Dennis A. Margosan

Control of citrus green mold by carbonate and bicarbonate salts and the influence of commercial postharvest practices on their efficacy

The toxicity to Penicillium digitatum and practical use of carbonate and bicarbonate salts to control green mold were determined. The effective dose (ED50) concentrations to inhibit the germination of P. digitatum spores of sodium carbonate (SC), potassium carbonate, sodium bicarbonate (SBC), ammonium bicarbonate, and potassium bicarbonate were 5.0, 6.2, 14.1, 16.4, and 33.4 mM, respectively. All were fungistatic because spores removed from the solutions germinated in potato dextrose broth. SC and SBC were equal and superior to the other salts for control of green mold on lemons and oranges inoculated 24 h before treatment. When sodium content and high pH must be minimized, SBC could replace SC. Furthermore, because a higher proportion of NaOCl would be present in the active hypochlorous acid at the lower pH of SBC compared to SC, sanitation of the SBC solution should be easier to maintain. NaOCl (200 μg/ml) added to SBC at pH 7.5 improved green mold control. Rinse water as high as 50 ml per fruit applied after SC did not reduce its effectiveness; however, high-pressure water cleaning after SC did. Conversely, high-pressure water cleaning of fruit before SC improved control of green mold. The risk of injury to fruit posed by SC treatment was determined by immersing oranges for 1 min in 3% (wt/vol) SC at 28, 33, 44, 50, 56, or 61°C (±1°C) and followed by storage for 3 weeks at 10°C. Rind injuries occurred only after treatment at 56 and 61°C. The risk of injury is low because these temperatures exceed that needed for control of green mold. SC was compatible with subsequent imazalil and biological control treatments.

Combination of Hot Water and Ethanol to Control Postharvest Decay of Peaches and Nectarines

Spores of Monilinia fructicola or Rhizopus stolonifer were immersed in water or 10% ethanol (EtOH) for 1, 2, 4, or 8 min at temperatures of 46 or 50°C to determine exposure times that would produce 95% lethality (LT95). EtOH reduced the LT95 by about 90%. Peaches and nectarines infected with M. fructicola were immersed in hot water alone or with EtOH to control decay. EtOH significantly increased the control of brown rot compared to water alone. Immersion of fruit in water at 46 or 50°C for 2.5 min reduced the incidence of decayed fruit from 82.8% to 59.3 and 38.8%, respectively. Immersion of fruit in 10% ethanol at 46 or 50°C for 2.5 min further reduced decay to 33.8 and 24.5%, respectively. Decay after triforine (1,000 μg ml-1) treatment was 32.8%. Two treatments, 10% EtOH at 50°C for 2.5 min and 20% EtOH at 46°C for 1.25 min, were selected for extensive evaluation. The flesh of EtOH-treated fruit was significantly firmer, approximately 4.4 N force, than that of control fruit among seven of nine cultivars evaluated. No other factor evaluated was significantly influenced by heated EtOH treatments. The EtOH content of fruit treated with 10 or 20% EtOH was approximately 520 and 100 μg g-1 1 day and 14 days after treatment, respectively.

Influence of pH and NaHCO3 on Effectiveness of Imazalil to Inhibit Germination of Penicillium digitatum and to Control Postharvest Green Mold on Citrus Fruit

