Molecular verification of dispersal of phytoseiid mites from groundcover plants to tree leaves in Japanese peach orchards

Biological Control 80 (2015) 143–155

Contents lists available at ScienceDirect

Biological Control journal homepage: www.elsevier.com/locate/ybcon

Molecular verification of dispersal of phytoseiid mites from groundcover plants to tree leaves in Japanese peach orchards David Wari a, Ken Funayama b, Hidenari Kishimoto c, Masatoshi Toyama d, Shoji Sonoda a,⇑ a

Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama 710-0046, Japan Akita Fruit-Tree Experiment Station, Yokote, Akita 013-0102, Japan c NARO Institute of Fruit Tree Science, Morioka, Iwate 020-0123, Japan d NARO Institute of Fruit Tree Science, Higashihiroshima, Hiroshima 739-2494, Japan b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A population survey of phytoseiid and

spider mites was conducted in peach orchards.  Phytoseiid mite species composition changed seasonally and varied among orchards.  Phytoseiid mite species of various feeding habits preferred Tetranychus to Panonychus.  Phytoseiid mites move from groundcover to tree leaves.

a r t i c l e

i n f o

Article history: Received 1 June 2014 Accepted 8 October 2014 Available online 16 October 2014 Keywords: Generalist predator Groundcover vegetation Species composition Spider mite Gut analysis

a b s t r a c t A population survey of phytoseiid mites and of spider mites on randomly selected trees and their groundcover plant Paederia foetida L. (Rubiaceae) was conducted in Japanese peach orchards that used different pesticide practices. An organic orchard with wild groundcover and no synthetic chemicals used for pest control and a conventionally managed orchard with bare ground had no trees on which spider mite density was beyond the control threshold density (one mite per leaf). On the other hand, spider mite densities in some trees at conventionally managed orchards with wild groundcover were temporary beyond the control threshold level. The phytoseiid mite species composition on peach leaves estimated by previously established method using quantitative sequencing changed during the survey period and varied among orchards. PCR amplification of the internal transcribed spacer (ITS) region of ribosomal genes of Tetranychus kanzawai Kishida and Panonychus mori Yokoyama from three phytoseiid mite species, Neoseiulus californicus (McGregor), Amblyseius eharai Amitai and Swirski, and Euseius sojaensis (Ehara), collected on peach leaves was conducted. Results showed that the feeding preference for the three phytoseiid mite species was greater for T. kanzawai than for P. mori in the field. PCR amplification of the ITS sequences of Petrobia harti (Ewing) inhabiting Oxalis corniculata L. (Oxalidaceae) showed that phytoseiid mites move from groundcover plants to peach leaves, possibly through ambulatory and aerial dispersal. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Phytoseiid mites have been recognized as potential biological control agents to suppress pests such as spider mites, thrips, ⇑ Corresponding author. Fax: +81 86 434 1249. E-mail address: [email protected] (S. Sonoda). http://dx.doi.org/10.1016/j.biocontrol.2014.10.002 1049-9644/Ó 2014 Elsevier Inc. All rights reserved.

whiteflies, and other arthropods (Croft and Jung, 2001; Helle and Sabelis, 1985; McMurtry and Croft, 1997; Nomikou et al., 2002; van Lenteren, 2001). The importance of some groundcover plants has been suggested to promote the occurrence of phytoseiid mites

144

Table 1 Location, area, and pest control of each study site. Latitud longitude

Area (m2)

Product applied

IRAC mode of action classificationa

Study site

Latitud longitude

Area (m2)

Product applied

IRAC mode of action classificationa

Organic/groundcover

N34° 350 05.900 E133° 390 36.700

2440

BT (Apr 17)

11A

Conventional III/ groundcover

N34° 350 02.400 E133° 390 37.800

2900

Tolfenpyrad (Apr 16)

21A

Thiacloprid (May 5)

4A

N34° 350 02.700 E133° 390 41.100

1500

Alanycarb, buprofezin (Jun 1)

1A, 16

Etoxazole (Jun 10) Acetamiprid (Jun 23) Thiacloprid, cyenopyrafen (Jul 12) Permethrin (Apr 17)

10B 4A 4A, 25

Alanycarb (Apr 29)

1A

Conventional I/groundcover

Conventional II/no groundcover

a

N34° 350 04.0’’ E133° 390 40.2’’

400

Adion (Apr 22)

3A

Buprofezin (May 14)

16

Chlorantraniliprole, etoxazole (Jun 5) Thiacloprid (Jun 16) Dinotefuran (Jun 25) Tolfenpyrad (Jul 5)

28, 10B

DMTP (Sep 6)

1B

4A 4A 21A

Conventional IV/groundcover

N34° 350 06.700 E133° 390 38.800

1400

3A

MEP(Oct12)

1B

Buprofezin (May 9)

16

Adion (Apr 28)

3A

Alanycarb (May 22)

1A

Chlorantraniliprole, etoxazole (Jun 6)

28, 10B

Acetamiprid, cyenopyrafen (Jun 4)

4A, 25

Thiacloprid (Jun 17) Dinotefuran (Jun 30) Tolfenpyrad (Jul 7) Acetamiprid (Jul 19) Flubendiamide (Aug 15)

4A 4A 21A 4A 28

Thiacloprid (Jun 14) Dinotefuran (Jun 28) Tolfenpyrad (early Jun) Acetamiprid (mid Jul) Flubendiamide (early Aug)

4A 4A 21A 4A 28

See IRAC (http://www.irac-online.org/teams/mode-of-action/).

