Naturwissenschaften (2009) 96:1169–1176 DOI 10.1007/s00114-009-0578-x

ORIGINAL PAPER

Recilia banda Kramer (: Cicadellidae), a vector of Napier stunt phytoplasma in Kenya

Evans Obura & Charles A. O. Midega & Daniel Masiga & John A. Pickett & Mohamed Hassan & Shinsaku Koji & Zeyaur R. Khan

Received: 12 November 2008 /Revised: 2 June 2009 /Accepted: 10 June 2009 /Published online: 4 July 2009 # Springer-Verlag 2009

Abstract Napier grass (Pennisetum purpureum)isthe oped characteristic stunt disease symptoms while 60% of most important fodder crop in smallholder dairy produc- R. banda samples were similarly phytoplasma tion systems in East Africa, characterized by small zero- positive. We compared the nucleotide sequences of the grazing units. It is also an important trap crop used in the phytoplasma isolated from R. banda,Napiergrasson management of cereal stemborers in maize in the region. which these were fed, and Napier grass infected by However, production of Napier grass in the region is R. banda, and found them to be virtually identical. The severely constrained by Napier stunt disease. The etiology results confirm that R. banda transmits Napier stunt of the disease is known to be a phytoplasma, 16SrXI phytoplasma in western Kenya, and may be the key vector strain. However, the putative insect vector was yet of Napier stunt disease in this region. unknown. We sampled and identified five and three planthopper species associated with Napier grass and Keywords Napier stunt disease . Phytoplasma . Vector . used them as candidates in pathogen transmission experi- Recilia banda . Western Kenya ments. Polymerase chain reaction (PCR), based on the highly conserved 16S gene, primed by P1/P6-R16F2n/ R16R2 nested primer sets was used to diagnose phyto- Introduction plasma on test plants and insects, before and after transmission experiments. Healthy plants were exposed In eastern Africa, most dairy farming is practiced by for 60 days to insects that had fed on diseased plants and smallholder farmers, in the high agricultural potential areas, acquired phytoplasma. The plants were then incubated for which are densely populated and farm holding sizes are small. another 30 days. Nested PCR analyses showed that 58.3% These conditions limit natural grazing and therefore cattle are of plants exposed to Recilia banda Kramer (Hemiptera: fed on crop residues, concentrates, and cultivated fodder, Cicadellidae) were positive for phytoplasma and devel- mainly Napier grass, Pennisetum purpureum Schumach, the highest ranked fodder type. The grass is also planted for environmental protection, to stabilize soils and act as windbreaks (Jones et al. 2004). Recently, a novel use of E. Obura : C. A. O. Midega : D. Masiga : M. Hassan : Z. R. Khan (*) Napier grass in management of the most injurious pests of International Centre of Insect Physiology and Ecology (ICIPE), cereals, stemborers, has been discovered and exploited in a P.O. Box 30772, Nairobi 00100, Kenya ‘push–pull’ strategy (Cook et al. 2007;Khanetal.2008). e-mail: [email protected] The strategy involves intercropping maize, Zea mays L., J. A. Pickett with a stemborer-repellent plant (push), with the attractant Harpenden, Napier grass planted as a border crop (pull) around this Hertfordshire AL5 2JQ, UK intercrop. The grass is more attractive to stemborer moths than maize for oviposition but supports only minimal S. Koji Kanazawa University, survival of larvae (Khan et al. 2006, 2007). Therefore, when Ishikawa 927-1462, Japan planted as a trap crop around maize it attracts more 1170 Naturwissenschaften (2009) 96:1169–1176 oviposition by stemborer pests than the main crop leading to parasitic plants (such as dodder (Cuscuta spp.)), grafting an increase in grain yields (Khan et al. 1997). of infected material (Dale and Kim 1969) and sap-sucking There has been an intensified cultivation of Napier grass insect vectors belonging to the families Cicadellidae (leaf- in the region in recent years as a result of increased hoppers) and Delphacidae (planthoppers; Hemiptera), commercial dairying and uptake of the ‘push−pull’ tech- which transmit the phytoplasma in a persistent propaga- nology. However, Napier grass faces a serious phytopath- tive manner (Banttari and Zeyen 1979;Grylls1979; ological constraint from a disease, visible in re-growth after Nielson 1979; Murray and Schleifer 1994; Weintraub and cutting or grazing of Napier grass called “Napier stunt Beanland 2006). Since there is no parasitic plant associ- disease” (Ns-disease), caused