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Abstract: A densely compacted soil layer has been a prevalent feature of the vegetable crop production region of Central Wisconsin, resulting in poor water drainage, increased susceptibility to tuber disease, and reduced yield and quality in potatoes. Subsoil tillage has been implemented on a wide-scale for management of problems associated with compacted soil layers. Pink eye is a tuber disorder that causes storage and processing losses. Periods of moisture stress and increased hill temperatures have been linked to pink eye incidence, but the cause of this disorder is unknown and few specific management recommendations exist. The goals of this work were to quantify the impact of compacted soils and subsoil tillage on development of potato pink eye. Small plot experiments were conducted to quantify response of soil conditions and potato pink eye to soil compaction and subsoil tillage. Compacted soils resulted in higher volumetric soil water content and extended periods of free water, especially after substantial precipitation events. Subsoil tillage allowed for more rapid movement of water following large precipitation events decreasing periods of free water. More compacted soils also had higher soil temperatures. Subsoil tillage delayed the development and progression of pink eye symptoms, but did not influence overall incidence. Increased soil moisture and temperature corresponded with more rapid development of pink eye in compacted and conventionally tilled soils. This research suggests subsoil tillage may be useful in reducing the severity of pink eye symptoms by alleviating compacted subsoil layers that limit water drainage. In addition, prevention of compaction reduced soil temperatures which limited pink eye development.

In response to increasing interest in diploid potato (Solanum tuberosum) breeding and the production of diploid inbred hybrid potato varieties, the Breeding and Genetics section of the Potato Association of America (PAA) organized a symposium on diploid breeding that took place during the 2021 PAA annual meeting. Proceedings from that symposium are documented in this manuscript. Speakers from academia, government and industry presented their unique perspectives. Presentations covered a wide range of topics. Potential advantages of diploid breeding were introduced, and reasons to be skeptical about diploid breeding were highlighted. The impact that diploid breeding might have on the potato seed industry was discussed. Advantages for genetics research were emphasized. Aspects of tomato breeding and production were reviewed and considered as potential models for diploid potato breeding and production activities. Lastly, an industry-centered view of diploid potato breeding was provided. Taken together, these presentations are a snapshot of how diploid potato breeding was viewed in the moment, a vision for how diploid breeding might be implemented, and a thoughtful reflection on how diploid breeding and inbred hybrid varieties might change the potato variety development process and impact the potato industry.

Soft rot bacteria are the main cause of early seed decay and stand losses in field grown potatoes. These bacteria are present in most surface water and colonize lenticels of early generation seed potatoes and cause disease when conditions are favorable for bacterial growth. Soft rot bacteria, formerly in the genus Erwinia, have been reclassified into the genera Dickeyaand Pectobacterium.Because of their ubiquitous association with potato tubers, it is assumed that the soft rot bacteria is spread during handling and planting of seed potatoes and can potentially cause seed decay and blackleg. A soft rot bacterium new to the United States, Dickeya dianthicolawas first recognized in 2015 (Secor, unpublished) as the cause of serious stand losses of potatoes in production sites in the eastern US. Replicated and commercial field trials using seed laboratory inoculated and naturally infected with D. dianthicolawere conducted in multiple locations to determine spread from infected seed potatoes to healthy seed potatoes during handling and cutting. There is no evidence of increased stand losses or blackleg after simultaneous handling and cutting of Dickeya infected seed potatoes with seed potatoes free of Dickeya. Laboratory trials with labeled soft rot bacteria demonstrated spread of bacteria by cutting knives. There may be other factors that prevent establishment and infection by soft rot bacteria that may spread during seed potato cutting that need to be investigated. We conclude that spread of Dickeyabacteria during handling and cutting of seed potatoes is not a major cause of subsequent seed decay and blackleg resulting in increased stand losses in the field.