D. Wari et al. / Biological Control 80 (2015) 143–155

Study site

D. Wari et al. / Biological Control 80 (2015) 143–155

as insectary plants providing refuges, alternate foods, plant resources such as nectar and pollen, and places to diapause, develop, and reproduce (Gravena et al., 1993). For example, Ageratum conyzoides L. (Asteraceae), Eupatorium pauciflorum Humboldt, Bonpland and Kunth (Asteraceae), Chloris gayana Kunth, and Festuca arundinacea Schreb. (Poaceae) have been reported as insectary plants to natural enemies including phytoseiid mites in citrus orchards (Aguilar-Fenollosa et al., 2011; Gravena et al., 1993; Liang and Huang, 1994; Smith and Papacek, 1991). Plants in the genera Malva and Raphanus harbored phytoseiid mites through the year, whereas plants in the genera Amaranthus, Commelina, Conyza, Digitaria, Eleusine, Ipomoea, and Sonchus harbored phytoseiid mites during the time when these mites were active on vine leaves (de Villiers and Pringle, 2007, 2011). Phytoseiid mites were also present on Trifolium spp., Taraxacum spp., and Solidago spp. in apple orchards (Bugg and Waddington, 1994; Coli et al., 1994). However, the contributions of those plants in vineyard and apple orchards harboring phytoseiid mites in spider mite control remain unclear. Previously, we examined quantities of phytoseiid mites on groundcover plants inhabiting Japanese peach orchards to select potential insectary plants (Wari et al., 2014). Phytoseiid mites were

145

the most abundant on Veronica persica Poir. (Plantaginaceae) in May and June. Paederia foetida L. (Rubiaceae) harbored the largest quantities of phytoseiid mites during July–September. In October, greater numbers of phytoseiid mites were detected on Persicaria longiseta (Bruijin) Kitag. (Polygonoideae) than on P. foetida. Oxalis corniculata L. (Oxalidaceae) also had numerous phytoseiid mites in June and September. Consequently, the wild plant species harboring numerous phytoseiid mites change seasonally. However, no information exists to indicate that these wild plants are the source of phytoseiid mites on peach leaves. We have shown that the phytoseiid mite species composition on peach leaves differs among Japanese peach orchards (Sonoda et al., 2012; Wari et al., 2014). At the organic peach orchard with wild groundcover and no synthetic pesticide application, Euseius sojaensis (Ehara) was dominant. Neoseiulus californicus (McGregor) was dominant at the peach orchard that was conventionally managed with pesticides including herbicides. At the conventionally managed peach orchards with wild groundcover, Amblyseius eharai Amitai and Swirski and N. californicus each accounted for large proportions. Phytoseiid mites are classified into four types based on differences in food utilization as follows: type I, specialized predators of Tetranychus species; type II, specialized predators of

Fig. 1. Seasonal fluctuation of phytoseiid mite species composition on peach leaves. The frequency of each species was normalized as the sum of the frequencies being 100%. Bars with gray, black, white, dots, vertical lines, and upward-diagonal lines respectively represent proportions of Euseius sojaensis, Neoseiulus californicus, Amblyseius eharai, Typhlodromus vulgaris, Amblyseius tsugawai, and Neoseiulus makuwa. Numbers represent the number of individuals examined per sampling date.

146

D. Wari et al. / Biological Control 80 (2015) 143–155

tetranychid mites including N. californicus; type III, generalist predators including A. eharai; type IV, specialized pollen feeders and generalist predators including E. sojaensis (McMurtry and Croft, 1997). N. californicus is a promising prospect for use as a biological control agent for spider mites and is commercially mass-produced for sale in various countries (Cooping, 2001). A. eharai is known as an effective predator of Tetranychus kanzawai Kishida (Inoue et al., 1983; Tanaka and Kashio, 1977) and has an impact on Panonychus species on tree crops (McMurtry, 1985). Wari et al. (2014) showed that E. sojaensis uses both Panonychus mori Yokoyama and T. kanzawai, both of which are the most abundant spider mites in peach orchards of Okayama Prefecture, western Japan, as prey. However, there is little information related to their preferences for spider mites in the field. In this study, we examined population dynamics of phytoseiid mites and spider mites on randomly selected trees and their groundcover plant P. foetida at the Japanese peach orchards that use different pesticide practices. Species composition was also examined for phytoseiid mites on peach leaves and P. foetida using quantitative sequencing (QS) (Sonoda et al., 2012; Wari et al., 2014). Subsequently we examined feeding preferences of phytoseiid mites for spider mites in the field using PCR. Finally, we used PCR to examine the dispersal of phytoseiid mites from groundcover plants to peach leaves to evaluate the usefulness of groundcover plants as a source for phytoseiid mites on peach trees.

2. Materials and methods 2.1. Study site The survey was conducted in five commercial peach orchards in Kurashiki City, Okayama Prefecture, western Japan (Organic/ groundcover, Conventional I/groundcover, Conventional II/no groundcover, Conventional III/groundcover, and Conventional IV/groundcover) (Wari et al., 2014). Information related to these orchards is presented in Table 1. Organic/groundcover is managed according to the Japanese Agricultural Standard for organic agricultural products, which allows no synthetic chemicals for pest control. The wild groundcover for Organic/groundcover, Conventional I/groundcover, Conventional III/groundcover, and Conventional IV/groundcover was managed using a mowing machine. Non-selective herbicides, glyphosate and paraquat dichloride, were applied at Conventional II/no groundcover. Sampling was conducted every week from May 2 to October 22, 2013 in all trees at the five peach orchards. 2.2. Sampling procedure Five, seven, four, nine, and five trees were selected, respectively, at Organic/groundcover, Conventional I/groundcover, Conventional II/no groundcover, Conventional III/groundcover, and Conventional