by a phytoplasma. The ated with Napier grass, either the plants arrived as infected disease has been reported in Kenya, Uganda, and Ethiopia, seedlings and/or insects carrying the phytoplasma infected posing a great threat to Napier grass production (Farrell et them. Although the causative organism of Ns-disease is al. 2002; Jones et al. 2004, 2006). Affected plants are known, the putative vector involved is not yet identified recognized by severe stunting, profuse tillering, and and this could be a critical starting point to screening for yellowing. Often the whole stool is affected with complete resistant Napier grass varieties and development of an loss in yield and eventual death (Jones et al. 2004, 2006). It integrated management approach for the disease. Use of therefore has a serious negative effect on smallholder dairy resistant varieties would be an environmentally friendly and cereal farmers in the region. way to contain the disease in smallholder dairy industry Phytoplasmas are wall-less, vascular colonizing plant (Orodho 2006). prokaryotes, belonging to the class Mollicutes (Sears With assistance from the laboratory of Entomology, and Kirkpatrick 1994). In plants, they are obligate Tokyo University of Agriculture in Japan and the National parasites restricted to the phloem sieve elements. Phyto- Museum of Wales in the UK, we identified five leafhopper plasmas were assigned a provisional taxonomic status of species (Glossocratus afzelii (Stäl), Cofana polaris Young, Candidatus (ICSB 1997), defined at 97.5% similarity of C. spectra (Distant), Recilia banda Kramer, and Cicadulina the 16S rDNA sequence. Currently, there are 15 16Sr mbila Naude) and three planthopper species (Leptodelphax groups, including at least one Ca. Phytoplasma species dymas Fennah, Thriambus strenuus Van Stalle, and Sogatella (Lee et al. 1998). Unlike most human and manetho Fennah) to be frequently found on Napier grass in mycoplasmas, phytoplasmas have never been cultured in addition to other grasses. Using Safranine dye technique vitro, their detection and characterization are therefore developed by Khan and Saxena (1984), we demonstrated based on molecular techniques; foremost among them is that all insects probed phloem and could potentially nested polymerase chain reaction (nPCR), based on the transmit Napier stunt phytoplasma. Considering the epide- conserved nature of ribosomal DNA across all prokaryotic miology of stunt disease and its economic importance, and organisms (Lee et al. 2000), using short synthetic the need to understand its transmission mechanism, by universal primers. The efficiency of nPCR has shown that adopting standard natural transmission assays, this study it can re-amplify the direct PCR product in dilutions of was carried out to determine whether these leafhopper 1:60,000 (Khan et al. 2004). and planthopper species could transmit the phytoplasma Phylogenetic analysis of 16S rDNA sequences of pathogen. Napier stunt phytoplasma (Ns-phytoplasma) in Kenya (Jones et al. 2004) and Uganda (Nielsen et al. 2007) showed the strains to be very closely related to phyto- Materials and methods plasma isolated from Bermuda grass wheat leaf, Sorghum grassy shoot, and sugarcane white leaf, members of Collection and rearing of insects 16SrXI, Candidatus Phytoplasma oryzae or rice yellow dwarf phytoplasma group. However, 16S rDNA sequen- The five leafhopper and three planthopper species ces of Ns-phytoplasma in Ethiopia had highest similarity mentioned above, collected from two sites in western to the African sugarcane yellow leaf phytoplasma, a Kenya, Mbita (0°25′ S, 34°12′ E) in Suba district and member of the 16SrIII, Candidatus Phytoplasma pruni Mabanga (0°45′ S, 34°34′ E) in Bungoma district, were (Jones et al. 2006). Therefore, the possibility of the reared on potted stunt-free 40–50-day-old Napier grass phytoplasma infecting crops and grasses other than Napier plants in the screen house, at 20–28°C and 65–70% RH, grass poses a great risk to the well being of communities in separate cages (25×25×60 cm) made of wooden in eastern Africa where rice, Sorghum, and sugarcane are frame. Top and side openings of the cages were covered major food and cash crops. with fine nylon mesh for aeration. Periodically, these Phytoplasma transmission is known to occur through colonies were infused with insects collected from the vegetative propagation of infected plant material, by field to minimize inbreeding. Naturwissenschaften (2009) 96:1169–1176 1171

