Twospotted Spider Mites (Tetranychus urticae) on Strawberry (Fragaria × ananassa) Transplants, and the Potential to Eliminate Them with Steam Treatment

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ABSTRACT

The twospotted spider mite (Tetranychus urticae) is a serious pest of strawberries. The objectives of this study were to determine incidence and severity of twospotted spider mite infestations on transplants and the potential for steam to eliminate them. Mites occurred on transplants of Florida cultivars from each of eight North American nurseries from which samples were obtained, but levels were usually low (59% of samples had zero mites). Estimated complete mortality of twospotted spider mite adult females and eggs on leaf discs using steam at 48°C was 2.7 and 1.9 h, respectively; 44°C and 46°C for up to 4 h killed 20% and 60% of the mites, respectively. Based on these results, eliminating twospotted spider mites on transplants is feasible and may be valuable for improving their management in fruit-production fields.

KEYWORDS:

• Vapor heat
• heat treatment
• nursery stock

Introduction

The twospotted spider mite (Tetranychus urticae Koch) is a common and often serious pest of many crops. Needlelike mouthparts remove contents from plant cells, resulting in chlorotic spots at the feeding sites and overall leaf bleaching or stippling that leads to leaf necrosis and senescence if mites are not controlled (Bensoussan et al., 2016; Fasulo and Denmark, 2009). In strawberry (Fragaria × ananassa) production, twospotted spider mites are considered one of the most economically important arthropod pests (Strand, 1994; Walsh et al., 1998), requiring frequent population monitoring and control. Twospotted spider mites primarily reside on the undersides of fully formed leaves, and field-monitoring is usually conducted by examining leaves with a hand-lens (Kovach et al., 1990). At low population levels, presence/absence sampling is accurate for making control decisions (Greco et al., 2004; Strand, 1994).

The twospotted spider mite is controlled in strawberry fields with judicious use of acaricides and timely releases of biological control agents, particularly predatory mites. Phytoseiulus persimilis and Neoseiulus californicus have the most impact at low twospotted spider mite populations, and some registered acaricides (e.g., acequinocyl, bifenazate, cyflumetofen) are compatible with these two predatory mites, having low toxicity to both species (Evans et al., 2018). The twospotted spider mite also requires control in open-field strawberry nurseries to produce clean, healthy transplants. Transplants are shipped from nurseries to growers in different geographic locations for fruit production. As such, acaricide application in nurseries may overlap with those in fruit-producing fields, potentially resulting in unintentional overexposure to specific acaricides if live twospotted spider mites are moved to fruit-production fields on transplants. The twospotted spider mite is notorious for developing acaricide resistance (Van Leeuwen et al., 2010). A non-chemical method to eliminate mites on transplants would be a valuable resistance management tool, resulting in cleaner transplants and possibly fewer acaricide applications in strawberry-producing fields.

Heat treatment of strawberry transplants has been investigated and is being used for eliminating pathogens, nematodes, and arthropods. Angular leaf spot, caused by Xanthomonas fragariae, was effectively reduced at 44°C for 4 h or 48°C for 2 h when transplants were bagged wet or dry and submersed in water, but flowering was adversely affected (Turechek and Peres, 2009). Short immersions of strawberry leaves in hot water (6, 2.5, and 1.25 min at 44°, 46°, and 48°C, respectively) eliminated cyclamen mite, Phytonemus pallidus (Hellqvist, 2002). Hot water treatment is recommended in California for cyclamen mite (Zalom et al., 2018), and in Europe for Aphelenchoides spp. nematodes (EPPO, 2012). Cyclamen mite and root knot nematode, Meloidogyne hapla, mortality was >99.7% when strawberry transplants were exposed to 50% CO₂ and 35°C for 20 h followed by 40°C for 20 h (van Kruistum et al., 2015). These controlled atmosphere temperature treatment (CATT) conditions are being used on a commercial scale in Europe for strawberry mother stock (van Kruistum et al., 2012). Heated air may be advantageous because some cultivars are sensitive to anoxic conditions in hot water, and using steam or vapor heat may mitigate any adverse effects of dry heat on transplants (Turechek and Peres, 2009).