Salicylic acid (SA) is a hormone that mediates systemic acquired resistance in plants. We demonstrated that SA can interfere with group behavior and virulence of the soft-rot plant pathogen Pectobacteriumspp. through quorum sensing (QS) inhibition. QS is a population density-dependent communication system that relies on the signal molecule acyl-homoserine lactone (AHL) to synchronize infection. P. parmentierimutants, lacking the QS AHL synthase (expI–) or the response regulator (expR–), were used to determine how SA inhibits QS. ExpI was expressed in DH5α, the QS negative strain of Escherichia coli, revealing direct interference of SA with AHL synthesis. Docking simulations showed SA is a potential ExpI ligand. This hypothesis was further confirmed by direct binding of SA to purified ExpI, shown by isothermal titration calorimetry and microscale thermophoresis. Computational alanine scanning was employed to design a mutant ExpI with predicted weaker binding affinity to SA. The mutant was constructed and displayed lower affinity to the ligand in the binding assay, and its physiological inhibition by SA was reduced. Taken together, these data support a likely mode of action and a role for SA as potent inhibitor of AHL synthase and QS.

Potato virus Y(PVY) causes one of the most serious and widespread diseases in North America. In recent years, the virus has become increasingly difficult to control. Durable dominant genes for resistance to PVY exist in potato germplasm and provide an effective control strategy. This paper describes a Solanum chacoenseclone (M19) that is homozygous for a PVY resistance gene. The gene is linked to a previously published marker for Rychcfound in Solanum chacoense. M19 was crossed with a diploid S. tuberosumclone to produce an adapted clone carrying the resistance gene. This hybrid clone is named M20. M20 tuberizes in the field, producing round tubers with white skin and flesh and moderate size. M20 is resistant to PVYO, PVYN:O, and PVYNTN. Both M19 and M20 are female and male fertile, so they are being released as sources of PVY resistance for breeding programs. El Virus Y de la Papa(PVY) causa una de las enfermedades más serias y de amplia distribución en Norteamérica. En años recientes, el virus ha aumentado la dificultad para su control. Los genes durables dominantes para resistencia al PVY existen en el germoplasma de papa y proporcionan una estrategia de control efectivo. Este artículo describe un clon (M19) de Solanum chacoenseque es homozigótico para un gen de resistencia al PVY. El gen esta ligado a un marcador previamente publicado para Rychcencontrado en Solanum chacoense. Se cruzó M19 con un clon diploide de S. tuberosumpara producir un clon adaptado llevando el gen de resistencia. A este clon híbrido se le llama M20. Este clon tuberiza en el campo, produciendo tubérculos redondos con piel y pulpa blancas, de tamaño moderado. M20 es resistente al PVYO, PVYN:Oy PVYNTN. Ambos M19 y M20 son fértiles como hembra y macho, de manera que están siendo liberados como fuentes de Resistencia al PVY para los programas de mejoramiento.

Potato virus Y(PVY) is the most important virus in North American seed potato (Solanum tuberosumL.) production. Planting virus-free minitubers in place of field-grown seed, which usually has a low PVY incidence, reduces initial PVY inoculum in the field. However, plants grown from minitubers are smaller and emerge later than those grown from conventional seed, which could make them more likely to become infected with PVY. We tested the effects of seed type of three potato cultivars (Dark Red Norland, Goldrush, and Red La Soda) on PVY incidence, tuber yield, and flowering time. The incidence of PVY in plants grown from minitubers did not differ from that of plants grown from conventional seed. Minituber-grown plants produced lower tuber yields than plants grown from conventional seed. Plants from minitubers also emerged and flowered later, but this did not increase their incidence of PVY. Cultivar-specific differences were observed in tuber yield and flowering times, suggesting that this variation may influence PVY incidence more than seed type. El virus Y de la papa (PVY) es el más importante en la producción de semilla de papa (Solanum tuberosumL.) en Norteamérica. La siembra de minitubérculos libres de virus en lugar de la semilla obtenida del campo, que generalmente tiene una baja incidencia de PVY, reduce el inóculo inicial de PVY en el campo. No obstante, las plantas a partir de minitubérculos son más pequeñas y emergen más tarde que aquellas cultivadas de semilla convencional, que las pudiera hacer más propensas a infectarse con PVY. Nosotros probamos los efectos del tipo de semilla de tres variedades de papa (Dark Red Norland, Goldrush, y Red La Soda) sobre la incidencia de PVY, rendimiento de tubérculo y tiempo de floración. La incidencia de PVY en las plantas cultivadas a partir de minitubérculos no difirió de las cultivadas de semilla convencional. Las plantas de minitubérculos produjeron rendimientos de tubérculo más bajos que las de semilla convencional. Las plantas de minitubérculos también emergieron y florecieron más tarde, pero esto no aumentó su incidencia de PVY. Las diferencias específicas por variedad se observaron en rendimiento de tubérculo y tiempos de floración, lo que sugiere que esta variación pudiera influenciar la incidencia de PVY más que el tipo de semilla.