Raising healthy and diseased Napier grass DNA was estimated by electrophoresis on ethidium- bromide stained, 0.8% agarose gel. DNA from the insects Napier grass (bana variety) accessions susceptible to Ns- was similarly extracted but the buffer contained 3% CTAB. disease were obtained from Mabanga, as above, a disease Nucleic acid, 20 ng/µl, was used for amplification by prevalent area. The plants were provisionally identified nPCR. PCR and nPCR reactions were performed according by foliar symptoms (as above), carefully removed from to published protocols (Lee et al. 1998), using primer pair the ground, potted, labeled, and maintained in separate P1/P6 (Deng and Hiruki 1991) and nPCR with R16F2n/ sections of similar insect-free screen houses as above. R16R2 (Lee et al. 1993) primers, in a PTC 100 program- Clean healthy plants (bana variety) were obtained from mable thermocycler (MJ Research). DNA amplified in PCR Kitale (1°0′ N, 35°7′ E), Trans Nzoia district, from where primed by P1/P7 was diluted 1:50 with sterile water and the incidence of Napier stunt disease has not been vortexed to mix, and 1µl used as a template in an nPCR. reported, and maintained in a different screen house PCR products were detected in a 1% ethidium-bromide from that of the cultured insects. All plants were tested stained agarose gel using 1× TAE (40 mM Tris acetate, for the presence of phytoplasma by nPCR. 1 mM EDTA pH8.0) as running buffer, and photographed.

Transmission tests Sequencing and phytoplasma classification

Using an aspirator, a total of ten gravid females of each Nested PCR products (1.2 kb in size) from infected Napier insect species were obtained from the healthy colonies in grass and insect vector species were purified on Qiagen’s cages and reared on diseased Napier grass for 30 days to QIAquick PCR purification kit (Qiagen, Valencia) accord- acquire stunt phytoplasma (acquisition feeding). The insects ing to manufacturer’s protocol and sequenced directly. oviposited on the diseased Napier grass and the emerging Sequencing was done in both directions using Dye nymphs were allowed to feed on the same plants and Terminator chemistry and a DNA automatic sequencer similarly acquire the phytoplasma. After the 30 days, four (SegoliLab, International Livestock Research Institute- pots of healthy Napier plants were introduced into the same ILRI, Nairobi Kenya). Sequences were assembled and cage and exposed to the adult insects and their nymphs for edited using FinchTv software version 4.0. Sequences of 60 days. Surviving insects were sampled from each cage phytoplasmas infecting grasses for comparison (Table 1) after 60 days and kept at −20°C, after which two insects were obtained from the EMBL GenBank database. Nearly were pooled at a time for phytoplasma detection by nPCR. full-length 16S rRNA gene sequences were aligned for The exposed plants were then pruned for the appearance of phylogenetic analysis using ClustalW2 software (Thompson re-growth and incubated for another 30 days. Their leaf- et al. 1994). Genetic distances among the nucleotide cuts were then sampled for DNA isolation and phytoplasma sequences were calculated using DNA neighbor-joining detection by nPCR. The plants were quarantined until the method (Saitou and Nei 1987) and the tree viewed using appearance of well-developed stunt symptoms. The exper- Java 6.0 software. iment was repeated three times for each insect species. Unexposed healthy plants were used as a control setup. Results DNA extraction and nested polymerase chain reaction DNA templates isolated from healthy plant samples before The presence of phytoplasma in plants and insects was transmission experiments were phytoplasma negative by assayed by conventional nPCR. Total DNA was extracted nPCR (Fig. 1). After 60 days of exposing healthy plants to from plant and insect tissue according to Doyle and Doyle phytoplasma transmitting insects, a 1.2 kb DNA fragment (1990) with slight modifications. Five grams of leaf tissue was amplified in seven out of 12 plants (58.3%) exposed to was powdered in liquid nitrogen, 600μl cetyltrimethylam- leafhopper R. banda and the positive controls (Fig. 2). monium bromide (CTAB) buffer at 65°C was added (CTAB These plants later simultaneously developed severe stunt- buffer, 2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM ing, profuse tillering, and lethal yellowing, stunt symptoms Tris–HCl, pH8.0, 0.2% 2-mercaptoethanol), then the identical to the known stunt-induced symptoms on Napier mixture incubated at 65°C for 30 min. DNA was extracted grass. All plants exposed to the seven other insect species with 1 vol of chloroform:isoamyl alcohol (24:1) and (G. afzelii, C. polaris, C. spectra, C. mbila, L. dymas, T. precipitated overnight in a −20°C freezer. Following strenuus,andS. manetho) remained negative by nPCR centrifugation at 14,000 rpm, the DNA pellet was rinsed (data not shown). All amplifications observed were only twice with 70% ethanol, dried and dissolved in 50μl Tris– after nPCR with templates previously amplified by P1/P6 EDTA pH8 buffer. The quantity and quality of the isolated primer pair. Similarly, 1.2-kb phytoplasma-specific DNA 1172 Naturwissenschaften (2009) 96:1169–1176

