Exp Appl Acarol (2009) 47:275–283 DOI 10.1007/s10493-008-9217-z
Evaluation of the predatory mite Phytoseiulus macropilis (Acari: Phytoseiidae) as a biological control agent of the two-spotted spider mite on strawberry plants under greenhouse conditions Hamilton Oliveira Æ Marcos Antonio Matiello Fadini Æ Madelaine Venzon Æ Daniela Rezende Æ Fernanda Rezende Æ Angelo Pallini
Received: 21 July 2008 / Accepted: 3 November 2008 / Published online: 19 November 2008 Ó Springer Science+Business Media B.V. 2008
Abstract The predatory mite Phytoseiulus macropilis is a potential biological control agent of the two-spotted spider mite (TSSM) Tetranychus urticae on strawberry plants. Its ability to control TSSM was recently assessed under laboratory conditions, but its ability to locate and control TSSM under greenhouse conditions has not been tested so far. We evaluated whether P. macropilis is able to control TSSM on strawberry plants and to locate strawberry plants infested with TSSM under greenhouse conditions. Additionally, we tested, in an olfactometer, whether odours play a role in prey-finding by P. macropilis. The predatory mite P. macropilis required about 20 days to achive reduction of the TSSM population on strawberry plants initially infested with 100 TSSM females per plant. TSSM-infested plants attract an average of 27.5 ± 1.0% of the predators recaptured per plant and uninfested plants attracted only 5.8 ± 1.0% per plant. The predatory mites were able to suppress TSSM populations on a single strawberry plant and were able to use odours from TSSM-infested strawberry plants to locate prey in both olfactometer and arena experiments. Hence, it is concluded that P. macropilis can locate and reduce TSSM population on strawberry plants under greenhouse conditions. Keywords Fragaria
Herbivore-induced plant volatiles Tetranychidae Tetranychus urticae
H. Oliveira M. A. M. Fadini (&) M. Venzon Agriculture and Livestock Research Enterprise of Minas Gerais (EPAMIG), Vila Gianetti 46, CEP 36570-000 Vic¸osa, Minas Gerais, Brazil e-mail:
[email protected] D. Rezende F. Rezende A. Pallini Department of Animal Biology/Entomology, Federal University of Vic¸osa, Av. PH Rolfs s/n., CEP 36571-000 Vic¸osa, Minas Gerais, Brazil
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Introduction The two-spotted spider mite (TSSM) Tetranychus urticae (Acari: Tetranychidae) is a polyphagous mite that feeds on parenchym cells of more than 600 plant species (Helle and Sabelis 1985). It is a major pest of horticultural crops, such as strawberry (van de Vrie et al. 1972; Garcia-Mari and Gonzalez-Zamora 1999; Sato et al. 2002). At high population densities, this species can cause a severe reduction in strawberry yield. Problems with TSSM are becoming increasingly serious as strawberry crops are grown under protected conditions, mainly in polyethylene tunnels, but also in greenhouses (Easterbrook et al. 2001; Sato et al. 2007). TSSM populations thrive under hot and dry conditions. In the early stages of an infestation the foliage of strawberry plants get a speckled appearance. As plants become heavily infested, foliage turns yellow and finally the leaves drop (Lourenc¸a˜o et al. 2000). In Brazil, control of TSSM populations is done mainly by pesticides, but in some cases pesticide sprays make the pest problem even worse because they negatively affect natural enemies (Liburd et al. 2007). Furthermore, strains of TSSM resistant to pesticides are selected, making chemical control more difficult (Sato et al. 2002). Considerable research effort has been targeted on finding an environmentally friendly way to control TSSM, e.g., biological control. Among several natural enemies of TSSM, phytoseiid mites are the most important biological control agents (McMurtry and Croft 1997). Several phytoseiid species have proven to be suitable for integrated pest management programs outdoors and in greenhouse crops (Van Lenteren and Woets 1988; Weintraub et al. 2007). Recently, the indigenous predatory mite from Brazil, Phytoseiulus macropilis (Acari: Phytoseiidae), was evaluated to control TSSM in lab tests and showed promising results (Oliveira et al. 2007). Predation and reproduction rates of this predator were as high as those of another phytoseiid, Phytoseiulus persimilis, a commercially available species that is widely used to control TSSM. However, laboratory tests implicate only local predator– prey interactions, as they are restricted to experimental arenas where predator and prey cannot display the full range of their behaviour. Thus, lab tests are only indicative of the outcome of a predator–prey system, and care should be taken when extrapolating lab results to larger scales (Kareiva 1989; Nomikou 2003). Under greenhouse and field conditions this relationship could be more complex, because other variables may interfere with the predator’s ability to control a prey population. Thus, a logical next step to follow when evaluating a biological control agent is to study local predator–prey dynamics at the scale of a plant (Nomikou 2003). Subsequently, larger scales should be evaluated. Another important trait to be considered when selecting a predator for biological control purpose is its ability to find prey-infested plants. During this searching process many predators can use volatiles that are produced by plants in response to herbivore injury (Dicke and Sabelis 1988; Dicke 1994; Turlings et al. 1990). It is known that TSSMinfested plants, such as cucumber, beans and many more, produce volatiles induced by herbivory (Pallini et al. 1997; Sabelis et al. 1998; Janssen 1999) and that the predatory mite P. persimilis use these volatiles for prey finding (Janssen 1999). However, it is not known yet whether volatiles produced by TSSM-infested strawberry plants can be used by P. macropilis for finding prey patches. Here, we tested whether P. macropilis is able to control TSSM population on single strawberry plants. Subsequently, we investigated the predator’s ability to locate strawberry plants infested with TSSM under greenhouse conditions. We also used a Y-tube olfactometer (e.g., Sabelis and van de Baan 1983) to test whether odours play a role in prey finding by P. macropilis.