AbstractLate and early maturing potato cultivars were grown under two organic management systems using straw mulch or mechanical cultivation for weed management. It was found that application of straw mulch at emergence consistently increased total and marketable A-size yields for the late season cultivar Freedom Russet, and shifted the profile of marketable A-size tubers toward larger sizes. Increased yields were seen for the early season cultivar Dark Red Norland in some years, and may be related to amelioration of environmental stress by straw mulch. Mulch provided more effective control than mechanical cultivation for some broadleaf weeds, especially late in the season as potato foliage senesced and weed numbers tended to increase in unmulched plots relative to mulched plots. Grass weed incidence did not differ between straw mulched and mechanically weeded plots. Mulch did not consistently impact abundance of pest, virus vector, or predatory insects, although potato leafhopper numbers tended to be lower in straw mulched plots than mechanically cultivated plots. Straw mulching did not impact PVY incidence. It was concluded that straw mulching is a viable management option for organic potato production, with potential benefits for broadleaf weed management and tuber yield.

Potato common scab is a widespread disease in which scab-like lesions develop on tubers. The disease is caused by pathogenic Streptomycesspecies, which synthesize the phytotoxin thaxtomin. The txtABoperon, responsible for thaxtomin production, can be used as a marker to identify pathogenic strains of the bacterium. Screening methods to assess scab susceptibility in breeding programs are time-consuming and can produce variable results. Management practices to control the disease vary and include crop rotation, tolerant varieties, monitoring soil pH, avoiding low soil moisture at tuber initiation, and application of soil- and/or seed-applied pesticides. There is a wide range in levels of tolerance among potato varieties. Many public research programs are committed to breeding for scab-tolerant varieties and evaluating management methods. Topics reviewed target readers focused on breeding and disease management objectives to reduce the incidence and severity of potato common scab. La roña común de la papa es una enfermedad ampliamente distribuida, que desarrolla lesiones tipo pústulas en los tubérculos. La enfermedad es causada por especies patogénicas de Streptomyces, que sintetizan la fitotoxina thaxtomin. El operón txtAB, responsable de la producción de thaxtomin, puede usarse como marcador para identificar variantes patogénicas de la bacteria. Los métodos de análisis para evaluar la susceptibilidad a la roña en los programas de mejoramiento se llevan mucho tiempo y pueden producir resultados variables. Las prácticas de manejo para controlar la enfermedad varían, e incluyen rotación de cultivos, variedades tolerantes, monitoreo del pH del suelo, evitando baja humedad en el suelo al principio de la tuberización, y la aplicación de plaguicidas al suelo y/o a la semilla. Existe una gran amplitud de niveles de tolerancia entre las variedades de papa. Muchos programas públicos de investigación están comprometidos al mejoramiento para variedades tolerantes a la roña y en la evaluación de métodos de manejo. Los tópicos revisados conducen a lectores enfocados en objetivos de mejoramiento y manejo de la enfermedad para reducir la incidencia y la severidad de la roña común de la papa.

