AEM Accepts, published online ahead of print on 13 July 2012

Appl. Environ. Microbiol. doi:10.1128/AEM.01439-12

Copyright 2012, American Society for Microbiology. All Rights Reserved.

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Candidates for Symbiotic Control of Sugarcane White Leaf Disease

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Jureemart Wangkeeree 1, Thomas A. Miller2 and Yupa Hanboonsong 1*

Entomology Division, Faculty of Agriculture, Khon Kaen University, Thailand,

Department of Entomology, University of California Riverside, Riverside, California, USA

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ABSTRACT

The leafhopper, Matsumuratettix hiroglyphicus (Matsumura), is the most important

vector of a phytoplasma pathogen causing sugarcane white leaf (SCWL) disease. The purpose of

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this study was to evaluate candidate bacterial symbionts for possible use vehicles in the control

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of the disease. 16S rRNA bacterial genes were amplified from whole bodies of M. hiroglyphicus

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and analyzed by cloning and sequencing. Two dominant groups were found; one belonging to

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Betaproteobacteria that did not closely match any sequences in the database, which was named

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Bacterium Associated with M. hiroglyphicus (BAMH). Another one found to be abundant in

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this leafhopper is Candidatus Sulcia muelleri in the order Bacteroidetes, which was previously

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reported in the insect members of the Auchenorrhyncha. Most M. hiroglyphicus carry both

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BAMH and Ca. Sulcia muelleri. Fluorescent in situ hybridization showed BAMH and Ca.

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Sulcia muelleri were co-localizing in the same bacteriomes. BAMH was present in the midgut

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and ovaries of the leafhopper and was found in all developmental stages including eggs, nymphs,

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and adults. Because BAMH appears specific for SCWL vector, we evaluated it as a candidate for

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symbiotic control of sugarcane white leaf disease.

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*Corresponding author: Yupa Hanboonsong

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Mailing address:

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Entomology Division

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Faculty of Agriculture

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Khon Kaen University

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Thailand 40002

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Phone/Fax: 66- 43362108.

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Email: yupa_han@kku.ac.th

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INTRODUCTION

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Sugarcane white leaf (SCWL) is one of the most destructive of known sugarcane diseases in

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the Asian region and has been reported in Taiwan, Sri Lanka, Lao PDR and Thailand (9, 18, 19,

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25, 46). This disease is caused by a phytoplasma, a bacterium without a cell wall belonging to

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the class Mollicutes that lives in the phloem of host plants and is transmitted by insect vectors

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(24). The SCWL phytoplasma belongs to the rice yellow dwarf group 16Sr XI (23). SCWL

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phytoplasma is transmitted by phloem feeding leafhoppers Matsumuratettix hiroglyphicus

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(Matsumura) belonging to the Cicadellidae in the suborder Auchenorrhyncha of the

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Deltocephalinae (9, 18, 19, 25). The infected plant shows leaf chlorosis and tiller proliferation

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(33, 46). So far there is no sugarcane-resistant variety or effective methods to control SCWL

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disease except for elimination of infected plants to decrease the amount of phytoplasma

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inoculums. Measures to limit the spread of the disease have focused on control of the insect

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vector. However, use of insecticides is unsustainable for vector control on large-scale

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commercial crops while other traditional control methods are lacking (44).

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A new biological control method for the plant disease treatment is known as the

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symbiotic control. In this method, symbiotic microorganisms are isolated, genetically modified

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and then reintroduced to express an anti-pathogen agent in the insect vectors (4). This approach

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has been used to control Chagas disease (4, 5, 6, 13), where a symbiotic bacterium,

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Rhodococcus rhodnii, was isolated from the vector Rhodnius prolixus and genetically

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transformed to express an anti-pathogen agent in the vector gut. The bacterial genus Asaia found

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in malarial mosquito vectors can also be cultivated, genetically manipulated and reintroduced to

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the insect host (14). Moreover, this same acetic acid bacterium is reported to be capable of cross-

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colonizing leafhopper Scaphoideus titatus, a vector of Flavescence doree, the yellow disease in

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grapevines which is caused by a phytoplasma (10). Symbiotic control is also being developed for

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control of Pierces disease in grapevines caused by the bacterial pathothen, Xylella fastidiosa,

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and transmitted by sharpshooters, principally, Homalodisca vitripennis (7). Wolbachia, has been

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reported to induce abnormal reproduction in insects (41, 48) and it is currently being used in

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symbiotic control of dengue fever (39). These studies have shown that there is a possibility to

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use symbiotic control as an alternative strategy for SCWL disease control.

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Therefore, the purpose of this study was to identify and characterize the candidate

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bacterial symbionts associated with leafhopper M. hiroglyphicus with the long-term goal of

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developing symbiotic control of SCWL.

