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Applied and Environmental Microbiology, May 2002, p. 2198-2208, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2198-2208.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Endophytic Colonizing Bacteria from Agronomic Crops and Prairie Plants

Denise K. Zinniel,^1,2 Pat Lambrecht,¹ N. Beth Harris,² Zhengyu Feng,² Daniel Kuczmarski,^1, Phyllis Higley,^1, Carol A. Ishimaru,^1,|| Alahari Arunakumari,^1,# Raúl G. Barletta,^2* and Anne K. Vidaver^1*

Departments of Plant Pathology,¹ Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583-0722²

Received 6 September 2001/ Accepted 4 February 2002

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log₁₀ CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log₁₀ CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log₁₀ CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log₁₀ CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.

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* Corresponding author. Mailing address: Department of Veterinary and Biomedical Sciences, University of Nebraska, 211 Veterinary Basic Sciences, Lincoln, NE 68583-0905. Phone: (402) 472-8543. Fax: (402) 472-9690. E-mail: rbarletta{at}unl.edu.

* Corresponding author. Mailing address: Department of Plant Pathology, University of Nebraska, 406C Plant Sciences Hall, Lincoln, NE 68583-0722. Phone: (402) 472-2858. Fax: (402) 472-2853. E-mail: avidaver1{at}unl.edu.

This is a contribution of the University of Nebraska Agricultural Research Division, Lincoln, Journal Series no. 13445.

Present address: Alex R. Masson Incorporated, Linwood, KS 66052.

Present address: 7320 Raven Circle, Lincoln, NE 68506.

^|| Present address: Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523-1177.

^# Present address: Bristol-Myers Squibb Company, Pennington, NJ 08534.

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Applied and Environmental Microbiology, May 2002, p. 2198-2208, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2198-2208.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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