Fig. 1 (continued)

D. Wari et al. / Biological Control 80 (2015) 143–155

IV/groundcover. Thirty leaves were sampled every week from each tree. The number of spider mites and phytoseiid mites collected in each sampling of 30 leaves was counted in a petri dish filled with 70% ethanol. Then, phytoseiid mites were separated from spider mites. Separated phytoseiid mites in each sampling were used for DNA extraction or stored in a glass container filled with 99.5% ethanol until DNA extraction. 2.3. DNA extraction DNA extraction was performed according to the method described in Wari et al. (2014). Briefly, phytoseiid mites in each sampling with more than five individuals were homogenized with 400 ll of extraction buffer (50 mM Tris–HCl pH 8.5, 10 mM EDTA, 100 mM NaCl, 2% SDS). The number of phytoseiid mites in each sampling are represented on the right side of columns in Figs. 1 and 2. The DNA was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated using 99.5% ethanol in the presence of 3 M sodium acetate (pH 5.2). The DNA pellet was washed with 70% ethanol and dissolved in 100 ll of TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). After treatment with RNase A, the DNA was purified once with phenol:chloroform:isoamyl alcohol (25:24:1), once with chloroform:isoamyl alcohol (24:1) and was precipitated using 99.5% ethanol in the presence of 3 M sodium

147

acetate (pH 5.2). The DNA pellet was washed with 70% ethanol and was dissolved in H2O. 2.4. Quantitative sequencing (QS) for the prediction of phytoseiid mite proportions For the prediction of phytoseiid mite proportions, QS was conducted according to the method described in a previous report (Sonoda et al., 2012). Briefly, species-specific polymorphic sites of the 28S ribosomal gene (728 bp) from N. californicus (GenBank/ EMBL/DDBJ accession Nos. AB618055, AB618056, and AB618057), Neoseiulus womersleyi (Schicha) (accession Nos. AB618060, AB618061, and AB618062), A. eharai (accession Nos. AB618058 andAB618059), Amblyseius tsugawai Ehara (accession No. AB618063), E. sojaensis (accession Nos. AB618064 and AB618065), Typhlodromus vulgaris (Ehara) (accession No. AB862881), Scapulaseius okinawanus (Ehara) (accession No. AB862880), and Neoseiulus makuwa (Ehara) (accession No. AB862882) were identified at nucleotides 492, 705, 624, 652, 465, 656, 475, and 479, respectively (see Fig. 1 in Wari et al., 2014). The genomic DNA fragments corresponding to the 28S ribosomal gene were amplified from each DNA sample using PCR with primer sets rD43 (50 -gacccgctgaacttaagcat-30 ) and rD13dp (50 -cgtgtttcaagacgggtcaaataact-30 ) as described in Sonoda et al. (2012). A series of PCR products covering the eight

Fig. 1 (continued)

148

D. Wari et al. / Biological Control 80 (2015) 143–155

phytoseiid mite species were cloned into pGEM-T East vector (Promega Corp., Madison, WI, USA). PCR was conducted using a plasmid containing the 28S ribosomal gene sequence of each species as templates to generate standard templates for QS. The PCR products derived from each species were purified using a PCR purification kit (QIAquick; Qiagen Inc., Hilden, Germany) and quantified. Purified PCR products of one species were mixed with those of the other species to produce the following molar ratios for the standard DNA template mixtures: 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1. The standard DNA template mixtures were sequenced directly with the internal primer rD25 (50 -gggaaagttgaaaagaactc-30 ) (Sonoda et al., 2012). The peak nucleotide sequence signal intensities of both nucleotides at each species-specific polymorphic site were measured from the sequence chromatogram using software (Photoshop CS3 ver. 10.0.1; Adobe Systems Inc., San Jose, CA, USA) (see Fig. 3 in Sonoda et al., 2012). Then the signal ratio was calculated. The signal ratios were assessed against the corresponding molar ratio. Standard regression equations were generated using software (SIGMA plot ver. 11.2; Systat Software, Inc., San Jose, USA) (See Fig. 2 in Wari et al., 2014). Regression equations to estimate the phytoseiid mite species composition in unknown samples collected on P. foetida were developed using the all eight phytoseiid mite species (Wari

et al., 2014). For unknown samples collected on peach leaves, regression equations were developed using N. californicus, N. womersleyi, A. eharai, A. tsugawai, E. sojaensis, and T. vulgaris (Wari et al., 2014). Quadratic equations which showed higher regression coefficients (r2) than linear equations for both cases were used in this study (See Table 2 in Wari et al., 2014). In the equations, x-axis and y-axis were nucleotide signal ratio [species-specific nucleotide signal/(species-specific nucleotide signal + the other nucleotide signal)] and expected proportion at species-specific polymorphic site, respectively. For signal ratios of 0 and 1.0, the resulting frequencies were regarded as 0% and 100%, respectively, without incorporating it into the prediction equations. PCR with primer set rD43 and rD13dp was conducted for unknown samples collected from peach leaves and P. foetida. Amplified fragments were sequenced directly using the primer rD25. The presence or absence of phytoseiid mite species in unknown samples was confirmed at their respective species-specific polymorphic sites. Then, the prediction of proportions for phytoseiid mite species was conducted using the quadratic equations mentioned above. It is noteworthy that the proportions of phytoseiid mite species estimated using the QS-based method cannot be a prediction of their number, but their biomass (Sonoda et al., 2012).

