From fox to fork? Toxocara contamination of spinach grown in the south of England, UK.

BACKGROUND: Toxocara canis and Toxocara cati are intestinal parasites of dogs, cats and foxes, with infected animals shedding eggs of the parasite in their faeces. If humans accidentally ingest embryonated Toxocara spp. eggs from the environment, severe clinical consequences, including blindness and brain damage, can occur. Previous work has demonstrated the presence of Toxocara spp. eggs on vegetable produce grown in the UK, but only in small-scale community gardens. The aim of this study was to determine whether Toxocara spp. eggs are also present on vegetables grown on commercial farms in the UK, which supply produce to a greater number of people. METHODS: A total of 120 samples (300 g each) of spinach (Spinacia oleracea) were collected across four farms in the south of England, UK. The samples were processed using a sieving approach followed by multiplex quantitative polymerase chain reaction analysis. RESULTS: Overall, 23.0% of samples were positive for T. canis (28/120; 95% confidence interval 16.7-31.7%) and 1.7% for T. cati (2/120; 95% confidence interval 0.5-5.9%). There was a statistically significant difference in the number of positive samples between farms (P = 0.0064). To our knowledge, this is the first report of the isolation of Toxocara spp. from vegetables grown on commercial farms in the UK. CONCLUSIONS: The results of this study highlight the requirement for the thorough washing of vegetables prior to their consumption, especially those such as spinach which may be eaten without first peeling or cooking, and effective farm biosecurity measures to minimise access to farmland by definitive host species of Toxocara spp.

Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay.

Pathogen contamination of fresh produce presents a health risk for consumers; however, the produce industry still lacks adequate tools for simultaneous detection of protozoan parasites. Here, a simple multiplex PCR (mPCR) assay was developed for detection of protozoan (oo)cysts and compared with previously published real-time PCR assays and microscopy methods. The assay was evaluated for simultaneous detection of Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii followed by parasite differentiation via either a nested specific PCR or a restriction fragment length polymorphism (RFLP) assay. Spiking experiments using spinach as a model leafy green were performed for assay validation. Leaf-washing yielded higher recoveries and more consistent detection of parasites as compared with stomacher processing. Lowest limits of detection using the nested mPCR assay were 1-10 (oo)cysts/g spinach (in 10 g samples processed), and this method proved more sensitive than qPCR for parasite detection. Microscopy methods were more reliable for visual detection of parasites in lower spiking concentrations, but are more costly and laborious, require additional expertise, and lack molecular confirmation essential for accurate risk assessment. Overall, the nested mPCR assay provides a rapid (<24 h), inexpensive ($10 USD/sample), and simple approach for simultaneous detection of protozoan pathogens on fresh produce.

African Nightshade and African Spinach Decrease Root-Knot Nematode and Potato Cyst Nematode Soil Infestation in Kenya.

Plant-parasitic nematodes, particularly root-knot nematodes (RKN: Meloidogyne spp.) and cyst nematodes (CN: Globodera and Heterodera spp.) cause severe yield reduction in most cultivated crops and are of high economic importance. African nightshade (Solanum spp.) and African spinach (Amaranthus spp.) are important African indigenous vegetables (AIV) and are rich sources of nutrition and income. However, their host status to plant-parasitic nematodes remains largely speculative. Therefore, a survey was conducted which revealed that S. villosum exhibited high root galling, whereas on S. scabrum, A. cruentus, and A. dubius root galling was rare or very low. Additionally, soil collected from the rhizosphere of S. villosum and S. scabrum contained few cysts of potato cyst nematodes (PCN), and no developing PCN females were observed on the roots of growing plants. Therefore, we studied the dynamics of RKN and PCN on A. dubius, A. cruentus, S. scabrum, and S. villosum over 2 years in a field experiment. The effects of AIV crop species on RKN and PCN soil infestation were evaluated using susceptible S. lycopersicum or S. tuberosum. After first, second, and third cultivation of A. dubius, A. cruentus, and S. scabrum, RKN infestation of the soil decreased by more than 85%, whereas S. scabrum and S. villosum decreased PCN densities by more than 80%. When cropping susceptible crops, after three seasons of successive cultivation of these AIV, galling index and number of developing PCN females measured on susceptible crops decreased by more than 75%. Wilting and RKN-PCN coinfection incidences also decreased significantly. Here, we present data that support the development of a novel cropping system including African spinach and African nightshade, which reveals a high potential to manage RKN and PCN in an environmentally friendly, effective, and productive way.