Table 1 Acronyms and GenBank accession numbers of phytoplasma 16 S rDNA sequences used for phylogenetic analyses

Name Acronym Phytoplasma strain designation Origin Accession numbers 16Sr group

Sugarcane yellow leaf Ca. P. solani Candidatus Phytoplasma solani India EU170474 16SrXII Barley deformation Ca. P. asteris Candidatus Phytoplasma asteris Lithuania AY734453 16SrI Rice orange leaf Ca. P. oryzae Candidatus Phytoplasma oryzae Japan AB052870 16SrXI Maize bushy stunt Ca. P. asteris Candidatus Phytoplasma asteris USA AY265208 16SrI Sorghum grassy shoot Ca. P. oryzae Candidatus Phytoplasma oryzae Australia AF509325 16SrXI Napier grass stunt Ca. P. oryzae Candidatus Phytoplasma oryzae Kenya AY377876 16SrXI Napier grass stunt Ca. P. oryzae Candidatus Phytoplasma oryzae Uganda EF012650 16SrXI Napier grass stunt Ca. P. pruni Candidatus Phytoplasma pruni Ethiopia DQ305977 16SrIII Brachiaria grass white leaf Ca. P. oryzae Candidatus Phytoplasma oryzae Japan AB052872 16SrXI Bermuda grass white leaf Ca. P. cynodontis Candidatus Phytoplasma cynodontis Iran EF444485 16SrIV Sugarcane white leaf Ca. P. oryzae Candidatus Phytoplasma oryzae Thailand AB052874 16SrXI Sugarcane grassy shoot Ca. P. oryzae Candidatus Phytoplasma oryzae India DQ459438 16SrXI Rice yellow dwarf Ca. P. oryzae Candidatus Phytoplasma oryzae Japan AB052873 16SrXI fragments were amplified from three out of five R. banda of phytoplasmas infecting grasses, obtained from GenBank templates (Fig. 3), showing a vector infection rate of 60%. (Table 1). The three sequences (GenBank acc. FJ862997, No phytoplasma-specific amplification (1.2 kb) was ob- FJ862998 & FJ862999) shared 99% identity between them, served in the templates of the other seven insect species, and 99% identity with Napier grass stunt phytoplasma implying that the pathogen was non-persistent in these isolated from Kenya (GenBank acc. AY377876), Bermuda insects. grass white leaf, Brachiaria grass white leaf, Sorghum grassy Date of exposing plants to the vector was known, shoot, sugarcane white leaf, Napier grass stunt (GenBank acc. therefore, we determined how long the plants stayed before AY377877 and EF012650), all members of Ca. Phytoplasma showing Napier stunt disease symptoms. All the plants that oryzae. They also shared 99-100% sequence identity with were positive for phytoplasma developed symptoms after Napier grass stunt phytoplasma Uganda (GenBank acc. 120 days from the date of exposure, showing that the EF012650), a member of Candidatus Phytoplasma oryzae disease incubates in the host plants for about 4 months or Rice yellow dwarf phytoplasma. before symptoms appear. nPCR however detected phyto- plasma in the exposed plants after 90 days, scoring its ability to diagnose Napier stunt pathogen before symptoms Discussion appear. The 1,190, 1,194, and 1,199 bp 16S rDNA sequences were Hemiptera or sap-feeding insects, probe phloem and obtained from R. banda, exposed Napier grass and diseased passively ingest phytoplasma cells with the phloem-sap Napier grass (source plant), and deposited in the EMBL from infected plants (Weintraub and Beanland 2006). The GenBank under accessions: FJ862999, FJ862998, and time required for an insect to acquire enough titre of FJ862997, respectively. A dendrogram (Fig. 5) was con- phytoplasma (acquisition access period, AAP) varies from structed from the two sequences and 12 additional sequences insect to insect. Although planthoppers require longer AAPs

Fig. 1 Ethidium-bromide stained M - + 1 2 3 4 5 6 7 8 9 10 11 12 agarose gel of nested PCR prod- ucts of healthy plants before exposure to Recilia banda. Lanes: M HyperLadder1 (Bioline), − negative control, + positive control, 1–12 healthy plants before exposure to R. banda 1.2 kb Naturwissenschaften (2009) 96:1169–1176 1173

