Brenda K. Schroeder

Interrelationships of Food Safety and Plant Pathology: The Life Cycle of Human Pathogens on Plants

Bacterial food-borne pathogens use plants as vectors between animal hosts, all the while following the life cycle script of plant-associated bacteria. Similar to phytobacteria, Salmonella, pathogenic Escherichia coli, and cross-domain pathogens have a foothold in agricultural production areas. The commonality of environmental contamination translates to contact with plants. Because of the chronic absence of kill steps against human pathogens for fresh produce, arrival on plants leads to persistence and the risk of human illness. Significant research progress is revealing mechanisms used by human pathogens to colonize plants and important biological interactions between and among bacteria in planta. These findings articulate the difficulty of eliminating or reducing the pathogen from plants. The plant itself may be an untapped key to clean produce. This review highlights the life of human pathogens outside an animal host, focusing on the role of plants, and illustrates areas that are ripe for future investigation.

Development of a Functional Genomics Platform for Sinorhizobium meliloti: Construction of an ORFeome

ABSTRACT The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.

A portal for rhizobial genomes: RhizoGATE integrates a Sinorhizobium meliloti genome annotation update with postgenome data

Sinorhizobium meliloti is a symbiotic soil bacterium of the alphaproteobacterial subdivision. Like other rhizobia, S. meliloti induces nitrogen-fixing root nodules on leguminous plants. This is an ecologically and economically important interaction, because plants engaged in symbiosis with rhizobia can grow without exogenous nitrogen fertilizers. The S. meliloti-Medicago truncatula (barrel medic) association is an important symbiosis model. The S. meliloti genome was published in 2001, and the M. truncatula genome currently is being sequenced. Many new resources and data have been made available since the original S. meliloti genome annotation and an update was needed. In June 2008, we submitted our annotation update to the EMBL and NCBI databases. Here we describe this new annotation and a new web-based portal RhizoGATE. About 1000 annotation updates were made; these included assigning functions to 313 putative proteins, assigning EC numbers to 431 proteins, and identifying 86 new putative genes. RhizoGATE incorporates the new annotion with the S. meliloti GenDB project, a platform that allows annotation updates in real time. Locations of transposon insertions, plasmid integrations, and array probe sequences are available in the GenDB project. RhizoGATE employs the EMMA platform for management and analysis of transcriptome data and the IGetDB data warehouse to integrate a variety of heterogeneous external data sources.

Evaluation of Onion Cultivars for Resistance to Enterobacter cloacae in Storage

Sixty-nine storage onion (Allium cepa) cultivars (seven white, five red, and 57 yellow cultivars) were evaluated in the Washington State University Onion Cultivar Trials in the semiarid Columbia Basin of central Washington in 2007-08 and/or 2008-09. Each cultivar was inoculated with Enterobacter cloacae, cured, stored under commercial storage conditions, and evaluated for bacterial storage rot symptoms approximately 4.5 months after storage. Noninoculated bulbs of each cultivar served as a control treatment in each experiment. In addition, bulbs injected with water served as a second control treatment in the 2008-09 experiment. Inoculation of onion bulbs with E. cloacae resulted in significantly higher incidence and severity of Enterobacter bulb decay compared to noninoculated bulbs and bulbs injected with sterile water. For bulbs inoculated with E. cloacae, mean severity of bacterial storage rot per cultivar ranged from 5 to 19% of the cross-section evaluated for each onion bulb in 2007-08 and from 9 to 29% in 2008-09. For noninoculated bulbs, mean severity ranged from 0 to 1% in 2007-08 and 0 to 3% in 2008-09. For bulbs injected with water in the 2008-09 experiment, severity of bulb rot ranged from 0 to 10% per cultivar, with four cultivars (OLYX05-26, RE-E, Redwing, and Talon) displaying bulb rot ratings significantly greater than 0%. For the 33 cultivars included in both experiments, a significant correlation in bulb rot severity ratings was detected for the 2007-08 versus 2008-09 experiments (r = 0.43 at P = 0.013). Redwing, Red Bull, T-433, Centerstone, and Salsa had low severity ratings in both experiments; whereas Montero, OLYS05N5, Caveat, and Granero had severe bulb rot ratings in both experiments. The results demonstrate that it should be possible to select for increased resistance to Enterobacter bulb decay in storage onion cultivars.

Complete genome of the onion pathogen Enterobacter cloacae EcWSU1

Previous studies have shown that the members of the Enterobacter cloacae complex are difficult to differentiate with biochemical tests and in phylogenetic studies using multilocus sequence analysis, strains of the same species separate into numerous clusters. There are only a few complete E. cloacae genome sequences and very little knowledge about the mechanism of pathogenesis of E. cloacae on plants and humans. Enterobacter cloacae EcWSU1 causes Enterobacter bulb decay in stored onions (Allium cepa). The EcWSU1 genome consists of a 4,734,438 bp chromosome and a mega-plasmid of 63,653 bp. The chromosome has 4,632 protein coding regions, 83 tRNA sequences, and 8 rRNA operons.