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MATERIALS AND METHODS

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Leafhopper collection and DNA extraction. M. hiroglyphicus adults were collected at

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light traps from 6 different sugarcane fields in the northeast region of Thailand during the rainy

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season (July-September 2008). They were kept in 95% EtOH at -20 C until DNA extraction.

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Twelve males and 12 females from 6 fields were selected for individual DNA extraction by

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using a phenol-chloroform protocol (3).

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microorganisms with 70% EtOH and 6% sodium hypochlorite for 3 min each and then washed 3

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times with sterilized water. Individual leafhoppers were ground up in DNA extraction buffer

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(200 mM Tris pH 8.0, 250 mM NaCl, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K) and

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incubated at 37 C for 24 h. DNA was extracted with an equal volume of phenol-chloroform-

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isoamyl alcohol (25:24:1), followed by 5 min of centrifugation at 13,000 rpm. The supernatants

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were transferred and an equal volume of chloroform-isoamyl alcohol (24:1) was added, followed

Insects were sterilized to remove surface

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by 5 min centrifugation at 13,000 rpm. The supernatants were precipitated with 3M sodium

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acetate and isopropanol. The pellet was washed with 70% EtOH, air dried, and re-suspended in

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TE buffer (10 mM Tris, 1 mM EDTA), before storage at 20 C until use.

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Bacteria screening by PCR, cloning and sequencing of 16S rRNA gene. Identification

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of the bacteria associated with M.hiroglyphicus was studied by amplification of the 16S rRNA

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genes with universal primers 27F and 1513R (45), Table 1. Polymerase chain reaction (PCR)

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amplification was performed in a final volume of 25 l containing 2 l DNA templates, 0.2 mM

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dNTPs, 0.3 M of each primer, 1 U of Taq DNA polymerase, 2.5 mM MgCl2 and 1X reaction

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buffer (Promega). Cycling conditions consisted of an initial denaturation (95C, 5 min) followed

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by 30 rounds of denaturation (95C, 1 min), annealing (55C, 1 min), extension (72C, 2 min),

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and a final extension step at 72C for 10 min. PCR products were analyzed by gel

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electrophoresis and the positives were cloned into the TOPO-TA vector (Invitrogen) following

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the manufacturer instructions. Five recombinant plasmid clones were randomly selected from

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each leafhopper DNA template for sequencing analysis. Nucleotide sequencing was done at the

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Genome Institute of the National Center of Engineering and Biotechnology, Thailand. All 120

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sequences obtained were compared with other 16S rRNA gene sequences by using nucleotides

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BLAST search at the National Center for Biotechnology Information (NCBI database).

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Specific primers and PCR reaction for BAMH and Ca. Sulcia muelleri 16S rRNA

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gene. A representative of the most common sequences was selected to design specific primers

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using Primer-Blast program at NCBI homepage. The forward primer (MHF) and reverse primer

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(MHR) specific for 16S rRNA gene of BAMH did not match with any sequences in NCBI

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database (Table 1). PCR was performed with MHF and MHR according the amplification

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program described above, except the annealing temperature was 58C. PCR products were

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checked by 1% agarose gel electrophoresis for an expected 1.3 kb gene size. The specific primer

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for 16S rRNA gene of Ca. Sulcia muelleri (SulF and SulR) of M. hiroglyphicus was also

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designed and PCR reaction was performed with the same program of BAMH detection.

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Phylogenetic analysis Almost the entire sequence of BAMH and Ca. Sulcia muelleri of M.

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hiroglyphicus were analyzed for phylogenetic relationship with other selected sequences from

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the NCBI database including closely related sequences and unrelated sequences. Sequence

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alignments were performed using the ClustalX program under default settings with a gap-

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opening and gap-extension (43). The aligned sequences were analyzed by using the Neighbor-

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Joining and Kimura 2 parameter with 1,000 bootstraps replications. Phylogenetic trees were

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generated by using a consensus program within the PHYLIP package version 3.6 (15). The trees

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were displayed by using the TREEVIEW program (37).

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Frequencies prevalence of bacterial symbionts and SCWL phytoplasma in M.

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hiroglyphicus. Leafhoppers were collected from three different sugarcane fields during 2010-

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2011. DNA was extracted from whole bodies by phenol: chloroform protocol for detection the

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BAMH and Ca. Sulcia muelleri with PCR methods. SCWL phytolasma was detected by nested

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PCR reaction with two sets of primers designed to amplify the 16S rRNA and 23S rRNA genes

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including spacer region as previously described (18, 19). The first set of primers are MLO-X and

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MLO-Y, the first PCR reaction was performed in 25 l containing 2 l leafhopper DNA, 0.2

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mM dNTPs, 0.25 M each primers, 1 U taq DNA, polymerase 1.5 mM MgCl2 and 1X reaction

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buffer (Promega). PCR cycling consisted of one cycle of 5 min at 94C for initial denaturation

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and 35 cycles of 1 min at 94C, annealing 30 s at 60 C, extension 1 min at 72C and a final

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extension step of 10 min at 72 C. The second round of PCR were carried out using 1 l of the

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first PCR product as template with the second sets of primers P1 and P2. A total of 40 cycles

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were performed under the same conditions except the annealing temperature was 68 C.

