Genome Sequence Resource for Colletotrichum scovillei, the Cause of Anthracnose Disease of Chili.

Colletotrichum species cause anthracnose disease on the economically important spice crop chili. A total of 24 Colletotrichum species are known to infect chili and cause anthracnose. C. scovillei belongs to the C. acutatum species complex, and it shows greater aggressiveness than other species, particularly in the case of inoculation onto the nonwounded fruits of chili plants. The current work introduces an initial Illumina-Nanopore hybrid draft genome for C. scovillei TJNH1 together with the related annotations. Knowledge of this genome sequence provides an important reference genome of C. scovillei and will help further understand the pathogenic mechanism of C. scovillei to plant.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Short-Read Genome Assembly Resource for Leveillula taurica Causing Powdery Mildew Disease of Sweet Pepper (Capsicum annuum).

Powdery mildew of sweet pepper (Capsicum annuum) is an economically important disease. It is caused by Leveillula taurica, an obligate biotrophic ascomycete with a partly endophytic mycelium and haustoria, i.e., feeding structures formed in the mesophyll cells of infected host plant tissues. The molecular basis of its pathogenesis is largely unknown because genomic resources only exist for epiphytically growing powdery mildew fungi with haustoria formed exclusively in epidermal cells of their plant hosts. Here, we present the first reference genome assembly for an isolate of L. taurica isolated from sweet pepper in Hungary. The short read-based assembly consists of 23,599 contigs with a total length of 187.2 Mbp; the scaffold N50 is 13,899 kbp and N90 is 3,522 kbp; and the average GC content is 39.2%. We detected at least 92,881 transposable elements covering 55.5 Mbp (30.4%). BRAKER predicted 19,751 protein-coding gene models in this assembly. Our reference genome assembly of L. taurica is the first resource to study the molecular pathogenesis and evolution of a powdery mildew fungus with a partly endophytic lifestyle.

Screening and Assessment of Potential Plant Growth-promoting Bacteria Associated with Allium cepa Linn.

Plant growth-promoting bacteria (PGPB) are beneficial microbes that increase plant growth and yield. However, limited information is currently available on PGPB in onion (Allium cepa Linn.). The aims of the present study were to isolate and identify PGPB in onion and examine the effects of isolated PGPB on germination and growth during the vegetative stage in onion, pak choy (Brassica chinensis), and sweet pepper (Capsicum annuum). Twenty-three strains of PGPB were isolated from the roots, bulbs, and rhizosphere soil of onion. All isolated bacterial strains showed one or more PGP traits, including indole acetic acid production, phosphate solubilization ability, and 1-aminocyclopropane-1-carboxylate deaminase and nitrogenase activities; most of these traits were derived from Bacillus sp., Microbacterium sp., and Pseudomonas sp. Eight bacteria that exhibited strong abilities to produce indole acetic acid were selected for a Petri dish trial, soil pot test, and vermiculate pot test. The Petri dish trial showed that strains ORE8 and ORTB2 simultaneously increased radicle and hypocotyl lengths in onion, but inhibited growth in sweet pepper after 7 d. The soil pot experiment on onion revealed that strains ORE5, ORE8, and ORTB2 strongly promoted growth during the vegetative stage with only a half dose of chemical fertilizer. The present results indicate that ORE8 (Bacillus megaterium) and ORTB2 (Pantoea sp.) are the most promising biofertilizers of onion and may simultaneously inhibit the seedling growth of other plants.

Assessment of Plant-Probiotic Performance of Novel Endophytic Bacillus sp. in Talc-Based Formulation.

Endophytic bacteria are considered to have a plethora of plant growth promoting and anti-phytopathogenic traits to live within the plants. Hence, they have immense promises for plant probiotic development. In the current study, plant probiotic endophytic Bacillus sp. CaB5 which has been previously isolated from Capsicum annuum was investigated for its performance in talc-based formulation. For this, CaB5 was made into formulation with sterile talc, calcium carbonate, and carboxymethyl cellulose. The viability analysis of the formulation by standard plate count and fluorescence methods has confirmed the stable microbial count up to 45 days. Plant probiotic performance of the prepared formulation was analyzed on cowpea (Vigna unguiculata) and lady's finger (Abelmoschus esculentus). The results showed the formulation treatment to have enhancement effect on seed germination as well as plant growth in both selected plants. The results highlight the potential of CaB5-based formulation for field application to enhance growth of economically important plants.

