Simultaneous infection of Leptodelphax maculigera (Hemiptera: Delphacidae) by Maize rayado fino virus (MRFV) and Maize bushy stunt phytoplasma (MPSP) detected in Rio Grande do Sul, Brazil.

Simultaneous infection of Leptodelphax maculigera (Hemiptera: Delphacidae) by Maize rayado fino virus (MRFV) and Maize bushy stunt phytoplasma (MPSP) detected in Rio Grande do Sul, Brazil. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Simultaneous infection of Leptodelphax maculigera (Hemiptera: Delphacidae) by Maize rayado fino virus (MRFV) and Maize bushy stunt phytoplasma (MPSP) detected in Rio Grande do Sul, Brazil. Glauber Renato Stürmer, Caroline Wesp Guterres, Henrique Pozebon, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4455424/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Aug, 2024 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted 5 You are reading this latest preprint version Abstract The corn stunt disease is a major threat to maize production in Brazil. In addition to the more common insect vector Dalbulus maidis (Hemiptera: Cicadellidae), the African species Leptodelphax maculigera (Hemiptera: Delphacidae) was recently found in the Brazilian states of Goiás, Paraná and Santa Catarina, raising concern regarding its potential ability to transmit stunt pathogens. We confirmed the presence of L. maculigera in the state of Rio Grande do Sul by conducting a population survey with yellow sticky traps and light traps in three different sites, although it corresponded to only 2.3% of the D. maidis population captured during the same period. Molecular analysis via RT-qPCR multiplex confirmed the simultaneous infection of L. maculigera by two corn stunt pathogens – Maize rayado fino virus (MRFV) and Maize bushy stunt phytoplasma (MBSP) – in 76.9% of the samples, while the remaining 23.1% showed single infection by MRFV. The confirmation of infectiveness by two stunt pathogens in L. maculigera will aid in the outlining of managment strategies for this insect vector. corn stunt disease phytoplasma spiroplasma Zea mays Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The corn stunt disease, caused by Maize rayado fino virus (MRFV), Maize bushy stunt phytoplasma (MBSP) and Spiroplasma kunkelii (Mycoplasmatales: Mycoplasmataceae) (CSS), is the main production drawback currently faced by maize ( Zea mays L.) (Poales: Poaceae) growers in Brazil. Yield losses equal or higher than 70% have been reported in susceptible maize hybrids subjected to high disease pressure (Pozebon et al. 2022 ). The corn stunt pathogens colonize the phloem tissues of maize plants and are transmitted in a persistent-propagative manner by Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae), which was long considered the only vector for these pathogens in Brazil (Oliveira and Frizzas 2022 ). However, the African leafhopper Leptodelphax maculigera (Hemiptera: Delphacidae) was recently found in Brazil, feeding on maize, signalgrass ( Brachiaria sp.), Napier grass ( Cenchrus purpureus ) and common bean ( Phaseolus vulgaris ) in the state of Goiás (Ferreira et al. 2023 ). The species was later reported in the states of Paraná (Bortolotto et al. 2023 ) and Santa Catarina (Canale et al. 2023 ), with positive results for infectiveness by corn stunt pathogens (Canale et al. 2024 ). However, this information is still lacking for the state of Rio Grande do Sul, an important maize producing region. Therefore, the aim of this study was to assess infectiveness by corn stunt pathogens in L. maculigera individuals captured in different sites of Rio Grande do Sul state, Brazil, during 2023/24 crop season. Infectiveness of D. maidis by the same pathogens was also assessed and used for comparison. Material and Methods The study was carried out as a cooperation among researchers from Cooperativa Central Gaúcha LTDA (CCGL), Universidade Federal do Rio Grande do Sul (UFRGS) and Agronômica Laboratory of Phytosanitary Diagnosis. Yellow sticky traps were set up in June, 2023 in maize and wheat fields, including areas with volunteer maize plants, in the municipalities of Passo Fundo, Santa Rosa and Cruz Alta (all located within Rio Grande do Sul state, Brazil). The traps were sampled weekly and taken to the CCGL Entomology Laboratory for counting and morphological identification of the collected specimens. Seven samples identified as L. maculigera were sent to Agronômica Laboratory to assess infectiveness by corn stunt pathogens using molecular tools. Figure 1 Yellow sticky trap used to capture corn leafhoppers. A 1,20 m tall light trap was also used in the experimental maize field of Cruz Alta (Fig. 2), which was sowed on 20/08/2023 and emerged on 07/09/2023 (maize hybrid AG9021 PRO3). The lamp was kept turned on once a week during night time, beginning on 15/09/2023. Sampling was carried out weekly from 15/10/2023 to 24/02/2024, followed by counting and morphological identification of the collected specimens in the CCGL Entomology Laboratory. The experimental maize field was harvested on 24/01/2024. Figure 2 Light trap used to capture corn leafhoppers. Infectiveness by stunt pathogens was assessed in both trials for 11 different sampling times, at Agronômica Laboratory, Porto Alegre (Rio Grande do Sul state) using RT-qPCR (reverse transcription quantitative real-time PCR), which is 10.000x more sensitive to corn stunt pathogens compared to detection by conventional PCR. Acid nucleic was extracted following Doyle ( 1991 ) adapted. Amplification of the pathogens DNA (MRFV, MBSP and CSS) by RT-qPCR