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Localization of bacterial symbionts in dissected organs. Eight leafhoppers were

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dissected with forceps under a light microscope. Dissected organs were fat bodies, ovaries from

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females, salivary glands, guts, and pairs of bacteriomes. DNA was extracted from each tissue

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with phenol: chloroform as described above. The selected bacterial symbionts, BAMH and Ca.

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Sulcia muelleri, were tested in each sample by using specific primers and PCR reaction.

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Localization of bacterial symbionts by fluorescent in situ hybridization. For whole

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leafhopper, live specimens were collected and their legs and wings were removed in 70% EtOH

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under a dissecting microscope. Their cuticle was pricked with a needle in several places to

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facilitate the infiltration of reagents (22). Specimen fixation and embedding followed standard

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protocols (3). The specimens were immediately fixed with 4% paraformaldehyde in PBS and

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kept at 4 C overnight. After fixation the samples are dehydrated by using an ethanol-xylene

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series and embedded in paraffin wax. Sample tissues were cut into 8 M section with a rotary

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microtome, the tissue ribbons were floated on diethylpyrocarbonate (DEPC)-treated water bath at

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40-42 C and collected on superfrost plus glass slides (Fisherbrand, USA). The slides were

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completely dried at room temperature, then dewaxed and rehydrated with an xylene- ethanol

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series. Then specimens were treated with 0.2 M HCl 20 min, proteinase K (10 gml-1) 30 min

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and refixed in 4% paraformaldehyde for 5 min. Slides were air dried before hybridization. A

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specific

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CCCGTTCGCCACTAGAATAA-3) and was labeled with FAM (6-carboxyfluorescein,

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excitation 495 nm and emission 520 nm). To determine the localization of the endosymbiont Ca.

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Sulcia muelleri, the probe CFB319 (5-TGGTCCGTGTCTCAGTAC-3), was used which

probe

was

designed

for

the

BAMH

16S

rRNA

gene,

MH95

(5-

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perfectly matched positions 319 to 336 of the 16S rRNA of Bacteroidetes species (31), including

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all of our sequences from Ca. Sulcia muelleri. CFB319 was labeled with Cy5 (Cyanine-5,

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excitation 646 nm and emission 669 nm). 200 l of a hybridization buffer (0.9 M NaCl, 20 mM

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Tris-HCl (pH 7.4, 0.01% SDS, 30% formamide) containing 100 pmol ml-1 of probes was added

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to the tissues on the slides. The slides were covered with parafilm and incubated in 46C

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overnight in humid chambers. Unhybridized probes were rinsed with washing buffer (0.2 M

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NaCl, 20 mM Tris-HCl (pH 7.4) , 0.01% SDS) at 46C for 20 min, and then rinsed with 1XPBS

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buffer and DEPC- treated water. They were air dried and mounted in prolong-anti fade reagent

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(Invitrogen) with cover slips. The slides were observed under confocal laser scanning

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microscopy (OLYMPUS FV1000) at the Center of Nanoimaging (CNI) Mahidol University,

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Thailand.

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The universal bacterial probe EUB338 (5-GCTGCCTCCCGTAGGAGT-3) (2) was

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labeled with Cy5 (Cyanine-5, excitation 646 nm and emission 669 nm) and used as a positive

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control. Slides without the probe and hybridization with sense strands probes were used as a

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negative control of the experiments.

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BAMH detection in each life stage of M. hiroglyphicus. Adult M. hiroglyphicus were

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collected at light traps from sugarcane fields in the northeast of Thailand during July-September

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2010. They were maintained on sugarcane plants that tested negative for the presence of the

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BAMH. The plants were prepared by cutting the first and last sugarcane buds from plant stocks

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and genomic plant DNA was extracted by the CTAB method (3). BAMH was detected by PCR

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and the stocks which tested negative were used for planting by using the rest of the buds. The

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leafhoppers were maintained in cages on this plant stock until they produced eggs and nymphs.

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Some of eggs and nymphs were collected and kept in 95% ethanol at -20C until DNA extraction

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and some of them were transferred to a new plant if the old plant became yellow. Some of the

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adults of first generation were transferred to a new plant and maintained until they produced their

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second generation offspring. DNA was extracted from individual specimens and the presence of

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target bacterial symbiont was assessed using PCR and specific primers, as described. The plants

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used for rearing the insects were rechecked for the target bacterial symbiont.

