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Bacteria and Yeast Associated with Sugar Beet Root Rot at Harvest in the Intermountain West

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Bacteria and Yeast Associated with Sugar Beet Root Rot at Harvest in the Intermountain West 1237 Bacteria and Yeast Associated with Sugar Beet Root Rot at Harvest in the Intermountain West Carl A. Strausbaugh, United States Department of Agriculture–Agricultural Research Service (USDA-ARS) NWISRL, Kimberly, ID 83341; and Anne M. Gillen, USDA-ARS CGPRU, Stoneville, MS 38776 MATERIALS AND METHODS ABSTRACT Survey. An assessment was made of Strausbaugh, C. A., and Gillen, A. M. 2008. Bacteria and yeast associated with sugar beet root bacterial root rot in recently harvested rot at harvest in the Intermountain West. Plant Dis. 92:357-363. roots delivered to piling grounds in Idaho and Oregon at the end of the 2004 and An undescribed wet rot of roots was observed in surveys of recently harvested sugar beet roots 2005 growing seasons. In all, 29 and 28 in Idaho and eastern Oregon in 2004 and 2005. Microorganisms isolated from 287 roots fell into piling grounds were visited from south- the following groups: A (41% of strains), B (29%), C (17%), D (11%), E (2%), and F (1%). eastern Idaho (American Falls, southeast- Groups A, B, C, and F were composed of bacteria while groups D and E were yeasts. Subgroup ern Idaho; Magic Valley, south-central A1 (80% of group A strains) included Leuconostoc mesenteroides subsp. dextranicum strains and subgroup A2 (20%) contained Lactobacillus strains. Group B was dominated by subgroup Idaho; and Treasure Valley, southwestern B1 (92% of strains), which included Gluconobacter strains. When only one organism was iso- Idaho) to southeastern Oregon in 2004 and lated from rotted roots, strains from subgroup A1 were isolated most frequently. Group C was 2005, respectively. We collected 120 roots composed of enteric bacteria. Strain B322 of L. mesenteroides subsp. dextranicum caused the in 2004 and 167 roots in 2005 with wet rot most severe rot on root slices and produced symptoms similar to those in harvested roots. Re- symptoms suggestive of bacterial causality. sults suggest that L. mesenteroides subsp. dextranicum is among the first bacterial species to To ensure an even sampling distribution, enter sugar beet roots, closely following fungal infections or entering directly through openings no more than 15 symptomatic roots were such as growth cracks. The bacterial rot leads to yield loss in the field but likely also leads to collected from a pile each year. Isolations storage and factory-processing problems. were conducted by removing 10-by-10- mm pieces of internal root tissue from the Additional keywords: acetic acid bacteria, fermentation, lactic acid bacteria margins between rotted tissue and white, healthy-appearing tissue. The pieces of tissue were surface sterilized, disinfested in 0.6% sodium hypochlorite for 60 s, and Bacterial vascular necrosis and rot in been identified (14,33,34) and incorpo- then rinsed in sterilized reverse osmosis sugar beet (Beta vulgaris L.) has been rated into commercial cultivars. Several water for 60 s. The surface areas of each attributed to Pectobacterium betavasculo- seed companies regularly screen germ- tissue piece then were removed and a 2-by- rum (Thomson et al.) Gardan et al. (syn. plasm to maintain resistance in commer- 2-mm piece was macerated in a drop of Erwinia carotovora (Jones) Bergey et al. cial cultivars. sterile well water. To isolate bacteria and subsp. betavasculorum Thomson et al.) Recent field observations and isolations yeasts, a loop of the suspension was (11,20,29,32) and Pseudomonas spp. revealed that other bacteria also might play streaked onto yeast extract-dextrose- (16,30). In the western United States, P. important roles in causing bacterial rot. In calcium carbonate agar (YDC) and King’s betavasculorum has been associated with Oregon and Idaho, a previously unde- Medium B (KMB) and the culture incu- root rot in sugar beet in Arizona, Califor- scribed root rot with wet symptoms that bated at 30°C (21). Representative colo- nia, Idaho, Montana, and Washington suggested the involvement of bacteria has nies from each root were restreaked onto (20,24,29,33,36). The bacterial rot in been observed. Roots with advanced YDC after 3 to 7 days. sugar beet can be variable from wet to dry symptoms had a vinegar-like fermented Identification. Organism strains ini- (29). Resistance to P. betavasculorum has smell, rotted out cavities, and viscous tially were characterized and grouped slime indicative of bacterial activity. Pre- based on colony morphology, color, gram liminary isolations revealed that the dis- stain reaction, cell shape, and size. To Corresponding author: C. A. Strausbaugh ease was associated not with Pectobacte- ensure that strains were grouped appropri- E-mail: [email protected] rium spp. but with a complex of other ately, carbon source utilization profiles Organism strains and GenBank accession numbers: bacteria instead. Observations from a sur- were determined on one-third