Hidemi Izumi

On-farm sources of microbial contamination of persimmon fruit in Japan.

Potential sources of microbial contamination for persimmon fruit during growing and harvesting in the 2005 season were investigated to provide a baseline to design the good agricultural practices program for persimmons in Japan. Microbial counts in the peel of persimmon fruit during production season were close to or below 2.4 log CFU/g for bacteria and 3.0 log CFU/g for fungi but were above these values on harvested fruit. The counts in the flesh were below the detection level with all fruit. Bacteria and molds isolated from peel and flesh of persimmons during growing were phytopathogenic and soilborne organisms such as bacteria genera Enterobacter and Bacillus and mold genera Fusarium and Cladosporium, which were found in soil, weeds, agricultural water, and pesticide solution throughout the production season. The agricultural water was one of the most important potential preharvest sources, because Escherichia coli O157:H7 was identified from agricultural water in May, and Salmonella was detected in agricultural water, pesticide solution containing the agricultural water for the mixture, and soil after application of the pesticide solution in June. Neither of the two pathogenic bacteria was detected in any of the fruit samples. Microbial counts and diversity in the peel of persimmons at harvest increased after contact with plastic harvest basket and container, which could be sources of contamination during harvesting. Therefore, monitoring and management on-farm should focus on agricultural water and harvest equipment as important control points to reduce microbial contamination on persimmons.

Potential Sources of Microbial Contamination of Satsuma Mandarin Fruit in Japan, from Production through Packing Shed

Potential sources of microbial contamination of satsuma mandarin fruit were investigated from production through the packing shed in the 2005 season. Microbial counts in the peel and flesh during the fruit development stage were below 2.4 log CFU/g for bacteria and 3 log CFU/g for fungi, except for the peel in August and September. In the field environment, the highest microbial counts were found in fallen leaves on the ground, followed by soil, organic fertilizer, and agricultural water. Only the pesticide solution collected in July was positive for Salmonella, while no verotoxin-producing Escherichia coli was detected from any of the samples. The bacterial and mold flora in the peel comprised phytopathogenic organisms such as bacteria genus Pantoea and mold genus Mycosphaerella and soilborne organisms such as bacteria genus Bacillus and mold genus Cladosporium, which were found in soil, fallen leaves, agricultural water, and cloth mulch throughout the production season. After fruit harvest and sorting, microbial counts of the peel increased, while those of the flesh remained below the lower limit of detection. Although some of the preharvest sources could also be postharvest sources, some packing shed equipment was assumed to be postharvest sources, because Bacillus cereus was not identified from the fruit in the production field but was detected on the peel after sorting and on equipment such as gloves, plastic harvest basket, and size sorter. These results suggest that using sanitizers for agricultural water and packing sheds to prevent cross-contamination would be useful in a good agricultural practices program of the satsuma mandarin in Japan.

Enumeration and Identification of Coliform Bacteria Injured by Chlorine or Fungicide Mixed with Agricultural Water

Chemical sanitizers may induce no injury (bacteria survive), sublethal injury (bacteria are injured), or lethal injury (bacteria die). The proportion of coliform bacteria that were injured sublethally by chlorine and fungicide mixed with agricultural water (pond water), which was used to dilute the pesticide solution, was evaluated using the thin agar layer (TAL) method. In pure cultures of Enterobacter cloacae , Escherichia coli , and E. coli O157:H7 (representing a human pathogen), the percentage of chlorine-injured cells was 69 to 77% for dilute electrolyzed water containing an available chlorine level of 2 ppm. When agricultural water was mixed with electrolyzed water, the percentage of injured coliforms in agricultural water was 75%. The isolation and identification of bacteria on TAL and selective media suggested that the chlorine stress caused injury to Enterobacter kobei . Of the four fungicide products tested, diluted to their recommended concentrations, Topsin-M, Sumilex, and Oxirane caused injury to coliform bacteria in pure cultures and in agricultural water following their mixture with each pesticide, whereas Streptomycin did not induce any injury to the bacteria. The percentage of injury was 45 to 97% for Topsin-M, 80 to 87% for Sumilex, and 50 to 97% for Oxirane. A comparison of the coliforms isolated from the pesticide solutions and then grown on either TAL or selective media indicated the possibility of fungicide-injured Rahnella aquatilis , Yersinia mollaretii , and E. coli . These results suggest the importance of selecting a suitable sanitizer and the necessity of adjusting the sanitizer concentration to a level that will kill the coliforms rather than cause sanitizer-induced cell injury that can result in the recovery of the coliforms.

Sanitation and Microbiological Quality in Production Field and Fruit-Packing Shed of Persimmon and Satsuma Mandarin in Japan

The effects of sanitation treatments including chlorination (ca 10 ppm available chlorine) of agricultural water and ethyl alcohol (70%) spraying on packing shed equipment on microbial contamination on fruits and the environment were determined and compared with those in conventionally managed field and packing shed in persimmon and satsuma mandarin orchards. Chlorinated water reduced the microbial counts to levels below the lower limit of detection (1.4 log CFU/ml for bacteria and 2.0 log CFU/ml for fungi) in most agricultural water samples. Microbial counts of pesticide solution, which contained the agricultural water or chlorinated water for the mixture, were lower in sanitary field than in control field in both fruit orchards. The number of bacterial and mold species detected in agricultural water, chlorinated water, and pesticide solution were almost proportional to microbial counts in each sample throughout the year. The chlorination treatment of agricultural water tended to reduce the counts of mesophiles and fungi on the peel of persimmon fruit during production season. The ethyl alcohol spray treatment on packing shed equipment resulted in a substantial microbial reduction on plastic harvest basket and container in persimmon orchard and plastic harvest basket and container, gloves, scissors, and size sorter in satsuma mandarin orchard. The spray application on packing shed equipment reduced the counts of mesophiles and fungi on the peel of persimmon fruit by >1 log CFU/g. The number of satsuma mandarin packing shed equipment containing the species found on fruit peel was higher in control than in sanitary packing shed. No human pathogens such as verotoxin-producing Escherichia coli and Salmonella were detected in any of the fruit and environmental samples. These results indicate that uses of sanitizers such as chlorine for agricultural water and ethyl alcohol for packing shed equipment would be useful in a good agricultural practices program of persimmons and satsuma mandarin.

