Phytoplasmas are wall-less bacteria, unculturable in vitro, and transmitted primarily by leafhoppers (Cicadellidae). Maize bushy stunt disease has been linked to phytoplasmas belonging to the16SrI-B subgroup and vectored by leafhoppers in the genus Dalbulus spp. (Hemiptera: Cicadellidae). The recent detection of maize bushy stunt affecting native corn, maize, in the southeast highlands of Mexico motivated the survey to determine which leafhoppers were associated with this crop during the 2013-2014 growing season. We detected 7 leafhopper genera in native corn cultivated 2,400 meters above sea level (masl), with 4 of these genera reported for the first time in corn. Based on external morphology and male genitalia, we identified Idiodonus wickhami (Ball) (Hemiptera: Cicadellidae), Amblysellus grex (Oman) (Hemiptera: Cicadellidae), Empoasca fabae (Harris) (Hemiptera: Cicadellidae), Macrosteles quadrilineatus (Forbes) (Hemiptera: Cicadellidae), and Dalbulus elimatus (Ball) (Hemiptera: Cicadellidae). We were not able to identify the leafhopper genera Graphocephala (Hemiptera: Cicadellidae) and Erythridula (Hemiptera: Cicadellidae) to species because of a lack of male leafhoppers. Nymphal stages of I. wickhami also were identified using taxonomic and molecular tools. The presence of adults and nymphs of I. wickhami in the crop suggest that native corn grown in the southeast highlands of Mexico is a feeding and reproductive host for I. wickhami. Moreover, I. wickhami was found infected with 16SrI-B strain maize bushy stunt-Ver while D. elimatus, a well-known maize bushy stunt phytoplasma vector, was found infected with the 16SrI-B strain maize bushy stunt-Pueb.
Maize bushy stunt is the most serious disease affecting corn in the Americas (Alvarez et al. 2014; Pérez-López et al. 2016). Maize bushy stunt has been associated in previous studies with phytoplasma strains related to ‘Candidatus Phytoplasma asteris’, which belongs to the 16SrI-B subgroup. Phytoplasmas are vectored by phloem-feeding insects, primarily leafhoppers (Hemiptera: Cicadellidae) and members of the genus Dalbulus have been identified as the vector of maize bushy stunt phytoplasma in maize. Dalbulus maidis (DeLong & Wolcott), D. elimatus (Ball), D. guevari (DeLong), D. quinquenotatus (DeLong & Nault), D. gelbus (DeLong) (Hemiptera: Cicadellidae), and D. tripsacoides (DeLong and Nault) (Hemiptera: Cicadellidae) transmit maize bushy stunt phytoplasma, with the corn leafhopper, D. maidis Delong, and the Mexican corn leafhopper, D. elimatus Ball, being the most efficient vectors (Madden & Nault 1983; Moya-Raygoza & Nault 1998; Weintraub & Beanland 2006). Dalbulus maidis can be found at low altitudes while D. elimatus is distributed at altitudes higher than 1,000 meters above sea level (masl) (Pinedo-Escatel & Moya-Raygoza 2015).
Along with maize bushy stunt phytoplasma, other pathogens are efficiently transmitted by leafhoppers from the genus Dalbulus, such as maize rayado fino virus and corn stunt spiroplasma Spiroplasma kunkeii (Whitcomb et al. 1986) (Entomoplasmatales: Spiroplasmataceae) (Moya-Raygoza et al. 2012). Together, the 3 pathogens form the corn stunt complex, which is a well-known cause of yield loss in corn production in Central and South America. The overlap in symptom expression caused by these pathogens has led to confusion and inaccurate disease diagnosis (Nault 1983).
Corn (Zea mays L. ssp. mays) (Poaceae) was first domesticated in Mexico around 10,000 years ago (Doebley 2004). Centuries of local selection and seed interchanges have led to the development of native corn varieties with unique genotypes (Serratos 2009). In the highlands of the state of Puebla, Mexico (> 2,400 masl), small agricultural communities use indigenous corn varieties that are adapted to the specific environmental conditions present at high altitudes (Perales & Golicher 2014). The recent detection of maize bushy stunt phytoplasma in native corn in the highlands of southeast Mexico could become a serious economic problem for the local subsistence farmers (Pérez-López et al. 2016). Phytoplasma diseases can be controlled with chemicals or cultural practices that target the insect vectors (Weintraub 2007). However, the maize bushy stunt phytoplasma vector(s) identity and biodiversity in crops of native corn grown at high elevations is not known.
