Growth assessment of Salmonella enterica multi-serovar populations in poultry rinsates with commonly used enrichment and plating media.

Isolation of Salmonella from enrichment cultures of food or environmental samples is a complicated process. Numerous factors including fitness in various selective enrichment media, relative starting concentrations in pre-enrichment, and competition among multi-serovar populations and associated natural microflora, come together to determine which serovars are identified from a given sample. A recently developed approach for assessing the relative abundance (RA) of multi-serovar Salmonella populations (CRISPR-SeroSeq or Deep Serotyping, DST) is providing new insight into how these factors impact the serovars observed, especially when different selective enrichment methods are used to identify Salmonella from a primary enrichment sample. To illustrate this, we examined Salmonella-positive poultry pre-enrichment samples through the selective enrichment process in Tetrathionate (TT) and Rappaport Vassiliadis (RVS) broths and assessed recovery of serovars with each medium. We observed the RA of serovars detected post selective enrichment varied depending on the medium used, initial concentration, and competitive fitness factors, all which could result in minority serovars in pre-enrichment becoming dominant serovars post selective enrichment. The data presented provide a greater understanding of culture biases and lays the groundwork for investigations into robust enrichment and plating media combinations for detecting Salmonella serovars of greater concern for human health.

The Sigatoka Disease Complex Caused by Pseudocercospora spp. and Other Fungal Pathogens Associated with Musa spp. in Puerto Rico.

Bananas (Musa spp.) are among the world's most economically important staple food crops. The most important fungal leaf diseases of Musa spp. worldwide are caused by the Sigatoka disease complex, which comprise black Sigatoka (Pseudocercospora fijiensis), yellow Sigatoka (P. musae), and Eumusae leaf spot (P. eumusae). Considering the rapid spreading rate of black Sigatoka in Puerto Rico after its first observation in 2004, a disease survey was conducted from 2018 to 2020 to evaluate the Sigatoka disease complex on the island. Sixty-one leaf samples showing Sigatoka-like symptoms were collected throughout the island for diagnosis by molecular approaches and fungal isolation. Molecular analysis using species-specific primers for P. fijiensis, P. musae and P. eumusae detected the presence of P. fijiensis in fifty leaf samples. Thirty-eight fungal isolates were collected and identified by morphology and genomic sequencing from various nuclear genes. The analysis identified 24 isolates as P. fijiensis, while the rest of the isolates belonged to the genus Cladosporium spp. and Cladosporium-like spp. (n=5), Neocordana musae (n=2), Zasmidium spp. (n=6), and Z. musigenum (n=1). The high frequency of P. fijiensis found in leaf samples and collected isolates suggest that black Sigatoka has displaced the yellow Sigatoka (P. musae) in Puerto Rico. Accurate identification of fungal species causing foliar diseases in Musa spp. will allow the establishment of quarantine regulations and specific management approaches in Puerto Rico.

First Report of Diaporthe tulliensis Causing Necrotic Spots and Leaf Blight on Rambutan in Puerto Rico.