The third most important food crop worldwide, potato (Solanum tuberosumL.) is a tetraploid outcrossing species propagated from tubers. Breeders have long been challenged by polyploidy, heterozygosity, and asexual reproduction. It has been assumed that tetraploidy is essential for high yield, that the creation of inbred potato is not feasible, and that propagation by seed tubers is ideal. In this paper, we question those assumptions and propose to convert potato into a diploid inbred line–based crop propagated by true seed. Although a conversion of this magnitude is unprecedented, the possible genetic gains from a breeding system based on inbred lines and the seed production benefits from a sexual propagation system are too large to ignore. We call on leaders of public and private organizations to come together to explore the feasibility of this radical and exciting new strategy in potato breeding.

AbstractA new leaf spot disease was observed on leaves of Rheum palmatum(Chinese rhubarb) in Northwest China (Gansu Province) starting in 2005. A Septoria-like fungus was isolated and completion of Koch’s postulates confirmed that the fungus was the casual agent of the leaf spot disease. Morphology and molecular methods were combined to identify the pathogen. The fungus produced conidiomata pycnidia and the conidia were 2~5 septate, 61.2~134.1 μm in length and 3.53~5.3 μm in width, which is much larger than the known Spetoriaspecies that infects Polygonaceae species. Phylogenic analysis of the internal transcribed spacer region confirmed that this Septoria-like fungus is within the Septoriagenus but distinct from known Septoriaspecies. Together, these morphological and phylogenetic data support that the R. palmatuminfecting Septoriastrain is a newlydescribed plant pathogenic species.

Potato virus Y(PVY) is responsible for the majority of seed potato lot rejections in North America. Some commercial cultivars and breeding lines have major genes for PVY resistance, but their use in North American breeding programs has been limited. Marker assisted selection of PVY resistance can increase the efficiency of identifying resistant plants and integrating resistance into new cultivars. We used markers RYSC3 and YES3-3B, linked to genes Ryadgand Rysto, respectively, to screen 46 breeding clones and cultivars, and identified 19 resistant clones. Resistant parents were crossed with susceptible parents with good market and agronomic traits and molecular marker analysis of each family showed a 1:1 segregation ratio. The persistence of this segregation ratio over 2 years of selection and the lack of major linkage drag as assessed by a breeder rating indicated that there was no evidence of an association of PVY resistance with undesirable traits. El virus Y de la papa (PVY) es responsable por la mayoría de los rechazos de los lotes de papa para semilla en Norteamérica. Algunas variedades comerciales y líneas de mejoramiento tienen genes mayores para resistencia al PVY, pero es limitado su uso en programas de mejoramiento de Norteamérica. La selección asistida con marcadores de resistencia al PVY puede aumentar la eficiencia en la identificación de plantas resistentes y en la integración de la resistencia a nuevas variedades. Nosotros usamos los marcadores RYSC3 y YES3-3B, ligados a los genes Ryadg y Rysto, respectivamente, para evaluar 46 clones y variedades, e identificamos 19 clones resistentes. Se cruzaron padres resistentes con susceptibles de buenas características agronómicas y de mercado, y el análisis de marcadores moleculares de cada familia mostró una relación de segregación de 1:1. La persistencia de esta relación de segregación en dos años de selección, y la carencia de ligamiento mayor de arrastre evaluada por calificación de un fitomejorador, indicaron que no hubo evidencia de una asociación de la resistencia del PVY con caracteres no deseables.