Fig. 2 Ethidium-bromide stained M + - 1 2 3 4 5 6 7 8 9 10 11 12 agarose gel of nested PCR on DNAfromplants90daysafter exposure to Recilia banda, Lanes: M HyperLadder1 (Bioline), + positive control, − negative control, 1–12 healthy plants after 1.2 kb exposure to R. banda.Notethe 1.2 kb specific amplification in seven out of 12 samples

than , 30 days AAP was optimal for stunt In this study, Napier stunt pathogen persisted in three pathogen acquisition in both insect groups (Obura 2008). out of five R. banda samples after multiplication feeding Therefore, rearing the test insects on diseased plants for and not in the other seven insect species, and there was 30 days allowed them to acquire phytoplasma to enable positive transmission in seven out of 12 healthy plants transmission. Once the insect has acquired the phytoplasma exposed to R. banda, as determined by nPCR assay after there is a latent period during which the phytoplasma must 90 days. These plants later developed stunt-characteristic cross the gut wall and hematocele and establish in the symptoms after 120 days. Persistence of stunt pathogen in salivary gland cells before being ejected with the saliva. R. banda and positive transmission of stunt pathogen to Since phytoplasma transmission is by persistent propagative healthy plants incriminate R. banda as a vector of Napier manner in the vector (Murray and Schleifer 1994), exposing stunt phytoplasma in Kenya. R. banda (Fig. 4)isa clean plants to the inoculated insects for 60 days was to leafhopper in the family Cicadellidae, subfamily Deltoce- ensure ample time for the insects to ‘pass’ the pathogen to phalinae, and tribe . It is a small leafhopper the healthy plants by stylet penetration and salivation during with triangularly produced vertex. Deltocephalini are feeding. A vector species is permissive to pathogen widely distributed in the world from the tropical to the establishment, significantly aiding increase of pathogen titre semi-polar regions, with 23 species having been reported levels even upon cessation of inoculum source. In transmission in Afrotropical regions (Satoshi 1999). They are mostly experiments, insects are reared on infected plants (acquisition phytophagous on grasses in the family Gramineae, such as feeding), then changed to healthy diet to allow the pathogen to rice, Sorghum,maize,wheat,sugarcane,inadditiontoa multiply (multiplication feeding) (Weintraub and Beanland variety of other wild grasses (Satoshi 1999). Some species 2006). As a standard procedure for confirming transmis- vector phytopathogenic organisms (viruses, micoplasmas, sion, plants were tested before and after transmission. We used nPCR and sequencing, based on the conserved nature of ribosomal DNA across all prokaryotic organisms, as a diagnostic tool to confirm the presence or absence of phytoplasma in the test plants and in the insects.

M +-1 2 345

Fig. 3 Ethidium-bromide stained agarose gel electrophoresis showing results of nested PCR on DNA isolated from R. banda samples used in the transmission experiment. Lanes: M 1 kb molecular weight marker (Fermentas), + positive control, − negative control, (1–5) R. banda Fig. 4 Dorsal view of Recilia banda (Aauchenorryncha: Cicadelli- samples dae), a vector of Napier grass stunt phytoplasma in Kenya 1174 Naturwissenschaften (2009) 96:1169–1176

Table 2 Percent identity between the 16S rDNA sequen- SeqA Name Len (nt) SeqB Name Len (nt) Percent ces of phytoplasmas infecting identity Napier grass, isolated from: exposed Napier grass 1 Ns. Exposed. plant. FJ862998 1,194 2 Ns. R. banda. FJ862999 1,190 99 (acc. FJ862998), Recilia banda 1 Ns. Exposed. plant. FJ862998 1,194 3 Ns. source. plant FJ862997 1,199 99 (acc. FJ862999), Napier Stunt 1 Ns. Exposed. plant. FJ862998 1,194 4 Napier. grass. stunt. Kenya 1,417 98 phytoplasma source grass (acc. FJ862997), and Napier 1 Ns. Exposed. plant. FJ862998 1,194 5 Napier. grass. stunt. Uganda 1,139 100 stunt phytoplasma in Kenya, 1 Ns. Exposed. plant. FJ862998 1,194 6 Napier. grass. stunt. Ethiopia 1,519 92 Uganda, and Ethiopia, obtained 2 Ns. R. banda FJ862999 1,190 3 Ns. source. plant FJ862997 1,199 99 from the EMBL GenBank 2 Ns. R. banda FJ862999 1,190 4 Napier. grass. stunt. Kenya 1,417 98 2 Ns. R. banda FJ862999 1,190 5 Napier. grass. stunt. Uganda 1,139 99 2 Ns. R. banda FJ862999 1,190 6 Napier. grass. stunt. Ethiopia 1,519 92 3 Ns. source. plant FJ862997 1,199 4 Napier. grass. stunt. Kenya 1,417 98 3 Ns. source. plant FJ862997 1,199 5 Napier. grass. stunt. Uganda 1,139 100 3 Ns. source. plant FJ862997 1,199 6 Napier. grass. stunt. Ethiopia 1,519 92 4 Napier. grass. stunt. Kenya 1,417 5 Napier. grass. stunt. Uganda 1,139 99 4 Napier. grass. stunt. Kenya 1,417 6 Napier. grass. stunt. Ethiopia 1,519 90 5 Napier. grass. stunt. Uganda 1,139 6 Napier. grass. stunt. Ethiopia 1,519 93