There are few studies evaluating high temperatures for elimination of the twospotted spider mite. Complete mortality of adult females and eggs was achieved on strawberry leaf discs in water at 47.5°C (the lowest temperature tested) in 20 and 10 min, respectively (Gotoh et al., 2013). Under altered atmospheric conditions (0.4% O₂ and 20% CO₂), 99% mortality of diapausing adult twospotted spider mites was achieved in 15.5 h at 40°C (the highest temperature tested) and 95% relative humidity (Whiting and van den Heuvel, 1995). As a post-harvest treatment, immersion in water at 44°C eliminated 99% of non-diapausing and diapausing adult twospotted spider mites on persimmons after 102 and 211 min (Lester et al., 1997). Similarly, 24 h of warm air between 43.2°C and 46°C eliminated non-diapausing twospotted spider mites on nectarines, whereas 15.5 h at 46.7°C and 34 h at 43.9°C eliminated diapausing mites (Waddell and Birtles, 1992).

The goal of our research is to develop innovative tools as alternatives to chemical pesticides for managing arthropod pests on strawberry transplants. Our objectives for these experiments were to determine the incidence and severity of twospotted spider mite infestations of strawberry transplants arriving in Florida, and to determine the efficacy of steam treatments for eliminating twospotted spider mites. By determining twospotted spider mite incidence on transplants, and evaluating practical methods to eliminate them, recommendations can be made for designing protocols that complement current research on steam treatment for disease control on transplants in North America.

Materials and Methods

Incidence and Severity on Strawberry Transplants

To assess the incidence and severity of twospotted spider mite infestations on strawberry transplants shipped to fruit producers in central Florida, batches (60–120 transplants) were collected within 2 days of arrival at grower facilities and stored in coolers (4 – 8°C) at the University of Florida, Gulf Coast Research and Education Center (GCREC) for up to 3 days until processing. The nursery source, cultivar, and transplant type were recorded for each batch of transplants.

Batches were processed by separating the 60–120 transplants into 20-plant samples and cutting each below the crown to remove roots and associated soil. Each sample was washed in water (10 L) with bleach (100 ml of 5% sodium hypochlorite) and liquid dish detergent (1 ml) in a plastic bucket (19 L). The plant material was washed by vigorously stirring and agitating the suspension with a large spoon for 3 min to dislodge any twospotted spider mites from the plants. The contents of the bucket were drained through stacked sieves (500 and 250 µm), with the coarser sieve filtering out larger debris, and the finer sieve collecting twospotted spider mites. Each sample was washed three times, with the particulate remaining on the finer sieve (250 µm) washed into containers (500 ml; Solo Cup Company, Lake Forest Illinois, USA) with ethanol (70%) and stored in a refrigerator. Samples were observed under a stereomicroscope at 25 – 30X magnification, and numbers of twospotted spider mite motiles were recorded.

Elimination with Steam Treatments

Laboratory Experiment

Twospotted spider mites were obtained from a non-diapausing colony maintained on green bush bean plants, Phaseolus vulgaris L. ‘Buffalo’ (Siegers Seed Co®, Holland MI, USA) reared in a growth room at GCREC at 25°C and 14 L:10D. Leaf discs (4 cm diameter) were cut from fully developed leaves from lima bean plants Phaseolus lunatus L. ‘Henderson’ (Seedway LLC, Hall NY, USA), disinfested by dipping in 1% bleach (5% sodium hypochlorite) solution and placed in small, glass Petri dishes (5.5 cm diameter) on cotton pads moistened with deionized water to maintain humidity in the dish and prevent mites from leaving the leaf disc. Adult females from the colony were transferred individually from infested colony leaves onto leaf discs with a fine camel-hair brush.

Experiments were conducted Jan to Apr 2018 in a modified growth chamber (1.85 × 0.77 m) (GCW30 Growth Chamber, Conviron®, Winnipeg MB, Canada) fitted with a steam generator (AK Generator, AMEREC, Woodinville WA, USA). Steam was emitted from small holes in a copper piping on the floor of the chamber. The steam generator was set at 37°C for 1 h (standard for acclimatizing transplants to heat) and then increased to testing temperatures: 44°C, 46°C, and 48°C. Actual temperatures in the growth chamber were monitored with four temperature sensors and recorded on data loggers. At each test temperature, leaf discs with twospotted spider mites in Petri dishes with a glass cover were treated for 0.5, 1, 2, or 4 h (10 replicates per time point). Five leaf discs with twospotted spider mites (adult females or eggs) were held at room temperature (~22°C) as a control.