Fig. 1 (continued)

D. Wari et al. / Biological Control 80 (2015) 143–155

2.5. Detection of spider mite DNA from phytoseiid mites In total, 17, 78, and 14 DNA samples of phytoseiid mites were predicted to contain only E. sojaensis, A. eharai, and N. californicus, respectively using QS-based method described above (Fig. 1 and Table S1). They were used to amplify the internal transcribed spacer (ITS) region of ribosomal genes of spider mites. PCR amplification for the ITS sequences from P. mori and T. kanzawai was conducted as described previously with minor modifications (Wari et al., 2014). First, PCR was conducted using primer set T/P-ITS-50 -2 (50 -cctgcggaaggatcattaac-30 ) and T/P-ITS-30 -2 (50 -ggtaattcgagtgatccacc-30 ) at PCR conditions of 40 cycles of 15 s at 94 °C, 30 s at 60 °C and 1 min at 72 °C with subsequent final extension of 72 °C for 7 min. The primer set was designed to amplify the ITS sequences of both spider mite species. The PCR products were

149

used for re-amplification using primer sets PM-ITS-50 (50 -atgcaggcacacataccgt-30 ) and PM-ITS-30 (50 -ccgtgggacttttattctc-30 ) and TK-ITS-50 (50 -caacatgattctatttgtg-30 ) and TK-ITS-30 (50 -gccaccgtgggacttttaa-30 ) at PCR conditions of 40 cycles of 15 s at 94 °C, 30 s at 50 °C, and 1 min at 72 °C with subsequent final extension of 72 °C for 7 min. The former and latter primer sets exclusively amplify PCR products from P. mori (415 bp) and T. kanzawai (323 bp), respectively (Wari et al., 2014). The PCR conditions amplified detectable amounts of spider mite DNA from 1.010 ng of genomic DNA of both spider mite species (data not shown). In addition, PCR amplification for the ITS sequences of Petrobia harti (Ewing) (Acari: Tetranychidae) from the phytoseiid mite DNA samples was also conducted. First PCR conducted using primer set PhITS-50 -1 (50 -gcataaattctgcgggtagc-30 ) and Ph-ITS-30 -1 (50 -ctgtggcatactctcccttg-30 ) was followed by second PCR using primer set

Fig. 2. Seasonal fluctuation of phytoseiid mite species composition on Paederia foetida. Bars with gray, black, white, dots, vertical lines, upward-diagonal lines, downwarddiagonal lines, and small checker board respectively represent proportions of Euseius sojaensis, Neoseiulus californicus, Amblyseius eharai, Typhlodromus vulgaris, Amblyseius tsugawai, Neoseiulus makuwa, Scapulaseius okinawanus, and Neoseiulus womersleyi. Numbers represent the number of individuals examined per sampling date.

150

D. Wari et al. / Biological Control 80 (2015) 143–155

Ph-ITS-50 -2 (50 -taccatccattagtgcggtg-30 ) and Ph-ITS-30 -2 (50 -caccgcttgtaggtgtatct-30 ). These primer sets for P. harti were designed based on the nucleotide sequences deposited in the DNA data bank (GenBank/EMBL/DDBJ accession No.GQ141935). The primer sets exclusively amplify PCR products from P. harti (416 bp) (data not shown). The PCR conditions were 30 cycles of 15 s at 94 °C, 30 s at 60 °C and 1 min at 72 °C, with subsequent final extension of 72 °C for 7 min. PCR products were size-fractionated on 1.0% agarose gel and were observed under ultraviolet light after staining with ethidium bromide.

Two trees with P. foetida as a groundcover plant were selected from Organic/groundcover (tree III and tree V), Conventional I/groundcover (tree VI and tree VII), and Conventional III/groundcover (tree II and tree III) to compare phytoseiid mite species composition between peach leaves and P. foetida. P. foetida harbored the largest quantities of phytoseiid mites for a longer period compared to the other wild plants (Wari et al., 2014). Phytoseiid mite species that were not detected on peach leaves were detected on P. foetida (Fig. 2). Nevertheless, dominant phytoseiid mite species on P. foetida were similar to those of peach leaves at respective sites (Figs. 1 and 2).

3. Results 3.1. Species composition of phytoseiid mites on peach leaves and P. foetida

3.2. Population dynamics of spider mites and phytoseiid mites on peach leaves and P. foetida

Wari et al. (2014) reported that E. sojaensis, N. californicus, and A. eharai are dominant, respectively, at Organic/groundcover, Conventional II/no groundcover, and Conventional III/groundcover. These observations at an orchard level were confirmed in this study with a few exceptions (tree VI and tree VII) at Conventional III/groundcover (Fig. 1). At Conventional I/groundcover, in general, N. californicus constituted a large share from June to early July and then A. eharai became dominant after late July (Fig. 1). Phytoseiid mite species composition varied among trees at Conventional IV/groundcover (Fig. 1). In addition to N. californicus and A. eharai, T. vulgaris (Ehara) showed a large share in some trees (tree III, tree IV, and tree V) at Conventional IV/groundcover (Fig. 1).