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper.

Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out. Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

ABSTRACT Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

A Risk Assessment Model for Bacterial Leaf Spot of Pepper (Capsicum annuum), Caused by Xanthomonas euvesicatoria, Based on Concentrations of Macronutrients, Micronutrients, and Micronutrient Ratios.

The phytopathogenic bacterium Xanthomonas euvesicatoria causes bacterial leaf spot (BLS) of pepper and has a worldwide distribution. BLS is difficult to control and an integrated management strategy that incorporates crop rotation, use of clean seed and clean plants, weed control, resistant varieties, applications of bactericides, biocontrol agents, and systemic acquired resistance (SAR) inducers is generally recommended. However, even with that arsenal of weapons, BLS can still be responsible for severe losses under favorable environmental conditions. Thus, additional tools need to be added to an overall integrated management strategy to combat BLS. In this article, we developed several models from 2012 to 2014 that were based on how macronutrients, micronutrients, and micronutrient ratios affect BLS severity. Factors used to select a model for validation included highly significant P values, high adjusted R2 values, low variance inflation factor values (<5), root mean square error, Mallow's Cp, and high Akaike's information criterion correction values. In addition, salicylic acid (SA) concentrations and relative expression of nonexpresser pathogenesis-related gene1 (NPR1) and pathogenesis-related protein 1 (PR1) in pepper tissues were also considered in model selection. A model (ECGA1) consisting of concentrations of copper, manganese, potassium, and the iron/zinc ratio as independent variables was used for validation in three different commercial pepper fields in Georgia: Colquitt County and Worth County in 2015 and Tift County in 2016. When area under the disease progress curve (AUDPC) values for two field sites (Colquitt and Worth Counties) in 2015 were pulled together and plotted against ECGA1-predicted values for both sites, the resulting relationship was highly significant (P = 0.0001) with an R2 value of 0.92. A significant relationship between observed AUDPC versus predicted values was also observed in Tift County in 2016 (P < 0.001; adjusted R2 = 0.98). Relative gene expression of both NPR1 and PR1 genes was significantly (P < 0.01) higher in pepper grown in predicted low-risk sites compared with pepper from high-risk sites in Colquitt, Worth, and Tift Counties. Although BLS severity will fluctuate depending on environmental conditions, the data indicate that the level of risk at a particular location may be influenced by how macronutrient and micronutrient concentrations affect plant disease resistance genes in the SAR pathway.

Genetic Lineages and Antimicrobial Resistance in Pseudomonas spp. Isolates Recovered from Food Samples.

Raw food is a reservoir of Pseudomonas isolates that could be disseminated to consumers. The presence of Pseudomonas spp. was studied in food samples, and the phenotypic and genotypic characterizations of the recovered isolates were analyzed. Two samples of meat (3%, turkey and beef) and 13 of vegetables (22%, 7 green peppers and 6 tomatoes) contained Pseudomonas spp. A total of 20 isolates were identified, and were classified as follows (number of isolates): P. aeruginosa (5), P. putida (5), P. nitroreducens (4), P. fulva (2), P. mosselli (1), P. mendocina (1), P. monteilii (1), and Pseudomonas sp. (1). These 20 Pseudomonas isolates were clonally different by pulsed-field-gel-electrophoresis, and were resistant to the following antibiotics: ticarcillin (85%), aztreonam (30%), cefepime (10%), imipenem (10%), and meropenem (5%), but were susceptible to ceftazidime, piperacillin, piperacillin-tazobactam, doripenem, gentamicin, tobramycin, amikacin, ciprofloxacin, norfloxacin, and colistin. Only one strain (Ps158) presented a class 1 integron lacking the 3' conserved segment. The five P. aeruginosa strains were typed by multilocus sequence typing in five different sequence-types (ST17, ST270, ST800, ST1455, and ST1456), and different mutations were detected in protein OprD that were classified in three groups. One strain (Ps159) showed a new insertion sequence (ISPa47) truncating the oprD gene, and conferring resistance to imipenem.