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Prevalence of bacterial symbionts and SCWL phytoplasma in different species of

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leafhoppers. Leafhoppers were collected at light traps from 3 different sugarcane fields in the

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northeast of Thailand during July-September 2010 and 2011. They were identified by species

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and sex, and kept in 95% EtOH at -20C until use. The leafhopper species identified were Recilia

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dorsalis (18 females), Recilia sp. nr. vetus (41 females), Exitianus indicus (36 females and 10

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males), Yamatotettix flavovittatus (48 females and 38 males), Bhatia olivacea (11 females and 10

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males), Balclutha sp. nr. impicta (40 females and 25 males). DNA was extracted from individual

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insects using the phenol: chloroform method. The selected bacterial symbionts, BAMH and Ca.

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Sulcia muelleri, were tested in each sample by using specific PCR as described above. SCWL

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phytoplasma was also tested in the same specimens by using specific nested PCR.

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Nucleotide accession number. Representatives of 16S rRNA bacterial genes sequenced

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from M. hirohiglyphicus were submitted to the GenBank database; the accession number are

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JF836007 for Bacterium Associated with M. hiroglyphicus (BAMH) and JQ898318 for Ca.

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Sulcia muelleri of M. hiroglyphicus.

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RESULTS

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16S rRNA bacterial gene sequencing and BLAST search. Our first experiment was to

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identify candidate bacterial symbionts in M. hiroglyphicus using non-culture methods. The DNA

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of 24 individual leafhoppers was extracted and PCR was performed using universal primers for

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the bacterial 16S rRNA gene, followed by cloning, sequencing and BLAST search. Nucleotide

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sequencing was performed on 5 randomly picked clones from each leafhopper DNA template

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using the forward primer of the plasmid vector. Sequencing results were obtained from 116

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clones, whereas 4 clones did not yield any sequence. The sequences were 500 to 750 bp long and

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separated into two main groups, based on the BLAST search. Results are presented in Table 2.

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The largest group was made up of 76 clones (66% of total sequenced clones); 36 clones from

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female and 40 clones from male leafhoppers. Sequences from this group showed low similarity,

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on average 85%, with tick Haemaphysalis longicornis-associated microorganism 16S rRNA

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gene (34) (accession no. AB001520; class Betaproteobacteria). These sequences were amplified

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from all 24 tested leafhoppers from different sugarcane fields. These data suggest they are

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dominant and stably associated with M. hiroglyphicus. Because of low similarity to the closest

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matching sequence in the database, these 76 clones are likely to come from an unknown species

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that was named Bacterium Associated with M. hiroglyphicus (BAMH) in this paper. Almost

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the entire sequence of BAMH (1,428 bp) was obtained by sequencing the entire inserts with

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forward and reverse primers of their plasmid vector. A phylogenetic tree of BAMH and other

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selected bacterial symbionts from three different classes including Alpha-, Beta-and Gamma-

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proteobacteria was generated with the Neighbor-Joining method. The BAMH sequence was

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ordered in Betaproteobacteria with 99% bootstrap value support within the group and is most

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closely related with the bacterium associated with the tick H. longicornis 100% bootstrap value.

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Moreover, BAMH has a phylogenetic position close to the Betaproteobacteria, an endosymbiont

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of leafhopper Hyalesthes obsoletus (accession no. FR686932 (17)) and Candidatus Zinderia

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insecticola (accession no. CP00261) living in spittlebug Clastoptera arizonana (27) (Fig 1).

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Second group comprising thirty-eight sequenced clones (20 from females and 18 from

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males) has 98% similarity with Ca. Sulcia muelleri (accession no. DQ066645, phylum

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Bacteroidetes, class Flavobacteria (32)). It was reported as a primary endosymbiont restricted to

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the bacteriome of insects in the suborder Auchenorrhyncha (32). This sequence was also found

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in all leafhoppers examined indicating that it is a common endosymbiont of this leafhopper. The

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entire sequence (1,479 bp) was obtained with the forward and reverse primers of the plasmid

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vector. Phylogenetic relationship analysis showed this sequence could be grouped into a branch

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of Ca. Sulcia muelleri of insect host in superfamily Membracoidea with 100% bootstrap. The

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phylogenetic position of Ca. Sulcia muelleri of M. hiroglyphicus was close (100% bootstrap

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value) to Ca. Sulcia of Ecultanus excultus (DQ066645) and Ecultanus sp. (DQ066644)

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belonging to the order Cicadellidae (Fig 2).

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In addition, one sequence from a female leafhopper shares 99% identity match with

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Burkholderia vietnamiensis (accession no. FJ436055), which is grouped in the

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Betaproteobacteria class. Also one clone from female leafhopper showed 90% similar to

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Rickettsia sp, (accession no. FN550103) belonging to the Alphaproteobacteria class.

