Performance of Entomopathogenic Fungal-Based Insecticides against the Citrus Mealybug (Hemiptera: Pseudococcidae) on Coleus (Lamiales: Lamiaceae) Plants under Greenhouse Conditions¹

Citrus mealybug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae), is an important insect pest of greenhouse-grown horticultural crops (McKenzie 1967, Pillai 2016), causing direct plant damage when feeding on plant leaves, stems, flowers, and fruits (Franco et al. 2009, Godfrey et al. 2002, McKenzie 1967). In addition, while feeding, they excrete honeydew that serves as a substrate for black sooty mold (Mani and Shivaraju 2016) inhibits plant photosynthesis and diminishes the aesthetic quality of the plants (Charles 1982, Mani and Shivaraju 2016).

One female citrus mealybug adult can produce more than 400 eggs during her lifetime (Copeland et al. 1985). As such, managing citrus mealybug populations by killing the nymphs before they can develop into adults is critical (Pillai 2016). Insecticides are typically used to manage citrus mealybug populations in greenhouse production systems (Franco et al. 2009, Parrella 1999, Pillai 2016).

Studies have demonstrated that systemic insecticides applied to the growing medium are not effective in managing citrus mealybug populations (Herrick and Cloyd 2017, Herrick et al. 2019). Therefore, greenhouse producers must rely on foliar applications of contact insecticides. However, contact insecticides have limited effectiveness against citrus mealybugs because (a) later instars and adults have a waxy covering that repels water, which protects them from exposure to insecticide sprays (Copeland et al. 1985, Gupta et al. 2021, McKenzie 1967, Venkatesan et al. 2016); (b) thorough coverage of aboveground plant parts (leaves and stems) with spray applications where citrus mealybugs feed is difficult; and (c) citrus mealybugs feed in locations on plants that reduce their exposure to insecticide spray applications (Charles 1982, Franco et al. 2009, Herrick and Cloyd 2017). Insecticides cause higher mortality on early-instar nymphs because early-instar nymphs do not have the waxy covering that is associated with later-instar nymphs and adults (Ahmed and Abd-Rabou 2010, Charles 1982, Pillai 2016, Venkatesan et al. 2016). Nonetheless, thorough coverage and application frequency are important to effectively manage citrus mealybug populations with contact insecticides (Dibble 1962, Martini et al. 2012, Radosevich and Cloyd 2021, Venkatesan et al. 2016).

Entomopathogenic fungi are the active ingredients in commercially available entomopathogenic fungal-based insecticides registered for use against aphids, whiteflies, and the citrus mealybug. These fungi include Beauveria bassiana strain GHA (BotaniGard^®, Laverlam International Corp., Butte, MT), B. bassiana strain PPRI 5339 (Velifer™, BASF Corp., Research Triangle Park, NC), and Isaria fumosorosea Apopka strain 97 (Ancora^®, OHP, Inc., Bluffton, SC). Commercial preparations of entomopathogenic fungi are contact microbial insecticides composed of formulated fungal conidia. Conidia can sporulate on the surface of host insects; penetrate the cuticle via the release of proteases, chitinases, and other enzymes; and enter and grow in the hemocoel and various tissues. Death of the host is usually attributed to mechanical damage, release of fungal toxins, or both (Clarkson and Charnley 1996, Ortiz-Urquiza and Keyhani 2013). Infection and mortality are longer (in days) than commonly used contact insecticides (Zhang et al. 2021).

There is no information on the effectiveness of entomopathogenic fungal-based insecticides against the citrus mealybug under greenhouse conditions. Therefore, the objective of our study was to determine the efficacy of spray applications of entomopathogenic fungal-based insecticides on the citrus mealybug feeding on coleus, Solenostemon scutellarioides (L.) Codd (Lamiales: Lamiaceae), plants under greenhouse conditions.

The citrus mealybugs used in our study were obtained from a laboratory colony maintained on butternut squash, Cucurbita maxima Duchesne (Cucurbitales: Cucurbitaceae), at 22–27°C, 50–60% relative humidity (RH), and constant light (Department of Entomology, Kansas State University, Manhattan). The study described herein consisted of four experiments conducted in a research greenhouse at Kansas State University from 2020 to 2022. Voucher specimens (number 254) are deposited in the Kansas State University Museum of Entomological and Prairie Arthropod Research. Coleus was used in the four experiments because this plant is susceptible to the citrus mealybug (Ghorbanian et al. 2011, Sridhar et al. 2016). The treatments and associated information for all four experiments are listed in Table 1. All the treatments used the label rate for the citrus mealybug.

