Microbial symbionts of the putative vector of coconut lethal yellowing (CILY) phytoplasma in Cote d'ivoire

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1 Elena Gonella, PhD DISAFA University of Turin largo Paolo Braccini Grugliasco (TO), Italy Tel Fax December 2016_Final Report Microbial symbionts of the putative vector of coconut lethal yellowing (CILY) phytoplasma in Cote d'ivoire A preliminary characterization of the microbial community associated with the putative phytoplasma vectors was carried out by means of 16S rrna gene libraries. Such study was then powered by Illumina sequencing of the regions V3-V4 of 16SrRNA gene. Moreover, qualitative PCR reactions with specific primers for major leafhopper symbiont clades were performed on whole insect and on specific body parts (head, thorax, and abdomen) to assess their presence, prevalence, and localization. Materials and methods Twenty whole insects (6 females and 14 males), as well as 18 body parts (head, thorax and abdomen from three females and three males) were subjected to DNA extraction according to Gonella et al. (2012). DNA samples from four of these specimens (two males and two females) were used to create 16S rrna gene libraries, whereas the DNA of all of the 20 whole leafhoppers was employed for Illumina sequencing. Moreover, based on the results obtained by Illumina sequencing, specific qualitative PCR reactions for the symbiont phyla, subphyla or orders majorly represented in the vector (Gammaproteobacteria, Bacteroidetes, Actinobacteria, Bacillales) were carried out on all of the samples. The primer pairs used for this study are listed in Table 1; whole insects were submitted to direct PCR reactions with specific primers, whereas for body parts the same specific primers were used for a nested PCR, using the Eubacterial 27F and 1492R primers for direct reactions. Table 1. Primers adopted in this work. Target symbiont Primer Sequence References 27F 5 -AGAGTTTGATCMTGGCTCAG-3 Suzuki and Eubacteria Giovannoni, 1492R 5 -GGTTACCTTGTTACGACTT Bacillales BLS342F 5 - CAGCAGTAGGGAATCTTC -3 Blackwood et

2 1392R 5 - ACGGGCGGTGTGTACA -3 al., 2005 Gammaproteobacteria Actinobacteria Bacteroidetes 1080γF 5 -TCGTCAGCTCGTGTYGTGA -3 Bacchetti et γ1202r 5 -CGTAAGGGCCATGATG -3 al., 2011 Act920F3 5 - TACGGCCGCAAGGCTA -3 Bacchetti et Act1200R 5 - TCRTCCCCACCTTCCTCCG -3 al., cfbF 5 - CRAACAGGATTAGATACCCT -3 Bacchetti et al., Cfb967R 5 - GGTAAGGTTCCTCGCGTAT Results For the 16S rrna gene clone library, a total of 60 clones per sample were sequenced, showing 7 different OTUs as a whole. All of these were referable to the genus Pectobacterium, suggesting that this bacterium is a major symbiont of the vector. The Illumina sequencing analysis allowed to obtain more than 100,000 sequences. The occurrence of many relevant bacterial phyla in the leafhopper was revealed, even though a certain variability among the samples has been reported (Table 2). Table 2. OTU tables obtained by the analysis of the sequences (OTUs 97%) indicating diversity indices (calculated using PAST). Column names refer to leafhopper specimens. C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C20 C21 OTUs Sequence Dominance_D Simpson_1-D Shannon_H Evenness_e^H/S Brillouin Menhinick Margalef Equitability_J Fisher_alpha Berger-Parker Chao By measuring the α-diversity in each sample and plotting it on a rarefaction curve, we confirmed the saturation of the bacterial diversity associated to the samples (Figure 1).

3 Figure 1. Diversity curves of the analysed samples. β-diversity, evaluated through principal coordinates analysis (PCoA), showed that no statistical differences were present among the bacterial communities associated to male and female individuals (PERMANOVA, p=0.4512) (Figure 2).

4 Figure 2. Principal coordinate analysis (PCoA) on the phylogenetic β-diversity matrix on the vector male and female samples. The two principal components explain 53.6% of the variation. Considering the bacterial community s composition, bacteria referable to 10 phyla, divided in 32 orders, have been found to be affiliated to the putative phytoplasma vector (Figure 3). The most abundant orders identified (i.e. those present at least in one of the samples with percentages >5) included Actinomycetales, Flavobacteriales, Bacillales, Lactobacillales, Clostridiales, Rhizobiales, Enterobacteriales, Pseudomonadales, and Xanthomonadales. Additional sequences emerged by sequencing analysis were referable to chloroplasts (namely, those included in the clade Streptophyta), probably due to the ingestion of plant DNA by leafhoppers while feeding into the palm phloem. A

5 Figure 3. Illumina 16S RNA gene sequencing describing bacterial communities, at phylum (A) and order (B) levels, associated with the vector. Histogram columns indicate the relative abundances in percentages of the identified clusters. The prevalence of the major symbiont groups was investigated by specific PCR reactions on whole insects and on body parts. All of whole leafhoppers showed to be infected by Gammaproteobacteria and Actinobacteria, whereas infection rates for Bacteroidetes and Bacillales were of 90% and 95%, respectively. For Bacteroidetes, the 83.33% of females and the 92.86% of males were positive, while for Bacillales, 100% of males and 83.33% of females showed infection. On the other hand, the main symbiont groups were widely distributed in all the insect body parts, as they were retrieved in 100% of heads, thoraxes and abdomens. Such high infection rates confirm the possible important role of these symbionts within the microbial community of the leafhopper.

6 References Bacchetti De Gregoris T, Aldred N, Clare AS, Burgess JG (2011) Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa. J Microbiol Methods 86: Blackwood C.B., Oaks A., Buyer J.S. (2005) Phylum- and class-specific PCR primers for general microbial community analysis. Appl. Environ. Microbiol. 71(10): Gonella, E. Crotti E., Rizzi A., Mandrioli M., Favia G., Daffonchio D., Alma A. (2012) Horizontal transmission of the symbiotic bacterium Asaia sp. in the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae). BMC Microbiol. 12:S4. Suzuki M.T. and Giovannoni S.J. (1996) Bias caused by template annealing in the amplification of mixtures of 16S rrna genes by PCR. Appl. Environ. Microbiol. 62(2):