Medicago sativa L. is one of the most important perennial forage crops and has been cultivated for more than 2,000 years in China. A previously unreported sprout wilt disease of M. sativa, affecting as much as 25% of the seedlings, was observed in northwest China (Gansu Province) in March 2011. First symptoms on the sprouts were dehydration and yellowing. Within 24 to 48 h, the sprouts stopped growing, wilted, turned brown, and bacteria began oozing from the material. Three symptomatic sprout samples collected from a grower with the wilt problem were processed for microbiological analysis. Bacteria isolated from symptomatic samples produced a pink, diffusible pigment in King's medium B and nutrient agar supplemented with glucose, sucrose, and maltose. The three isolates were negative for gram stain reaction, oxidase, production of hydrogen sulfide and indole, growth in KCN broth, arginine dihydrolase activity, hydrolysis of casein, hydrolysis of gelatin, and acid production from L-arabinose, dulcitol, glycerol, lyxose, and starch. In contrast, they were positive for catalase, nitrate reduction, Voges-Proskauer, hydrolysis of aesculin, acid production from D-ribose, maltose and sucrose, assimilation of adonitol, L-lactate, mannitol, Myo-inositol, erythritol, sorbitol, and sucrose, and growth in 5% NaCl at 36°C. The 16S rDNA of three isolates (Cp1, Cp2, and Cp3) was amplified using the 7F (5′-CAGAGTTTGATCCTGGCT-3′) and 1540R (5′-AGGAGGTGATCCAGCCGCA-3′) primers. The sequences for the 1,428-bp amplicon from the isolates were identical (GenBank Accession No. JN900058) and had 99% sequence identity with 16S rDNA of Erwinia persicinus strains (including the type strain LMG 11254 [GenBank Accession No. Z96086.1], GS 04 [GenBank Accession No. DQ365580.1], LPPA 373 [GenBank Accession No. AJ937837.1], LPPA 408 [GenBank Accession No. AM294946.1], LMG 2691 [GenBank Accession No. AJ001190.1], and HK 204 [GenBank Accession No. NR_026049.1]). The three isolates were also evaluated in pathogenicity tests. Bacterial suspensions (108 CFU/ml) were spray inoculated on 7-day-old M. sativa sprouts of cv. Algonquin. The inoculated sprouts were placed onto 2% water agar in petri dishes (five sprouts per 9-cm dish) with four dishes for each bacterial isolate and control. The dishes were sealed with Parafilm for 2 days and held in an incubator at 25°C with a 12-h photoperiod. Assays were repeated twice. Symptoms that developed within 7 days were similar to those originally observed, whereas symptoms did not occur on control sprouts sprayed with sterile distilled water. Bacteria sharing the characteristics of the inoculated isolates were recovered from symptomatic sprouts, hence fulfilling Koch's postulates. E. persicinus has been isolated previously from Phaseolus vulgaris (1), Pisum sativum (2), tomato, banana, and cucumber (3). To our knowledge, this is the first report of E. persicinus from M. sativa.
References: (1) A. J. González et al. Plant Dis. 89:109, 2005. (2) A. J. González et al. Plant Dis. 91:460, 2007. (3) M. V. Hao et al. Int. J. Syst. Bacteriol. 40:379, 1990.