Adult female (12–15 per leaf disc) mortality was assessed 24 h after steam treatment ended (Petri dishes kept at 25°C) under a stereomicroscope at 25–30X magnification. Mites were scored as alive if they moved at least one body length when prodded with a camel-hair brush. Numbers of alive and dead females on the leaf disc and cotton were recorded.

For egg mortality rates, adult females (7 per leaf disc) were removed from leaf discs after 24 h at 25°C, and eggs (30–40 per leaf disc) were counted. Numbers of hatched nymphs were counted under a stereomicroscope at 25–30X magnification 96 h after steam treatment. Unhatched eggs were considered dead.

Field Experiment

Pieces (4 × 4 cm) of infested bean leaves from the twospotted spider mite laboratory colony were placed on strawberry transplants (cv. ‘Florida Radiance’) in plug trays held in the rearing growth room (1 piece per transplant). After 3 days, control transplants (no steam treatment) were set in field plots 11 Dec 2017, and treated transplants were subjected to steam heat (same procedure as described above for the laboratory experiment) at 44°C for 4 h or 48°C for 0.5 h and set the following day. Twospotted spider mite levels were assessed on trifoliates from five non-treated transplants using a mite-brushing machine.

Field plots (14–15 plants each) at the GCREC were arranged in a randomized complete block design in five empty rows × 18 m of raised, plastic-mulched beds at 38 cm in row spacing that were fumigated with Telone® C-35 (740 kg ha⁻¹) at bed formation in early Aug 2017. Plants were fertilized with 0.27 and 0.34 kg N per day from Nov to mid-Jan and mid-Jan through Apr, respectively. Chateau® and Prowl® + Round-up® were applied in early Sep to control weeds. Plots were separated by at least 6 m within rows/blocks, and blocks (four replications) were positioned to maximize distances from surrounding strawberry research plots and from each other.

Twospotted spider mite densities were assessed 19, 26 Dec 2017, and 2, 16, 23, and 30 Jan 2018 by removing an open trifoliate from 10 plants per plot and using a mite-brushing machine to estimate the numbers of motile stages and eggs. Total leaf area was measured with a LI-3100 C area meter (LI-COR_®, Lincoln NE, USA). On 15 Mar 2018, numbers of surviving plants, flowers, and fruits per plot were counted (and removed), and plant biomass per plot was measured by drying above-ground material at 80°C for 48 h, cooling at room temperature and weighing.

Data Analysis

Twospotted spider mite means (±SD) per batch were calculated, and the count frequency distribution was fit to discrete probability models (Poisson, binomial). To determine the accuracy of using 20-plant samples to determine the likelihood of finding at least one twospotted spider mite, a binomial outcome of (Y > 0 mites vs. N = 0 mites) for each sample (n = 84) was regressed on the batch mean (n = 18) from which the sample was taken. One batch of samples was removed from the analysis due to the extraordinarily high number of twospotted spider mites on one sample.

Non-linear, predictive modeling was used to estimate the minimum time required to achieve 99–100% twospotted spider mite adult female and egg mortality at 48°C (data from 44°C to 46°C not analyzed because 100% mortality was not reached within 4 h). Models were compared using AICc, RMSE and R² criteria and visualizations with the data. A three-parameter exponential model provided the best fit to the data. JMP® software (SAS, 2018) was used for analyses.

Results

Incidence and Severity on Strawberry Transplants

We collected 19 batches of strawberry transplants from 10 Florida growers representing eight nurseries. Thirty-seven percent (7/19) of the batches were from California, 32% (6/19) from Nova Scotia, and 21% (4/19) were from North Carolina (Table 1). All but three of the batches were ‘Florida Radiance’ (84%), and 79% were bare-root as opposed to plug transplants (Table 1). At least one batch from all eight nurseries had twospotted spider mites, but most batches (63%) had low numbers (0.3–8.3 per 20 transplants) (Table 1). From one nursery (G), twospotted spider mite numbers were low on transplant batches sampled from four Florida growers (5–7, 9), but high on a batch from a fifth grower (8) (Table 1). Similarly, another nursery (E) had high twospotted spider mite numbers on one of two cultivars (Table 1).