In vitro, spores of Penicillium digitatum germinated without inhibition between pH 4 and 7, but were inhibited at higher pH. Estimated concentrations of imazalil (IMZ) in potato-dextrose broth-Tris that caused 50% reduction in the germination of spores (ED50) of an IMZ-sensitive isolate M6R at pH 4, 5, 6, and 7 were 0.16, 0.11, 0.015, and 0.006 μg/ml, respectively. ED50 IMZ concentrations of an IMZ-resistant isolate D201 at pH 4, 5, 6, and 7 were 5.9, 1.4, 0.26, and 0.07 μg/ml, respectively. The natural pH within 2-mm-deep wounds on lemon was 5.6 to 5.1 and decreased with fruit age. IMZ effectiveness to control green mold and its residues increased with pH. The pH in wounds on lemon fruit 24 h after immersion in 1, 2, or 3% NaHCO3 increased from pH 5.3 to 6.0, 6.3, and 6.7, respectively. NaHCO3 dramatically improved IMZ performance. Green mold incidence among lemon fruit inoculated with M6R and treated 24 h later with IMZ at 10 μg/ml, 1% NaHCO3, or their combination was 92, 55, and 22%, respectively. Green mold among lemon fruit inoculated with D201 and treated 24 h later with water, IMZ at 500 μg/ml, 3% NaHCO3, or their combination was 96.3, 63.0, 44.4, and 6.5%, respectively. NaHCO3 did not influence IMZ fruit residue levels.

Evaluation of Heated Solutions of Sulfur Dioxide, Ethanol, and Hydrogen Peroxide to Control Postharvest Green Mold of Lemons

Lemon fruit were inoculated with spores of Penicillium digitatum and immersed in solutions of ethanol. sulfur dioxide, or hydrogen peroxide to control postharvest green mold. Green mold incidence and fruit injury were assessed after treatments employing various combinations of concentration, duration of treatment, temperature, and post-treatment rinses. Heating of the solutions was needed to attain acceptable efficacy. Sulfur dioxide and ethanol controlled green mold without injury to fruit, whereas hydrogen peroxide did not effectively control green mold and caused unacceptable injury to fruit. Treatments selected for extensive evaluation were immersion in 10% ethanol at 45 degrees C for 150 s without rinsing, or in 2% sulfur dioxide at 45 degrees C for 150 s followed by two fresh water rinses. These treatments were compared with two existing decay control methods: immersion in 3% sodium carbonate at 45 degrees C for 150 s followed by two fresh water rinses, or in 1,000 microgram/ml imazalil at 25 degrees C for 60 s. Lemons were inoculated at 20 degrees C then incubated for 12, 24, 48, or 60 h before treatments were applied. Efficacy of sulfur dioxide and ethanol treatments was comparable to that of sodium carbonate and imazalil. Sulfur dioxide and ethanol did not injure the fruit and their residues were low. The sulfur dioxide content of lemons immediately after treatment was less than 1 microgram/ml. The ethanol content of lemons analyzed immediately after ethanol treatment was 58.6 (plus or minus 9.6) microgram/ml and 24.4 (plus or minus 11.7) microgram/ml after storage for 7 days at 20 C. The ethanol content of untreated fruit was 3.3 microgram/ml.

Effect of Chitosan Dissolved in Different Acids on Its Ability to Control Postharvest Gray Mold of Table Grape

Chitosan is a natural biopolymer that must be dissolved in an acid solution to activate its antimicrobial and eliciting properties. Among 15 acids tested, chitosan dissolved in 1% solutions of acetic, L-ascorbic, formic, L-glutamic, hydrochloric, lactic, maleic, malic, phosphorous, and succinic acid. To control gray mold, table grape berries were immersed for 10 s in these chitosan solutions that had been adjusted to pH 5.6. The reduction in decay among single berries of several cultivars (Thompson Seedless, Autumn Seedless, and grape selection B36-55) inoculated with Botrytis cinerea at 1 x 10(5) conidia/ml before or after immersion in chitosan acetate or formate, followed by storage at 15 degrees C for 10 days, was approximately 70%. The acids alone at pH 5.6 did not control gray mold. Decay among clusters of two cultivars (Thompson Seedless and Crimson Seedless) inoculated before treatment was reduced approximately 60% after immersion in chitosan lactate or chitosan acetate followed by storage for 60 days at 0.5 degrees C. The viscosity of solutions was 1.9 centipoises (cp) (ascorbate) to 306.4 cp (maleicate) and the thickness of chitosan coating on berries was 4.4 microm (acetate) to 15.4 microm (ascorbate), neither of which was correlated with solution effectiveness. Chitosan acetate was the most effective treatment which effectively reduced gray mold at cold and ambient storage temperatures, decreased CO2 and O2 exchange, and did not injure the grape berries.