Prevalence, level and distribution of Salmonella in shipments of imported capsicum and sesame seed spice offered for entry to the United States: observations and modeling results.

In response to increased concerns about spice safety, the United States Food and Drug Administration (FDA) initiated research to characterize the prevalence and levels of Salmonella in imported spices. 299 imported dried capsicum shipments and 233 imported sesame seed shipments offered for entry to the United States were sampled. Observed Salmonella shipment prevalence was 3.3% (1500 g examined; 95% CI 1.6-6.1%) for capsicum and 9.9% (1500 g; 95% Confidence Interval (CI) 6.3-14%) for sesame seed. Within shipment contamination was not inconsistent with a Poisson distribution. Shipment mean Salmonella level estimates among contaminated shipments ranged from 6 × 10(-4) to 0.09 (capsicum) or 6 × 10(-4) to 0.04 (sesame seed) MPN/g. A gamma-Poisson model provided the best fit to observed data for both imported shipments of capsicum and imported shipments of sesame seed sampled in this study among the six parametric models considered. Shipment mean levels of Salmonella vary widely between shipments; many contaminated shipments contain low levels of contamination. Examination of sampling plan efficacy for identifying contaminated spice shipments from these distributions indicates that sample size of spice examined is critical. Sampling protocols examining 25 g samples are predicted to be able to identify a small fraction of contaminated shipments of imported capsicum or sesame seeds.

Resistance to the novel fungicide pyrimorph in Phytophthora capsici: risk assessment and detection of point mutations in CesA3 that confer resistance.

Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC(50) value of 1.4261 (± 0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1 × 10(-4). The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC(50) values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations.

Microbial quality of some vegetables sold in ED DueimTwon, Sudan.

This study was probably the first research carried out to investigate the microbiological quality of some vegetables sold in ED DueimTwon, Sudan. Four species of vegetables were used, Arugula (Eruca sativa), Mloukhia (Corchorus olitorius), Tomato (Lycopersicon esculentum) and Green pepper (Capsicum annuum). The samples were collected and examined according to standardized methods for total viable bacteria, coliforms and fecal coliform count. The average of total viable count ranged from 1.2 x 105-5.6 x 105 CFU mL(-1) for Arugula; 2.1 x 105-2.8 x 107 CFU mL(-1) for Mloukhia; 3.4 x 105-4.8 x 105 for Tomato and 2.3 x 105-8.0 x 106 CFU mL(-1) for Green pepper. However, the maximum level of total and fecal coliform were (93, 21); (28, 11); (75, 15) and (150, 20) MPN 100 mL(-1), respectively. Twelve bacteria belonging to five genera were isolated. Staphylococcus (33%) was the most predominant isolated followed by Enterobacteriaceae (25%), Bacillus (17%) and Streptococcus (17%). Micrococcus (8%) was the least dominant isolated. The results of microbial counts of these vegetable samples in this study indicate that, the agricultural practices, harvesting, hygiene, transporting and selling points are poor and therefore, the higher microbial load could be risked for public health.

Presence of some indicator bacteria and diarrheagenic E. coli pathotypes on jalapeño and serrano peppers from popular markets in Pachuca City, Mexico.

The chili pepper is a very important crop in Mexico. Diarrheagenic E. coli pathotypes (DEPs) are important foodborne pathogens in different countries including Mexico. No data exists on DEPs presence on fresh jalapeño and serrano pepper and little data have been published on the microbiological quality of these peppers. The frequencies of coliform bacteria (CB), thermotolerant coliforms (TC), E. coli and DEPs were determined for jalapeño and serrano peppers. Of 100 serrano samples, CB, TC, E. coli and DEPs were identified in 100, 90, 58 and 36%, respectively. Of 100 jalapeño samples, CB, TC, E. coli and DEPs were identified in 100, 88, 38 and 14%, respectively. Identified DEPs included enterotoxigenic E. coli (ETEC) and Shiga toxin-producing E. coli (STEC). STEC were isolated from 36% of serrano samples and 14% of jalapeño samples. ETEC were isolated from 12% of serrano samples and 2% of jalapeño samples. Both STEC and ETEC were identified in 14 serrano samples and 2 jalapeño samples. No E. coli O157:H7 were detected in any STEC-positive samples. Jalapeño and serrano peppers could be an important factor contributing to the endemicity of DEPs-caused gastroenteritis in Mexico.