Pectobacteriumspecies cause soft rot, blackleg, and stem rot in potato and a wide range of other vegetable crops and ornamental plants. Diseases caused by Pectobacteriumare controlled mainly through use of healthy planting material, sanitation and copper sprays. Environmental factors, such as temperature, moisture and soil oxygen concentration, have a large effect on development of diseases caused by Pectobacteriumand disease incidence can be unpredictable. The pathogen is spread by various mechanisms including water, seed, equipment and insects. Little is understood about plant resistance to soft rot bacterial pathogens and no commercial potato cultivars are resistant to soft rot, although some have tolerance and some wild potato species are resistant. Pectobacteriumis a diverse genus, with multiple species capable of infecting potato. Multiple Pectobacteriumspecies may be found in the same field and even on the same plant. Pectobacteriumstrains vary in aggressiveness and the virulence genes they encode but there are many commonalities across the genus. Over the past decade, genomic studies have provided new insights into Pectobacteriumbiology. For example, some Pectobacteriumstrains may elicit plant cell death to promote disease in leaves. Strains of the pathogen also produce an orange pigment and volatile compounds that increase virulence and that may act as insect kairomones. Recent work with a supervised machine learning program has identified several novel target genes likely to contribute to plant-microbe interactions, suggesting that there is still much to learn about how soft rot bacteria cause disease. Las especies de Pectobacteriumcausan pudriciones blandas, pierna negra y pudrición del tallo en papa y en una gran amplitud de otros cultivos hortícolas y plantas ornamentales. Las enfermedades causadas por Pectobacteriumse controlan principalmente mediante el uso de material de siembra sano, prácticas sanitarias y aspersiones de cobre. Los factores ambientales tales como la temperatura, la humedad y la concentración del oxígeno en el suelo, tienen un gran efecto en el desarrollo de enfermedades causadas por Pectobacteriumy la incidencia de la enfermedad puede ser impredecible. El patógeno se dispersa por varios mecanismos incluyendo agua, semilla, equipo e insectos. Se ha entendido poco sobre la resistencia de la planta a los patógenos bacterianos de pudrición blanda, y no hay variedades comerciales de papa que sean resistentes a la pudrición blanda, aunque algunas tienen tolerancia y algunas especies de papa silvestre son resistentes. El género Pectobacteriumes diverso, con múltiples especies capaces de infectar a la papa. Se pueden encontrar múltiples especies de Pectrobacteriumen el mismo campo y aun en la misma planta. Las cepas de Pectobacteriumvarían en su agresividad y en los genes de virulencia que codifican, pero hay muchas cosas en común en todo el género. En la década pasada, estudios genómicos han proporcionado nueva información de la biología de Pectobacterium. Por ejemplo, algunas variantes de este grupo de patógenos pueden inducir muerte de la célula vegetal para promover la enfermedad en hojas. Las cepas de este patógeno también inducen un pigmento color naranja y compuestos volátiles que aumentan la virulencia y que pudieran actuar como kairomonas de insectos. El trabajo reciente con una máquina supervisada con un programa de aprendizaje ha identificado varios genes nuevos como objetivos con probabilidades de contribuir en las interacciones planta-microbio, sugiriendo que aún hay mucho por aprender acerca de cómo las bacterias de pudrición suave causan enfermedad.

Helminthosporium solaniis a slow-growing fungal pathogen belonging to the family Massarinaceae. It causes blemishes on potato tubers, affecting processing and fresh market trade. Despite its worldwide distribution, little is known about the biology of H. solani. Here we report the generation of a draft genome sequence of H. solaniwith an estimated genome size of ~35 megabases, for use in phylogenetic and pathogenicity studies of this fungal pathogen. This sequence is also the first reference genome within the family Massarinaceae. We identified a large suite of genes in the H. solanigenome that encode putative cell wall degrading enzymes. Based on comparison with other genomes, we speculate that H. solaniis a hemibiotroph or necrotroph. The presence of a large number of genes in the glycoside hydrolase (GH) 10 and 43 families, which aid in the hydrolysis of glucoronoarabinoxylan, also suggests that H. solanimay be able to survive on grass hosts and indicates the need to re-examine the life cycle and host range of this pathogen. Helminthosporium solani, es un hongo patógeno de lento crecimiento que pertenece a la familia Massarinaceae. Causa manchas en los tubérculos de papa, afectando el mercado en fresco y de procesamiento. A pesar de su distribución mundial, se sabe poco de la biología de H. solani. Aquí reportamos la generación de una secuencia preliminar del genomio de H. solani, con un tamaño genómico estimado de ~35 megabases, para uso en estudios filogenéticos y de patogenicidad de este patógeno fúngico. Esta secuencia también es la primera referencia genómica dentro de la familia Massarinaceae. Identificamos un gran conjunto de genes en el genómio de H. solanique codifican supuestamente para enzimas degradadoras de pared celular. Con base en la comparación con otros genomios, especulamos que H. solanies un hemibiótrofo o necrótrofo. La presencia de un gran número de genes en las familias 10 y 43 de glicósido hidrolasa (GH), que ayudan en la hidrólisis de glucoronoarabinoxilano, también sugiere que H. solanipudiera ser capaz de sobrevivir en pastos hospedantes o en desechos, e indica la necesidad de reexaminar el ciclo biológico y la amplitud de hospederos de este patógeno.

