Colony morphology: The pathogen is difficult to isolate directly from plant material and seed due to slow growth of bacterium and overgrowth by other organisms. Contaminants may also be of a yellow color (e.g., Pantoea agglomerans and Xanthomonas-like saprophytic bacteria). Isolation from symptomatic material should be completed as soon as possible after collection of samples. Plant parts showing fresh symptoms preferably with bacterial exudates should be selected for isolation if available. See EPPO (2007) for additional detail about pathogen isolation from plant material and seeds.
Isolation of Xanthomonas from symptomatic material can be performed using peptone sucrose agar (PSA), nutrient broth yeast extract medium (NBY), growth factor (GF) agar, modified Wakimoto's agar, and various semi-selective media (Agarwal et al., 1989; Mew and Mistra, 1994; Sakthivel et al., 2001). Growth is very slow on nutrient agar (EPPO, 2007).
Ming et al. (1991) developed a semi-selective medium, called XOS, to isolate both Xanthomonas oryzae pathovars from rice seed.
Yuan (1990) describes a new culture medium for culturing both Xanthomonas oryzae pv. oryzae from rice seed.
Gnanamanickam et al. (1994) tested three strains for growth on TZC, WF-P, YCM, YAT, MXO, and XOS semi-selective media. Results varied for each isolate used, but worked best when using monoclonal antibodies to confirm the genus and pathovar.
2. Serological: An ELISA test is available for Xanthomonas at the genus level for primary screening. A positive does not indicate Xanthomonas oryzae pv. oryzae. The ID must be confirmed by other methods.
Monoclonal antibodies: Genus and pathovar specific antibodies can be used in an ELISA reaction on presumptive positives (Alvarez et al., 1985; Benedict et al., 1989).
In the early stage of disease, the symptoms are similar to narrow brown leaf spot.
At the later stage, when the streaks have coalesced, symptoms of bacterial blight and bacterial leaf streak are similar. The shape of the edges of the lesions differs; straight in leaf streak and wavy in leaf blight.
X. oryzae pv. oryzicola may be distinguished from X. oryzae pv. oryzae by colony morphology in typical isolates, strong starch and gelatin hydrolysis, and by biochemical and molecular methods.
In Progress / Literature-based Diagnostics:
Pathogenicity: Isolates can be tested for pathogenicity on susceptible rice cultivars. For X. oryzae pv. oryzae use 30 to 45 day-old IR24 or IR8 (International Rice Institute) or local popular varieties with known susceptibility to bacterial blight. Leaf clipping and spray inoculation methods are available for inoculations (Kauffman et al., 1973; Cottyn et al., 1994b; EPPO, 2007). Nino-Liu et al. (2005) inoculated plants by dipping them in bacterial mixture and incubating in a growth chamber. Symptoms developed a 6-day period.
Fatty Acid Profiles: Fatty acid profiles allow identification at the genus level only (Swings et al., 1990), so this analysis is not recommended a diagnostic method.
Molecular: PCR/BIO-PCR: Vera Cruz et al. (1995) and Vera Cruz et al. (1996) compared Rep-PCR using repetitive DNA sequences with Restriction Fragment Length Polymorphism (RFLP).
Leach et al. (1990) used a repetitive DNA sequence (pJEL101) to distinguish X. oryzae pv. oryzae from other pathovars and species of Xanthomonas.
PCR assays using primers that amplify internal fragments IS 1112 and IS 1113 of X. oryzae pv. oryzae (Cottyn et al., 1994; Alvarez et al., 1997) have been used as well.
Sakthivel et al. (2001) developed a PCR technique to detect X. oryzae pv. oryzae in rice seed. A combined biological and enzymatic amplification (BIO-PCR) technique was used to detect the pathogen in naturally infected seed.
Gonzalez et al. (2007) found that X. oryzae pv. oryzae African and Asian strains were genetically different from each other by using RFLP, fluorescent amplified fragment length polymorphism (FAFLP) and repetitive sequence-based polymerase chain reaction.
Real-time PCR: Zhao et al. (2007) developed a real-time PCR to detect X. oryzae pv. oryzae and can distinguish it from X. oryzae pv. oryzicola.
Liao et al. (2003) developed a real-time PCR that can distinguish the two pathovars.
Computational Genomics/Multiplex PCR: Lang et al. (2010) used a computational genomics pipeline to compare sequenced genomes of Xanthomonas species to identify regions for development of highly specific diagnostic markers. A suite of primers were selected to monitor diverse loci and to distinguish the rice bacterial blight and leaf streak pathogens. A subset of primers were combined into a multiplex PCR to accurately distinguish the two rice pathogens in a geographically diverse collection from other xanthomonads and other plant pathogenic and plant-or seed associated bacteria.