Rambutan (Nepehelium lappaceum) is a tropical exotic fruit belonging to the Sapindaceae family. Several pathogens have been identified in rambutan causing different diseases on fruits, inflorescences, and branches (Serrato-Diaz et al., 2015, 2017, 2020) but few on leaves. From 2015 to 2021, a disease survey was conducted in one greenhouse in Mayaguez, Puerto Rico and experimental rambutan field orchards of the USDA-ARS Tropical Agriculture Research Station located at Isabela, Corozal, Santa Isabel, and Adjuntas, Puerto Rico (Latitude: 18°12'28"N, 18°34'10'' N, 18°00'47''N, 18°16'35''N and Longitude: 67°08'17"W, 66°31'74'' W, 66°38'98''W, 66°72'32''W, respectively). Varieties Benjai, Gula Batu, Jitlee, R-134, R-156Y, R-162, R-167 and Rongren were sampled. Necrotic spots and leaf blight were commonly observed with a disease incidence of 80%. Diseased leaves showed necrosis starting from the apex and spreading through the lamina. Ten diseased leaves were collected from each location and sections of symptomatic tissue (5mm2) were disinfected and plated on potato dextrose agar (PDA) and oatmeal agar (OA). Two representative isolates of Diaporthe tulliensis, A3 and A4, were obtained, purified, and identified morphologically and by PCR amplifications of three nuclear genes of the Internal Transcribed Spacer ITS1-5.8S-ITS2 region of the ribosomal DNA primers ITS5/ITS4, portions of the ß tubulin (BT) primers Bt2a/Bt2b and the translation elongation factor 1-α (TEF1-α) primers EF1728F/EF1986R. On PDA and OA colonies of isolates A3 and A4 were initially white and flat with sparse mycelia that turned yellowish-white to grey with age. Pycnidia were black with cream to pale yellow conidial droplets that exuded from ostioles. Hyaline, unicellular alpha conidia were oval to cylindrical, rounded at apex and obconically truncate at base. Alpha conidia (n = 50) for isolates of D. tulliensis were 4.9 to 5.9 µm long by 2.2 - 2.5 µm wide. DNA sequences of the ITS region and partial sequences of TEF1-α and BT genes were compared by BLASTN with Diaporthe sequences deposited in GenBank. ITS, BT and EF1-α sequences of isolates A3 and A4 (OP219651 and OP161553 for ITS region; OP222137 and OP168832 for TEF1-α; OP222136 and OP168831 for BT, respectively) were grouped to the holotype BRIP 62248a (Bootstrap BS=100) of Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan. Pathogenicity tests were conducted on six of six-months-old rambutan seedlings of R-167 variety. Three unwounded healthy non-detached leaves were inoculated per isolate with one 5mm mycelial disk from pure cultures grown on PDA. Rambutan seedlings were kept in a humid chamber using plastic bags for 8 days under greenhouse conditions. Two of six seedlings were used as controls and inoculated with PDA disks only. Eight and 14 days after inoculation (DAI), D. tulliensis isolates caused necrotic spots and leaf blight, on leaves. Diseased leaves turned from light to dark brown starting from the apex and spreading through the lamina with necrotic lesions ranging in size from 5 - 10 mm. Untreated controls showed no symptoms, and no fungi were re-isolated from tissue. D. tulliensis was re-isolated from diseased leaves, fulfilling Koch's postulates. D. tulliensis has been reported in Taiwan causing Diaporthe leaf spot in Boston Ivy (Huang, C. et al., 2021) and Bodhi trees (Li, K.Y. et al., 2022), as well as Jasmin stem canker (Ching Hsu, C. et al., 2022). It has been reported as causing leaf blight of coffee (Gong, J. L., et al., 2019), kiwifruit stem canker in China (Bai et al., 2017), and most recently causing cacao pod rot in Puerto Rico (Serrato-Diaz, L.M. et al., 2022). To our knowledge, this is the first report of Diaporthe tulliensis causing necrotic spots and leaf light on rambutan in Puerto Rico and often associated with a potassium deficiency in many parts of the world. It will be important to establish an adequate and effective control management of this disease in rambutan producing countries worldwide. References and doi hyperlinks: 1. Huang, C. et al. Plant Dis. 105:2718, 2021 https://doi.org/10.1094/PDIS-12-20-2652-PDN 2. Li, K.Y. et al. Plant Dis. 0:ja, 2022 https://doi.org/10.1094/PDIS-01-22-0211-PDN 3. Ching Hsu, C. et al. Plant Dis. 0:ja, 2022 https://doi.org/10.1094/PDIS-09-21-1908-PDN 4. Gong, J. L., et al. Plant Dis. 104:570, 2019 https://doi.org/10.1094/PDIS-09-19-1833-PDN 5. Bai et al. Plant Dis. 101:508, 2017 https://doi.org/10.1094/PDIS-10-16-1445-PDN 6. Serrato-Diaz L.M., et al. 2015. Plant Dis. 99: 1187. https://doi.org/10.1094/PDIS-09-14-0923-PDN 7. Serrato-Diaz L.M. et al. 2017. Plant Dis. 101: 1043. https://doi.org/10.1094/PDIS-11-16-1557-PDN 8. Serrato-Diaz, L.M., et al. 2020. Plant Dis. 104: 105-115. https://doi.org/10.1094/PDIS-02-19-0295-RE 9. Serrato-Diaz, L.M. et al. 2022 Plant Dis. 106: 2530. https://doi.org/10.1094/PDIS-12-21-2634-PDN.

Salmonella enterica Serovar Diversity, Distribution, and Prevalence in Public-Access Waters from a Central California Coastal Leafy Green-Growing Region from 2011 to 2016.

Prevalence and serovar diversity of Salmonella enterica were measured during a 5-year survey of surface waters in a 500-mi2 agricultural region of the Central California Coast. Rivers, streams, lakes, and ponds were sampled bimonthly resulting in 2,979 samples. Overall prevalence was 56.4% with higher levels detected in spring than in fall. Small, but significant, differences in prevalence were detected based on sample locations. Detection of Salmonella was correlated positively with both significant rain events and, in some environments, levels of generic Escherichia coli. Analysis of 1,936 isolates revealed significant serovar diversity, with 91 different serovars detected. The most common isolated serovars were S. enterica subsp. enterica serovars I 6,8:d:- (406 isolates, 21.0%, and potentially monophasic Salmonella Muenchen), Give (334 isolates, 17.3%), Muenchen (158 isolates, 8.2%), Typhimurium (227 isolates, 11.7%), Oranienburg (106 isolates, 5.5%), and Montevideo (78 isolates, 4%). Sixteen of the 24 most common serovars detected in the region are among the serovars reported to cause the most human salmonellosis in the United States. Some of the serovars were associated with location and seasonal bias. Analysis of XbaI pulsed field gel electrophoresis (PFGE) patterns of strains of serovars Typhimurium, Oranienburg, and Montevideo showed significant intraserovar diversity. PFGE pulsotypes were identified in the region for multiple years of the survey, indicating persistence or regular reintroduction to the region. IMPORTANCE Nontyphoidal Salmonella is among the leading causes of bacterial foodborne illness, and increasing numbers of outbreaks and recalls are due to contaminated produce. High prevalence and 91 different serovars were detected in this leafy green growing region. Seventeen serovars that cause most of the human salmonellosis in the United States were detected, with 16 of those serovars detected in multiple locations and multiple years of the 5-year survey. Understanding the widespread prevalence and diversity of Salmonella in the region will assist in promoting food safety practices and intervention methods for growers and regulators.