The sensitivity of B. cinerea conidia to SO2 increases two- to fourfold for every 10°C increment between 0 and 32°C, because of the effect of temperature on SO2 absorption on the fruit, fungi and surrounding packaging . SO2 has mainly been used to control gray mold disease of table grapes, but the low application rates applied as part of the total utilization technique have not prevented the emergence and spread of brown spot during cold storage . The objectives of our two-part study were to evaluate, in vitro, the effect of temperature and SO2 concentration over time applications on fungal colony growth of three Cladosporium species , and to determine the efficacy of different SO2 concentrations over time in inhibiting the growth of Cladosporium species on artificially inoculated Redglobe berries.Cladosporium species were isolated from brown spot–symptomatic berries grown in Delano, California, from 2013 to 2015. Species isolates were obtained from symptomatic tissue placed on 150 by 15 mm petri plates with 3% rose bengal potato dextrose agar amended with 500 ppm tetracycline and 300 ppm streptomycin. Cladosporium colonies were hyphal tipped after 7 days to produce pure cultures for species identification. PCR amplification of the actin gene using primers ACT-512 and ACT- 783 and DNA sequencing confirmed the identity of species .
Species isolates were maintained in Dr. W. Douglas Gubler’s laboratory, Department of Plant Pathology, UC Davis, square pot as of June 2016. Pathogenicity was demonstrated when species isolates were inoculated and re-isolated from lesions on Crimson Seedless berries following standard protocols . Asymptomatic, nonwounded berries were removed from clusters with the pedicel still intact by clipping with sterile scissors. Berries were then vigorously washed in a 0.5% potassium chloride and 0.1% Tween 20 solution to remove surface debris. Berries were surface disinfected first in a 70% ethanol solution for 30 seconds, then in a 10% bleach solution for 5 minutes, and dried in a sterile laminar flow hood . Dry berries were aseptically distributed in triplicate on sterilized polyethylene chambers on sterilized polyethylene grids at 2°C with high relative humidity . RH was obtained with paper towels moistened with sterile deionized water and placed below a plastic grid. RH was measured with a humidity sensor .To determine the effect of temperature and SO2 on fungal growth, potato dextrose agar plates were inoculated with one of three species: C. ramotenellum, C. cladosporioides or C. limoniforme. Inoculum was grown on 2% PDA at 23°C for 5 to 7 days. Plugs of colonized PDA obtained with a sterile 4-mm cork borer were placed on a 2% PDA plate, spore side down. The baseline for all measurements was 4 mm. Inoculated plates were incubated in a polyethylene chamber at 23°C for 13 hours. The petri plates were moved to a fumigation chamber and exposed to the three SO2 treatments while still lidded.
SO2 concentrations were measured using passive colorimetric dosimeter tubes and a portable SO2 detector that continuously measured SO2 concentration inside the fumigation chambers . Dosimeter tubes were taped to chamber walls opposite the SO2 flow as well as inside of a petri plate. Untreated controls were inoculated as previously described, received no SO2 exposure but were treated and incubated identically. For each species, three petri plates were inoculated and treated in triplicate and were incubated at 2°C and −2°C for up to 32 days after treatment. Radial measurements of fungal colonies were taken to assess the effect of SO2 concentration over time on mycelial growth. Colony measurements were taken from the reverse side of the lidded petri plate. Using a polyethylene ruler, two perpendicular diameter measurements were made and averaged to determine overall colony size. The diameter of the mycelium plug inoculum was included as the minimal measurement for all treatments. Measurements were taken every 6 to 8 days during incubation at 2°C and −2°C.Inoculated berries were not wounded prior to inoculation. Inoculum was prepared with 14-day-old cultures grown on PDA. Spores were suspended in 0.5% potassium chloride and 0.1% Tween 20 in sterile distilled water. Spore density was determined using a hemocytometer, and the suspension was adjusted to 1 × 107 spores/mL by the addition of sterile deionized water. The shoulder of each berry was inoculated by placing 10 μL of spore suspension within a 4-mm Vaseline ring, to prevent the inoculum from moving. Berries were positioned with the inoculated shoulder facing up. Untreated controls consisting of berries inoculated with sterile deionized water were included for each temperature and SO2 treatment. Berry lesions on unwounded berries were measured after 28 days of storage at 2°C.Twenty-four hours after berries were inoculated with one of the three Cladosporium species, they were exposed to three concentration-over-time treatments of gaseous SO2: 100 ppm-h , 200 ppm-h or 400 ppm-h . SO2 concentrations were measured as previously mentioned. Dosimeter tubes were taped to chamber walls opposite the SO2 flow. For both studies, fumigation chambers were Sterilite containers modified to allow for a rubber tube to flow SO2 into the chamber.