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Materials and methods Rearing methods Strawberry plants were grown in 2.5 l pots in a greenhouse (13–38°C, 40–60% RH and 13:11 L:D). The greenhouse conditions were tuned to mimic the abiotic conditions in the field. Colonies of TSSM were established in the greenhouse and in the laboratory. In the greenhouse, strawberry plants infested with TSSM were kept inside wooden frame cages (l 9 w 9 h = 1.2 9 0.8 9 1.0 m) covered with nylon gauze. In the laboratory, detached TSSM-infested strawberry leaves, from greenhouse colonies, were placed on moistened cotton pads on top of sponges (3.0 cm thick) in plastic boxes (15 9 25 9 5 cm). Water was added to the rearing units when necessary to keep the cotton wet. When leaves started to deteriorate, they were taken out of the rearing unit and placed on top of a new arena to allow the mites to move onto this arena. To rear predators, females of P. macropilis were released on rearing units with TSSM on all stages. The predator culture started from mites collected from strawberry fields of Caldas and Barbacena, State of Minas Gerais, Brazil. When over-population of predators was observed, they were transferred to new arenas infested with TSSM. Predator and TSSM rearings were kept each inside a separate incubator at 25 ± 1°C, 60 ± 5% RH and 13:11 L:D, corresponding to the average conditions in the region where mites were collected. Capacity of Phytoseiulus macropilis to control TSSM in strawberry plants Potted strawberry plants, with three to four compound leaves, were infested with 100 adult females of TSSM. Plants were kept inside wooden frame cages in a greenhouse. Females were allowed to oviposit for 7 days, enough for the establishment of TSSM colonies on infested plants. Subsequently, the number of TSSM mobile stages was assessed. Next, one mated female of P. macropilis was released on each strawberry plant. All stages of prey and predators were counted weekly. Predators were released in four cages, and each cage had four infested plants. Four other cages contained predator-free plants infested with TSSM, that were used as a control treatment. The experiment was terminated pffiffiffiffiffiffiffiffiffiffiffi when numbers of prey and predatory mites were too low to persist. Data were x þ 1-transformed and subsequently analyzed using ANOVA. Release-recapture experiment A tray consisting of a wooden frame (1.74 9 1.74 9 1.70 m) covered with plastic was placed on a table in a greenhouse compartment. The tray was filled with soil and three potted TSSM-infested strawberry plants and three potted clean plants were put inside. Plants had three compound leaves. Pots were put into the soil with the rim just below the soil surface (Pallini et al. 1997). Four replicates were done and in each replicate, uninfested and infested plants occupied alternating positions in a circle with a diameter of 100 cm. In two replicates, the three uninfested plants were placed in positions 1, 3, and 5 of the circle, whereas TSSM-infested plants were placed in these positions during the other two replicates. This was done to correct for any directionality in the walking behaviour of the predatory mites. Plants were supported by three small wooden sticks so as to prevent the leaves from touching the soil. Thus, the only objects sticking out of the soil were the plant stems and these sticks.