Detection of Latent Peronospora effusa Infections in Spinach.

Downy mildew disease of spinach, caused by Peronospora effusa, is managed in conventional fields by a combination of host resistance and scheduled fungicide applications. Fungicides are currently applied to prevent downy mildew epidemics regardless of the infection status of spinach crops. A more streamlined approach would be to develop methods to target either latent infections for fungicide application in conventional production systems or to hasten harvest in organic production. In this study, conventional polymerase chain reaction (PCR) was applied to detect P. effusa DNA in symptomless spinach leaves in three spatially and temporally separated field plots, each containing four 2-m beds, 35 m in length. Spinach leaves were sampled weekly at 3-m intervals at 48 locations throughout each plot. Initial samples were asymptomatic and yet PCR enabled detection of P. effusa DNA extracted from sampled spinach leaves. Detection of latent downy mildew infection in spinach leaves was confirmed by PCR as early as 7 days prior to symptom development. The limit of pathogen DNA detection in spinach leaves was calculated at 10 pg using the conventional PCR approach. Quantitative PCR with TaqMan methodology revealed the presence of inhibitors from spinach leaf DNA extracts and affected amplification efficiencies, but not when diluted, enabling detection of P. effusa DNA at a concentration of <0.1 pg. In conclusion, detection of latent infections may enable management decisions for earlier-than-normal harvest of infected, symptomless organic crops, and for timing fungicide applications on symptomless plants in conventional production.

Identification of Key Root Volatiles Signaling Preference of Tomato over Spinach by the Root Knot Nematode Meloidogyne incognita.

The root knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood, is a serious pest of tomato ( Solanum lycopersicum) and spinach ( Spinacea oleracea) in sub-Saharan Africa. In East Africa these two crops are economically important and are commonly intercropped by smallholder farmers. The role of host plant volatiles in M. incognita interactions with these two commodities is currently unknown. Here, we investigate the olfactory basis of attraction of tomato and spinach roots by the infective second stage juveniles (J2s) of M. incognita. In olfactometer assays, J2s were attracted to root volatiles from both crops over moist sand (control), but in choice tests using the two host plants, volatiles of tomato roots were more attractive than those released by spinach. Root volatiles sampled by solid phase microextraction (SPME) fiber and analyzed by gas chromatography/mass spectrometry (GC/MS) identified a total of eight components, of which five (2-isopropyl-3-methoxypyrazine, 2-(methoxy)-3-(1-methylpropyl)pyrazine, tridecane, and α- and ß-cedrene) occurred in the root-emitted volatiles of both plants, with three (δ-3-carene, sabinene, and methyl salicylate) being specific to tomato root volatiles. In a series of bioassays, methyl salicylate contributed strongly to the attractiveness of tomato, whereas 2-isopropyl-3-methoxypyrazine and tridecane contributed to the attractiveness of spinach. M. incognita J2s were also more attracted to natural spinach root volatiles when methyl salicylate was combined than to spinach volatiles alone, indicating that the presence of methyl salicylate in tomato volatiles strongly contributes to its preference over spinach. Our results indicate that since both tomato and spinach roots are attractive to M. incognita, identifying cultivars of these two plant species that are chemically less attractive can be helpful in the management of root knot nematodes.

Genome Sequences of Three Races of Peronospora effusa: A Resource for Studying the Evolution of the Spinach Downy Mildew Pathogen.

Downy mildew disease, caused by the obligate oomycete pathogen Peronospora effusa, is the most important economic constraint for spinach production. Three races (races 12, 13, and 14) of P. effusa have been sequenced and assembled. The draft genomes of these three races have been deposited to GenBank and provide useful resources for dissecting the interaction between the host and the pathogen and may provide a framework for determining the mechanism by which new races of the pathogen are rapidly emerging.

Characterization of Foliar Web Blight of Spinach, Caused by Pythium aphanidermatum, in the Desert Southwest of the United States.