Experiment 1. Thirty coleus (‘Redhead’) plugs (e.g., young plants as either seedlings or cuttings grown as single units in modular trays) were obtained from Ball Horticultural Co. (West Chicago, IL), with plugs supplied by Tagawa Greenhouses, Inc. (Brighton, CO). Upon receipt, plugs were transplanted into 15.2-cm-diameter containers with a growing medium (Berger^® BM1, Saint-Modeste, Quebec, Canada) composed of 75–85% course sphagnum peat moss, perlite, vermiculite, and a wetting agent. Plants were grown for 84 d and fertilized 75 d after transplanting with Miracle-Gro^® Water Soluble All Purpose Plant Food (Scotts Miracle-Gro Products, Inc., Marysville, OH) consisting of 24–3.5–13.2 (N–P–K) at a rate of 15 g/3.8 L. Plants were irrigated with approximately 500 ml of tap water as needed.

Treatments were arranged in a randomized complete block design, with blocks accounting for different numbers of spray applications (one or two). Treatments were BotaniGard 22WP, Ancora, and a water control (Table 1), with one or two applications (blocks) made at weekly intervals and five replications per treatment. Coleus plants (n = 30) were artificially infested with approximately 10 second- to early-third instar citrus mealybug nymphs obtained from the laboratory colony on 6 February 2020. Spray applications of the treatments were applied to the coleus plants (20.5 ± 0.7 cm [mean ± SEM] in height, with 40.4 ± 1.2 leaves) on 14 and 21 February 2020. The spray solutions were prepared in 946 ml of tap water with approximately 55 ml applied to the upper and lower leaf surfaces and the stems of each coleus plant. The spray solution volume thoroughly covered all aboveground plant parts.

The environmental conditions in the greenhouse during the experiment were 19–53°C, 0–69% RH, and natural daylight. Whole plants were destructively sampled 7 d after each application, and the numbers of live and dead citrus mealybugs were recorded. Citrus mealybugs that did not move after prodding with a dissecting probe were considered dead. The presence of male pupae, which indicated that citrus mealybug nymphs completed development, or females that produced egg masses were counted as alive.

Percent citrus mealybug mortality was calculated by dividing the number of dead citrus mealybugs on each coleus plant by the total number associated with each coleus plant and then multiplying by 100. Data were analyzed using analysis of variance (ANOVA, P=0.05), with treatment as the main effect. Individual treatment means were separated using Tukey's honestly significant difference test when the ANOVA indicated a significant treatment effect (SAS Institute 2012).

Experiment 2. The protocol for Experiment 2 was similar to that described for Experiment 1. Fifty ‘Mariposa’ coleus plants were used. The plugs were obtained from Ball Horticultural Co., with the source being Kube-Pak Corp. (Allentown, NJ). After transplanting, plants were grown for 54 d before use in the experiment.

Treatments were arranged in a completely randomized design, with 10 treatments and five replications per treatment. The treatments were Velifer with and without the surfactant CapSil, afidopyrofen (Ventigra^®) with and without Capsil, sesame oil (Organocide Bee Safe 3-in-1 Garden Spray) without Capsil, cyclaniliprole (Sarisa^®) without Capsil, Sarisa+flonicamid (Pradia^®) without Capsil, Capsil alone, a water control, and an untreated control (Table 1). Coleus plants (n= 50) were artificially infested with citrus mealybug nymphs, as previously described, on 22 December 2020. Spray applications of nine of the treatments were made to the coleus plants (22.4 ± 0.5 cm in height, with 20.2 ± 0.6 leaves) on 24 December 2020. Approximately 53 ml of the spray solution for each treatment was applied to the upper and lower leaf surfaces and stems of each coleus plant. The environmental conditions in the greenhouse during the experiment were 20–39°C, 0–30% RH, and natural daylight. Data collection and analysis were the same as for Experiment 1.