This study is a preliminary survey of leafhoppers associated with native corn grown in “Sierra Norte de Puebla,” at the high-altitude community of Las Trancas located in southeast Mexico. The objective of this research was to identify the leafhopper species present in native corn crops and to determine their maize bushy stunt phytoplasma infection status.
Materials and Methods
STUDY AREA AND LEAFHOPPER COLLECTION
The field survey was conducted in 2014 in native corn fields, located in the municipality of Ejido Las Trancas in the region of Zaragoza of Puebla in Mexico (19.7293°N, 97.8634°W; 2,400 masl). Fields were cropped mainly with white and blue varieties (Pérez-López et al. 2016). Leafhoppers were sampled in 2 corn fields 5 kilometers apart, once in Jul 2014 and once in Nov 2014 (4 and 8 mo after seeding). The 2 cornfields and their direct surroundings were never treated with insecticide. Insects were collected with a sweep net (diam 38 cm) and 10 sweeps per field were taken along a short transect of 10 footsteps, starting 10 m from the border of the field.
Leafhoppers collected in this study and their phytoplasma infection status.
Leafhopper adults were counted and identified in the laboratory using a binocular microscope. Species were keyed according to several features such as length, morphology, color, and genitalia, using figures and data referenced in DeLong (1946, 1931), Forbes (1885), Oman (1949), Hepner (1978) and Young (1977).
Molecular tools were used to identify the leafhopper nymphs. Total DNA was extracted from 5 insects using a modified CTAB method (Pérez-López et al. 2016). DNA extracts were amplified using the mitochondrial cytochrome c oxidase 1 (CO1, cox1) specific primers Uni-MinibarR1/Uni-MinibarF1, following the protocol previously described (Meusnier et al. 2008). PCR products were examined using 1% agarose gel electrophoresis and ethidium bromide-stained products were visualized using a GelDoc (BioRad, Mississauga, Ontario, Canada). CO1 amplification generated an approximately 150bp DNA fragment when observed under an ultraviolet (UV) transiluminator at 365 nm (AIML 26, Alpha Innotech Corp., San Leandro, California, USA).
Leafhoppers were grouped by genus or species and DNA was extracted from 2 or more individuals randomly selected, using a modified CTAB method (Pérez-López et al. 2016). DNA extracts were diluted 1:10 with 10mM Tris-Cl, pH 8.5, and used as a template in PCR to amplify the 16S rRNA-encoding gene F2nR2 fragment with primers R16F2n/R16R2 and cpn60 Universal Target (cpn60 UT) sequence with primers H279p/H280p: D0317/D0318 (1:7 ratio) (Gundersen & Lee 1996; Dumonceaux et al. 2014). PCR products were examined using 1% agarose gel electrophoresis with ethidium bromide staining and visualized using a GelDoc. F2nR2 and cpn60 UT amplifications produced 1.2kb and 604 bp DNA fragments, respectively.
DNA SEQUENCING AND PHYLOGENETIC ANALYSES
The 16S, cpn60 UT, and cox1 mini-barcode amplicons were purified using a QIAquick® PCR Purification Kit (QUIAGEN, Mississauga, Ontario, Canada), and directly sequenced using the corresponding primers.
Phylogenetic analyses were conducted using the neighbor-joining method with MEGA v6.0 (Tamura et al. 2013), with 1,000 bootstrap replicates. All the sequences obtained were assembled using the Staden package (Bonfield & Whitwham 2010), and compared with reference sequences from GenBank through the BLAST program ( http://www.ncbi.nlm.nih.gov). Acholeplasma laidlawii (Edward and Freund 1970) (Acholeplasmatales: Acholeplasmataceae) strain PG-8A (U14905) was used as outgroup to root the tree generated for F2nR2 and cpn60 UT, and a member of the family Membracidae (GU013584) was used as outgroup to root the tree generated with cox1 mini-barcode.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSES
The 1.2 kb of F2nR2 sequence obtained from phytoplasma-positive leafhoppers were digested with endonucleases AluI, BstUI, HaeIII, HinfI and Tsp509I (Thermo Scientific, Mississauga, Ontario, Canada), and the restriction fragment length polymorphism (RFLP) pattern compared between them and with the previously recorded RFLP pattern (Lee et al. 2004). Reactions with AluI, BstUI, HaeIII, and HinfI were incubated at 37 °C, while reaction with Tsp509I was incubated at 65 °C according to the manufacturer's recommendations (Thermo Scientific, Mississauga, Ontario, Canada). Once digested, the samples were observed through electrophoresis using 4% UltraPureTM Agarose 1000 gel (Invitrogen, Mississauga, Ontario, Canada) stained with ethidium bromide (Pérez-López et al. 2016).