The seasonal fluctuations of phytoseiid mites and spider mites on peach leaves are presented in Fig. 3. Wari et al. (2014) reported that the spider mite appearance was preceded by that of phytoseiid mites at Organic/groundcover. Similar preceded appearance of phytoseiid mites was observed at all trees at Organic/groundcover in this study. At Conventional I/groundcover, Conventional III/ groundcover, and Conventional IV/groundcover, phytoseiid mites generally appeared in response to the occurrence of spider mites at an orchard level (Wari et al., 2014). In this study, Conventional I/groundcover was the only site where the phytoseiid mite appearance responded to that of spider mites in all trees surveyed. Such phytoseiid mite appearance in response to spider mites was

Fig. 3. Seasonal fluctuation in the number of phytoseiid mites and spider mites collected on peach leaves and on Paederia foetida. Solid, dotted, and broken lines respectively represent the number of phytoseiid mites, Panonychus mori, and Tetranychus kanzawai. Bars at Organic/groundcover (tree III and tree V), Conventional I/groundcover (tree VI and tree VII), and Conventional III/groundcover (tree II and tree III) show seasonal fluctuation in the number of phytoseiid mites on P. foetida.

D. Wari et al. / Biological Control 80 (2015) 143–155

151

observed in some trees at Conventional III/groundcover and Conventional IV/groundcover. At Conventional II/no groundcover, no such clear response of phytoseiid mites against spider mites was observed in any trees surveyed in this study. No data show the prey for E. sojaensis and N. californicus on peach leaves at Organic/groundcover and Conventional II/no groundcover, respectively, when spider mites were not collected. Conventional I/groundcover, Conventional III/groundcover, and Conventional IV/groundcover had trees for which the spider mite densities were temporarily beyond the control threshold density for spider mites in Japanese peach orchards (one mite per leaf) (tree IV, tree V, and tree VII at Conventional I/groundcover; tree I, tree II, tree VIII, and tree IX at Conventional III/groundcover; tree I, tree II, and tree V at Conventional IV/groundcover) (Fig. 3). In contrast, at Organic/groundcover and Conventional II/no groundcover, spider mite densities were below the control threshold in all trees during the survey period (Fig. 3). The seasonal fluctuations of phytoseiid mites were compared between peach leaves and P. foetida at selected trees at Organic/ groundcover (tree III and tree V). Conventional I/groundcover (tree VI and tree VII), and Conventional III/groundcover (tree II and tree III). Similar trends of the seasonal fluctuations of phytoseiid mites were observed between peach leaves and P. foetida in respective trees (Fig. 3).

sequences of P. mori and T. kanzawai. All E. sojaensis samples were derived from Organic/groundcover. Samples for A. eharai and N. californicus were derived respectively from four sites (Conventional I/groundcover, Conventional II/no groundcover, Conventional III/ groundcover, and Conventional IV/groundcover) and two sites (Conventional II/no groundcover and Conventional III/groundcover). The total number of individuals for T. kanzawai and P. mori collected at Organic/groundcover was, respectively, 87 and 257 (data not shown). The total number of individuals for both spider mites collected at Conventional I/groundcover, Conventional II/no groundcover, Conventional III/groundcover and Conventional IV/ groundcover was similar (1132 for T. kanzawai; 1374 for P. mori) (data not shown). Actually, P. mori was collected more (total 758 individuals) than T. kanzawai (total 132 individuals) at Conventional II/no groundcover and Conventional III/groundcover (data not shown). The ITS sequences of T. kanzawai were amplified more frequently than those of P. mori for E. sojaensis (Tables 2 and S1). More frequent amplification of the ITS sequences of T. kanzawai was also observed for A. eharai (Tables 2 and S1). The ITS sequences of both spider mite species were amplified with similar frequencies for N. californicus (Tables 2 and S1). However, this was observed in the condition at which P. mori shared a higher proportion, as described above. Therefore, N. californicus might also show prey preference on T. kanzawai.

3.3. Detection of spider mite DNA from phytoseiid mites collected on peach leaves

3.4. Detection of P. harti DNA from phytoseiid mites collected on peach leaves

A total of 109 DNA samples of phytoseiid mites with more than five individuals collected on peach leaves and predicted to contain only one species (Fig. 1) were used for amplification of the ITS

Amplification of the ITS sequences of P. harti was conducted from 109 DNA samples as described above. Results showed that the ITS sequences of P. harti were amplified from two E. sojaensis

Fig. 3 (continued)

152

D. Wari et al. / Biological Control 80 (2015) 143–155

samples out of 17, three A. eharai samples out of 79, and two N. californicus samples out of 14 (Tables 2 and S1). 4. Discussion 4.1. Determinants of phytoseiid mite species composition on peach leaves At Conventional I/groundcover and Conventional II/no groundcover, which had similar pesticide practices, up to early July, N. californicus showed a large share in phytoseiid mite population (Fig. 1 and Table 1). Reportedly, the dominant existence of N. californicus is often observed in those orchards that received more pesticides applications (Amano et al., 2004; Kishimoto, 2002; Kishimoto et al., 2007). Pesticide sprays were performed intensively up to harvest of peach (late July to early August) at the study sites, as shown in Table 1. Therefore, the large share of N. californicus in phytoseiid mite population observed by early July at both sites might be mainly attributable to the intensive pesticide applications. Nevertheless, the dominance of N. californicus was more prominent at Conventional II/no groundcover. AguilarFenollosa et al. (2011) reported that the predatory phytoseiid mites might be affected by increased competition with generalist phytoseiid mites in clementine mandarin orchards with wild groundcover. N. californicus, A. eharai, and E. sojaensis, belonging respectively to type II (selective predators of tetranychid mites), type III (generalist predators), and type IV (specialized pollen feeders/generalist predators) phytoseiid mites (McMurtry and Croft, 1997), were detected at Conventional I/groundcover with wild groundcover. At Conventional I/groundcover, the advantage of