Influence of Concentration of Soda Ash, Temperature, and Immersion Period on the Control of Postharvest Green Mold of Oranges

Oranges were inoculated with spores of Penicillium digitatum, the citrus green mold pathogen, and immersed 24 h later in heated soda ash (Na2CO3, sodium carbonate) solutions to control postharvest citrus green mold. Oranges were immersed for 1 or 2 min in solutions containing 0, 2, 4, or 6% (wt/vol) soda ash heated to 35.0, 40.6, 43.3, or 46.1°C. After 3 weeks of storage at 10°C, the number of decayed oranges was determined. Soda ash significantly controlled green mold in every test. The most effective control of green mold was obtained at 40.6 or 43.3°C with 4 or 6% soda ash. The concentration of soda ash greatly influenced efficacy, whereas the influences of temperature or immersion period on soda ash efficacy were small. Solutions of 4 and 6% soda ash were similar in efficacy and provided superior control of green mold compared with 2% soda ash. The control of green mold by soda ash solutions heated to 40.6 or 43.3°C was slightly superior to control by solutions heated to 35.0 or 46.1°C. The control of green mold by 1-min immersion of inoculated oranges in heated soda ash solutions was inferior to immersion for 2 min, but the magnitude of the difference, particularly with 6% soda ash, was small. A second-order response surface model without interactions was developed that closely described the influence of soda ash concentration, temperature, and immersion period on efficacy. The efficacy of soda ash under commercial conditions was better than that predicted by the model, probably because under commercial conditions the fruit were rinsed less thoroughly with water after treatment than in laboratory tests. The primary finding of this work was that soda ash controlled 24-h-old green mold infections at commercially useful levels using shorter immersion periods and lower temperatures than those recommended by other workers for the use of soda ash on lemons. The oranges were not visibly injured in any test.

Improved Control of Green Mold of Citrus with Imazalil in Warm Water Compared with Its Use in Wax

The effectiveness of imazalil for the control of citrus green mold (caused by Penicillium digitatum) improved significantly when fruit were treated with heated aqueous solutions of the fungicide as compared with the current commercial practice of spraying wax containing imazalil on fruit. When applied at less than 500 μg·ml-1 in solutions heated to 37.8°C, control of postharvest green mold of citrus was significantly superior to applications of 4,200 μg·ml-1 imazalil in wax sprayed on fruit at ambient temperatures. The improvement in imazalil efficacy was obtained with a decrease in fungicide residues on the fruit. Residues of about 3.5 μg·g-1 imazalil deposited by the application of imazalil in wax reduced the incidence of green mold on lemons from 94.4% among untreated controls to 15.1%, whereas an equal residue deposited by passing fruit through heated aqueous imazalil reduced green mold incidence to 1.3%. Similar differences were found in tests with oranges. Residues of 2 and 3.5 μg·g-1 imazalil were needed to control the sporulation of P. digitatum on oranges and lemons, respectively. The mode of application of imazalil did not influence control of sporulation. The influence of immersion time, imazalil concentration, and solution temperature on imazalil residues on oranges and lemons was determined in tests using commercial packing equipment, and a model that describes residue deposition was developed. Residues after a 30- or 60-s treatment in heated aqueous imazalil were sufficient to control sporulation, but residues after 15-s treatments were too low and required an additional application of 1,070 μg·ml-1 imazalil in wax to deposit an amount of imazalil sufficient to control sporulation. An imazalil-resistant isolate of P. digitatum was significantly controlled by heated aqueous imazalil. The incidence of green mold of navel oranges was reduced from 98.8 to 17.4% by treatment in 410 μg·ml-1 imazalil at 40.6°C for 90 s. However, control of the resistant isolate required imazalil residues on the fruit of 7.9 μg·g-1, which is within the U.S. tolerance of 10 μg·g-1 but above the 5 μg·g-1 tolerance of some countries that import citrus fruit from the United States.

