148 bank accession numbers in Table 1) or from complete genome sequences (Caballero et al. 2014; Zhao et al. 2014). These were trimmed and aligned using ClustalW (Larkin et al. 2007) and Mega5 (Tamura et al. 2011), respectively. Bayesian phylogenetic analyses were performed in MrBayes using the general time-reversible model, with inverse-gamma rates of evolution for 500,000 generations, and a burn-in equal to 0.25 (Huelsenbeck and Ronquist 2001). Erwinia piriflorinigrans was used as the outgroup. Pathogenicity Studies One-year-old potted northern red, pin, and Shu- mard oaks were grown in a shade house at Colo- rado State University, and used to confirm microbe pathogenicity. Trees were inoculated with a north- ern red oak isolate of L. quercina subsp. quercina (NCCB100490) collected in Boulder, Colorado. Two experiments were conducted with different inoculation sites: emerging leaf whorls and one- year-old stems. A single colony was streaked onto full-strength nutrient agar, incubated for two days at 26°C, then diluted in 1 mL of sterile water for each inoculation experiment. In the first experi- Sitz et al.: Drippy Blight, a Disease of Red Oaks in Colorado, US. ment, five trees of each species were used, and each tree was inoculated three times (n = 45). Emerg- ing leaf whorls were mechanically wounded with a 1 cc syringe needle as trees broke dormancy, then 50 µl of the 5x108 CFU bacterial suspen- sion was injected between the layered whorls. In a second experiment, three trees of each species were inoculated three weeks post budbreak with 5 µl of a bacterial suspension injected into one- year-old stems. Each tree was inoculated twice (n = 18). In each experiment, control inoculations using sterile water were also made twice, slightly (3–4 cm) above the inoculation sight, or on a dif- ferent twig. Each site was wrapped (Parafilm®, Bemis Company, Inc., Oshkosh, Wisconsin, U.S.). The canker lengths were recorded and the pathogen was re-isolated from cankers on nu- trient agar five to fiſteen days aſter inoculation. Data were analyzed using JMP soſtware (11.1.0v; SAS Institute). For each inoculation experiment, a mixed model was fit using can- ker lesion length as the response variable with tree species as a fixed effect. Tukey’s HSD was used to obtain pairwise comparisons between lesion lengths on the different tree species. Table 1. GenBank Accession Numbers. The species, isolate number, and GenBank accession numbers are provided for the species used to compare the pathogen Lonsdalea quercina subsp. quercina to closely related Enterobacteriaceae species. The species comparison was based off of several loci, including 16S ribosomal RNA, DNA gyrase subunit B genes (gyrB), beta subunit of ATP synthase (atpD), and translational initiation factor genes (infB) sequences. Species Isolate number 16S ribosomal DNA Cronobacter sakazakii CMCC 45402 Erwinia piriflorinigrans CFBP 5882 E. toletana Lonsdalea quercina subsp. britannica retrieved from genome: NC_023032 LMG 24162 LMG 26267T LMG 26268 LMG 26269 R-43661 LMG 6054 Lonsdalea quercina subsp. iberica Lonsdalea quercina subsp. quercina LMG 26264T LMG 26265 LMG 26266 R-43277 NCCB100490 NCCB100489 ATCC 29281 GQ405203 FN547375 JF311442 JF311443 JF311444 JF311445 JF311446 JF311441 JF311439 JF311440 JF311438 KX537747 KX537748 KX537749 DNA gyrase subunit B genes (gyrB) retrieved from genome: NC_023032 JF311583 EU145274 JF311666 JF311667 JF311669 JF311671 JF311672 JF311665 JF311662 JF311663 JF311661 retrieved from genome: JIBQ01000000 retrieved from genome: JIBP01000000 retrieved from genome: JIBO01000000 Beta subunit of ATP synthase (atpD) retrieved from genome: NC_023032 JF311470 EU145258 JF311553 JF311554 JF311556 JF311558 JF311559 JF311552 JF311549 JF311550 JF311548 retrieved from genome: JIBQ01000000 retrieved from genome: JIBP01000000 retrieved from genome: JIBO01000000 Translational initiation factor genes (infB) retrieved from genome: NC_023032 JF311696 EU145290 JF311779 JF311780 JF311782 JF311784 JF311785 JF311778 JF311775 JF311776 JF311774 retrieved from genome: JIBQ01000000 retrieved from genome: JIBP01000000 retrieved from genome: JIBO01000000 ©2018 International Society of Arboriculture
May 2018
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