Experiment 3. The procedures and materials for Experiment 3 were similar to the previous two experiments. Fifty ‘PS EL Brighto’ coleus plants were obtained from Family Tree Nursery (Kansas City, KS). These plants were grown for 37 d before use in the experiment. Treatments were arranged in a completely randomized design, with 10 treatments (Table 1) and five replications per treatment. Treatments were BotaniGard with and without Capsil, Ancora with and without Capsil, Velifer with and without Capsil, Capsil alone, flupyradifurone (Altus™), vinegar+70% isopropyl alcohol+blue Dawn Ultra^® dishwashing liquid, and a water control. Coleus plants (n = 50) were artificially infested with citrus mealybug nymphs, as described for Experiment 1, on 14 April 2021. Spray applications of the 10 treatments were made to the coleus plants (21.0 ± 0.2 cm in height, with 55.2 ± 0.9 leaves) on 16 April 2021. Approximately 47 ml of the spray solution from each treatment was applied to the upper and lower leaf surfaces and stems of each coleus plant. The environmental conditions in the greenhouse during the test were 19–53°C, 0–69% RH, and natural daylight. Data collection and analysis were the same as described previously.

Experiment 4. The procedures, materials, and the coleus cultivar used in Experiment 4 were similar to those of Experiment 2. Treatments were arranged in a completely randomized block design, with blocks accounting for one or two spray applications. The five treatments were BotaniGard, Ancora, Velifer, Altus, and a water control (Table 1), with five replications for each treatment. Coleus plants (n= 50) were artificially infested with citrus mealybug nymphs, as described for Experiment 1, on 25 January 2022. The first spray application of the treatments was made to the coleus plants (28.8 ± 0.9 cm in height, with 6.7 ± 0.3 leaves) on 31 January 2022, and a second application was made to the 25 remaining coleus plants 7 d after the first application on 7 February 2022. Approximately 50 ml of the spray solution from each treatment was applied to the upper and lower leaf surfaces and stems of each coleus plant. Water served as the control solution. The environmental conditions in the greenhouse during the experiment were 12–39°C, 0–47% RH, and natural daylight. Data collection and analysis were the same as described previously.

Experiment 1. Citrus mealybug mortality was <25% (n = 230) across all treatments; in addition, there were significant differences in percent citrus mealybug mortality across the treatments (F = 3.74; df = 2, 9; P = 0.042), with the first application of BotaniGard resulting in 24.7% citrus mealybug mortality, which was significantly greater than mortality observed in the water control (Fig. 1). A second application yielded no significant differences among the treatments (P > 0.05).

Experiment 2. Citrus mealybug mortality across all the treatments was <50% (n = 447). Mortality was significantly greater (F = 8.17; df = 9, 36; P < 0.0001) in the Ventigra, Ventigra + CapSil, Sarisa, and Pradia treatments than in the water control and the untreated control (Fig. 2). Mortality after the application of the B. bassiana strain PPRI 5339 (Velifer) treatment, with or without the surfactant, did not differ significantly from the water control or untreated control. The addition of the surfactant did not significantly affect mortality associated with the treatments Ventigra or Velifer.

Experiment 3. In all but two treatments, citrus mealybug mortality was <50% (n = 345) (Fig. 3). Treatments associated with Altus, vinegar + alcohol + dishwashing liquid, BotaniGard, and Ancora yielded percent mortality levels that were statistically greater (F = 19.72; df = 9, 36; P < 0.0001) than that of the water control. Mortality after application of Altus and the vinegar + alcohol + dishwashing liquid mixture was >70%, but there was no significant difference (P > 0.05) between the treatments (Fig. 3).

Experiment 4. There was no significant difference (P > 0.05) in citrus mealybug mortality between one or two spray applications of the treatments. Consequently, the data were pooled by treatment. There was a significant treatment effect. The flupyradifurone (Altus) at 1.03 ml/946-ml treatment was the only treatment that resulted in a significantly higher (F= 21.52; df = 4, 36; P < 0.0001) percent mortality of citrus mealybug than the water control (Fig. 4). None of the three entomopathogenic fungal-based insecticide treatments (BotaniGard, Ancora, and Velifer) differed significantly from the water control (Fig. 4).