Silver scurf, caused by Helminthosporium solani,and black dot, caused by Colletotrichum coccodes,cause tuber blemishes on potato (Solanum tuberosumL.) which affect processing and fresh market trade. Tubers from ten cultivars were collected at harvest from three organic farms in Wisconsin and categorized as symptomatic or asymptomatic based on visual symptoms of silver scurf and black dot and/or signs of H. solaniand C. coccodes. Tuber incubation and PCR assays were performed on asymptomatic tubers to detect H. solaniand C. coccodes. Tuber incubation and PCR assays were in slight to fair agreement (kappa coefficient <0.4) for detecting both pathogens. Most asymptomatic tubers tested were positive by one or both assays for H. solani(75 %) or C. coccodes(94 %). Minituber inoculation assays were also performed to screen potato lines for resistance to silver scurf. Of the 14 lines tested, a diploid interspecific hybrid, C287, had consistently low sporulation, suggesting it has partial resistance to silver scurf. Since the majority of tubers harvested are already infected with one or both pathogens further research should focus on organically acceptable management practices that may inhibit disease development in field and in storage.

Vegetative propagation of potato leads to virus accumulation, resulting in significant yield losses and reduced quality. Virus identification is critical for developing disease management strategies and measuring seed lot health. The most widely used method of virus diagnosis in seed potatoes is a post-harvest test, for which the enzyme-linked immunosorbent assay (ELISA) is often used. ELISA was previously modified by substituting microtiter plates with membranes to develop a more flexible and inexpensive assay. We optimized a dot-blot immunoassay with viral proteins bound to a polyvinylidene fluoride (PVDF) membrane and detection of the proteins with alkaline phosphatase-labeled antibodies and a chemiluminescence reagent. The assay was tested for detection of viruses of seven genera. We have also altered the assay by spotting an antibody array onto a PVDF membrane and tested it for its potential uses as a diagnostic tool for Potato virus Y, Hosta virus X, and Potato leafroll virus.

Abstract: A densely compacted soil layer has been a prevalent feature of the vegetable crop production region of Central Wisconsin, resulting in poor water drainage, increased susceptibility to tuber disease, and reduced yield and quality in potatoes. Subsoil tillage has been implemented on a wide-scale for management of problems associated with compacted soil layers. Pink eye is a tuber disorder that causes storage and processing losses. Periods of moisture stress and increased hill temperatures have been linked to pink eye incidence, but the cause of this disorder is unknown and few specific management recommendations exist. The goals of this work were to quantify the impact of compacted soils and subsoil tillage on development of potato pink eye. Small plot experiments were conducted to quantify response of soil conditions and potato pink eye to soil compaction and subsoil tillage. Compacted soils resulted in higher volumetric soil water content and extended periods of free water, especially after substantial precipitation events. Subsoil tillage allowed for more rapid movement of water following large precipitation events decreasing periods of free water. More compacted soils also had higher soil temperatures. Subsoil tillage delayed the development and progression of pink eye symptoms, but did not influence overall incidence. Increased soil moisture and temperature corresponded with more rapid development of pink eye in compacted and conventionally tilled soils. This research suggests subsoil tillage may be useful in reducing the severity of pink eye symptoms by alleviating compacted subsoil layers that limit water drainage. In addition, prevention of compaction reduced soil temperatures which limited pink eye development.