Evolutionary and experimental assessment of novel markers for detection of Xanthomonas euvesicatoria in plant samples.

BACKGROUND: Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. METHODOLOGY: In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. SIGNIFICANCE: This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.

Assessment of hot peppers for aflatoxin and mold proliferation during storage.

Aflatoxin contamination and mold proliferation in three hot pepper hybrids (Sky Red, Maha, and Wonder King) were studied during 5 months of storage at three temperatures (20, 25, and 30°C) and under different packaging conditions (low-density polyethylene bags and jute bags). The presence of aflatoxins in hot pepper samples was determined by high-performance liquid chromatography with a UV-Vis detector. Sampling for analysis of aflatoxins, total mold counts, and Aspergillus counts was carried out at 0, 50, 100, and 150 days of storage. Hot peppers packed in jute bags were more susceptible to aflatoxin contamination than those packed in polyethylene bags; aflatoxin concentrations were 75% higher in peppers stored in jute bags. The effect of storage temperature resulted in aflatoxin concentrations that were 61% higher in hot peppers stored at 25 and 30°C than in those stored at 20°C. Of the three pepper hybrids, Wonder King was more susceptible to aflatoxin contamination, with a maximum of 1.50 µg/kg when packed in jute bags and stored at 25°C for 150 days. However, no sample exceeded the maximum permitted level for total aflatoxins in spices established by European Union regulations (10 µg/kg). Total mold counts and Aspergillus counts increased with storage duration, but all counts were significantly lower in peppers stored in polyethylene bags. A gradual increase in temperature during prolonged storage of hot peppers in combination with aeration may be the main reasons for increases in fungal biomass and Aspergillus proliferation with the subsequent aflatoxin production.

Bacterial wilt resistance in tomato, pepper, and eggplant: genetic resources respond to diverse strains in the Ralstonia solanacearum species complex.

Bacterial wilt, caused by strains belonging to the Ralstonia solanacearum species complex, inflicts severe economic losses in many crops worldwide. Host resistance remains the most effective control strategy against this disease. However, wilt resistance is often overcome due to the considerable variation among pathogen strains. To help breeders circumvent this problem, we assembled a worldwide collection of 30 accessions of tomato, eggplant and pepper (Core-TEP), most of which are commonly used as sources of resistance to R. solanacearum or for mapping quantitative trait loci. The Core-TEP lines were challenged with a core collection of 12 pathogen strains (Core-Rs2) representing the phylogenetic diversity of R. solanacearum. We observed six interaction phenotypes, from highly susceptible to highly resistant. Intermediate phenotypes resulted from the plants' ability to tolerate latent infections (i.e., bacterial colonization of vascular elements with limited or no wilting). The Core-Rs2 strains partitioned into three pathotypes on pepper accessions, five on tomato, and six on eggplant. A "pathoprofile" concept was developed to characterize the strain clusters, which displayed six virulence patterns on the whole set of Core-TEP host accessions. Neither pathotypes nor pathoprofiles were phylotype specific. Pathoprofiles with high aggressiveness were mainly found in strains from phylotypes I, IIB, and III. One pathoprofile included a strain that overcame almost all resistance sources.

Real-time reverse-transcriptase--polymerase chain reaction for Salmonella enterica detection from jalapeño and serrano peppers.