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Prevalence of BAMH, Ca. Sulcia muelleri and SCWL phytoplasma. Infection

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frequencies of two bacterial symbionts and the SCWL phytoplasma were measured in individual

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leafhopper from three different fields during two years by PCR and nested PCR diagnosis. The

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results are presented in Table 3. BAMH was present in 97% (135 out of 142) of the females

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tested while it was shown to be present in 92% (79 out of 86) of male leafhoppers. Ca. Sulcia

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muelleri tested positive in 96% (137 out of 142) of females and 93% (80 out of 86) of male

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leafhoppers. The test for the SCWL phytoplasma in leafhoppers showed positive in an average of

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61% of females (87 out of 142) and 53% of males (45 out of 86). In addition, 95% (83 out of 87)

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of female and 91% (41 out of 45) of male leafhoppers infected with SCWL phytoplasma are

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also infected with BAMH.

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Localization of BAMH and Ca. Sulcia muelleri. To determine the location of BAMH

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and Ca. Sulcia muelleri in leafhoppers, selected organs were dissected from female leafhoppers.

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DNA was extracted and analyzed by PCR using specific primers. BAMH and Ca. Sulcia

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muelleri were detected in all of the dissected organs except the salivary glands of the leafhopper.

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These two bacterial symbionts were present in the same dissected organs and the percentage of

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infection was identical in bacteriomes 100% (8/8) and in ovaries 88% (7/8). BAMH was found in

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50 % of the guts and 50 % of the fat bodies which is slightly different from the infection of Ca.

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Sulcia muelleri which was found to be 88% in the guts and 75% in fat bodies (Table 4).

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A more detailed localization of BAMH and Ca. Sulcia muelleri was revealed by the

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fluorescent in situ hybridization experiment on tissue sections of adult females and fourth instar

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nymphs. Oligonucleotide probe MH95 was designed to target the 16S rRNA gene sequences for

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BAMH and a CBF319 probe specific for the 16S rRNA of Bacteroidetes species was used to

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detect Ca. Sulcia muelleri of M. hiroglyphicus. As shown in Fig 3A-C, hybridization signals

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from both MH95 and CBF319 specific probes were found in the same bacteriome of the adult

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leafhopper. The MH95 signals were found all over the bacteriome (Fig3A), while the signal of

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the CBF319 probe for Ca. Sulcia muelleri was detected only at the margin of the bacteriome (Fig

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3B). There is co-localization of BAMH and Ca. Sulcia muelleri at the margin of the bacteriome

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(Fig 3C). The bacteriome is located on each side of the abdomen. It is attached to the leafhopper

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cuticle, and is egg shaped, 0.2-0.3mm and yellow in color (Fig 3D). No BAMH hybridization

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signals were detected in the salivary glands, thorax or muscle of the leafhopper, consistent with

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the PCR results

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Hybridization signals of MH95 probe were observed in other parts of nymphs and adults.

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They appeared in the entire midgut of the nymphs (Fig 3F) whereas in the adults, they appeared

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only in the anterior midgut (Fig 3E). The signals in the guts of both nymphs and adults were non-

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uniform but appeared close to the internal wall of the gut. The signals was also present in the

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abdominal area near the reproductive tissues and were present in parts of the ovarioles of female

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leafhoppers (Fig 3G). No BAMH hybridization signals were detected in the salivary glands of

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the leafhopper, consistent with the PCR results. The CBF319 probe for detect Ca. Sulcia

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muelleri signal was also found in reproductive tissues near the ovipositor, oviduct and ovarioles

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(Fig 3H- I).

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bacteriome area or the other parts that showed a signal with probe for the two bacterial

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symbionts BAMH and Ca. Sulcia muelleri, since this probe does not match with the 16S rRNA

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gene of BAMH and Ca. Sulcia muelleri of M. hiroglyphicus. However, a signal was obtained

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with the universal probe EUB338 in the anterior midgut of other leafhopper that do not harbor

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BAMH (Fig 3J). Moreover, no hybridization signals were obtained from tissues without probe

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and sense strands probes control.

No hybridization signal with the universal probe EUB338 was found in the

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Prevalence of BAMH in developmental stages of leafhopper M. hiroglyphicus. The

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leafhopper was reared on sugarcane plant stocks that test negative for presence of the BAMH.

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Insects from each stage were collected and DNA was extracted from single individuals. BAMH

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was detected in all developmental stages of the subsequent first and second generation by

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specific PCR. The results showed that BAMH was found in eggs, all nymph stages and adults of

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the first generation. The percentage of infection was 46% in eggs, 55 %, 63 %, 90 %, 100 % and

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100 % in the first to fifth nymphal instars respectively and 100% in both female and male adults.

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Furthermore, BAMH was also found in all nymph stages of second generations from first to fifth

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nymphal instars (90%, 83%, 96%, 100% and 100% respectively) (Table 5).

