Aswathy Sreedharan

Global gene expression changes in Candidatus Liberibacter asiaticus during the transmission in distinct hosts between plant and insect

Huanglongbing (HLB) or citrus greening disease is a destructive disease of citrus worldwide, which is associated with Candidatus Liberibacter asiaticus. This phloem-limited fastidious pathogen is transmitted by the Asian citrus psyllid, Diaphorina citri, and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The molecular basis of the interaction of this pathogen with its hosts is not well understood. We hypothesized that, during infection, Ca. L. asiaticus differentially expresses the genes critical for its survival and/or pathogenicity in either host. To test this hypothesis, quantitative reverse transcription-polymerase chain reaction was performed to compare the gene expression of Ca. L. asiaticus in planta and in psyllid. Overall, 381 genes were analysed for their gene expression in planta and in psyllid. Among them, 182 genes were up-regulated in planta compared with in psyllid (P < 0.05), 16 genes were up-regulated in psyllid (P < 0.05) and 183 genes showed no statistically significant difference (P ≥ 0.05) in expression between in planta and in psyllid. Our study indicates that the expression of the Ca. L. asiaticus genes involved in transcriptional regulation, transport system, secretion system, flagella assembly, metabolic pathway and stress resistance are changed significantly in a host-specific manner to adapt to the distinct environments of plant and insect. To our knowledge, this is the first large-scale study to evaluate the differential expression of Ca. L. asiaticus genes in a plant host and its insect vector.

Control of Salmonella Cross-Contamination between Green Round Tomatoes in a Model Flume System

Tomato Best Management Practices require Florida packers to treat tomatoes in a flume system containing at least 150 ppm of free chlorine or other approved sanitizer. However, research is needed to determine the ability of these sanitizers to prevent the transfer of pathogens from contaminated to uncontaminated tomatoes, particularly under realistic packinghouse conditions. The goal of this research was to assess the minimum levels of sanitizer needed to prevent Salmonella cross-contamination between tomatoes in a model flume system under clean conditions and conditions where organic matter was added. Inoculated tomatoes (ca. 8.3 log CFU per tomato) were treated along with uninoculated tomatoes in a model flume system containing 0, 10, or 25 ppm of hypochlorous acid (HOCl) under organic loading conditions of 0, 500, or 4,000 ppm of chemical oxygen demand (COD). In the absence of HOCl, uninoculated tomatoes were highly contaminated (ca. 5 log CFU per tomato) by 15 s. No contamination was detectable (<2 log CFU per tomato) on uninoculated tomatoes when HOCl was present, except with 10 ppm at 4,000 ppm of COD, suggesting failure of 10 ppm of HOCl as a sanitizer under very high organic loading conditions. In the presence of HOCl or peroxyacetic acid, Salmonella was undetectable (<1 log CFU/ml) in the model flume water samples after 2 and 30 s, respectively. Upon enrichment, none of the uninoculated tomatoes treated with 25 ppm of HOCl for 120 s were positive for Salmonella, even in the presence of organic loading at 500 ppm of COD. Based on these findings, 25 ppm of HOCl may be adequate to prevent cross-contamination when the concentration is properly maintained, COD does not exceed 500 ppm, and tomatoes are treated for at least 120 s. Further validation in a larger commercial setting and using higher organic loading levels is necessary because managing HOCl at this low concentration is difficult, especially in a recirculating system. The use of less sanitizer by packers could reduce chemical and disposal costs.

Determination of Optimum Sanitizer Levels for Prevention of Salmonella Cross-Contamination of Mature Round Tomatoes in a Laboratory Model Flume System

Salmonella has been reported to be involved in several foodborne illness outbreaks, many of which resulted from consumption of raw tomatoes. This research aimed to optimize and evaluate the concentration of free chlorine (hypochlorous acid [HOCl]) used as a sanitizer to prevent cross-contamination of tomatoes inoculated with a cocktail of five rifampin-resistant Salmonella enterica serovars in a laboratory-based model flume system. Organic load, prepared using sterilized Scotts Premium Topsoil, was added in different quantities to the flume wash water to simulate real-world packinghouse conditions. In a typical packinghouse operation utilizing a recirculating flume system, the organic matter washed from tomato surfaces accumulates over time. In this study, different concentrations (0, 25, 50, 75, and 100 ppm) of HOCl were used as sanitizers under three organic load conditions (0, 650, and 1,000 mg/L chemical oxygen demand). Results showed that 100 ppm of HOCl was necessary to prevent Salmonella cross-contamination of uninoculated tomatoes in the model flume system in the presence of organic loading. Also, when treated with 100 ppm of HOCl, Salmonella levels were reduced by >4.5 log CFU per tomato from inoculated tomatoes in the presence of organic load. At 75 ppm of HOCl, Salmonella cross-contamination was prevented, but only in the absence of organic loading. In studies in which plate counts were negative, whole tomato enrichment studies were performed. No cross-contamination of uninoculated tomatoes was recorded when 100 ppm of HOCl was used, even in the presence of high organic load (1,000 mg/L chemical oxygen demand). Although sanitizer application reduces contamination on tomato surfaces, the primary function of sanitizers in the wash water is to prevent cross-contamination.