First Report of Enterobacter cloacae Causing Onion Bulb Rot in the Columbia Basin of Washington State

In August of 2006, onion plants of cv. Redwing exhibiting premature dieback and bulb rot were obtained from a commercial onion crop under center pivot irrigation in the Columbia Basin of Washington State. High temperatures during the summer were similar to those in 2004, which preceded significant outbreaks of Enterobacter rot of onion bulbs in storage. Fungal pathogens of onion were not observed. Bacteria from infected bulb tissue were isolated and purified on nutrient broth yeast extract (NBY) agar, and 537 isolates were evaluated for the ability to ferment glucose anaerobically. Of the facultative anaerobes (~50% of all isolates), 48 isolates were arginine dihydrolase positive, indole negative, and unable to degrade pectin, i.e., characteristics typical of the genus Enterobacter (2), which includes Enterobacter cloacae, a bacterial pathogen reported to cause onion bulb rot in California and Colorado (1,3). Sixteen of the putative Enterobacter isolates, along with four strains of E. cloacae known to be pathogenic on onion (1) (ATCC 23355 and ATCC 13047, 310 (H. F. Schwartz, Colorado State University), and E6 (J. Loper, USDA ARS), were tested for pathogenicity on onion bulbs (8 to 10 cm in diameter; cv. Tamara). The isolates were grown overnight in NBY broth at 28°C, harvested by centrifugation and resuspended to an OD600 = 0.3 (~108 CFU/ml) in sterile distilled water. After the outermost fleshy scale of each bulb was removed, each bulb was surface disinfected in 0.6% NaOCl for 2 min, dipped in sterile distilled water, and then dipped in 95% ethanol. Each bulb was air dried before a 0.5-ml aliquot of bacterial suspension was injected into the shoulder of the bulb with a 20-gauge needle. Three bulbs were inoculated for each isolate, placed in individual plastic bags, sealed, and incubated at 30°C in the dark. Three bulbs injected with water and three noninjected bulbs served as controls. After 14 days, each bulb was sliced through the center and rated for rot. Thirteen isolates induced rot symptoms on the inner fleshy scales of all inoculated bulbs. Of these, seven also caused tan-to-brown discoloration of the inner fleshy scales; similar symptoms were caused by the four pathogenic reference strains of E. cloacae (1). No symptoms were observed in any of the controls. Symptoms were not observed when the bacteria, prepared as described above, were infiltrated into onion leaves. Bacteria were reisolated from the symptomatic inoculated bulb tissue and confirmed to be Enterobacter spp. by the above physiological tests. In addition, an isolate designated ECWSU2 and the corresponding strain recovered from one of the inoculated symptomatic bulbs, along with the four reference strains, were evaluated for anaerobic growth on a variety of carbon sources by using API 50 CHE test strips (bio Mérieux Vitek, Inc., Hazlewood, MO). The physiological test data along with sequence analysis of a portion of the 16S rRNA gene of each isolate confirmed all of these isolates to be E. cloacae (4; Ribosomal Database Project [ http://rdp.cme.msu.edu/ ]). To our knowledge, this is the first report of E. cloacae causing a bulb rot of onion in Washington State. References: (1) A. L. Bishop and R. M. Davis. Plant Dis. 74:692, 1990. (2) J. G. Holt et al. Bergeys Manual of Determinative Bacteriology. Williams and Wilkins, Baltimore, MD, 1994. (3) H. F. Schwartz and K. Otto. Plant Dis. 84:808, 2000. (4) L. Verdonck et al. Int. J. Syst. Bacteriol. 37:4, 1987.

Effects of Postharvest Onion Curing Parameters on Enterobacter Bulb Decay in Storage

Enterobacter bulb decay is a recently described storage disease of onion (Allium cepa) bulbs caused by Enterobacter cloacae. The disease is generally considered minor but, on occasion, can cause significant losses for onion producers. The impact of postharvest curing temperature and duration on Enterobacter bulb decay of onion was evaluated by inoculating bulbs of the cultivars Redwing and Vaquero with E. cloacae after harvest, curing the bulbs at 25, 30, 35, or 40°C for 2 or 14 days, and storing the bulbs at 5°C for 1, 2, or 3 months. Noninoculated bulbs and bulbs injected with sterile water served as control treatments. The trial was completed using bulbs harvested from commercial onion crops grown in the semi-arid Columbia Basin of central Washington in each of 2008-09 (center-pivot irrigated crop) and 2009-10 (drip irrigated crop). Severity of bulb rot was assessed by cutting each bulb down the center from the neck to the basal plate, and rating the percentage of cut surface area with bacterial rot symptoms. Bulb rot severity was negligible for noninoculated bulbs (mean of 0.3% in the 2008-09 storage trial and 1.0% in the 2009-10 storage trial) and bulbs injected with water (0.8% in the 2008-09 trial and 1.3% in the 2009-10 trial) compared to bulbs inoculated with E. cloacae (15.3% in 2008-09 and 23.3% in 2009-10). Severity of Enterobacter bulb decay was affected significantly (P < 0.05) by season (trial), cultivar, curing temperature, curing duration, and storage duration, with significant interactions among these factors. Enterobacter bulb decay was significantly more severe for bulbs cured at 40°C than for bulbs cured at 25, 30, or 35°C. This effect was even greater when bulbs were cured for 14 days versus 2 days prior to cold storage, and in bulbs stored for 2 or 3 months after curing compared to bulbs stored for 1 month. The increase in bulb rot severity caused by curing bulbs at 40°C for 14 days compared to lower temperatures and shorter durations was greater for Vaquero than Redwing, particularly in the 2008-09 trial. The results suggest that curing temperatures ≤35°C should significantly reduce the risk of Enterobacter bulb decay in storage for these cultivars. If higher curing temperatures are used in order to dry onion necks for long-term storage and reduce the risk of fungal diseases such as neck rot (caused by Botrytis spp.), a shorter curing duration may be necessary to minimize the risk of Enterobacter bulb decay in storage.