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Prevalence of BAMH, Ca. Sulcia muelleri and SCWL phytoplasma in others

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leafhopper. We tested for the presence of two bacterial symbionts and the SCWL phytoplasma

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in different species of leafhoppers by specific PCR and nested PCR, respectively. As

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summarized in table 6, BAMH was present in 2 out of 6 species tested which are R. dorsalisa

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(100% positive) and Recilia sp. nr. vetus (88% positive). On the other hand, Ca. Sulcia muelleri

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was present in all 6 species tested, with percentage of infection ranging from 80-100%. SCWL

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phytoplasma were found in all 6 species of leafhopper tested, with a variation in the rate of

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infection in each species ranging from 17- 100%.

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DISCUSSION

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We found two dominant bacterial species in the leafhopper M. hiroglyphicus. The largest

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group of the bacterial clones was made up from 66% of total clones with no close match with

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any sequences in the databases. The low identity (85%) with the closest match an

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uncharacterized bacterial symbiont of tick (34), suggests it is a new Betaproteobacteria that has

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not been reported before and was named as BAMH. Ca. Sulcia muelleri was found in 33% of all

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clones sequenced and belongs to the phylum Bacteroidetes. Ca. Sulcia muelleri is an

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evolutionarily ancient endosymbiont of sap feeding insects and plays an important role in the

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hosts by synthesizing essential amino acids, missing from their food sources (26, 27, 30, 32, 47).

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It has been reported as the primary endosymbiont, located inside the bacteriome and is often

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associated with another type of bacterial symbiont of insects in suborder Auchenorrhyncha such

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as cicadas, planthoppers and leafhoppers. For instance, Ca. Sulcia muelleri and

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gammaproteobacterium Ca. Baumannia cicadellinicola are co-localized in the bacteriome of

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sharpshooter (31, 32), Ca. Sulcia is associated with the alphaproteobacterium Ca. Hodkinia

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cicadicola of cicadas (28) and Ca. Sulcia muelleri was reported to be associated with

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betaproteobacterium Ca. Zinderia insetticola in the bacteriome of spittlebugs (27). One exception

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is Ca. Sulcia muelleri in the leafhopper Hyalesthes obsoletus which was observed in organs like

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guts, salivary glands and female and male gonads (17). Our approach was to identify the most

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prevalent bacterial species associated with the insects. No plant-associated bacteria were found.

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If we sequenced a larger number of clones, we could have identified additional bacterial species,

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including plant bacteria. The clones in each group of BAMH and Ca. Sulcia muelleri showed

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high similarity (97%-100%), but were not identical. These minor differences in nucleotide

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sequences may reflect population to population variation. However, the variation of nucleotides

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could not separate these sequences into a different group.

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Our finding showed that BAMH and Ca. Sulcia muelleri bacteria were found at high

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infection rates and similar prevalence in natural populations of M. hiroglyphicus. PCR assays

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have found BAMH in a variety of tissues co-prevalent with the Ca. Sulcia muelleri, including fat

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bodies, ovaries, intestines and bacteriomes in the same individual leafhoppers. With FISH

324

experiments, BAMH and Ca. Sulcia muelleri were found to be co-localized in the same

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bacteriome. We did not observe co-existence of BAMH and Ca. Sulcia muelleri in other parts of

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the body. However, ovariole stained by MH95 or CBF319 probes, but not in the same

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leafhopper.

16
328

BAMH was found in all the developmental stages including eggs, nymphs and adults of

329

first and second generation of leafhoppers that were reared on BAMH-free sugarcane plant

330

stocks, suggesting that it is transmitted vertically in the same way as the SCWL phytoplasma

331

(18). BAHM was found in all individuals tested from nymphal instars 4-5 to adults but only in a

332

fraction of eggs and nymphal instars 1-3. The lack of detection in all early stage individuals may

333

be due to the sensitivity of the method, as very little material was obtained from individual eggs

334

and early stages of nymphal development. These data indicated a strong association between

335

BAMH and the leafhopper, M. hiroglyphicus. An alternative explanation could be horizontal

336

transmission. However, BAMH is not transmitted from the insect host to sugarcane plants

337

because BAMH-free plants on which the insect was reared remained free of this bacterium. We

338

believe that BAMH is a candidate bacterial symbiont of the leafhopper M. hiroglyphicus based

339

on the following: i) it is a predominant bacterium, representing 66 % of the clones; ii) it is

340

associated with the Ca. Sulcia muelleri endosymbiont in the bacteriome; and iii) it is highly

341

prevalent in the leafhopper population and passed from generation to generation.

342

The role of BAMH and its beneficial effect on the leafhopper host remains to be

343

described. BAMH resides in the bacteriome suggesting it is an obligate primary endosymbiont. It

344

is present throughout the life cycle of the leafhopper suggesting BAMH is essential for

345

leafhopper growth or part of growth development. In addition to the bacteriome, BAMH is also

346

found in the midgut. It is conceivable that at this location the symbiont provides an essential

347

nutrient to the host, and therefore interfering with BAMH would impact the viability of the

348

insect. It has been reported that primary endosymbionts which cannot be cultured outside host,

349

are necessary for its normal growth and reproduction (1, 8, 11, 12, 16, 42). In addition some

350

reports showed that intracellular bacteria can increase the heat tolerance of their host (29, 40) or

17
351

provide the host with resistance to parasitoid wasps (35, 36). In our case we speculate that

352

BAMH may provide some benefit or play an important role in the biology of specific hosts.

