Brevipalpus mites (Acari: Tenuipalpidae) carrying citrus leprosis virus are considered serious quarantine pests. The objective of this research was to clarify the effectiveness of commonly used fruit cleaners, soaps, waxes, and mechanical brushing techniques (alone and in combination) on removal and/or mortality of mites (percentage of density reduction) from infested citrus fruits. Six bioassays were conducted with infested lemons, Citrus limon (L.) Burm.f. (Sapindales: Rutaceae), using non-virulent Brevipalpus yothersi Baker as a model species. In each bioassay, all stages (eggs, nymphs, and adults) of B. yothersi were recorded before and after treatment. Results indicated that none of the treatments provided 100% reduction of all stages of mites, as would be required for quarantine treatments. In general, mite reduction following single treatments (soap rinse, brushing, or waxing alone) was not significantly different from reduction obtained with a water drench control. However, several combination treatments were successful in achieving ~90% reduction of mites, particularly those that included application of a food-grade wax coating. Therefore, a combination of treatments, including a soap wash and mechanical brushing followed by a wax coating, may be the most effective method to achieve significant reduction of all stages of Brevipalpus mites from infested citrus.
The Brevipalpus (Acari: Tenuipalpidae) flat mites are highly polyphagous mites with a broad range of hosts, including citrus, grapes, and many ornamental plants (Childers & Rodrigues 2005). Their role as vectors of citrus leprosis virus, CiLV (Bastianel et al. 2006), has greatly increased their worldwide importance as quarantine pests. Symptoms of citrus leprosis include chlorotic lesions at the mite feeding sites on leaves, twigs, and fruit. In the absence of vector control, the local lesions can coalesce, girdle, and kill leaves and twigs, resulting in severe losses of production. CiLV is widespread in the Caribbean and Central and South America; in North America, it has been detected in Mexico (SINAVEF 2012) but not the United States (Childers et al. 2003). Because Mexico is a major supplier of fresh limes (Spreen 2000), including Persian lime Citrus latifolia (Tanaka ex Yu. Tanaka) Tanaka and key lime Citrus aurantifolia (Cristm.) Swingle (Sapindales: Rutaceae), there is concern that CiLV may enter the United States. Therefore, the United States Department of Agriculture (USDA) National Plant Disease Recovery System has identified citrus leprosis as a critical threat to U.S. agricultural production, particularly sweet orange Citrus sinensis (L.) Osbeck (Sapindales: Rutaceae), and has prepared a recovery plan for this high-priority disease (Hartung et al. 2013).
The standard post-harvest procedures for handling commercial citrus fruits include washing and waxing (Porat et al. 2000; Bosquez-Molina et al. 2004), but there is a current effort to find alternative treatments (e.g., environmentally friendly soaps) that can be used effectively against quarantine mites. Several of these treatments have been considered effective in post-harvest situations (Vincent et al. 2003). For instance, coatings with Primafresh 31® (Agro Pro Central America), Sta-Fresh 360HS® and Sta-Fresh 600® (Bornnet Corporation), and NatureSeal® (Mantrose-Haeuser, Co.), have been shown to cause approximately 90% mortality of Caribbean fruit fly Anastrepha suspensa (Loew) (Diptera: Tephritidae) larvae infesting grapefruits Citrus paradisi Mcfad. (Sapindales: Rutaceae) (Hallman et al. 1994). Gould & McGuire (2000) found that coating Persian limes with petroleumbased oils (AMPOL®, Caltex Australia, Sydney, New South Wales; and Sunspray Ultra-Fine Spray Oil®, Sunoco, Philadelphia, Pennsylvania, USA), a natural vegetable oil (Natural Organic oil, Custom Chemicides, Fresno, California, USA), and a soap (Mpede) achieved up to 94% mortality of 2 mealybugs, Planococcus citri (Risso) and Pseudococcus odermatti Miller & Williams (Hemiptera: Pseudococcidae). This result was considered effective as a post-harvest dip treatment but insufficient to provide quarantine security. Specific data are lacking regarding the effects of these procedures on citrus fruit infested with Brevipalpus mites as mitigating measures to reduce the risk of introduction. Therefore, the objective of this research was to evaluate the efficacy of commonly used fruit cleaners, soaps, waxes, and mechanical brushing techniques for removal and mortality of each life stage of Brevipalpus mites on citrus, using non-virulent B. yothersi Baker as a model.
