- Fruits: citrus
- Pest Management: biorational pesticides, botanical pesticides, chemical control, physical control
Stem-end rot (SER), caused by Lasiodiplodia theobromae, is often the most important postharvest disease of citrus fruit in warm and humid regions such as Florida. This disease is exacerbated by commercial degreening practices used to improve peel color of early season fruit. Currently, use of synthetic fungicides are the main method to control this disease. However, consumer concerns with pesticide usage and the potential for pathogen resistance to these fungicides limits their availability and use in many citrus-producing regions and is a concern for the southeast region. An alternate strategy to control SER on citrus fruit is needed. Heat treatments can be used to control postharvest decay by inhibiting pathogen growth and/or inducing fruit defense mechanism. The treatment is commercially attractive because fungicides are not involved. Essential oils are aromatic oily liquids obtained from plant organs and are usually considered safe and environmentally friendly. Many have been used for plant disease control. This one-year project will evaluate the efficiency of natural compounds combined with heat treatment to control SER on grapefruit. In vitro assays will screen essential oil compounds at different concentrations. Promising compounds will then be combined with a heat treatment and applied to fruit inoculated with L. theobromae or on naturally infected fruit to evaluate treatment efficacy and their effects on overall fruit quality during simulated commercial storage and marketing. Findings from this study are expected to provide an alternate method to control SER on grapefruit during postharvest handling and marketing.
Project objectives from proposal:
Objective 1. Screen essential oil compounds for their efficacy against L. theobromae in vitro. Study the effects of promising compounds to inhibit mycelium growth and conidial germination of L. theobromae.
Antunes and Cavaco (2009) demonstrated that the antifungal activity of essential oils is due to the specific action of their main component, like thymol in thyme, carvacrol in oregano, and eugenol in clove. In this project, we will test pure compounds from the following groups: aldehyde (benzaldehyde, cinnamaldehyde, and trans-cinnamaldehyde), phenol (thymol, carvacrol, and eugenol), and terpenoid (menthol, eucalyptol, and citral). All of them have been reported to inhibit postharvest decay caused by Penicillium spp., Alternaria spp., Botrytis cinerea, or Monilinia fructicola (Antunes and Cavaco, 2010).
Mycelial growth inhibition assay. Different essential oil compounds will be added to molten potato dextrose agar (PDA) before pouring into petri dishes. Mycelium plugs (5 mm in diameter) from actively growing area of the fungal colony will be transferred to the center of each plate. Unamended media will be used as control. Four replicates will be used for each compound. Plates will be incubated at 28 °C and colony diameter will be measured after 5 days.
Conidial germination inhibition assay. Conidial germination inhibition assays will be conducted for effective compounds. Conidial suspension will be prepared by flooding cultures with sterile distilled water, and conidial concentration will be adjusted to 106 ml-1 using a Hemocytometer. 100 μl of conidial suspension will be transferred to 50 ml of potato dextrose broth (PDB) with different concentrations of compounds and incubated on a shaker at 28 °C for 4 days. Unamended media will be used as the control. Conidia will be observed under a microscope to determine the germination rate. Four replicates will be used for each compound (Liu et al., 2010).
Objective 2. Using the most effective essential oil compounds from objective 1, evaluate their ability in both ambient and heated solutions to control SER on inoculated grapefruit and on naturally infected fruit.
The most effective essential oil compounds from objective 1 will be tested on inoculated fruit and naturally infected fruit. Previous reports demonstrated that short-duration (< 2min) hot water treatments (56 to 62 °C) can effectively reduce some types of postharvest decay (esp. Penicillium species). This project will test three time/temperature treatments combinations for control of L. theobromae on both inoculated and naturally infected fruit. The time/temperature treatments will be: 56 °C for 2 min, 59 °C for 1 min, and 62 °C for 30 seconds.
Fruit inoculation. Grapefruit of uniform size and color will be selected and inoculated by injecting 75 μl of conidial suspension into the fruit cavity using a pipette. The inoculated frit will be incubated at 28 °C and 80% relative humidity for 24 hours to allow infection development. Fruit inoculated with sterile distilled water will be used as the control (Brown and Burns, 1998).
Dip treatment. Dip treatment and evaluation will be done separately on inoculated fruit and naturally infected fruit. Fruit will be dipped into solutions within stainless steel tanks holding 40 liter of rapidly stirred solution. Heating will be conducted by using a gas burner with the temperature varying by ± 1°C. For each treatment, fruit will be placed in perforated plastic crates that allow solutions to circulate around the fruit. After treatment, the fruit will be air dried and degreened with 5 ppm ethylene at 28 to 29 °C and 90-95% relative humidity for two days. The fruit will then be stored at 10 °C with 90 % relative humidity. Incidence of SER will be observed during degreening and storage. Fruit dipped in water (25 °C) will be used as the control. Each treatment will have four replicates with 30 fruit each (John-Karuppiah et al., 2004).
Objective 3. Test the effects of essential oil compounds in ambient or heated solutions on fruit internal and external quality during postharvest handling and simulated marketing conditions.
Previous reports suggested hot water treatments can enhance fruit resistance to chilling injury and maintain overall fruit quality during storage. However, high temperature and high concentrations of essential oils may also result in peel injury/scalding. This objective will test treatment effects on fruit quality during postharvest handling and simulated marketing.
Quality evaluation. For quality evaluation, intact fruit will be treated as above and stored at 10 °C and 90 % relative humidity. Each treatment will have four replicates with 100 fruit each. Weight losses, peel color, peel puncture resistance (PPR), total soluble solids (TTS), and titratable acid (TA) will be measured every two weeks.
Peel color will be measured at three evenly spaced locations around the equator of each fruit using a chromameter. Color will be reported as a*/b*ratios where a* measures green (negative) to red (positive) and b* measures blue (negative) to yellow (positive). Peel puncture resistance (Newton) will be measured at three evenly spaced locations around the equator of each fruit using a texture analyzer with a 2 mm diameter, flat-tipped, cylindrical probe. The probe will be set to travel at a speed of 8 mm/s, and the maximum force exerted to puncture the peel will be recorded. Juice TSS (°Brix) will be measured using a refractometer. Juice TA (% citric acid) will be measured by titrating juice to PH 8.3 with sodium hydroxide (NaOH) using an automatic titrimeter (John-Karuppiah et al., 2004).
Statistical analysis All data will be collected and analyzed with one-way analysis of variance (ANOVA) using the statistical software of SAS 9.3 for windows. The effect of essential oil compounds, temperature, exposure duration and interactions between factors will be evaluated with a mixed model. Treatment means will be separated using the Least Significant Difference (LSD).