Table 1. Summary of twospotted spider mites (Tetranychus urticae) (TSSM) extracted from three Florida strawberry transplant cultivars collected 22 Sep–22 Oct 2017 in Florida from eight North American (NA) nursery sources. Each sample was 20 transplants subdivided from a three-to-six sample batch provided by the Florida grower.

Florida grower	NA nursery	Nursery location		Cultivar	Transplant Type	No. samples	Mean TSSM per sample	SD
1	A	Ontario	Canada	FL Radiance	Bare-root	4	5.5	2.4
2	B	Nova Scotia	Canada	FL Radiance	Plug	5	0.0	0.0
2	C	North Carolina	USA	FL Radiance	Bare-root	4	1.5	1.9
2	C	North Carolina	USA	FL Radiance	Bare-root	4	0.5	1.0
3	D	Quebec	Canada	FL Radiance	Bare-root	4	0.3	0.5
4	E	California	USA	FL Beauty	Bare-root	5	19.2	11.5
4	E	California	USA	FL Radiance	Bare-root	6	1.7	3.6
4	F	Nova Scotia	Canada	Sensation™ FL-127	Bare-root	5	0.4	0.9
4	F	Nova Scotia	Canada	FL Beauty	Bare-root	5	1.0	1.2
4	F	Nova Scotia	Canada	FL Radiance	Bare-root	5	0.0	0.0
5	G	California	USA	FL Radiance	Bare-root	3	7.0	8.9
5	H	North Carolina	USA	FL Radiance	Bare-root	4	0.0	0.0
6	B	Nova Scotia	Canada	FL Radiance	Plug	6	8.3	5.6
6	G	California	USA	FL Radiance	Bare-root	4	0.3	0.5
7	G	California	USA	FL Radiance	Bare-root	5	0.0	0.0
8	G	California	USA	FL Radiance	Bare-root	4	48.8	94.8
9	G	California	USA	FL Radiance	Plug	5	1.4	2.6
9	F	Nova Scotia	Canada	Sensation™ FL-127	Bare-root	5	0.0	0.0
10	H	North Carolina	USA	FL Radiance	Plug	4	1.5	1.3

Over half (59%) of the samples had no twospotted spider mites, and the distribution of counts did not fit a Poisson or binomial distribution (Figure 1). Mean numbers of twospotted spider mites per sample in a batch were related to the probability of finding at least one mite versus no mites in a sample (Figure 2). There was a 15.2% probability of finding at least one twospotted spider mite at a mean of 0.0001 (−2.08–0.76, 95% CI) mites per 20 transplants. Conversely, there was a 99.0% probability of finding at least one mite at a mean of 8.04 (5.21–20.72, 95% CI) mites per 20 transplants.

Figure 1. Distribution of twospotted spider mite (Tetranychus urticae) counts on samples (n = 87) of 20 strawberry transplants collected after shipment from North American nurseries to Florida growers in autumn 2017.

Figure 2. Probability of finding at least one twospotted spider mite (Tetranychus urticae) in a sample of 20 strawberry transplants based on the mean (dotted lines, 95% CI) number of mites per sample (n = 84) in 3–6 sample batches. Batches of transplants (n = 18) were collected after shipment from North American nurseries to Florida growers in autumn 2017.

Elimination with Steam Treatments

Laboratory Experiment

Mortality of adult female and egg-stage twospotted spider mites reached 100% at 2 and 4 h at 48°C, but only reached about 15% and 70% for females at 44°C and 46°C and 20% and 60% for eggs at 44°C and 46°C, respectively (Figure 3a,b). A three-parameter, non-linear model predicted 99.9% adult female mortality at 2.74 ± 1.22 h (95% CI) (Figure 4a) and 97% egg mortality at 1.91 ± 3.35 (95% CI) (Figure 4b). For eggs, the model reached an asymptote at ~97.4% mortality. Recorded temperatures in the steam chamber were 43.4 ± 0.59°C, 45.4 ± 0.68°C, and 47.2 ± 0.51°C during adult female tests and 43.6 ± 0.47°C, 45.5 ± 0.44°C, and 47.5 ± 0.31°C during egg tests (means ± 1 SD).