N. californicus with higher tolerance to pesticides might be diminished by increased competition with the other phytoseiid mites such as E. sojaensis and A. eharai. However, at Conventional II/no groundcover with bare ground and less competition with the other phytoseiid mites, the dominance of N. californicus might be more stable. After mid-August, A. eharai became dominant at Conventional I/groundcover and increased its share at Conventional II/no groundcover, possibly because of the application of less pesticide. Inter-specific interactions among phytoseiid mites at peach orchards with different pesticide practices and ground cover managements must be examined in greater detail in future studies. 4.2. Cause of successful suppression of spider mites at organic/ groundcover and conventional II/no groundcover In this study, no temporal local outbreak of spider mites was observed at Organic/groundcover, where E. sojaensis was dominant (Figs. 1 and 3). McMurtry (1992) reported that generalist phytoseiid mites play a vital role when spider mite densities are low. Ozawa and Yano (2009) showed in the laboratory that E. sojaensis can prey on adults of T. kanzawai as long as the predator settled on a plant before the prey. The successful suppression of spider mites by the generalist phytoseiid mite at Organic/groundcover might be attributable to the earlier settlement of E. sojaensis (Figs. 1 and 3). No temporal local outbreak of spider mites was also observed at Conventional II/no groundcover, where N. californicus was dominant during the survey period (Figs. 1 and 3). However, at Conventional I/groundcover, Conventional III/groundcover, and Conventional IV/groundcover where A. eharai showed a large share, temporary local outbreaks were observed in some trees (Figs. 1 and 3).

Fig. 3 (continued)

D. Wari et al. / Biological Control 80 (2015) 143–155

153

Fig. 3 (continued)

Table 2 Detection of ribosomal ITS sequences of spider mites from phytoseiid mites.

a b c

Phytoseiid mite species

na

P. harti

T. kanzawai

P. mori

Bothb

Nonec

Euseius sojaensis Amblyseius eharai Neoseiulus californicus

17 78 14

2 3 2

5 24 0

1 3 3

11 49 11

0 2 0

Number of samples of phytoseiid mite species examined. Detection of both T. kanzawai and P. mori DNA. No detection of both T. kanzawai and P. mori.

Type II phytoseiid mites, including N. californicus, have a stronger impact on tetranychid infestations than do type III phytoseiid mites such as A. eharai (McMurtry and Croft, 1997). Total numbers of pesticide application up to harvest at Conventional I/groundcover and Conventional III/groundcover were equal to that at Conventional II/no groundcover (Table 1). No temporary local outbreak observed at Conventional II/no groundcover might be attributable to differences in phytoseiid mite species composition (Fig. 1). Reportedly, Tetranychus urticae Koch populations developed more quickly and with higher densities in trees over wild groundcover than bare ground in peach orchards (Meagher and Meyer, 1990). Wild plants belonging to Vicia, Geranium, Lamium, and Lepidium hosted high densities of spider mites during early spring and might have become the source for later peach-tree infestation (Meagher and Meyer, 1990). Previously, we showed that Vicia sativa L. and Lamium purpureum L. hosted greater numbers of spider mites in peach orchards (Wari et al., 2014). Therefore, the

temporary local outbreaks of spider mites might also be attributable to quantitative differences in the wild plants harboring larger quantities of spider mites. 4.3. Feeding preferences of phytoseiid mites for Tetranychus and Panonychus spider mites Croft et al. (1998) showed that fecundity of N. californicus fed with Panonychus ulmi was lower than that fed with T. urticae. Miya et al. (2004) reported that the fecundity of N. californicus was slightly higher on T. urticae eggs than on P. mori eggs. Feeding on P. ulmi eggs caused slightly reduced consumption and developmental rates and shorter post-oviposition period of N. californicus than those of the eggs of the other spider mites including T. urticae, T. kanzawai, and Panonychus citri (Gotoh et al., 2006). The survival rate in immature stages of Typhlodromus annectens De Leon is significantly lower when fed on P. citri than when fed on Tetranychus pacificus McGregor (Badii et al., 1990). The survival, reproduction, and development rates of Neoseiulus fallacis (Garman) are higher when held with Tetranychus species than when held with other genera of tetranychid mites including P. ulmi and P. citri (Pratt et al., 1999). P. citri eggs were not suitable for generalist phytoseiid mite species, Typhlodromus laurentii Ragusa et Swirski and Typhlodromus rhenanoides AthiasHenrio compared to T. urticae eggs (Tsolakis et al., 2013). Results in this study showed that N. californicus, A. eharai, and E. sojaensis prefer to feed T. kanzawai rather than P. mori in the field (Tables 2 and S1). Although, reportedly, N. californicus can grow and oviposit by preying on P. citri eggs no less than T. urticae (Katayama et al., 2006), Panonychus species might be less preferred by phytoseiid