Materials and Methods
MITE STOCK COLONY
Maintenance of B. yothersi populations in the laboratory followed the method of Campos & Omoto (2002) used for rearing of Brevipalpus phoenicis (Geijskes) on store-bought lemons C. limon (L.) Burm.f. (‘Meyer') (Sapindales: Rutaceae). Fruits were washed with distilled water, and after drying, the stylar area was dipped in heated wax, leaving an area of approximately 79 cm2 to confine the mites. Then, 20 to 30 adult mites, fieldcollected from infested leaves and branches of Viburnum odoratissimum Ker-Gawl. (Dipsacales: Adoxaceae), were transferred per fruit with the aid of a fine brush. Each mite population was kept at 20 to 25 individuals per fruit. The rearing room was maintained at 26 ± 2 °C and 75 to 80% RH, with a photoperiod of 12:12 h L:D. Fruits were renewed every 30 to 35 d.
Products and compounds tested in bioassays were obtained either directly from the manufacturers or from retail stores (Table 1). Six bioassays were conducted at different times of the year, and each bioassay was designated with a number. All stages (eggs, nymphs, and adults) of B. yothersi infesting lemons were counted under a dissecting microscope before treatment. Fifteen lemons were dipped in a 5,400 mL aqueous solution containing each product and dose listed in Table 2. The solution was kept under constant manual agitation for 60 s, and lemons were removed afterwards. Water temperature was maintained at 22 °C and pH at 7.0 (verified each time before lemons were placed into a solution). A control treatment consisted of 15 lemons dipped for 60 s in water. To determine if any mites were dislodged into the water after treatment, each solution was divided into 15 parts, sieved individually through a P4 18.5 cm Fisher® filter paper, and mite density and life stages determined under a microscope. Additional treatments for a bioassay were either manual brushing using bottle brushes (25 cm2) for 1 min or placing each lemon under the brushes of a commercial mite brushing machine (2836 SM, BioQuip Products, California, USA). Mites dislodged were collected in a Petri dish and counted afterwards.
List of products used in bioassays of post-harvest treatments against Brevipalpus yothersi.
Additionally, some treatments included application of Decco Wax® and Shellac® (Table 1) to the fruits using a paint brush. Each lemon was placed on a drying rack thereafter and held in a climatic chamber at 25 °C and approximately 75 to 80% RH. All B. yothersi stages remaining on the lemons were counted under a microscope at 1, 2, 3 and 5 d posttreatment to verify survival and reproduction. Adult and motile immature stages were considered to be dead after treatment if they did not walk when prodded with the tip of a fine brush. Because the effect of treatments on adults and nymphs included dislodging mites from the fruit as well as mite mortality, the data obtained for these 2 stages were expressed as the mean percentage of reduction of mites achieved with each treatment. The data obtained for eggs were expressed as the percentage of eggs dislodged after each treatment. For each bioassay and development stage, data were analyzed separately by 1-way analysis of variance (ANOVA) using a general linear model (SAS v. 9.3 SAS Institute, Inc., Cary, North Carolina, USA); significant ANOVAs were then followed by Fisher least significance difference (LSD) mean separation (P = 0.05). Percentage data were arcsine transformed before statistical analysis to correct for non-linearity of percentages.
Application of most soap and foam solutions (Mold Strip, Saf Foam, Fit, Rebel Green, and Veggie Wash) alone, fruit brushing alone, and mixed treatments with soaps followed by brushing did not result in significant reduction of adult mites 1 d after treatment (Table 2; Bioassays 1–4). However, significant reduction (P = 0.0001; F = 5.96; df = 29, 98) was obtained with 3 fruit treatments in Bioassay 5: application of Shellac alone (91% reduction), dipping in Environne soap solution followed by brushing and Decco Wax application (90% reduction), and dipping in Environne followed by brushing and Shellac coating (88% reduction). In Bioassay 6, there were significant differences among treatments (P = 0.007; F = 1.09; df = 20, 84), with moderate reductions obtained with Clorox alone (77%) and Clorox plus brushing (66%) and greater reductions obtained following treatments with Clorox plus brushing and Decco Wax (87%) and Environne plus brushing and Shellac (100%). Further reduction in adult densities recorded 2 to 5 d after treatment varied between 2 and 48%, but the results were not statistically significant for most treatments as compared with the untreated controls (water immersion) (Table 2).
Density reduction (mean % ± SE) of Brevipalpus yothersi adults on fruits dipped in soap, foam, and wax solutions with additional fruit brushing.