of the strains B8, EU196380; B9,EU196381; B14, EU196382; vey of root rots in the field and fungal from each group using GN2 MicroPlates B20, EU196372; B22, EU196371; B31, isolations (unpublished data) suggested (Biolog Inc., Hayward, CA) and protocols EU196373; B71, EU196374; B82, EU196375; that fungal root rots were frequently but recommended by the manufacturer for B86, EU196376; B92, EU196377; B100, not always present with the wet rot symp- gram-negative enterics. Although designed EU196378; B106, EU196379; B122, EU196397; B138, EU196394; B139, EU196396; B147, toms. Roots in fields with heavy soils and for gram-negative bacteria, the Biolog EU196386; B150, EU196384; B154, EU196385; excessive soil moisture also were observed plates gave reproducible profiles with B176, EU196383; B194, EU196398; B208; to have the wet decomposition problem. gram-positive bacteria and yeast as well. EU196395; B225, EU196390, B231, EU196391; Because filamentous fungi and oomycetes When there was variability in carbon B247, EU196389; B268, EU196392; B322, were not always associated with the wet source utilization detected among mem- EU196393; B409, EU196388; B410, EU196387. symptoms suggestive of bacterial rot, this bers of a group, the remaining members of Accepted for publication 11 October 2007. study focused on the bacteria always asso- that group also were tested. ciated with these symptoms. Therefore, the Selected strains from each bacterial objective of this study was to identify the group were submitted to Microbial ID doi:10.1094/ PDIS-92-3-0357 bacteria possibly responsible for this wet (MIDI Labs, Newark, DE) for 16S rRNA This article is in the public domain and not copy- root rot problem in sugar beet, as well as to rightable. It may be freely reprinted with custom- sequencing (first 500 bp). The 16S rRNA ary crediting of the source. The American Phyto- establish their distribution and relative gene was polymerase chain reaction (PCR) pathological Society, 2008. pathogenicity. amplified from genomic DNA isolated Plant Disease / March 2008 357 from pure cultures. The primers used were sterile well water. A hole (2 mm in diame- ole, 3 = discoloration extends three- 16S universal primers 5F and 531R. Se- ter by 3 mm deep) was made in the center quarters of the way up the petiole, 4 = lected strains from each of the yeast of the slice with a sterile tooth pick. Bacte- inoculated petioles dead, 5 = adjacent peti- groups were submitted to Microbial ID for ria, yeast, or both were applied to the hole oles starting to show wilting, 6 = half of sequencing of 28S rRNA variable D2 re- using a sterile toothpick that had been the adjacent petioles dead, 7 = three- gion (approximately 300 bp). The primers dipped into a 48-h-old culture grown on quarters of the adjacent petioles dead, 8 = used corresponded to positions 3,334 and YDC at 30°C. Slower-growing bacterial only center whorl still green, and 9 = dead 3,630 in the Schizosaccharomyces japoni- strains were cultured for several additional plant. The scale was not used categorically cus large subunit rRNA gene. Sequence days prior to inoculation. The controls but was used as a guide to generate frac- analysis was performed using MicroSeq were mock inoculated with sterile tooth- tional ratings. Fresh weight of tops clipped (Applied Biosystems, Foster City, CA) picks. The petri dishes containing inocu- immediately above the crown was deter- microbial analysis software and database. lated root slices then were placed into mined 2 weeks after inoculation. Isolations Sequence comparisons also were con- plastic bags, arranged by block, and incu- were conducted on any petiole rated 2 or ducted using BLASTn to search the Gen- bated at 30°C. The root slices for each higher by streaking onto YDC and incubat- Bank database via the National Center for block came from the same root. Thus, each ing cultures at 30°C. Biotechnology Information website (www. experiment had a randomized complete Data analysis. Proc Univariate (SAS; ncbi.nlm.nih.gov/blast). block design with three replications per SAS Institute Inc., Cary, NC) was used to Root rot tests. Two experiments were treatment. The diameter of rotted area was test the normality of the data. Bartlett’s test conducted in which strains of bacteria and measured at both 24 and 48 h after inocu- was utilized to test for homogeneity of yeast were inoculated into sugar beet root lation. After the 48-h reading, streaks were variance between experiments. Data from slices to determine pathogenicity. Because made onto YDC from the outer edge of the multiple repetitions of an experiment were most of the strains isolated came from the rot and the cultures were incubated at 30°C pooled when possible and analyzed using bacterial subgroup (A1) containing Leu- to confirm the presence of all inoculated Proc GLM (SAS), and Fisher’s protected conostoc spp. or were found in association organisms and the absence of contamina- least significant difference was used for with strains in this subgroup, the first tion. Both experiments were repeated mean comparisons.
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