Outbreaks of Salmonella linked to fresh produce emphasize the need for rapid detection methods to curb the spread of foodborne pathogens. Reverse-transcriptase-polymerase chain reaction (RT-PCR) detects the presence of mRNA (shorter half-life than DNA), with greater potential of detecting viable pathogens. Real-time RT-PCR eliminates the need for gel electrophoresis and significantly enhances the speed of detection (<1 day) compared with traditional methods (>5 days). The objectives of this research were to apply real-time SYBR Green I-based RT-PCR to detect Salmonella from jalapeño and serrano peppers spiked with low and high inocula of Salmonella. Inoculated and uninoculated peppers were rinsed with water and dried under ultraviolet light for 10 min. Approximately 25 g peppers was inoculated with 10(8) to 10(1) colony forming units (CFU) of Salmonella enterica serovar Typhimurium in a stomacher bag and hand massaged in sterile 0.05 M glycine-0.14 M saline buffer (0.05% Tween, 3% beef extract) for optimal recovery of bacteria. A short preenrichment step of 6 h in buffered peptone water was needed for the detection of low inocula (10(4) CFU/25 g). One-milliliter portions of the extracts were serially diluted, plated on XLT4 agar, and used for RNA extraction with the Qiagen RNeasy Mini Kit. RT-PCR was carried out using SYBR Green I one-step RT-PCR with previously described invA gene primers and an internal amplification control. Detection limits were 10(4) CFU/25 g (approximately 10(2) CFU/g) and 10(7) CFU/25 g (approximately 10(5) CFU/g) Salmonella from enriched and unenriched inoculated peppers, respectively. Even though this method included a 6-h incubation period, the results were still obtainable in 1 day. This method shows promise for applications in routine surveillance and during outbreaks.

Assessment of root-associated paenibacillus polymyxa groups on growth promotion and induced systemic resistance in pepper.

Twenty-nine P. polymyxa strains isolated from rhizospheres of various crops were clustered into five genotypic groups on the basis of BOX-PCR analysis. The characteristics of several plant growth-promoting factors among the isolates revealed the distinct attributes in each allocated group. Under gnotobiotic conditions, inoculation of pepper roots with P. polymyxa isolates significantly increased the biomass in 17 of total 29 treated plants with untreated plants. Experiments on induced systemic resistance (ISR) against bacterial spot pathogen Xanthomonas axonopodis pv. vesicatoria in pepper by P. polymyxa strains were conducted and only one isolate (KNUC265) was selected. Further studies into ISR mediation by the KNUC265 strain against the soft-rot pathogen Erwinia carotovora subsp. carotovora in tobacco demonstrated that the tobacco seedlings exposed to either bacterial volatiles or diffusible metabolites exhibited a reduction in disease severity. In conclusion, ISR and plant growth promotion triggered by P. polymyxa isolates were systemically investigated on pepper for the first time. The P. polymyxa KNUC265 strain, which elicited both ISR and plant growth promotion, could be potentially used in improving the yield of pepper and possibly of other crops.

Application of microbial risk assessment to the development of standards for enteric pathogens in water used to irrigate fresh produce.

Microbial contamination of the surfaces of cantaloupe, iceberg lettuce, and bell peppers via contact with irrigation water was investigated to aid in the development of irrigation water quality standards for enteric bacteria and viruses. Furrow and subsurface drip irrigation methods were evaluated with the use of nonpathogenic surrogates, coliphage PRD1, and Escherichia coli ATCC 25922. The concentrations of hepatitis A virus (HAV) and Salmonella in irrigation water necessary to achieve a 1:10,000 annual risk of infection, the acceptable level of risk used for drinking water by the U.S. Environmental Protection Agency, were calculated with a quantitative microbial risk assessment approach. These calculations were based on the transfer of the selected nonpathogenic surrogates to fresh produce via irrigation water, as well as previously determined preharvest inactivation rates of pathogenic microorganisms on the surfaces of fresh produce. The risk of infection was found to be variable depending on type of crop, irrigation method, and days between last irrigation event and harvest. The worst-case scenario, in which produce is harvested and consumed the day after the last irrigation event and maximum exposure is assumed, indicated that concentrations of 2.5 CFU/100 ml of Salmonella and 2.5 x 10(-5) most probable number per 100 ml of HAV in irrigation water would result in an annual risk of 1:10,000 when the crop was consumed. If 14 days elapsed before harvest, allowing for die-off of the pathogens, the concentrations were increased to 5.7 x 10(3) Salmonella per 100 ml and 9.9 x 10(-3) HAV per 100 ml.