353

To determine if BAMH is specific for the leafhopper, M. hiroglyphicus, we investigated

354

its presence in 6 other species of leafhoppers in the order Cicadellidae. We found BAMH in

355

Recilia dorsalis and Recilia sp. nr. vetus but not in Exitianus indicus, Yamatotettix flavovittatus,

356

Bhatia olivacea and Balclutha sp. nr. impicta. Interestingly these two insect species (Y.

357

flavovittatus and R. dorsalis) are vectors that transmit phytoplasma to sugarcane and rice,

358

respectively (19, 20, 21, 38), whereas three of the four species that do not carry it are not vectors

359

for phytoplasma. Ca. Sulcia muelleri was found in all species tested including the non- vector

360

species whereas BAMH appears to be restricted to vector hosts. Therefore it constitutes a more

361

specific potential target for control.

362

It is tempting to speculate that BAMH or a related species is required for effective

363

transmission of phytoplasma from insect to plant. The one insect species Y. flavovittatus that is

364

known vector for sugarcane white leaf phytoplasma and does not carry BAMH may carry

365

another related bacteria species. Further research is required to elucidate the precise role of

366

BAMH in the insect vectors. If it is confirmed that BAMH is essential for host growth or for

367

transmission of the sugarcane white leaf phytoplasma, this may lead to novel approaches for

368

sugarcane white leaf disease control.

369
370

ACKNOWLEDGMENTS

18
371

This research is supported by the Royal Golden Jubilee Ph.D. program, grant no. 6.E.KK/49/B.1

372

of the Thailand Research Fund and the Research Center of Agricultural Biotechnology for

373

Sustainable Economy, Khon Kaen University, Thailand

374
375

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376

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498

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499

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500

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501

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504

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505

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506

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24
507

Table 1. Nucleotide primers sequences used in the study

508

Target organisms

Name

Eubacteria

27F:

16S rRNA

5-AGAGTTTGATCMTGGCTCAG-3

1513R:

16S rRNA

5-ACGGYTACCTTGTTACGACTT-3

MHF:

16S rRNA

5TTATTCTAGTGGCGAACGGG3

MHR:

16S rRNA

5GCGGTGTGTACAAGACCAGT3

Target gene

Sequences

Reference

509
510
511
512

BAMH

513
514

Ca. Sulcia muelleri of

SulF:

16S rRNA

5AAACGCTAGCGGAGGGCTTAAC3

515

M. hiroglyphicus

SulR:

16S rRNA

5CTTACCGCGACTGCTGGCAC3,

516

SCWL phytoplasma

MLO-X:

16/23S rRNA

5-GTTAGGTTAAGTCCTAAAACGAGC-3

MLO-Y :

16/23S rRNA

5-GTGCCAAGGCATCCACTGTATGCC-3

P1:

16/23S rRNA

5-GTCGTAACAAGGTATCCCTACCGG-3

16/23S rRNA

5-GGTGGGCCTAAATGGACTTGAACC-3.

517
518
519
520
521
522

SCWL phytoplasma

P2

45

in this study

in this study

18,19

18,19

25
523

Table 2. The sequencing and BLAST search results of 16S rRNA bacterial gene from leafhopper Matsumuratettix hiroglyphicus

524

(Matsumura).

525

No. of positive clones

526

/total clones

Most similar known bacteria

Class

Similarity (%)

No. of positive leafhopper tested

/ No. of total leafhopper

527
528

76/116

529

Bacterium associated with tick

Betaproteobacteria

85

24/24

Haemaphysalis longicornis

530

38/116

Candidtus Sulcia muelleri

Flavobacteria

98

24/24

531

1/116

Burkholderia sp.

Betaproteobacteria

99

1/24

532

1/116

Rickettsia sp.

Alphaproteobacteria

90

1/24

533

26
534

Table 3. Infection frequency of BAMH, Ca. Sulcia muelleri and SCWL phytoplasma in natural

535

population of M. hiroglyphicus.