SINGLE NUCLEOTIDE POLYMORPHISM ANALYSIS
The cpn60 UT sequences obtained from the phytoplasma-positive leafhoppers were aligned using ClustalW (Thompson et al. 1994) with 20 publicly available cpn60-encoding genes and cpn60 UT sequences from ‘Ca. P. asteris’-related strains. Sequences were then trimmed to the 552 bp cpn60 UT for phytoplasmas, using the Staden package (Bonfield & Whitwham 2010; Dumonceaux et al. 2014). The single nucleotide polymorphisms (SNP) were noted as previously described (Pérez-López et al. 2016).
LEAFHOPPER COLLECTION AND IDENTIFICATION
A total of 80 leafhopper (Hemiptera: Cicadellidae) specimens in different developmental stages was collected during the 2 surveys (Table 1). Based on their external morphology and male genitalia characteristics, the following leafhopper species were identified: Idiodonus wickhami (Ball), Amblysellus grex (Oman), Empoasca fabae (Harris), and Dalbulus elimatus (Ball) (Figs. 1, 2). Female specimens only were found in the genera Macrosteles, Graphocephala, and Erythridula. Specimens belonging to the genus Macrosteles were classified as Macrosteles quadrilineatus (Forbes) (Hemiptera: Cicadellidae) through the measurement of the wing ratio (Saguez et al. 2015). No species identification could be conducted for Graphocephala and Erythridula.
One type of nymph, with a ‘red speckled' pattern, was collected (Table 1). Red speckled nymphs showed red spots similar to the spots observed on the body of I. wickhami. The phylogenetic tree generated for cox1 from I. wickhami (GenBank accession no. KU722543) and from the red speckled nymphs (GenBank accession no. KU722544) showed that both sequences formed a well-supported independent phylogenetic group (bootstrap value 95 %) (Fig. 3). Both sequences showed a 93% or higher nucleotide sequence identity with Cicadellidae sp. (GenBank accession no. HF968661), and 100 % between them, suggesting that the red speckled nymphs are the nymphal stages of I. wickhami.
PHYTOPLASMA DETECTION AND IDENTIFICATION
Leafhoppers in 2 of the 7 genera collected tested positive for the presence of phytoplasma DNA. The sequence fragments of about 1.2 kb F2nR2 and about 605 bp of cpn60 UT, were amplified from DNA extracts obtained from I. wickhami and D. elimatus. The F2nR2 sequences obtained from both leafhopper species (GenBank accession no. KU722546 and KU722545 for I. wickhami and D. elimatus, respectively) through direct sequencing showed 99% nucleotide identity with maize bushy stunt phytoplasma strain Puebla and Veracruz (maize bushy stunt-Pueb and maize bushy stunt-Ver) (GenBank accession no. KT444670 and KT444671, respectively). The cpn60 UT sequence obtained from I. wickhami (GenBank accession no. KU722542) showed 100% nucleotide identity with the strain maize bushy stunt-Pueb (GenBank accession no. KT444672), and the cpn60 UT sequence obtained from D. elimatus (GenBank accession no. KU722541) showed 100% nucleotide identity with the strain maize bushy stunt-Ver (GenBank accession no. KT444673). The F2nR2 sequences obtained from I. wickhami and D. elimatus showed 99% nucleotide identity between them. Similarly, the cpn60 UT sequences obtained from I. wickhami and D. elimatus also showed 99% nucleotide identity between them. Maize bushy stunt-Pueb and maize bushy stunt-Ver are members of 16SrIB subgroup, ‘Ca. P. asteris’-related strains. The phylogenetic tree derived from the analysis of F2nR2 sequences and cpn60 UT sequences obtained from the leafhoppers were consistent between them and showed that the sequences clustered with strains within the 16SrI-B subgroup (Figs. 4, 5).