154

D. Wari et al. / Biological Control 80 (2015) 143–155

mites than Tetranychus species, irrespective of their feeding types. No data are available to explain the growth stages of spider mites at which phytoseiid mite species mostly prey in the peach orchards. Predatory performance of the phytoseiid mite species on various growth stages of T. kanzawai and P. mori remains to be examined to ascertain whether the preference depends on the food quality or on the defense strategy of the prey stage. 4.4. Dispersal of phytoseiid mites from groundcover to peach leaves Kawashima and Jung (2010) investigated the overwintering sites of N. californicus in mandarin orchards in Korea. They reported that most N. californicus were collected on herbaceous plants on the ground rather than on mandarin trees or woody plants surrounding the orchards. That observation seems reasonable considering that N. californicus on the peach leaves dispersed from the groundcover in this study. However, in persimmon orchards, a considerable number of overwintering A. eharai were collected on the trees (Kawashima, unpublished data). Overwintering sites for E. sojaensis have not been reported. Reportedly, in peach orchards of northern Greece, Euseius finlandicus Oudemans overwinters in various sites on the trees (Broufas et al., 2002). Therefore, it remained unclear if A. eharai and E. sojaensis on the peach leaves were supplied by the groundcover. In the present study, we detected P. harti DNA from phytoseiid mites including E. sojaensis and A. eharai collected on peach leaves at Organic/groundcover, Conventional I/groundcover, and Conventional III/groundcover. Also, P. harti DNA was detected from N. californicus collected on peach leaves at Conventional II/no groundcover. Actually, distribution of P. harti is worldwide, where they are usually found on Oxalis spp. (Dubitzki and Gerson, 1987), but they have never been observed on peach leaves (Sonoda, unpublished data). Reportedly, the walking speed of phytoseiid mites differs among species depending on their feeding types (Jung and Croft, 2001). The specialist predator N. fallacis (Garman), an ‘active’ mite, requires ca. 10 h of continuous walking to go 1 m in a linear distance (Croft and Jung, 2001). That rate seems reasonable considering that the ambulatory and aerial dispersal of the phytoseiid mites occurred from O. corniculata to peach trees as reported by Jung and Croft (2001). These results suggest that some groundcover plants such as O. corniculata, a host of P. harti, promote the occurrence of phytoseiid mites by providing food resources. For that reason, they can be inferred as the source of phytoseiid mites on peach leaves as insectary plants. Evaluation of other wild plants including V. persica, P. foetida, and P. longiseta harboring numerous phytoseiid mites (Wari et al., 2014) as insectary plants remains as a subject for future investigations. Acknowledgments This work was financially supported by the Ministry of Agriculture, Forestry and Fisheries, Japan through a research project entitled ‘Development of technologies for mitigation and adaptation to climate change in Agriculture, Forestry and Fisheries’ and the Ohara Foundation for Agricultural Research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocontrol.2014. 10.002. References Aguilar-Fenollosa, E., Ibáñez-Gual, M.V., Pascual-Ruiz, S., Hurtado, M., Jacas, J.A., 2011. Effects of ground-cover management on spider mites and their phytoseiid