536

No. positive

537

Year

Locationa

538

2010

field 1

16F/17 M

16F/15M

16F/15 M

9F/14M

9F/13M

539

field 2

21F/11M

18 F/10M

19F/11M

6F/6M

5F/6M

540

field 3

20F/10M

18F/9M

20F/8M

15F/3M

13F/3M

field 1

36F/20M

35F/20 M

34F/18M

25F/8M

25F/6M

542

field 2

23F/14 M

23F/12 M

23F/14M

14F/8M

14F/7M

543

field 3

26F/14M

25F/13M

25F/14M

18F/6M

17F/6M

142F/86M

135F/79M

137F/80M

87F/45M

83F/41M

541

2011

544

Total

545

546

No. testedb

BAMH

Ca. Sulcia muelleri

Phytoplasma

BAMH+ phytoplasma

Field 1 and 2 located in Udonthai province and field 3 located in Khon Kaen province.
Number of individual tested; F: female leafhopper, M: male leafhopper

547
548

Table 4. PCR assays of BAMH and Ca. Sulcia muelleri in dissected organs of leafhopper M.

549

hiroglyphicus .

550

No. positive / No. tested

551

Dissected organsa

Gut

Salivary gland

552

BAMH

4/8

0/8

7/8

4/8

8/8

553

Ca. Sulcia

7/8

0/8

7/8

6/8

8/8

554

555

All dissected organs are from female leafhopper.

Ovary

Fat body

Bacteriome

27
556

Table 5. Results of PCR amplification of DNA from BAMH in different developmental stages

557

of the leafhopper.

558

Stages

559

First Generation
No. positive / No. tested (%)

Second Generation
No. positive / No. tested (%)

560

Egg

13/28 (46)

None tested

561

Nymph 1

24/44 (55)

9/10 (90)

562

Nymph 2

21/33 (63)

15/18 (83)

563

Nymph 3

36/40 (90)

27/28 (96)

564

Nymph 4

29/29 (100)

10/10 (100)

565

Nymph 5

26/26 (100)

10/10 (100)

566

Female

20/ 20(100)

None tested

567

Male

7/ 7 (100)

None tested

568
569
570

28
571

Table 6. Results of PCR amplification of DNA from BAMH and Ca. Sulcia muelleri and

572

nested PCR assays of SCWL phytoplasma in different species of leafhopper.

573

Leafhopper

574

Species

575

Recilia dorsalis

18F

18F

18F

12F

12F

576

Recilia sp. nr. vetus

41F

36F

36F

28F

26F

577

Exitianus indicus

36F/10M

0F/0M

36F/9M

16F/8M

0F/0M

578

Bhatia olivacea

11F/10M

0F/0M

11F/10M

9F/6M

0F/0M

579

Balclutha sp. nr. impicta

40F/25M

0F/0M

38F/23M

28F/14

0F/0M

580

Yamatotettix flavovittatus 48F/38M

0F/0M

46F/36M

43F/31M

0F/0M

581

582

No. positive
No. testeda

BAMH

Ca. Sulcia muelleri

Phytoplasma

Number of individual tested; F: female leafhopper, M: male leafhopper

BAMH+Phytoplasma

29
583
584
585
586
587
588
589
590
591
592
593

Fig 1. Phylogenetic relationship of bacterium associated with M. hiroglyphicus with other 11

594

selected 16S rRNA bacterial gene sequences class in Alpha-, Beta- and Gamma- proteobacteria.

595

Numbers at each node indicate the percentage of 1,000 bootstraps replication. Bacillus

596

thuringiensis GU201858 was defined as an outgroup of the tree. The bar represents 0.1

597

substitutions per site.

598

30
599
600
601
602
603
604
605
606
607
608
609

Fig 2. Phylogenetic relationship of Ca. Sulcia muelleri in M. hiroglyphicus and other selected

610

Ca. Sulcia muelleri from three superfamily of insect hosts. Numbers at each node indicate the

611

percentage of 1,000 bootstraps replication. Bacteroides stercoris X83953 was defined as an

612

outgroup of the tree. The bar represents 0.1 substitutions per site.

613

31
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628

Fig 3. Localization of bacterial symbionts BAMH and Ca. Sulcia muelleri of M. hiroglyphicus

629

in longitudinal tissue sections of the leafhopper. (A-C) Fluorescent in situ hybridization (FISH)

630

and confocal laser scanning microscopy image of bacteriome part, (A) Hybridization signal of

631

MH95 labeled with FAM (blue) specific for BAMH, (B) Hybridization signals of CBF319

32
632

labeled with Cy5 (red) specific for Ca. Sulcia , (C) Merged images of BAMH and Ca. Sulcia

633

hybridization signals. (D) Photography of bacteriome (ba) on each side of leafhopper abdomen,

634

(E-G) The arrows indicate signal of MH95 probe specific for BAMH. (E) in anterior midgut

635

(mg-1) of adult leafhopper. (F) entire midgut (mg) of nymph leafhopper. (G) reproductive tissue

636

(rt) and ovariole (ov), (H and I) The arrows indicate signal of CBF319 specific probe for Ca.

637

Sulcia, in reproductive tissue (rt), ovariole (ov). (J) The arrow indicates the signal from the

638

universal bacterial probe EUB338 (positive control) in anterior midgut (mg-1) of adult

639

leafhopper. Scale bar show 100m.

640