Abstract: Potato plantlets initiated into tissue culture must be tested for numerous viruses prior to propagation for seed potato production. Ideally, one plantlet is tested for all pathogens of concern and, if found pathogen-free, this plantlet is propagated for production of seed potatoes. Commercially available ELISA kits are generally used for the pathogen tests, but the commercial kits have some limitations. For example, the protocols differ for different viruses, so multiple extractions must be completed, increasing the time and expense of testing. This is a significant problem with tissue culture plantlets, for which there is limited material available to test and an ever-increasing number of pathogens that must be tested for, including viruses in the potyvirus, carlavirus, potexvirus, luteovirus, pomovirus, tobravirus, tospovirus, alfamovirus, and tymovirus groups. We have optimized a non-radioactive nucleic acid hybridization (NASH) assay for the simultaneous detection of carlavirusPotato virus S (PVS), potexvirusPotato virus X (PVX) and potyvirusPotato virus Y (PVY) in potato tissue culture plantlets. This assay requires a single extraction from a small portion of a tissue culture plantlet for the detection of viruses from three different families.

Contamination of produce by bacterial pathogens is an increasingly recognized problem. In March 1999, 72 patrons of a Nebraska restaurant were infected with enterohemorrhagic Escherichia coli(EHEC) O157:H7, and shredded iceberg lettuce was implicated as the food source. We simulated the restaurant's lettuce preparation procedure to determine the extent of possible EHEC cross-contamination and growth during handling. EHEC inoculation experiments were conducted to simulate the restaurant's cutting procedure and the subsequent storage of shredded lettuce in water in the refrigerator. All lettuce pieces were contaminated after 24 h of storage in inoculated water (2 × 109CFU of EHEC per 3 liters of water) at room temperature or at 4°C; EHEC levels associated with lettuce increased by >1.5 logs on the second day of storage at 4°C. All lettuce pieces were contaminated after 24 h of storage in water containing one inoculated lettuce piece (~105CFU of EHEC per lettuce piece) at both temperatures. The mixing of one inoculated dry lettuce piece with a large volume of dry lettuce, followed by storage at 4°C or 25°C for 20 h resulted in 100% contamination of the leaves tested. Microcolonies were observed on lettuce stored at 25°C, while only single cells were seen on leaves stored at 4°C, suggesting that bacterial growth had occurred at room temperature. Three water washes did not significantly decrease the number of contaminated leaves. Washing with 2,000 mg of calcium hypochlorite per liter significantly reduced the number of contaminated pieces but did not eliminate contamination on large numbers of leaves. Temperature abuse during storage at 25°C for 20 h decreased the effectiveness of the calcium hypochlorite treatment, most likely because of bacterial growth during the storage period. These data indicate that storage of cut lettuce in water is not advisable and that strict attention must be paid to temperature control during the storage of cut lettuce.

At least 14 separate outbreaks of food poisoning attributed to either Salmonella entericaor Escherichia coliO157:H7 have been traced to sprouts in the past decade. Seeds contaminated with human pathogens caused most of these outbreaks, thus many sprout growers are now treating alfalfa seeds with the sanitizing agent, calcium hypochlorite (Ca[OCl]2), prior to sprouting. The efficacy of alfalfa seed sanitation varies between seed lots and between seeds within each lot. Alfalfa seeds from different seed lots were sorted by type in an effort to determine if certain seed types carry more aerobic bacteria than other seed types. Seeds with a wrinkled type, characteristic of lygus bug damage, had significantly higher levels of culturable aerobic bacteria and were more difficult to sanitize than smooth, healthy seeds. After sanitation, wrinkled alfalfa seeds that had been inoculated with S. entericaser. Newport carried significantly higher levels of SalmonellaNewport than smooth seeds. If S. entericais present on wrinkled seeds in naturally contaminated seed lots, it may be difficult to chemically sanitize the seed lot. Removal of the wrinkled alfalfa seeds from the seed lots, perhaps by adapting color sorting equipment similar to that used to sort rice grains and other seeds, should reduce the level of aerobic bacteria in seed lots and may result in lower levels of human pathogens on contaminated alfalfa seeds.

