Phytoseiid mite species composition in Japanese peach orchards estimated using quantitative sequencing

Abstract

We attempted a population survey of spider mites and phytoseiid mites in Japanese peach orchards with different pesticide practices; however, we had difficulty discriminating phytoseiid mites. To estimate phytoseiid mite species composition, ribosomal gene fragments were amplified from genomic DNA of five phytoseiid mite species using PCR. Cloning and nucleotide sequencing of amplified fragments identified species-specific polymorphic sites. Newly amplified fragments from recombinant plasmids were mixed in various ratios to produce standard DNA template mixtures. After direct sequencing, the signal ratios between two nucleotides at each species-specific polymorphic site were calculated and shown against the corresponding expected ratios. Quadratic regression equations were used to estimate the phytoseiid mite species composition. Results showed that the phytoseiid mite species composition changed during the survey period and varied among study sites.

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

“…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.…”

“…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 (5 0 -gacccgctgaacttaagcat-3 0 ) and rD13dp (5 0 -cgtgtttcaagacgggtcaaataact-3 0 ) as described in Sonoda et al (2012). A series of PCR products covering the eight Fig.…”

“…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 (5 0 -gggaaagttgaaaagaactc-3 0 ) (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.…”

“…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.…”

“…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.…”