Evaluation of verticillium wilt resistance in Mentha arvensis and M. longifolia genotypes.

Verticillium wilt, caused by Verticillium dahliae, is a major constraint to mint (Mentha spp.) production in the United States, and the use of resistant cultivars is an important component of Verticillium wilt management. Two Mentha arvensis and four M. longifolia genotypes were evaluated for resistance to Verticillium wilt in the greenhouse using V. dahliae isolates obtained from different hosts and belonging to different vegetative compatibility groups. Isolates of V. dahliae obtained from peppermint (M. × piperita) caused significantly higher disease severity, plant mortality, and yield reduction than isolates obtained from other hosts. Disease severity, plant mortality, and pathogen incidence in aboveground stems were higher and yields lower in peppermint, the susceptible standard, compared with the resistant standard, native spearmint (M. spicata). Root-dip inoculations of M. arvensis and M. longifolia with isolates of V. dahliae obtained from peppermint produced severe symptoms; however, both species displayed the ability to recover from infection and produce asymptomatic growth from rhizomes. Both M. arvensis cultivars exhibited lower mean disease severity ratings following cutback and regrowth and were not significantly different than native spearmint. The restriction of pathogen movement in aboveground tissue and ability to recover from infection may be important components of V. dahliae resistance in perennial mint cropping systems.

Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome

ABSTRACT The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.

First report of pink seed of pea caused by Erwinia rhapontici in the United States.

In March 2001, the USDA Federal Grain Inspection Service sent for analysis to USDA-ARS, Pullman, WA, 12 discolored seeds of field pea (Pisum sativum L.) from northeastern Montana. Symptoms consisted of pale pinkish brown-to-bright pink discoloration throughout the seed coat. Unlike the pink coloration resulting from application of pink pesticidal seed treatments, the coloration was permanent and could not be removed by extended washing. Ten discolored seeds were disinfested in 0.5% NaOCl for 1 to 2 min and rinsed in sterile distilled water. Five seeds were placed on malt extract agar amended with streptomycin sulfate and tetracycline hydrochloride at 50 mg per liter each, and five seeds were placed on nutrient broth yeast extract agar (NBY) (3) and incubated under ambient lab conditions. No organisms were isolated from peas on the antibiotic agar, but pale pink bacterial colonies were recovered from each of the five peas on NBY. The pink colonies were streaked for selection of single colonies, stored at 4°C, and revived for growth in shaken (120 rpm) NBY broth to optical density (OD) = 0.1 at 640 nm (≈108 CFU/ml). Three pea plants and one control plant were grown in the greenhouse until pods were sufficiently developed to be syringe-inoculated at the suture with 100 μl of bacterial culture. The experiment was repeated using a second bacterial isolate. In each experiment, pods on inoculated plants, but not the control plant, exhibited pale pinkish areas and later produced seeds discolored in shades of pink matching those from the original sample. Some symptomatic seeds were shriveled or aborted as well as discolored. Discolored seeds from the two experiments were plated to NBY and pale pinkish colonies were recovered as before, but no such colonies were recovered from the controls. Two isolates from the original sample and two recovered from the experimental inoculations were tested for anaerobic and aerobic growth using API 50 CHE and API 20 NE tests (bio Mérieux Vitek, Inc., Hazlewood, MO). Known strains of Erwinia rhapontici and E. rubrifaciens from the collection of Dennis Gross (Texas A&M University) were identically analyzed for comparison. All four isolates from pea were gram-negative, facultatively anaerobic, motile rod-shaped bacteria that produced a diffusible pink pigment on NBY. Tests indicated that these strains were E. rhapontici, because the results agreed with previously published data (1,2). Results with the known strain of E. rhapontici were congruent with those from the pea strains. The strain of E. rubrifaciens also produced a pink pigment on NBY, but unlike the other strains, it grew well at 37°C and was negative for acetoin production, nitrate reduction, esculin hydrolysis, and maltose, rhamnose, inositol, and melibiose fermentation and did not assimilate citrate aerobically. Pink seed of pea has been previously reported from Alberta, Canada (2). References: (1) J. G. Holt et al. Bergeys Manual of Determinative Bacteriology, Williams & Wilkins, Baltimore, MD, 1994. (2) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (3) A. K. Vidaver. Appl. Microbiol. 15:1523, 1967.