Enumeration and Identification ofEthanol-Injured Coliform BacteriaFound on Harvest Equipment andits Cross-Contamination withCabbage

Enumeration and Identification of Ethanol-Injured Coliform Bacteria Found on Harvest Equipment and its Cross-Contamination with Cabbage Although disinfection by sanitizers used to produce packing sheds which helps in preventing cross-contamination between farm equipment and produces, sanitizing agents like ethanol are capable of causing sanitizer-injured bacteria. Using the thin agar layer (TAL) method, the proportion of ethanol-injured coliform bacteria resulting from the use of an alcohol agent containing 1.6% and 47% ethanol was evaluated in pure cultures and in a harvest environment of cabbage, respectively. In pure cultures of Enterobacter cloacae, Escherichia coli, and E. coli O157:H7, ethanol-injured cells were observed at a range of 68-95% by treatment with an alcohol agent. When an alcohol agent was sprayed on harvest equipment including knife, container and gloves, 60% of the total coliforms detected on the knife were injured, but no injured coliforms were detected on the container and gloves. The isolation and identification of coliforms on selective and TAL media from the knife suggested that the ethanol stress caused injury to Enterobacter amnigenus, E. asburiae, and E. kobei. Injured bacteria were not detected on cabbage after harvest using the alcohol spray-treated knife because the species of bacteria found in cabbage (14 species belonging to 11 genera) were different from those on the knife. These results indicate that the sanitizer-injured coliforms would not be present on cabbage when transfer of the bacteria from harvest equipment to the cabbage is prevented or when a suitable sanitizer is used that causes death of the bacteria rather than sanitizer-induced cell injury.

Viability of sublethally injured coliform bacteria on fresh-cut cabbage stored in high CO 2 atmospheres following rinsing with electrolyzed water

The extent of sublethally injured coliform bacteria on shredded cabbage, either rinsed or not rinsed with electrolyzed water, was evaluated during storage in air and high CO2 controlled atmospheres (5%, 10%, and 15%) at 5°C and 10°C using the thin agar layer (TAL) method. Sublethally injured coliform bacteria on nonrinsed shredded cabbage were either absent or they were injured at a 64-65% level when present. Rinsing of shredded cabbage with electrolyzed water containing 25ppm available chlorine reduced the coliform counts by 0.4 to 1.1 log and caused sublethal injury ranging from 42 to 77%. Pantoea ananatis was one of the species injured by chlorine stress. When shredded cabbage, nonrinsed or rinsed with electrolyzed water, was stored in air and high CO2 atmospheres at 5°C for 7days and 10°C for 5days, coliform counts on TAL plates increased from 3.3-4.5 to 6.5-9.0 log CFU/g during storage, with the increase being greater at 10°C than at 5°C. High CO2 of 10% and 15% reduced the bacterial growth on shredded cabbage during storage at 5°C. Although injured coliform bacteria were not found on nonrinsed shredded cabbage on the initial day, injured coliforms at a range of 49-84% were detected on samples stored in air and high CO2 atmospheres at 5°C and 10°C. Injured cells were detected more frequently during storage at both temperatures irrespective of the CO2 atmosphere when shredded cabbage was rinsed with electrolyzed water. These results indicated that injured coliform bacteria on shredded cabbage, either rinsed or not rinsed with electrolyzed water, exhibited different degrees of injury during storage regardless of the CO2 atmosphere and temperature tested.

Changes in bacterial flora of Japanese cabbage during growth and potential source of flora.

Bacterial flora of cabbage were identified and enumerated during various stages of growth, and the potential sources of contamination in the field were determined. Bacterial counts increased from below the level of detection (2.4 log CFU/g) on seeds to 2.5 to 5.7 log CFU/g on seedlings. After transplanting, the counts of mesophilic aerobic bacteria on leaves decreased and then increased to 5.7 log CFU/g on outer leaves, 5.0 log CFU/g on middle leaves, and 3.0 log CFU/g on inner leaves at the harvesting stage. Counts of coliforms were below the level of detection during the growing period of the leaves. Bacteria isolated from cabbage seeds, seedlings, and leaves were soilborne organisms such as Bacillus, Curtobacterium, and Delftia and phytopathogenic organisms such as Pseudomonas, Pantoea, and Stenotrophomonas. These bacteria were found frequently in seeding machines, potting soil mix, soil, agricultural water, pesticide solutions mixed with the agricultural water, liquid fertilizers, and chemical fertilizers. Contamination from these environmental sites occurred throughout the cabbage growing period rather than only at the harvesting stage. These results indicate that use of clean water for irrigation and for mixing with pesticides and amendments from seeding to the harvesting stage is an important part of a good agricultural practices program for cabbage in Japan.