Figure 3. Mean (±SE) percent mortality of twospotted spider mite (Tetranychus urticae) (a) adult females and (b) eggs on bean leaf discs exposed to steam heat in a modified growth chamber at 44°C, 46°C, and 48°C for 0.5–4 h.

Figure 4. Predicted (lines) versus actual (circles) mortality of twospotted spider mite (Tetranychus urticae) (a) adult females and (b) eggs on bean leaf discs exposed to steam heat in a modified growth chamber at 48°C for 0.5–4 h.

Field Experiment

One week after transplanting (19 Dec 2017), there was no effect of treatment on numbers of twospotted spider mite motiles (F_2,6 = 2.1; P = 0.21) or eggs (F_2,6 = 3.1; P = 0.12) on 10 trifoliates or when converted to motiles (F_2,6 = 1.4; P = 0.32) or eggs (F_2,6 = 2.0; P = 0.22) per cm² of leaf. There were also no effects of treatments on twospotted spider mites for assessments after 19 Dec (P > 0.05 for total motiles, eggs, motiles per cm² and eggs per cm² for all assessment dates). No leaf damage due to treatments was observed and by 15 Mar 2018, there was no effect of treatment on flowers + fruit per plant (F_2,6 = 0.1; P = 0.91) or on plant biomass (F_2,6 = 0.3; P = 0.75).

Discussion

The twospotted spider mite can be a difficult pest to manage effectively in strawberry as well as in other crops. Our results from one season showed that there was a high incidence of the twospotted spider mite arriving in Florida on strawberry transplants but that the severity was relatively low. The consequences of high twospotted spider mite incidence, but low severity, on transplants for effectiveness of seasonal management actions is not known. However, their presence suggests that there would be a benefit in subjecting transplants from all sources to a pre-plant treatment to eliminate mites. We found that steam treatment at 48°C but not 44°C or 46°C killed all twospotted spider mite adult females and eggs. Future research will need to address effectively eliminating arthropod pests, fungal diseases and possibly nematodes on various cultivars using a single, steam treatment protocol.

Screening showed that the twospotted spider mite arrived on transplants from every North American nursery source at every Florida grower from which samples were obtained. However, twospotted spider mite distribution among samples was highly variable, as mean mites per sample for all batches differed from 0 to almost 50, and the majority of batches (9/19) had a standard deviation greater than the mean. These observations suggest that incoming transplants reflect patchy distributions of twospotted spider mites in nursery fields, as is known to occur in fruit-producing fields (Greco et al., 1999). Strawberry nursery fields are actively managed with acaricides, which appears to be effective for achieving nearly, but not entirely, clean transplants.

The significant relationship between the probability of finding ≥1 twospotted spider mite in a sample and the mean number of mites per batch suggests that 20-transplant samples are adequate for assessing twospotted spider mites. In spite of this, inferences from these data are limited by a low number of samples from some nursery sources and by just a single season of sampling. Twospotted spider mite populations are temporally unstable (Krainacker and Carey, 1990; Nachman, 1981), and the severity of infestation may be highly variable within or between seasons for any particular nursery. Establishment of twospotted spider mite populations in Florida fields from individuals on transplants is also affected by environmental factors (temperature, rainfall) or management practices, as some growers always use prophylactic early-season acaricide applications without scouting for mites. Few mites of other species, including predators, were observed in samples, but specimens were not recorded or identified.