Effect of overhead spray and brush roller treatment on the survival of Pectobacterium and Salmonella on tomato surfaces.

Overhead spray and brush roller (OSBR) treatment has been shown to remove significantly more Salmonella from tomato surfaces than flume treatment. However, OSBR is not widely used in tomato packing facilities compared with other commodities, and little is known about whether brushing causes microabrasions or other physical damage. Bacteria such as Pectobacterium, a soft rot-producing plant pathogen, and Salmonella, a human pathogen, show increased survival and growth on damaged tomato surfaces. This study evaluated whether OSBR treatment had a negative effect on the safety and/or marketability of tomatoes by examining its effect on Pectobacterium and Salmonella survival. Pectobacterium survival was evaluated on inoculated tomatoes that were OSBR treated with water or sanitizer (100 ppm of NaOCl, 5 ppm of ClO2, or 80 ppm of peracetic acid). A 15-s OSBR treatment using water or sanitizer achieved a 3-log CFU/ml reduction in Pectobacterium levels. Survival of Pectobacterium and Salmonella on OSBR-treated, untreated, and puncture-wounded tomatoes stored at 25°C and 75 to 85 % relative humidity for 7 days was also assessed. Both Pectobacterium and Salmonella populations declined rapidly on OSBR-treated and untreated tomatoes, indicating that brushing does not damage tomato fruit to the extent of promoting better pathogen survival. In contrast, the survival of both organisms was significantly (P ≤ 0.05) higher on artificially wounded fruit. These results indicate that OSBR treatment does not increase the survival and growth of Pectobacterium or Salmonella on tomato surfaces and that it is effective in reducing Pectobacterium levels on the surface of inoculated tomatoes. These results suggest that, if used properly, an OSBR system in packinghouses is effective in removing surface contamination and does not affect tomato quality or safety.

Salmonella transfer potential onto tomatoes during laboratory-simulated in-field debris removal.

Florida Tomato Good Agricultural Practices (T-GAPs) mandate the removal of dirt and debris from tomatoes during harvest but do not provide any specific regulations or guidance; thus, the current practice of using cloths needs to be evaluated. This study examined Salmonella transfer from inoculated green tomatoes to uninoculated cloths and from inoculated cloths to uninoculated tomatoes, upon single and multiple touches. Tomatoes were spot inoculated with a rifampin-resistant Salmonella cocktail (10(7) CFU per tomato) and were touched with cloth (clean, dirty-dry, dirty-wet) at 0, 1, or 24 h postinoculation. Salmonella was enumerated on tryptic soy agar, followed by enrichments when necessary. The transfer direction was then reversed by touching freshly inoculated cloths with uninoculated tomatoes. Transfer coefficients (TCs) were then calculated. Salmonella TCs from inoculated tomato and cloth were highest when the inoculum was wet (0.44 ± 0.13 to 0.32 ± 0.12), regardless of the condition of the cloth. Although Salmonella TCs from inoculated tomato to uninoculated cloth decreased significantly when the inoculum was dried (0.17 ± 0.23 to 0.01 ± 0.00), low levels of Salmonella were detected on cloth even after 24 h of drying. Inoculated dirty cloth did not transfer more Salmonella compared with inoculated clean cloth, and Salmonella survival was not higher on dirty cloth. When inoculated clean cloth (wet) was touched with 25 tomatoes, significantly higher levels of Salmonella were transferred to the first, second, and fourth tomatoes (0.03 ± 0.10 to 0.09 ± 0.02). However, inoculated dirty-wet (below limit of detection) and dirty-dry (0.00 to 0.04 ± 0.01) cloths transferred similar levels of Salmonella to all 25 tomatoes. Results indicate a low risk of potential Salmonella contamination when the same cloth is used multiple times for debris removal, especially under high moisture levels. Results also show that the use of dirty cloths did not increase the risk of Salmonella cross-contamination.