Chambers were placed inside a biological safety cabinet during treatment. Chamber lids were opened once a treatment was concluded and the inoculated berries on a grid/mycelial plugs on petri plates were placed into polyethylene chamber to be stored in cold storage.Radial growth of Cladosporium colonies was analyzed using a linear mixed model approach for all three species using the R package lme4 . The mixed model used a Gaussian error distribution and consisted of SO2 treatments, temperature and species as fixed effects and replicate as a random effect. We calculated the estimated marginal means and computed all pairwise comparisons using Tukey’s honestly significant difference test. For the berry study, we analyzed the proportion of infected berries with a linear mixed model approach using a Gaussian error distribution for all species, with SO2 treatments and species as fixed effects and replicate as a random effect and computed all pairwise comparisons using Tukey’s HSD test.Radial colony growth of the three Cladosporium species on petri plates with PDA was significantly reduced by the 400 ppm-h SO2 treatment, as seen in figures 2 and 3. The 400 ppm-h concentration was most effective against C. cladosporioides when petri plates were incubated at −2°C, which resulted in no radial growth. The 400 ppm-h was also effective in slowing C. cladosporioides colony growth at 2°C; radial growth grew only from 4 to 9 mm on average. Radial colony growth of C. limoniforme and C. ramotenellum was also slowed down at 400 ppm-h,growing from 4 to 5 mm and 4 to 6 mm, respectively, at −2°C. However, the same treatment was less effective at 2°C, where, on average, fungal radial growth reached 10 mm for C. limoniforme and 12 mm for C. ramotenellum after 30 days. At the lower SO2 ppm-h , there was no significant difference in radial colony growth between any of the treated species and the untreated controls by 30 days at 2°C. All species after 10 days at −2°C had reduced colony growth for all concentrations compared with those incubated at 2°C. The colony growth of C. cladosporioides at −2°C was slower than that of the other species; and it was also slower than the colony growth of C. cladosporioides incubated at 2°C, illustrating the effect of temperature on rate of growth.In the berry study, all Cladosporium species caused disease in untreated control berries: C. cladosporioides caused disease in 65% of berries, C. limoniforme in 55% berries and C. ramotenellum in 75% of berries . By contrast, SO2 treatments significantly reduced disease incidence by Cladosporium species on inoculated Redglobe berries. The 100 ppm-h treatment reduced disease incidence to less than 25% of berries for all species. The 200 ppmh treatment was the most effective, blueberries in containers in that it eliminated disease for all three Cladosporium species tested. In the 400 ppm-h treatment, there was less than 5% disease incidence in berries inoculated with C. ramotenellum and no infection occurred in berries inoculated with C. cladosporioides or C. limoniforme. Nevertheless, there was no statistically significant difference in the proportion of infected berries between the 100, 200 and 400 ppm-h SO2 treatments of the three Cladosporium species tested .
Our results demonstrate that a single application before cold storage of at least 200 ppm-h SO2 at cold storage temperatures may be an effective tool for reducing brown spot disease incidence on grape berries, although the ability of mycelium of Cladosporium species to grow on PDA growth medium in petri plates with an SO2 application was somewhat variable. We have demonstrated SO2 of 200 ppm-h and 400 ppm-h essentially prevent disease development in Redglobe berries from the three Cladosporium species tested. C. cladosporioides exhibited tolerance at 100 ppm-h, whereas 100 ppm-h was effective against C. ramotenellum and C. limoniforme. While mycelial growth on PDA was reduced with a treatment of 400 ppm-h SO2, mycelial growth was not completely eliminated. Additionally, C. ramotenellum grows faster than the other species on a petri plate with PDA, regardless of temperature or SO2 treatment. Unsurprisingly, in the berry study, inoculation of C. ramotenellum resulted in the highest disease incidence in the untreated control berries. Our study also confirmed the critical importance of maintaining table grapes below 0°C and above their freezing point to maximize their storage life potential .These results are promising for the control of brown spot during long-term storage and export of Redglobe table grapes. Although not much is known about the natural berry infection by Cladosporium, we do know that infection can occur on the surface through the epidermis and may also be a latent infection where infection starts from within the berry. Berries used in our experiments were surface sterilized before inoculation so we could be sure that infection occurred through the epidermis with our inoculum; thus, we can conclude the SO2 application prevented disease from of the Cladosporium surface inoculum. However, SO2 may not be adequate to prevent infection resulting from latent infection, causing internal rot of the berry and inoculum production, but may inhibit the resulting spread if applied after the emergence of fungal growth . This work demonstrates a potential management strategy of brown spot post-harvest. In practice, multiple factors can influence the amount of gaseous SO2 that comes into contact with the berry surface, including air flow in storage, sorption of packing materials and the amount of condensation on berries, which can absorb gasses. Additionally, a single application of SO2 is typically not as effective as multiple SO2 applications, but multiple applications may not be feasible during transport . Total utilization technique focused on gray mold control applies SO2 at 100 to 150 ppm-h, which may be too low to be effective against brown spot, especially under cold storage conditions. Future studies should be done to improve sampling and the detection of brown spot in the field prior to harvest as well as during post harvest, so this research can be applied to known compromised lots in transport and better determine the efficacy of this practice under commercial conditions.Macrotunnel production has been increasing in coastal counties of California and is poised for expansion due to its recent adoption as a standard practice by the U.S. Department of Agriculture . In high tunnel production, crops are grown within plastic-covered structures to enhance crop performance, extend production seasons and to protect crop quality. While most caneberries, some strawberries, cut flowers, herbs and leafy greens are widely grown under plastic in California, contributing $1 billion to the state’s economy, in other states small fruits, melons and nuts are also grown in high tunnel systems. This interest in plasticulture tunnels is driven by many factors: increased production due to season expansion; reduced exposure to deleterious weather events; consumer demand for fresh, local produce; and national interest in reducing transportation-related greenhouse gas emissions, amongst other concerns. Unfortunately, it comes at a time when climate-induced weather pattern changes, particularly shorter-duration, higher-frequency storm events, are expected to become the norm . The plastic covering hoop structures can reduce the available permeable surface of a field’s production area by over 90%, which increases the volume of water likely to run off a field in a storm event . During rains, water intercepted by plastic covers is channeled into post rows , accelerating soil erosion, especially on slopes, which ultimately degrades surface water quality. In California, surface water quality is regulated by the State Water Resources Control Board through the Irrigated Lands Regulatory Program .