High Prevalence of Pantoea spp. in Microbiota Associated with the Sorghum Plant Bug Stenotus rubrovittatus (Heteroptera: Miridae)

The sorghum plant bug, Stenotus rubrovittatus (order Heteroptera: family Miridae), is a notorious insect pest in Japan that causes pecky rice. In the present study, we sampled this insect pest in the northern part of Honshu Island in Japan and investigated its associated microbiota. The results obtained showed that Pantoea dominated the associated microbiota and was the sole genus detected in all samples. The dominant Pantoea were phylogenetically close to rice pathogens. The present results suggest that the sorghum plant bug needs to be regarded and controlled not only as a notorious insect pest, but also as a potential vector of rice pathogenic Pantoea spp.


Supplementary Materials and Methods
Insect samples: Sampling was performed at 24 locations over seven prefectures (29 samples in total) in the northern part of Honshu Island in Japan (Supplementary Table S2).Adult insects were collected by sweeping for weeds in rice fields with an insect net.
DNA extraction: DNA extraction from the whole insect body was performed with Wizard Genomic DNA Purification Kit (Promega), according to the manufacturer's instructions.The success of DNA extraction was confirmed by measuring DNA concentration with QuatiFluor ONE dsDNA System (Promega) and performing PCR amplification of the insect cytochrome oxidase 1 gene with TaKaRa EX Taq Hot Start Version (Takara Bio) (Supplementary Table S1 and Table S2).The composition of the PCR reaction mixture (total 10 µl) was as follow: 1 µl of 10´Ex Taq Buffer (Mg2+ plus) (20 mM), 0.8 µl of dNTP Mixture (2.5 mM each), 0.4 µl of primer mixture (10 µM each of forward and reverse primers), 1 µl of DNA sample, 0.05 µl of TaKaRa Ex Taq HS (5 U/µl), and 6.75 µl of distilled water.The temperature profile for the PCR was as follows: 94˚C for 2 min; and 30 cycles of 98˚C for 10 sec, 45˚C for 30 sec, and 72˚C for 1 min.
Clone library analysis: For the clone library analysis, a 1.5-kb fragment of bacterial 16S rRNA gene was amplified with TaKaRa EX Taq Hot Start Version (Supplementary Table S1).The composition of the PCR reaction mixture (total 10 µl) was as follow: 1 µl of 10´Ex Taq Buffer (Mg2+ plus) (20 mM), 0.8 µl of dNTP Mixture (2.5 mM each), 0.4 µl of primer mixture (10 µM each of forward and reverse primers), 1 µl of DNA sample, 0.05 µl of TaKaRa Ex Taq HS (5 U/µl), and 6.75 µl of distilled water.The temperature profile for the PCR was as follows: 94˚C for 2 min; 35 cycles of 98˚C for 10 sec, 52˚C for 30 sec, and 72˚C for 2 min; and 72˚C for 10 min.Cloning of the PCR products, Sanger sequencing of the clones, and assembling of the sequences were performed as described previously (Takeshita et al., 2015).Clone sequences were classified into operational taxonomic units (OTUs) with vsearch 2.15.0 with a 99% identity threshold (Rognes et al., 2016).The representative sequences of each OTU were subjected to a BLASTN search (Camacho et al., 2009) against the NCBI 16S ribosomal RNA BLAST database (downloaded in March 2021).
Culture of the associated bacteria: Two adult females collected in the Akita campus, Akita prefectural university, Japan in Sep.2020, were surface-sterilized by sinking in 70% ethanol for about 1 min, washed with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4 [pH 7.4]) adequately, and dissected in PBS with micro-scissors and micro-tweezers under a dissection microscope (S9 D, Leica Microsystems).The dissected digestive tract was photographed with a digital microscope camera (MC170 HD, Leica Microsystems).Each component of the digestive tract was separately homogenized in PBS, and then the diluted homogenates were plated on R2A (R2A broth "DAIGO", FUJIFILM Wako Pure Chemical Corp.) -agar and LB (1% Tryptone, 1% NaCl, 0.5% yeast extract) -agar plates.These plates were incubated at room temperature for 1-2 days.Emerging yellowish colonies, which is a characteristic of Patnoea spp.(Walterson and Stavrinides, 2015), were randomly picked up, and cultured in R2A or LB media at room temperature for 1 day.Stocks of them were stored in 20% glycerol at -80˚C.The 16S rRNA sequences of the isolates were determined and their homology search was performed, as described above.