Strawberry production in Florida is unique in that transplants are grown during the summer in a few areas of the USA and Canada, dug in early-autumn and shipped fresh with fully expanded leaves. Transplants destined for other growing regions are usually shipped as crowns without mature leaves and may be cold-stored or in transport for a longer period than transplants going to Florida. Therefore, there is a greater chance that twospotted spider mites on transplants become a founding population in Florida fields than in fruit-producing fields elsewhere, although mites occurring in Florida fields may also have dispersed from neighboring crop fields or other vegetation (e.g., Brandenburg and Kennedy, 1982; B. Evans, unpublished data). The establishment of founding populations from transplants that have been exposed to acaricides in nursery fields poses a potential management risk. These populations have faced selective pressure from commonly used acaricides, and they represent a population bottleneck comprised individuals potentially carrying a high proportion of acaricide-resistant alleles (Khalejehali et al., 2011; Pascual-Ruiz et al., 2014). For example, outbreaks of the twospotted spider mite occurred in Florida in 2016 in fields with transplants from a particular nursery that reportedly used twice as many acaricide applications than in a typical season due to high summer-time mite populations.

Results with the temperatures and durations tested against twospotted spider mites were comparable to results in previous studies. In this study, steam killed all adult females and eggs in 2.7 and 1.9 h, respectively, at 48°C, but dipping infested strawberry leaflets in 47.5°C water killed all females and eggs in 20 and 10 min (Gotoh et al., 2013), suggesting more rapid heating of mites in water than air, but not necessarily anoxic conditions (Suzuki et al., 2015), may have accelerated mortality. In this study, leaf discs with twospotted spider mites were contained in loosely covered Petri-dishes, which may have increased mortality times due to slower heating of dish interiors than the steam chamber itself. Lower temperatures, 40 – 46°C, can eliminate diapausing and non-diapausing twospotted spider mite adults on other plants or fruits (Lester et al., 1997; Waddell and Birtles, 1992; Whiting and van den Heuvel, 1995), even though in this study there was only a slight increase in mortality from 2 to 4 h at 44°C and 46°C. However, if the equation for a line between 2 and 4 h at 46°C is used, estimated mortality at 24 h is greater than 100%, similar to 100% mortality on persimmons after 24 h of warm air between 43.2°C and 46°C (Lester et al., 1997). Overall, lethal times at 40 – 48°C for twospotted spider mite adults and eggs appear to be considerably less in water than in steam, dry air, with or without altered atmospheric conditions.

Evaluation of heat for reducing disease on strawberry transplants has found that plants that were in sealed bags prior dipping in hot water were less damaged that plants that were directly dipped in water (Turechek and Peres, 2009; Wang et al., 2017). Angular leafspot infection rates were greatly reduced in water at 44°C for 4 h or 48°C for 2 h when transplants were directly dipped or bagged wet or dry and dipped, but 48°C for 2 h reduced survival, runner production, and flowering (Turechek and Peres, 2009). Based on modeling our results, 2.7 h at 48°C was predicted as the time needed for complete twospotted spider mite adult elimination, a duration that may be too detrimental to transplants. However, two data points at 1 h for 48°C resulted in low (<40%) mortality and are responsible for increasing the predicted time, as 100% of mites died at 2 h at 48°C. Further testing (at intervals between 1 and 2 h) or removal of these two data points will (likely) result in a model predicting 100% mortality of adults between 1 and 2 h. Our field results confirmed our leaf disc assay, as 0.5 h at 48°C and 4 h at 44°C had no effect on twospotted spider mite densities (times and temperatures chosen before completion of leaf disc assay and with only limited field space for plantings) but, as expected, with no effect on strawberry plant size or flowering/fruiting. Using steam heat at 48°C for 1–2 h for angular leaf spot will need to be tested, but if effective and not detrimental to cultivars currently grown in Florida, should be the basis for a protocol to eliminate at least one pathogen and one arthropod from transplants.

Florida growers are uniquely positioned to produce strawberries during winter months, when the climate in most other USA and all Canadian regions prevents field production. However, Florida production is dependent on clean, healthy transplants grown in other regions. Unfortunately, we showed that most transplants contain at least low levels of twospotted spider mites, and these mites could pose a risk if founding populations resistant to acaricides establish in Florida fields. Encouragingly, though, twospotted spider mites can be eliminated with steam treatments at 48°C, which may be compatible with conditions needed to reduce disease-causing pathogens while not compromising transplant growth and production.

Acknowledgments

The authors thank Ryan Batts, Shashan Devkota, Marc Santos and Darsy Smith for technical assistance and Florida strawberry growers for providing transplant samples.

Additional information

Funding

Funding was from the North American Strawberry Growers Association and the Florida Strawberry Research and Education Foundation.