The RFLP profiles obtained after the digestion of the F2nR2 sequences amplified from DNA extracts of I. wickhami and D. elimatus were identical between them (Fig. 6). The pattern observed was identical to the RFLP pattern described for 16SrI-B strains (Lee et al. 2004). The SNP analysis of cpn60 UT sequences confirmed the previous results showing that the cpn60 UT sequence obtained from D. elimatus is identical to the strain maize bushy stunt-Ver and maize bushy stunt-Col (GenBank accession no. AB599712) while the sequence amplified from I. wickhami is identical to the strain maize bushy stunt-Pueb (Fig. 7).
Maize bushy stunt disease has been detected throughout Latin America and the southern United States, with D. maidis and D. elimatus as vectors. All genera found in this study have been described as Nearctic leafhoppers with a distribution in the southern USA and throughout Mexico (Dmitriev & Dietrich 2009; Feil et al. 2000).
Species D. elimatus, A. grex, E. fabae, and M. quadrilineatus have been reported in corn previously, although maize has been described as a non-preferred host for M. quadrilineatus (Kunkel 1946; Madden & Nault 1983; Hammond & Stinner 1987; Zhou et al. 2003). However, the presence of I. wickhami, Graphocephala sp., and Erythridula sp. in corn crops has not been reported. Native corn is usually grown in mixed cultures with other crops such as potato, amaranth or common beans (Waddington et al. 1990. In the Sierra Norte de Puebla community, corn crops also are weedy because most crops are grown without herbicide treatments. The study area was surrounded by potato plants, and we caught more female E. fabae than male (Table 1), which suggests that the potato leafhopper may have immigrated into corn from the neighboring potato crop. This same explanation also can apply to M. quadrilineatus, A. grex, Graphocephala sp., and Erythridula sp. because more female leafhoppers were caught than males (Table 1). An excess of female leafhoppers as evidence of immigration previously is described for M. quadrilineatus, E. fabae, and D. elimatus (Drake & Chapman 1965; Emmen et al. 2004; Moya-Raygoza et al. 2012). The collection dates also may influence the differences between the number of females and males (Pinedo-Escatel & Moya-Raygoza 2015). To our knowledge, this is the first report of I. wickhami, Graphocephala sp., and Erythridula sp. in corn fields, and this is the first report of the association of A. grex, E. fabae, I. wickhami, Graphocephala sp., and Erythridula sp. in native corn fields grown at altitudes of 2,400 masl.
Based on nucleotide identity, phylogenetic analysis, and morphological similarities, we suggested that the red speckled nymphs are the progeny of I. wickhami. This morphological characteristic is a wellknown feature of the species I. wickhami (DeLong 1946). The presence of a high number of red speckled nymphs in the samples suggests that this leafhopper species is reproducing on native corn, but further experiments must be performed in order to confirm these results.
Maize bushy stunt phytoplasmas DNA was detected in D. elimatus and adult I. wickhami. The detection of maize bushy stunt phytoplasma in D. elimatus has been reported previously (Esau et al. 1976; Nault 1980, 1983). Interestingly, maize bushy stunt phytoplasma also was detected in DNA extracts of I. wickhami, a leafhopper not previously identified as a corn feeder or a phytoplasma vector. The most intriguing finding was the identification through the SNP of cpn60 UT sequences of strain maize bushy stunt-Ver from D. elimatus and strain maize bushy stunt-Pueb from I. wickhami. The strain maize bushy stunt-Ver is closely related to the strain maize bushy stunt Colombia, while maize bushy stunt-Pueb is different from previously identified maize bushy stunt phytoplasmas strains, based on cpn60 UT sequences (Pérez-López et al. 2016).
In conclusion, in this study maize bushy stunt phytoplasma was detected in 2 abundant leafhoppers, D. elimatus and I. wickhami. Also, native corn was identified as a probable new host for I. wickhami. This study is the first step towards identifying the vectors of maize bushy stunt phytoplasma in indigenous corn varieties produced in small agricultural communities in southern Mexico. Further surveys to characterize leafhopper populations and transmission bioassays are necessary in order to develop management strategies that are sustainable for those rural communities.
This work was supported by the Genomic Research and Development Initiative for the shared priority project on quarantine and invasive species. Edel Pérez-López thanks CONACYT for his PhD scholarship (CVU: 517835), and the Canadian government and Her Majesty the Queen for hosting EPL at the Saskatoon Research Centre of Agriculture and Agri-Food Canada. We also thank Adilson Pinedo-Escatel from CUCBA, Universidad de Guadalajara, and Christopher Dietrich from the Illinois Natural History Survey, Prairie Research Institute, University of Illinois, for their support identifying the leafhoppers collected in this study.
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