A unique foliar disease of spinach, determined to be caused by Pythium aphanidermatum, was observed on spinach in Yuma County, AZ and Imperial County, CA desert spinach production areas in both 2015 and 2016. The foliar symptoms of the disease included water-soaked foliage, rapid collapse of young plants, and white, aerial, cottony mycelia. The disease was associated with hot (27 to 42°C) and wet conditions associated with overhead irrigation under high-density plantings (>8.0 million seeds/ha). Isolations were performed on symptomatic tissue, and DNA was recovered from pure culture of the isolates recovered and sequenced using the internal transcribed spacer (ITS) ribosomal DNA (rDNA) primers ITS1/ITS4 and gene cytochrome oxidase I (COXI) primers FM55 and FM59. BLAST searches in GenBank indicated that the isolates were P. aphanidermatum based on 99 to 100% homology of ITS rDNA. Moreover, the DNA sequences of the ITS and COXI were identical for the five representative isolates. The objective of this research was to determine whether P. aphanidermatum recovered from symptomatic spinach tissue was able to cause foliar web blight and damping-off of spinach and other crops. In addition to spinach, other hosts evaluated included cotton, soybean, pepper, tomato, cucumber, melon, squash, lettuce, corn, wheat, and rice in greenhouse trials. Inoculations were performed by either foliar inoculations or infesting the soil with plugs of potato dextrose agar colonized by the P. aphanidermatum. Web blight symptoms were severe on spinach and all other dicotyledonous hosts tested, except lettuce. No web blight symptoms were observed on corn or rice, and only minor symptoms were observed on 10-day-old seedlings of wheat. P. aphanidermatum caused severe preemergence damping-off of all dicotyledonous plant species tested but only caused limited seedling size reduction in corn and wheat. Mefenoxam treatment of spinach seed provided complete protection against preemergence damping-off of spinach at both low (0.15 g a.i./kg of seed) and high (0.70 g a.i./kg of seed) rates of application, and the high rate of the application resulted in complete protection against web blight of spinach for 10 to 20 days after planting.

New Races and Novel Strains of the Spinach Downy Mildew Pathogen Peronospora effusa.

Downy mildew disease, caused by Peronospora effusa (=P. farinosa f. sp. spinaciae [Pfs]), is the most economically important disease of spinach. Current high-density fresh-market spinach production provides conducive conditions for disease development, and downy mildew frequently forces growers to harvest early owing to disease development, to cull symptomatic leaves prior to harvest, or to abandon the field if the disease is too severe. The use of resistant cultivars to manage downy mildew, particularly on increasing acreages of organic spinach production, applies strong selection pressure on the pathogen, and many new races of Pfs have been identified in recent years in spinach production areas worldwide. To monitor the virulence diversity in the Pfs population, downy mildew samples were collected from spinach production areas and tested for race identification based on the disease reactions of a standard set of international spinach differentials. Two new races (designated races 15 and 16) and eight novel strains were identified between 2013 and 2017. The disease reaction of Pfs 15 was similar to race 4, except race 4 could not overcome the resistance imparted by the RPF9 locus. Several resistance loci (RPF1, 2, 4, and 6) were effective in preventing disease caused by Pfs 15. The race Pfs 16 could overcome several resistance loci (RPF2, 4, 5, 9, and 10) but not others (RPF1, 3, 6, and 7). One novel strain (UA1014) could overcome the resistance of spinach resistant loci RPF1 to RPF7 but only infected the cotyledons and not the true leaves of certain cultivars. A new set of near-isogenic lines has been developed and evaluated for disease reactions to the new races and novel strains as differentials. None of the 360 U.S. Department of Agriculture spinach germplasm accessions tested were resistant to Pfs 16 or UA1014. A survey of isolates over several years highlighted the dynamic nature of the virulence diversity of the Pfs population. Identification of virulence diversity and evaluation of the genetics of resistance to Pfs will continue to allow for a more effective disease management strategy through resistance gene deployment.

Season-Long Dynamics of Spinach Downy Mildew Determined by Spore Trapping and Disease Incidence.

Peronospora effusa is an obligate oomycete that causes downy mildew of spinach. Downy mildew threatens sustainable production of fresh market organic spinach in California, and routine fungicide sprays are often necessary for conventional production. In this study, airborne P. effusa spores were collected using rotating arm impaction spore trap samplers at four sites in the Salinas Valley between late January and early June in 2013 and 2014. Levels of P. effusa DNA were determined by a species-specific quantitative polymerase chain reaction assay. Peronospora effusa was detected prior to and during the growing season in both years. Nonlinear time series analyses on the data suggested that the within-season dynamics of P. effusa airborne inoculum are characterized by a mixture of chaotic, deterministic, and stochastic features, with successive data points somewhat predictable from the previous values in the series. Analyses of concentrations of airborne P. effusa suggest both an exponential increase in concentration over the course of the season and oscillations around the increasing average value that had season-specific periodicity around 30, 45, and 75 days, values that are close to whole multiples of the combined pathogen latent and infectious periods. Each unit increase in temperature was correlated with 1.7 to 6% increased odds of an increase in DNA copy numbers, while each unit decrease in wind speed was correlated with 4 to 12.7% increased odds of an increase in DNA copy numbers. Disease incidence was correlated with airborne P. effusa levels and weather variables, and a receiver operating characteristic curve analysis suggested that P. effusa DNA copy numbers determined from the spore traps nine days prior to disease rating could predict disease incidence.