Impact of Postharvest Hot Water or Ethanol Treatment of Table Grapes on Gray Mold Incidence, Quality, and Ethanol Content

The influence of brief immersion of grape berries in water or ethanol at ambient or higher temperatures on the postharvest incidence of gray mold (caused by Botrytis cinerea) was evaluated. The incidence of gray mold among grape berries that were untreated, or immersed for 1 min in ethanol (35% vol/vol) at 25 or 50°C, was 78.7, 26.2, and 3.4 berries/kg, respectively, after 1 month of storage at 0.5°C and 2 days at 25°C. Heated ethanol was effective up to 24 h after inoculation, but less effective when berry pedicels were removed before inoculation. Rachis appearance, epicuticular wax content and appearance, and berry shatter were unchanged by heated ethanol treatments, whereas berry color changed slightly and treated grape berries were more susceptible to subsequent infection. Ethanol and acetaldehyde contents of grape berries were determined 1, 7, and 14 days after storage at 0.5°C following treatment for 30 or 90 s at 30, 40, or 50°C with water, or 35% ethanol. Highest residues (377 μg/g of ethanol and 13.3 μg/g of acetaldehyde) were in berries immersed for 90 s at 50°C in ethanol. Among ethanol-treated grape berries, the ethanol content declined during storage, whereas acetaldehyde content was unchanged or increased. Untreated grape berries initially contained ethanol at 62 μg/g, which then declined. Acetaldehyde content was 0.6 μg/g initially and changed little during storage.

Reduction of Microbial Populations on Prunes by Vapor Phase Hydrogen Peroxide

Vapor-phase hydrogen peroxide (VPHP) was used to disinfect prunes. Concentrated hydrogen peroxide solution (35%, wt/wt) was volatilized into a stream of dried air to approximately 3.1 mg/l (wt/vol) of hydrogen peroxide. Dried prunes obtained from commercial dehydrators were treated with VPHP and compared to untreated prunes. Microbial populations were determined for treatment comparisons. Untreated dried prune microbial populations were 155, 107, and 111 CFU/g of prunes on aerobic plate count agar, potato dextrose agar, and dichloran rose bengal agar, respectively. In contrast, VPHP-treated prune microbial populations were reduced to near zero on all media after 10 minutes of VPHP exposure. The color of prunes exposed for 20 min or longer, however, showed oxidation damage. No hydrogen peroxide residues were detected 90 days after treatment.

Mutation at β-Tubulin Codon 200 Indicated Thiabendazole Resistance in Penicillium digitatum Collected from California Citrus Packinghouses

Thiabendazole (TBZ) is commonly applied to harvested citrus fruit in packinghouses to control citrus green mold, caused by Penicillium digitatum. Although TBZ is not used before harvest, another benzimidazole, thiophanate methyl, is commonly used in Florida and may be introduced soon in California to control postharvest decay of citrus fruit. Isolates from infected lemons and oranges were collected from many geographically diverse locations in California. Thirty-five isolates collected from commercial groves and residential trees were sensitive to TBZ, while 19 of 74 isolates collected from 10 packinghouses were resistant to TBZ. Random amplified polymorphic DNA analysis indicated that the isolates were genetically distinct and differed from each other. Nineteen TBZ-resistant isolates and a known TBZ-resistant isolate displayed a point mutation in the β-tubulin gene sequence corresponding to amino acid codon position 200. Thymine was replaced by adenine (TTC → TAC), which changed the phenylalanine (F) to tyrosine (Y). In contrast, for 49 TBZ-sensitive isolates that were sequenced, no mutations at this or any other codon positions were found. All of the isolates of P. digitatum resistant to TBZ collected from a geographically diverse sample of California packinghouses appeared to have the same point mutation conferring thiabendazole resistance.