For motile immature stages, applications of Saf Foam, Rebel Green, and Veggie Wash (alone or in combination with brushing) did not result in significant reduction of infestation 1 d after treatment (Table 3; Bioassays 1, 2, and 4). Mold Strip plus brushing was effective (79% reduction) at a dilution of 1:2,000 (Bioassay 2; P = 0.0006; F = 2.94; df = 19, 70), but not at a dilution of 1:3,000 (Bioassay 1). Other treatments effective at reducing immature stages included Fit soap plus brushing (91%) and brushing alone (74%) in Bioassay 3 (P = 0.001; F = 16.82; df = 12, 42), and Environne plus brushing followed by coating with Decco Wax (59%) or Shellac (60%) in Bioassay 5 (P = 0.01; F = 3.45; df = 20, 84). In Bioassay 6, moderate reductions were observed following treatments with Shellac alone (79%) and Clorox plus brushing plus wax coating (60%); however, these results were not statistically different from the water control treatment, which had a fairly large percentage of reduction (37 ± 12%). Subsequent mortality of the immature stages 2 to 5 d after treatment was negligible.
Mortality (mean % ± SE) of Brevipalpus yothersi immature stages on fruits dipped in soap, foam, and wax solutions with additional fruit brushing.
Examination of eggs 1 d after treatment indicated that Mold Strip (Table 4, Bioassays 1 and 2) and Fit soap (Bioassay 3) were not effective treatments for dislodging eggs, even when combined with manual brushing. However, percentage of egg reduction was significant for a variety of treatments tested in Bioassay 4 (P = 0.001; F = 3.72; df = 20, 84), Bioassay 5 (P = 0.001; F = 3.89; df = 20, 84), and Bioassay 6 (P = 0.0001; F = 4.61; df = 21, 98). The greatest reduction was obtained with Environne plus brushing and Shellac coating (91%, Bioassay 6), followed by Rebel Green plus brushing (89%, Bioassay 4) and Environne plus brushing and Decco Wax (89%, Bioassay 5).
Number of eggs dislodged after treatment of mite-infested lemons with soaps, waxes, and brushing
Fruit washing, waxing, and brushing are currently among the treatments used for citrus imported from Mexico. The objectives of this study were to determine whether fruit washing combined with brushing and use of soaps could reduce numbers of B. yothersi from citrus fruits. In our study, a combination of treatments that involved using the soap Environne (1:1,000 dilution) followed by brushing and coating with Shellac wax caused 88 to 100% reduction in adult density. The general trend observed was that mechanical brushing alone caused 40 to 50% reduction, but the addition of a wax coating improved efficacy up to 60 to 91% reduction.
One day after treatment, the percentage of density reduction obtained for adults ranged between 28 and 85%, whereas the ranges for immature stages and eggs were between 23 and 50% and between 28 and 59%, respectively. The use of soaps and brushing, or soaps by themselves, reduced adult mite densities by only 14 to 46%, as compared with the water control. The concentrations of soaps and foams (1:1,000 dilution) used in this study were actually higher than those typically used in packing houses (1:3,000 dilution), underscoring the poor level of mite control provided by standard fruit washing methods.
Another objective of this study was to determine the efficacy of brushing in mite removal. The results obtained in the different bioassays using only water drenches followed by brushing showed that brushing improved mite removal by only 3 to 27% over the water drench alone. The study also investigated the efficacy of waxing in mite reduction. In general, mite densities were low after waxing was performed in all the bioassays. One outcome of this study was that a combination of different treatments, i.e., soaps followed by brushing and then waxing, resulted in fewer mites left on fruits compared with the water drench, which was considered the control. Consequently, a combination of treatments may be most effective to dislodge or cause a significant reduction of all developmental stages.
In a time of increased awareness among consumers that many of the chemical treatments of fruit and vegetables used to control insects, diseases, and physiological disorders are potentially harmful to humans, there is an urgent need to develop effective, non-damaging physical treatments for insect disinfection and disease control in fresh horticultural products (Fallik 2003). The treatments tested here did not provide 100% reduction of all stages of mites as is required for quarantine treatments; therefore, additional treatments should be evaluated, such as radiation (Hallman 1998), controlled atmosphere treatments (Shijum & Mitcham 1998), and fumigant activity of essential oils (Lim et al. 2011) and ethyl formate (Simpson et al. 2007). These treatments have been shown to provide effective control of tetranychid mites and should be evaluated for efficacy on Brevipalpus mites.
We thank A. Roda (APHIS-PPQ), Paul Kendra (USDA-ARS), and J. C. Rodrigues-Verle (University of Puerto Rico) for their suggestions to improve this manuscript. This work was supported by an APHIS PPQ grant to J. E. P. Note: Mention of proprietary products does not constitute endorsement for their exclusive use by the University of Florida and USDA.