 to 30 days in a lighted dew chamber (Model I-60DLM; Percival Scientific, Inc. Perry, IA) at 30/23°C day/night, symptoms were recorded. Cultures of sample S-1 (northern Somalia) from lime were phenotypically atypical of X. citri, PCR negative, and nonpathogenic. However, cultures of samples 3 to 7 (southern Somalia) from grapefruit were typical of X. citri and PCR positive; cultures 3 and 4 were tested for pathogenicity and produced erumpent lesions on lime. Isolations onto YDC agar resulted in typical mucoid, convex, yellow, PCR-positive colonies. To our knowledge, this is the first report of X. citri on citrus plants in Somalia. Strains S3 and S4 have been deposited in ICPB at Ft. Detrick, MD as ICPB 11650 and 11651, respectively. References: (1) J. F. Bradbury. Guide to Plant Pathogenic Bacteria. CAB International, Egham, UK, 1986. (2) N. W. Schaad et al. Xanthomonas. Page 175 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al. eds. American Phytopathological Society, St. Paul. MN. 2001. (3) N. W. Schaad et al. Syst. Appl. Microbiol. 29:690, 2006. (4) A. K. Vidaver. Appl. Microbiol. 15:1523, 1967.