ABSTRACTAlthough enteric human pathogens are usually studied in the context of their animal hosts, a significant portion of their life cycle occurs on plants. Plant disease alters the phyllosphere, leading to enhanced growth of human pathogens; however, the impact of human pathogens on phytopathogen biology and plant health is largely unknown. To characterize the interaction between human pathogens and phytobacterial pathogens in the phyllosphere, we examined the interactions between Pectobacterium carotovorumsubsp. carotovorumand Salmonella entericaor Escherichia coliO157:H7 with regard to bacterial populations, soft rot progression, and changes in local pH. The presence of P. carotovorumsubsp. carotovorumenhanced the growth of both S. entericaand E. coliO157:H7 on leaves. However, in a microaerophilic environment, S. entericareduced P. carotovorumsubsp. carotovorumpopulations and soft rot progression by moderating local environmental pH. Reduced soft rot was not due to S. entericaproteolytic activity. Limitations on P. carotovorumsubsp. carotovorumgrowth, disease progression, and pH elevation were not observed on leaves coinoculated with E. coliO157:H7 or when leaves were coinoculated with S. entericain an aerobic environment. S. entericaalso severely undermined the relationship between the phytobacterial population and disease progression of a P. carotovorumsubsp. carotovorum budBmutant defective in the 2,3-butanediol pathway for acid neutralization. Our results show that S. entericaand E. coliO157:H7 interact differently with the enteric phytobacterial pathogen P. carotovorumsubsp. carotovorum. S. entericainhibition of soft rot progression may conceal a rapidly growing human pathogen population. Whereas soft rotted produce can alert consumers to the possibility of food-borne pathogens, healthy-looking produce may entice consumption of contaminated vegetables.IMPORTANCESalmonella entericaand Escherichia coliO157:H7 may use plants to move between animal and human hosts. Their populations are higher on plants cocolonized with the common bacterial soft rot pathogen Pectobacterium carotovorumsubsp. carotovorum, turning edible plants into a risk factor for human disease. We inoculated leaves with P. carotovorumsubsp. carotovorumand S. entericaor E. coliO157:H7 to study the interactions between these bacteria. While P. carotovorumsubsp. carotovorumenhanced the growth of both S. entericaand E. coliO157:H7, these human pathogens affected P. carotovorumsubsp. carotovorumfundamentally differently. S. entericareduced P. carotovorumsubsp. carotovorumgrowth and acidified the environment, leading to less soft rot on leaves; E. coliO157:H7 had no such effects. As soft rot signals a food safety risk, the reduction of soft rot symptoms in the presence of S. entericamay lead consumers to eat healthy-looking but S. enterica-contaminated produce.

Salmonella has been associated with a number of recent outbreaks of foodborne illness involving sprouted seeds. In this research, we addressed the design of sensor components intended fop" use in a fully automated detection system. A real-time PCR assay was developed to detect Salmonella in sprout irrigation water. Specific detection was achieved by targeting a region of the invasion gene, invA. The detection limit for Salmonella in sterile water was approximately 400 CFU, and in sprout irrigation water the detection limit was approximately 200 CFU. Components for an automated sensing system were designed, including a thermal cycler and a .fluorescent optical sensor. The thermal cycler utilized a thermoelectric module for heating and cooling the sample block and a heat sink and fan to remove heat from the module during cooling. Sample temperature was controlled to within about 1°C at each of the PCR setpoints (94°C, 55°C, and 72°C). The optical sensor used a laser diode (635 nm) for excitation and a bandpass interference filter (700 ± 20 nm) coupled with a photodiode for fluorescence detection. The DNA dye TO-PRO-3 (642 nm excitation; 661 nm emission) was chosen to match the excitation wavelength of the laser diode. Calibration of the optical sensor with calf thymus DNA showed detection down to 0.01 µg mL-1, demonstrating the potential to detect down to 1 CFU if used in conjunction with PCR. When the sensor components were used to implement the real-time assay, S. Newport was detected to approximately 7.3 x 104 CFU in sterile water and approximately 1.5 x 106 CFU in sprout irrigation water. Further optimization of the assay in the sensor will be needed to improve the detection limits. [ABSTRACT FROM AUTHOR]