natural enemies in clementine mandarin orchards (II): top-down regulation mechanisms. Biol. Control 59, 171–179. Amano, H., Ishii, Y., Kobori, Y., 2004. Pesticide susceptibility of two dominant phytoseiid mites, Neoseiulus californicus and N. womersleyi, in conventional Japanese fruit orchards (Gamasina: Phytoseiidae). J. Acarol. Soc. Japan 13, 65–70. Badii, M.H., McMurtry, J.A., Johnson, H.G., 1990. Comparative life-history studies on the predaceous mites Typhlodromus annectens and T. porresi (Acari: Mesostigmata: Phytoseiidae). Exp. Appl. Acarol. 10, 129–136. Broufas, G.D., Koveos, D.S., Georgatsis, D.I., 2002. Overwintering sites and winter mortality of Euseius Finlandicus (Acari: Phytoseiidae) in a peach orchard in northern Greece. Exp. Appl. Acarol. 26, 1–12. Bugg, R.L., Waddington, C., 1994. Using cover crop to manage arthropod pests of orchards: a review. Agric. Ecosyst. Environ. 50, 11–28. Coli, W.M., Ciurlino, R.A., Hosmer, T., 1994. Effect of understory and border vegetation composition on phytophagous and predatory mites in Massachusetts commercial apple orchards. Agric. Ecosyst. Environ. 50, 49–60. Croft, B.A., Jung, C., 2001. Phytoseiid dispersal at plant to regional levels: a review with emphasis on management of Neoseiulus fallacis in diverse agroecosystems. Exp. Appl. Acarol. 25, 763–784. Croft, B.A., Monetti, L.N., Pratt, P.D., 1998. Comparative life histories and predation types: are Neoseiulus californicus and N. Fallacis (Acari: Phytoseiidae) similar type II selective predators of spider mites? Environ. Entomol. 27, 531–538. Cooping, L.G. (Ed.), 2001. The BioPesticide Manual, second ed. British Crop Protection Council, Surrey, p. 528. de Villiers, M., Pringle, K.L., 2007. Seasonal occurrence of vine pests in commercially treated vineyards in the Hex River Valley in the Western Cape Province, South Africa. Afr. Entomol. 15, 241–260. de Villiers, M., Pringle, K.L., 2011. The presence of Tetranychus urticae (Acari: Tetranychidae) and its predators on plants in the ground cover in commercially treated vineyards. Exp. Appl. Acarol. 53, 121–137. Dubitzki, E., Gerson, U., 1987. The natural history of Petrobia (Tetranychina) harti (Ewing) and Petrobia (Mesotetranychus) tunisiae Manson (Acari: Tetranychidae) in the laboratory. Exp. Appl. Acarol. 3, 91–94. Gravena, S., Coletti, A., Yamamoto, P.T., 1993. Influence of green cover with Ageratum conyzoides and Eupatorium pauciflorum on predatory and phytophagous mites in citrus. Bull. IOBC-SROP 16, 104–114. Gotoh, T., Tsuchiya, A., Kitashima, Y., 2006. Influence of prey on developmental performance, reproduction and prey consumption of Neoseiulus californicus (Acari: Phytoseiidae). Exp. Appl. Acarol. 40, 189–204. Helle, W., Sabelis, M.W., 1985. World crop pests. Spider Mites: Their Biology Natural Enemies and Control, vol. 1B. Elsevier, Amsterdam, The Netherlands. Inoue, K., Ashihara, W., Osakabe, M., Hamamura, T., 1983. Predator fauna on windbreaks around citrus groves. Jpn. J. Appl. Entomol. Zool. 35, 49–56 (in Japanese with English summary). Jung, C., Croft, B.A., 2001. Ambulatory and aerial dispersal among specialist and generalist predatory mites (Acari: Phytoseiidae). Environ. Entomol. 30, 1112– 1118. Katayama, H., Masui, S., Tsuchiya, M., Tatara, A., Doi, M., Kaneko, S., Saito, T., 2006. Density suppression of the citrus red mite Panonychus citri (Acari: Tetranychidae) due to the occurrence of Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) on Satsuma mandarin. Appl. Entomol. Zool. 41, 679–684. Kawashima, M., Jung, C., 2010. Overwintering sites of the predacious mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) in Satsuma mandarin orchards on Jeju Island, Korea. Appl. Entomol. Zool. 45, 191–199. Kishimoto, H., 2002. Species composition and seasonal occurrence of spider mites (Acari: Tetranychidae) and their predators in Japanese pear orchards with different agrochemical spraying programs. Appl. Entomol. Zool. 37, 603–615. Kishimoto, H., Teshiba, M., Kondoh, T., Miyazaki, T., Sugiura, N., Toda, S., Yamasaki, R., Wakatsuki, H., Motoyama, H., Horie, H., 2007. Occurrence of Neoseiulus californicus (Acari: Phytoseiidae) on citrus in Kyushu district, Japan. J. Acarol. Soc. Japan 16, 129–137 (in Japanese with English summary). Liang, W., Huang, M., 1994. Influence of citrus orchards ground cover plants on arthropod communities in China: a review. Agric. Ecosyst. Environ. 50, 29–37. Meagher Jr., R.L., Meyer, J.R., 1990. Influence of ground cover and herbicide treatments on Tetranycus urticae populations in peach orchards. Exp. Appl. Acarol. 9, 149–158. Miya, M., Kishimoto, H., Adachi, I., 2004. Evaluation of the resident time of the predatory mite Neoseiulus californicus (Acari: Phytoseiidae) in prey patches of different spider mite species. Annual Report of the Kanto-Tosan Plant Protection Society 51, 129–132 (in Japanese). McMurtry, J.A., 1985. Citrus. In: Helle, W., Sabelis, M.W. (Eds.), Spider Mites: Their Biology, Natural Enemies and Control, vol. 1B. Elsevier, Amsterdam, pp. 339–347. McMurtry, J.A., 1992. Dynamics and potential impact of ‘generalist’ photoseiids in agroecosystems and possibilities for establishment of exotic species. Exp. Appl. Acarol. 14, 371–382. McMurtry, J.A., Croft, B.A., 1997. Life-styles of phytoseiid mites and their role in biological control. Annu. Rev. Entomol. 42, 291–321. Nomikou, M., Janssen, A., Schraag, R., Sabelis, M.W., 2002. Phytoseiid predators suppress populations of Bemisia tabaci on cucumber plants with alternative food. Exp. Appl. Acarol. 27, 57–68. Ozawa, M., Yano, S., 2009. Pearl bodies of Cayratia japonica (Thunb.) Gagnep. (Vitaceae) as alternative food for a predatory mite Euseius sojaensis (Ehara) (Acari: Phytoseiidae). Ecol. Res. 24, 257–262. Pratt, P.D., Schausberger, P., Croft, B.A., 1999. Prey-food types of Neoseiulus fallacis (Acari: Phytoseiidae) and literature versus experimentally derived prey-food estimates for five phytoseiid species. Exp. Appl. Acarol. 23, 551–565.

D. Wari et al. / Biological Control 80 (2015) 143–155 Smith, D., Papacek, D.F., 1991. Studies of the predatory mite Amblyseius victoriensis (Acarina: Phytoseiidae) in citrus orchards in south-east Queensland: control of Tegolophus australis and Phyllocoptruta oleivora (Acarina: Eriophyidae), effect of pesticides, alternative host plants and augmentative release. Exp. Appl. Acarol. 12, 195–217. Sonoda, S., Kohara, Y., Siqingerile, Toyoshima, S., Kishimoto, H., Hinomoto, N., 2012. Phytoseiid mite species composition in Japanese peach orchards estimated using quantitative sequencing. Exp. Appl. Acarol. 6, 9–22. Tanaka, M., Kashio, T., 1977. Biological studies on Amblyseius largoensis Muma (Acarina: Phytoseiidae) as a predator of the citrus red mite, Panonychus citri (McGregor) (Acarina: Tetranychidae). Bull. Fruit Trees Res. Station D1, 49–67.

155

Tsolakis, H., Palomero, R.J., Ragusa, E., 2013. Predatory performance of two Mediterranean phytoseiid species, Typhlodromus laurentii and Typhlodromus rhenanoides fed on eggs of Panonychus citri and Tetranychus urticae. Bull. Insectol. 66, 291–296. van Lenteren, J.C., 2001. A greenhouse without pesticides: fact or fantasy? Crop Prot. 19, 375–384. Wari, D., Yamishita, J., Kataoka, Y., Kohara, Y., Hinomoto, N., Kishimoto, H., Toyoshima, S., Sonoda, S., 2014. Population survey of phytoseiid mites and spider mites on peach leaves and wild plants in Japanese peach orchard. Exp. Appl. Acarol. 63, 313–332.