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The day after putting the plants in the greenhouse, well-fed P. macropilis females were collected from the culture by sucking them into a pipette tip that was connected to the vacuum network and sealed by mite-proof gauze at the wide end (Oomen 1988). After collecting about 160 adult females in a tip, it was closed with Parafilm at the narrow end. After 1 h, the tip was opened and the mites were gently shaken into a Petri dish (8 cm diameter), which was then placed in the centre of the circle of plants, in the greenhouse. Subsequently, the predators were allowed to walk to any of the plants and move onto them. There was almost no wind in the greenhouse, so the predators could not disperse on air currents and had to reach the plants by walking over the soil. Ideally, the mites should have been observed after release to see how they reach plants and how they move up the plants. However, the complex structure of the soil hides the small mites from view, making direct observations impossible. Starting 1 h after the release, all plants were sampled once per hour, during a total period of 6 h. At each check, all mites found on the plants were removed so that the arrestment effect was minimised. Therefore, the main cause of the presence of predators on plants is likely to be attraction rather than arrestment (Janssen 1999). Frequent sampling was also done to ensure that the first predators attracted to the plants would not influence (i.e., elicit avoidance of attraction of) the predators that were still searching for plants (Janssen et al. 1997, 1998). The next day, all plants were sampled again, but only a few predators were recaptured. The temperature in the greenhouse was between 25 and 30°C. The results of the experiments were analyzed by a multifactor ANOVA of arcsine-transformed fractions of predators recaptured per plant, with the treatment of the plants and the position within the circle as factors. Olfactometer experiments A Y-tube olfactometer was used to study the response of adult predatory mite females to volatiles emanating from TSSM-infested strawberry plants (Sabelis and van de Baan 1983; Pallini et al. 1997). The olfactometer consists of a glass tube in the form of a Y, with a white Y-shaped metal wire in the middle to convey the mites. The odour sources consisted of strawberry plants that were infested with *1,400 spider mites for 48 h prior to the test and of strawberry plants without mite infestation. Potted strawberry plants with 8–12 leaves were placed in a tray (l 9 w 9 h = 50 9 35 9 42 cm) that was then put inside another water-containing tray (60 9 38 9 4 cm). Subsequently, a Plexiglass container was placed over the plants so that it rested in the outer water-containing tray. In this way a water barrier was created to prevent spider mites from escaping. Furthermore, the water served as an airtight seal. Each Plexiglass container had an air inlet and outlet (4 cm diameter) in opposite walls and these were covered with mite-proof gauze. The base of the tube was connected to an air pump that produced an airflow from the arms of the tube to the base. Airflow through both arms of the Y-tube was measured with a digital anemometer. Wind speeds were calibrated, using valves that were inserted into the hoses connecting the containers (one with three clean plants and one with three infested plants), and kept at 0.4 m/s in both arms. Predatory mites were starved for 1 h prior to the experiments and after this time they were introduced one at a time by disconnecting the pump and putting one female on the metal wire at the base of the Y-tube. After the pump was reconnected, the female started moving upwind to the junction of the wire, where she had to choose one of the two arms. Each individual was observed until she had reached the end of the arm or for a maximum of 5 min and was subsequently removed (Janssen 1999). Depending on the mite supply, we tested 18–23 predators per replicate. After five mites had been tested, the odour sources
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were switched to the opposite arm of the olfactometer, to correct for any unforeseen asymmetry in the experimental set-up. Mites used in this experiment were of the same age. We tested the number of responding predatory mites of each replicate against an expected 50:50 distribution with a G-test. Four replicates were done, using a different set of plants and group of predators for each replicate. The results of the different treatments were compared with a generalized linear model with binomial error distributions (R Development Core Team 2004).
Results The predatory mite P. macropilis required about 20 days to achive reduction of the TSSM population on strawberry plants initially infested with 100 TSSM females per plant (i.e., *10 TSSM females per leaf) (F = 122.4; P \ 0.001) (Fig. 1). After 28 days from predator release, TSSM populations went extinct. The highest predator population on plants, an average of 16 individuals per plant, was found 27 days after their release (Fig. 2).
No. two-spotted spider mites
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Fig. 1 Average numbers of mobile stages of two-spotted spider mites (TSSM) as a function of time (days) on strawberry plants with and without Phytoseiulus macropilis. The arrow represents the predatory mite release 20 18 16 14 12 10 8 6 4 2 0 0
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Fig. 2 Average numbers of mobile stages of Phytoseiulus macropilis as a function of time on strawberry plants infested with all stages of Tetranychus urticae
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Mean no. predatory mites/plant
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Fig. 3 Average percentages of Phytoseiulus macropilis recaptured per uninfested strawberry plants and per two-spotted spider mite (TSSM) infested strawberry plants in a greenhouse experiment. Means ? SD per plant of four independent replicates are shown. *P \ 0.05
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Fig. 4 Cumulative percentage of predatory mites recaptured on strawberry plants as a function of time (h)
Greenhouse experiments revealed that a significantly larger proportion of P. macropilis was recaptured on TSSM-infested plants compared with uninfested plants. In total, 35.8% of all mites released were recaptured. TSSM-infested plants attract an average of 27.5 ± 1.0% of the predators recaptured per plant (hence, 82.8 ± 3.0% of all predator recaptured were found on infested plants) and uninfested plants attracted only 5.8 ± 1.0% per plant (17.1 ± 3.0% of all recaptured mites) (F = 298.8; P \ 0.001) (Fig. 3). No significant effect of plant position was found in the percentage of mites recaptured per plant (F = 1.82; P = 0.16). Most of the predatory mites were recaptured 48 h after their release (Fig. 4). When the predatory mites were offered a choice between plants with TSSM and uninfested plants in the Y-tube olfactometer, plants infested with TSSM were significantly more attractive to P. macropilis than uninfested strawberry plants in all four replicates (Fig. 5). The results were not significantly heterogeneous (GH = 0.17; P = 0.79). Pooled results of the four replicates showed 79.2 ± 1.2% of tested predatory mites were attracted to TSSM-infested plants (GP = 23.07; P \ 0.001). Discussion The predatory mite P. macropilis was able to suppress TSSM populations on a single strawberry plant when released at a predator : prey ratio of 1:100. At this ratio, it took
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Uninfested plant P