Fig. 5 Dendrogram of nearly full-length 16S rDNA sequences from representative phytoplasma strains affecting grasses. Sequences were aligned with ClustalW2 and the tree viewed with Java 6.0 software. Abbreviations are specified in Table 1 Naturwissenschaften (2009) 96:1169–1176 1175 and spiroplasmas) (Chalam and Rao 2005). The zigzag disseminating plant pathogens. R. banda is phytophagous on leafhopper Recilia dorsalis (Motschulsky) is a vector of the family gramineae, and based on 16S sequence analysis, rice dwarf phytoreovirus (Takata 1985), rice gall dwarf there is similarity between Napier stunt phytoplasma and phytoreovirus (Brunt et al. 1990), and rice orange leaf those pathogenic to important grasses, raising the possibility phytoplasma (16SrXI group; Rivera et al. 1963)inAsia. of this phytoplasma to infect economically important grass Rice orange leaf and Napier stunt phytoplasmas (Table 1) species in East Africa. Vector identification is primary to are all members of Ca. Phytoplasma oryzae or the rice development of disease management strategies. Studies on yellow dwarf phytoplasma. Another of Recilia, vector biology and vector–pathogen interactions are being Recilia mica has been reported to vector blast disease in conducted in addition to evaluations of the other leafhoppers oil palm seedlings in West Africa (Desmier de Chenon and plant hoppers associated with Napier grass for any 1979). Ritthison (2004) detected 210-bp phytoplasma additional vector species. Since phytoplasmas are uncultur- DNA fragment associated with sugarcane white leaf able in vitro, the new vector shall act as natural inoculum disease (16SrXI) in Recilia distinctus and R. dorsalis, source in the screening for sources of Napier stunt-disease showing intricate interaction between this genus with resistance in the region. members of 16SrXI phytoplasma. Since phytoplasmas are assigned taxonomic status of Acknowledgements We acknowledge the taxonomic assistance Candidatus (ICSB 1995), defined at 97.5% similarity of the from Dr. Mike Wilson of the National Museum of Wales, UK and 16S rDNA sequence, phytoplasma isolated from source Dr. Tadashi Ishikawa of Tokyo University of Agriculture, Japan. We thank the staff at Molecular Biology and Biotechnology Department plant (diseased Napier grass), R. banda, and exposed of icipe for their valuable support and input and SegoliLab, Napier grass had 99% sequence identity, and were most International Livestock Research Institute, Nairobi Kenya for DNA related to Napier stunt Uganda (99%) and Kenya (98%). sequencing. The work was supported by funds from the Kilimo Trust, The similarity in sequences between Kenyan and Ugandan East Africa and the Gatsby Charitable Foundation, UK, and was conducted in collaboration with Rothamsted Research, UK, which phytoplasma strains should encourage the search for R. receives grant-aided support from BBSRC. banda in Uganda. If found, there is a strong likelihood that it would be associated with transmission the disease in the country (Table 2). References Phytoplasma infecting grasses are diverse, four groups have been identified to infect grasses; 16SrI, III, IV, and XI. Banttari EE, Zeyen RJ (1979) Interactions of mycoplasmalike However, most of these phytoplasma belong to 16SrXI organisms and viruses in dually infected leafhoppers, plan- group (Table 1). Phytoplasma GenBank accessions, thoppers, and plants. In: Maramorosch K, Harris KF (eds) FJ862998, FJ862997, and FJ862999, were most similar to Leafhopper vectors and plant disease agents. 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