Leaf anatomical and photosynthetic acclimation to cool temperature and high light in two winter versus two summer annuals.

Acclimation of foliar features to cool temperature and high light was characterized in winter (Spinacia oleracea L. cv. Giant Nobel; Arabidopsis thaliana (L.) Heynhold Col-0 and ecotypes from Sweden and Italy) versus summer (Helianthus annuus L. cv. Soraya; Cucurbita pepo L. cv. Italian Zucchini Romanesco) annuals. Significant relationships existed among leaf dry mass per area, photosynthesis, leaf thickness and palisade mesophyll thickness. While the acclimatory response of the summer annuals to cool temperature and/or high light levels was limited, the winter annuals increased the number of palisade cell layers, ranging from two layers under moderate light and warm temperature to between four and five layers under cool temperature and high light. A significant relationship was also found between palisade tissue thickness and either cross-sectional area or number of phloem cells (each normalized by vein density) in minor veins among all four species and growth regimes. The two winter annuals, but not the summer annuals, thus exhibited acclimatory adjustments of minor vein phloem to cool temperature and/or high light, with more numerous and larger phloem cells and a higher maximal photosynthesis rate. The upregulation of photosynthesis in winter annuals in response to low growth temperature may thus depend on not only (1) a greater volume of photosynthesizing palisade tissue but also (2) leaf veins containing additional phloem cells and presumably capable of exporting a greater volume of sugars from the leaves to the rest of the plant.

Use of a common laboratory glassware detergent improves recovery of Cryptosporidium parvum and Cyclospora cayetanensis from lettuce, herbs and raspberries.

The success of any protocol designed to detect parasitic protozoa on produce must begin with an efficient initial wash step. Cryptosporidium parvum and Cyclospora cayetanensis oocysts were seeded onto herbs, lettuces and raspberries, eluted with one of four wash solutions and the recovered number of oocysts determined via fluorescent microscopy. Recovery rates for fluorescein thiosemicarbazide labeled C. parvum oocysts seeded onto spinach and raspberries and washed with de-ionized water were 38.4 ± 10.1% and 34.9 ± 6.2%, respectively. Two alternative wash solutions viz. 1M glycine, pH 5.5 and a detachment solution were tested also using labeled C. parvum seeded spinach and raspberries. No statistically significant difference was noted in the recovery rates. However, a wash solution containing 0.1% Alconox, a laboratory glassware detergent, resulted in a significant improvement in oocyst recovery. 72.6 ± 6.6% C. parvum oocysts were recovered from basil when washed with 0.1% Alconox compared to 47.9 ± 5.8% using detachment solution. Also, C. cayetanensis oocysts were seeded onto lettuces, herbs and raspberries and the recovery using de-ionized water were compared to 0.1% Alconox wash: basil 17.5 ± 5.0% to 76.1 ± 14.0%, lollo rosso lettuce 38.3 ± 5.5% to 72.5 ± 8.1%, Tango leaf lettuce 45.9 ± 5.4% to 71.1 ± 7.8% and spring mix (mesclun) 39.8 ± 0.7% to 80.2 ± 11.3%, respectively. These results suggest that the use of Alconox in a wash solution significantly improves recovery resulting in the detection of these parasitic protozoa on high risk foods.

Mitochondrial phylogeny reveals intraspecific variation in Peronospora effusa, the spinach downy mildew pathogen.