Phylogenetic analysis:
The sequences of the representative from each of the five Pantoea OTU, the seven Pantoea isolates, those of the related Pantoea symbionts from other insects, and those of type strains of the representative Pantoea spp., as well as outgroup sequences, were subjected to phylogenetic analysis based on the maximum likelihood method.Multiple alignment was constructed by using SINA v1.7.2 (Pruesse et al., 2012), and gap-including and ambiguous sites in the alignment were then removed.Phylogenetic relationships based on maximum likelihood method were reconstructed with RAxML v8.2.12 using the general time reversible model with gamma distribution (GTR+Γ) (Stamatakis, 2014).The bootstrap values of 1,000 replicates were calculated with a rapid bootstrapping algorithm (Stamatakis et al., 2008).Phylogenetic reconstruction and bootstrap analysis based on neighbor joining method were performed with MEGAX using the Tamura-Nei model with gamma distribution (TN+Γ) (Stecher et al., 2020).
Quantitative PCR: For estimating the total number of bacteria associated with the insect host, qPCR targeting a 300-bp fragment of the bacterial 16S rRNA gene was performed (Supplementary Table S1).The composition of qPCR reaction mixture (total 20 µl) was as follow: 10 µl of Luna Universal qPCR Master Mix (New England Biolabs), 4 µl of DNA sample, 1 µl of primer mixture (10 µM each of forward and reverse primers), and 5 µl of distilled water.The temperature profile for qPCR was as follows: 95˚C for 2 min; and 40 cycles of 95˚C for 15 sec, 54˚C for 10 sec, and 60˚C for 30sec.For calculating the absolute copy number, 10-fold serial dilutions of the target PCR product of Escherichia coli DH5a were applied.
The temperature profile for the PCR was as follows: 94˚C for 2 min; 35 cycles of 94˚C for 30 sec, 55˚C for 30 sec, and 72˚C for 30 sec; and 72˚C for 5 min.Library preparation and amplicon sequencing with Illumina MiSeq platform (2´300 bp) were conducted at Bioengineering Lab.Co., Ltd.
Adapter sequencing were removed from raw sequence reads with Cutadapt Version 3.4 (Martin, 2011).Merging the paired reads and quality filtering was performed with fastp 0.20.1 (Chen et al., 2018).Low-quality reads (Q < 30, length < 100 bp) and short merged sequences (length < 400 bp) were removed before and after merging, respectively.Chimeric sequences were removed with chimera.uchime of mothur v.1.40.4 (Schloss et al., 2009).The resulting sequences were taxonomically assigned at the genus level with RDP Classifier 2.13 with 80% confidence threshold (Wang et al., 2007).The sequences assigned into Chloroplast were removed from the subsequent analyses.
for rapid assignment of rRNA sequences into the new bacterial taxonomy.Appl Environ Microbiol 73: 5261-5267.S1 a Major 34 genera of which relative abundance is > 1% in at least two samples, as well as Rickettia and "unclassified_Bacteria," are listed.

Supplementary Table
b This was non-target sequences, therefore we removed from Fig. 2. c Rickettsia includes "unclassified_Rickettsiaceae" and "unclassified_Rickettsiales." d The sum of the number of sequences of minor OTUs.
Sample Name

.
Primers used in this study.Adapter and random sequences are not shown. a

Table S2 .
Insect samples and summary statistics of the 16S rRNA amplicon sequencing.
c Accession No. d Qualified Bacteria f Pantoea Relative Abundance of Pantoea e AKT-F1a The samples were also used for clone library analysis.bNumber of 16S rRNA gene copies per insect determined by qPCR is shown.cAccessionnumbers of nucleotide sequences determined in the clone library analysis.dAccessionnumbers of raw reads produced in the 16S rRNA amplicon sequencing.eSummarystatistics on the 16S rRNA amplicon sequencing.fSequencesnot assigned as bacteria were assigned as Eukaryota or not done into any domains (unclassified).bSupplementary

Table S3 .
Numbers of 16S rRNA gene clones assigned in each OTU.
a Accession number of the representative clone sequence of the OTU.b The result of BLASTN search against the NCBI 16S rRNA BLAST database.

Table S4 .
The list of cultured isolates from S. rubrovittatus .
a The result of BLASTN search against the NCBI 16S rRNA BLAST database.b,c Letters indicate the isolates from the same individual.d These sequences show 100% identity of each other.e These strains are identical.