Our study demonstrated that the entomopathogenic fungal-based insecticides that we tested were not effective in managing citrus mealybug populations on coleus plants under greenhouse conditions. The main factor attributed to these results is that the waxy covering protects citrus mealybugs by preventing the conidia of the entomopathogenic fungi from attaching to the cuticle, thereby inhibiting infection. Anecdotal information suggests that adding a surfactant to a spray solution improves coverage and penetration through the waxy covering of citrus mealybugs (Pillai 2016), which may improve the ability of an insecticide to adhere to and be absorbed by the target surface, thereby increasing insecticidal activity (Marutani and Edirveeasingam 2006). However, in our study, adding the surfactant CapSil to spray solutions of all three entomopathogenic fungal-based insecticides did not increase citrus mealybug mortality. It is possible that the surfactant enhanced the spreading ability of the spray solution, resulting in runoff, which would have reduced fungal conidia contact with the citrus mealybugs (Gatarayiha et al. 2010), or that the surfactant killed the conidia.

Gupta et al. (2021) evaluated the effects of the bacterium Serratia marcescens (Bizio) (Enterobacterales: Yersiniaceae) in degrading the waxy covering of the citrus mealybug, along with combining with half application rates of the insecticide chlorpyrifos, to determine whether there were any synergistic or additive effects. Although citrus mealybug mortality was increased by 54% above that of chlorpyrifos alone, the level of mortality would not be sufficient to manage citrus mealybug populations in ornamental crop production systems.

The periodic nymphal molts could have removed fungal conidia attached to the exuviae before fungal infection occurred, which has been reported with western flower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), and twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) (Gatarayiha et al. 2010, Shipp et al. 2003, Vestergaard et al. 1995). Studies should be conducted to investigate whether adding an insect growth regulator, such as azadirachtin, to a spray solution increases the efficacy of entomopathogenic fungal-based insecticides against the citrus mealybug. Azadiracthin may delay or slow down the molting process, thereby allowing the entomopathogenic fungus to penetrate the cuticle and initiate an infection (Akbar et al. 2005, Hernandez et al. 2012).

Environmental conditions such as temperature and RH have a substantial effect on the performance of entomopathogenic fungi (Demirci et al. 2011). Germination of entomopathogenic fungal conidia occurs when the RH is 90% (Ramoska 1984). In fact, B. bassiana conidia cannot germinate at a RH <92% (Ferron 1977). The temperature and RH associated with the four greenhouse experiments in our study ranged from 12 to 53°C and from 0 to 69% RH. These fluctuating conditions are probably not conducive for entomopathogenic fungi infection, which likely contributed to the <50% mortality of citrus mealybug populations on the coleus plants treated with the entomopathogenic fungi. The environmental conditions in the research greenhouse where we conducted our study were more representative of production greenhouses than artificially established conditions in the laboratory (Demirci et al. 2011).

We targeted second- to early-third-instar nymphs in our study. These nymphal stages may be less susceptible to infection than earlier-stage nymphs. However, under production greenhouse conditions, overlapping generations with different age structures and life stages are usually present simultaneously (Cloyd 2011). Consequently, the insecticide must be effective against a range of life stages to sufficiently manage target insect pest populations.

The nonfungal-based insecticide flupyradifurone (Altus) provided >70% mortality of citrus mealybugs in Experiments 3 and 4. Flupyradifurone has demonstrated activity against sucking insect pests of horticultural crops, including aphids and whiteflies, by interacting and binding to the nicotinic acetylcholine receptors (Nauen et al. 2014). In previous studies, we found flupyradifurone to be one of the most effective insecticides in managing citrus mealybug populations on coleus plants (unpubl. data).

The three-way mixture of distilled white vinegar + isopropyl alcohol + blue Dawn Ultra dishwashing liquid was tested because anecdotal information indicated efficacy against the citrus mealybug. We found that the three-way mixture provided >70% mortality of citrus mealybug populations. Our preliminary results indicate that the blue Dawn Ultra dishwashing liquid may be the component of the three-way mixture responsible for mortality of citrus mealybug (unpubl. data).

In summary, in our study we found that the entomopathogenic fungal-based insecticides were not effective in managing citrus mealybug populations on coleus plants. The use of entomopathogenic fungal-based insecticides may be an option in greenhouse vegetable production systems as long as temperature and RH are optimal for sporulation and infection to occur. In addition, vegetable crops can sustain some damage from insect pests as long as yield is not affected (Singh and Kaur 2020). However, in ornamental crop production systems, the entire plant is sold; consequently, plants cannot sustain damage from insect pests because marketability is affected (Sridhar et al. 2016).

The authors thank Mary Beth Kirkham (Department of Agronomy, Kansas State University, Manhattan) for reviewing an initial draft of the manuscript.