Since about two hundred years, downy mildew caused by Peronospora effusa is probably the most economically important disease of spinach (Spinacia oleracea). However, there is no information on the global phylogeographic structure of the pathogen and thus it is unclear whether a single genotype occurs worldwide or whether some local genetic variation exists. To investigate the genetic variability of this pathogen, a sequence analysis of two partial mitochondrial DNA genes, cox2 and nad1, was carried out. Thirty-three specimens of Peronospora effusa from four continents were analyzed, including samples from Australia, China, Japan, Korea, Mexico, Russia, Sweden, and the USA. Despite the potential anthropogenic admixture of genotypes, a phylogeographic pattern was observed, which corresponds to two major groups, an Asian/Oceanian clade and another group, which includes American/European specimens. Notably, two of six Japanese specimens investigated did not belong to the Asian/Oceanian clade, but were identical to three of the specimens from the USA, suggestive of a recent introduction from the USA to Japan. As similar introduction events may be occurring as a result of the globalised trade with plant and seed material, a better knowledge of the phylogeographic distribution of pathogens is highly warranted for food security purposes.

Spinacia oleracea L. leaf stomata harboring Cryptosporidium parvum oocysts: a potential threat to food safety.

Cryptosporidium parvum is a cosmopolitan microscopic protozoan parasite that causes severe diarrheal disease (cryptosporidiosis) in mammals, including humans and livestock. There is growing evidence of Cryptosporidium persistence in fresh produce that may result in food-borne infection, including sporadic cases as well as outbreaks. However, drinking and recreational waters are still considered the major sources of Cryptosporidium infection in humans, which has resulted in prioritization of studies of parasite etiology in aquatic environments, while the mechanisms of transmission and parasite persistence on edible plants remain poorly understood. Using laser scanning confocal microscopy together with fluorescein-labeled monoclonal antibodies, C. parvum oocysts were found to strongly adhere to spinach plants after contact with contaminated water, to infiltrate through the stomatal openings in spinach leaves, and to persist at the mesophyll level. These findings and the fact that this pathogenic parasite resists washing and disinfection raise concerns regarding food safety.

Interactions between food-borne pathogens and protozoa isolated from lettuce and spinach.

The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.

Seasonal prevalence and susceptibility to agrochemicals of Tyrophagus similis (Acari: Acaridae) in spinach buds and agricultural soil under greenhouse conditions.

Seasonal prevalence of Tyrophagus similis was investigated from 1997 to 1998 in two spinach greenhouses in central Japan. Susceptibility of T. similis to agrochemicals was also tested in the laboratory. Tyrophagus similis density in the soil was low during the high temperature period from May to August. The density rapidly increased in late autumn and remained at a high level during the cool season from December to February. The number further increased in April and then rapidly decreased in May. The high temperatures in the greenhouses from spring to early autumn are considered the main causes of population decrease. Mites on spinach buds increased the number after those on and in the soil increased. Mites attacked spinach buds mostly in late autumn and early spring. Dichlorvos did not reduce the number of mites in either greenhouse even though it was highly toxic under laboratory condition. This discrepancy suggests that the mites in both the soil and spinach buds had little direct contact with the chemicals. These data suggest that once crop damage by mites is detected, it is usually too late to use chemicals, and that mites that live in the buds are protected from agrochemicals.

Interactions between Spinacia oleracea and Bradysia impatiens: a role for phytoecdysteroids.

Plant produced insect molting hormones, termed phytoecdysteroids (PEs), are thought to function as plant defenses against insects by acting as either feeding deterrents or through developmental disruption. In spinach (Spinacia oleracea), 20-hydroxyecdysone (20E) concentrations in the roots rapidly increase following root damage, root herbivory, or methyl jasmonate (MJ) applications. In this inducible system, we investigated the plant defense hypothesis by examining interactions of roots, 20E concentrations, and larvae of the dark-winged fungus gnat (Bradysia impatiens). Root herbivory by B. impatiens larvae resulted in a 4.0- to 6.6-fold increase in root 20E concentrations. In paired-choice tests, increases in dietary 20E stimulated B. impatiens feeding deterrency. B. impatiens larvae preferred control diets, low in 20E, to those constructed from induced roots and those amended with 20E (25 to 50 micro g/g wet mass). When confined to 20E-treated diets, concentrations as low as 5 micro g/g (wet mass) resulted in significantly reduced B. impatiens survivorship compared to controls. The induction of root 20E levels with MJ resulted in a 2.1-fold increase in 20E levels and a 50% reduction in B. impatiens larval establishment. In a paired-choice arena, untreated control roots were damaged significantly more by B. impatiens larvae than MJ-induced roots that contained 3-fold greater 20E levels. Based on dietary preference tests, the 20E concentrations present in the MJ-induced roots (28 micro g/g wet mass) were sufficient to explain this reduction in herbivory. Interactions between spinach roots and B. impatiens larvae demonstrate that PEs can act as inducible defenses and provide protection against insect herbivory.