multiplex was carried out using master mix GoTaq® Enviro RT-qPCR System (Promega, Madison) in a QuantStudio 5 Real-Time PCR System (Applied Biosystems) thermocycler, using specific primers for the three pathogens, according to method developed by Agronômica Laboratory (2023) in the regions: coat protein, cpn60 and spiralin lipoprotein, respectively. The resulting data were analysed using QuantStudio™ Design & Analysis Software (Applied Biosystems). Results and Discussion The presence of L. maculigera was confirmed in the municipalities of Cruz Alta, Passo Fundo and Santa Rosa from samples collected in 03/08/2023, 12/09/2023 and 01/10/2023, respectively. However, only the specimens captured in Cruz Alta were infected by corn stunt pathogens, showing simultaneous infection by MRFV and MBSP (Fig. 3). This is the first report of infection of L. maculigera by both pathogens in the state of Rio Grande do Sul, diverging from the results obtained by Obura et al. ( 2010 ), who found the species incapable of acquiring and transmiting MBSP to Napier grass in Kenya. Figure 3 Presence of Leptodelphax maculigera and infectiveness by corn stunt pathogens in Rio Grande do Sul state, Brazil, in the 2023/24 crop season. The confirmation of simultaneous MRFV and MBSP infection in L. maculigera raises an alert for the potential threat that this novel species represents to maize production in the Rio Grande do Sul state, and in Brazil as a whole. The morphological closeness between L. maculigera and the more common D. maidis is also of concern, as field samples could be easily misidentified and lead to underrated population estimates. Certain morphological traits are unique to L. maculigera , however, and can be used to correctly identify the species. Leptodelphax maculigera is straw colored, hyaline winged and black eyed, and is about 0,4 cm smaller than D. maidis. It also displays a single dark spot in the clypeus (i.e. lower face), whereas D. maidis has two characteristic dark spots in the upper face (Fig. 4). The antennae of L. maculigera are also distinct, with a visible dilated pedicel near its insertion in the insect’s head, whereas D. maidis’ pedicels are not dilated. Leptodelphax maculigera and other Delpachidae leafhoppers also present tibiae with movable apical spurs in the third pair of legs, which are absent in D. maidis (Fig. 4). The correct identification of corn leafhoppers is the first step in establishing adequate management measures for each detected species, ensuring that infesting populations are controlled before reaching economic injury level in maize and other crops. Figure 4 Morphological differences between Dalbulus maidis and Leptodelphax maculigera . Photos and drawings by Glauber R. Stürmer The population density of Cicadellidae and Delphacidae leafhoppers was previously assessed in two Brazilian regions during three crop seasons by Oliveira et al. ( 2013 ), who found that D. maidis made up 90.1% of the collected samples. The authors reported nine species of Delphacidae among the samples, three of them for the first time in Brazil, and raised concern about the possibility of these species acting as potential vectors for viruses and mollicutes in maize. In our study, the highest population of leafhoppers (653 individuals) was captured in the lamp trap between 26/11/2023 and 20/01/2024, with D. maidis representing 87% of the population (569 individuals). During this period the corn plants progressed from growth stage V4 (four leaves) to R5 (dent) (Ritchie and Hanway 1989 ). Only 2.3% of the samples collected in the same period were identified as L. maculigera (15 individuals), most of them captured between 14/01/2024 and 20/01/2024 (10 individuals), when maize plants were at growth stage R5 (Fig. 5). Figure 5 Population level of Dalbulus maidis and Leptodelphax maculigera during 2023/24 crop season in Cruz Alta, RS Assessment of infectiveness by stunt pathogens showed positive results for 72.7% of the D. maidis samples and 50.0% of the L. maculigera samples. The positive D. maidis samples showed 2.8% of single MBSP infection, 16.8% of single MRFV infection, 76.7% of simultaneous MRFV + MBSP infection and 3.7% of simultaneous MRFV + MBSP + CSS infection. As for the positive L. maculigera samples, 23.1% were of single MRFV infection and 76.9% of simultaneous MRFV + MBSP infection (Fig. 6). Transmission efficiency of MBSP and CSS is dependent on temperature, with MBSP being more tolerant to lower temperatures (Sabato et al. 2020 ). In this experiment, triple infection (MRFV + MBSP + CSS) was only detected in the samples collected between 31/12/2023 and 06/01/24, whereas single infection by MBSP was detected since the second sampling week (22/10/2023 to 28/10/2023). Vector competence of Cicadellidae and Delphacidae leafhoppers to transmit MBSP has been documented by Obura et al. ( 2009 ). Figure 6 Percentual amount of Dalbulus maidis and Leptodelphax maculigera samples detected with each corn stunt infection (MRFV, MBSP and CSS) during 2023/24 crop season, in Cruz Alta, RS The Delphacidae family includes several leafhoppers of economic importance, which can transmit up to 25 different viruses to maize and other grasses, such as sugarcane ( Saccharum officinarum ), millet ( Panicum miliaceum ), sorghum ( Sorghum bicolor ), rice ( Oryza sativa ), wheat ( Triticum aestivum ), oat ( Avena sativa ) and Bermuda grass ( Cynodon dactylon ) (Wilson 2005 ). The species Delphacodes kuscheli , for instance, is the main vector of MRCV (Mal de Río Cuarto virus) in Argentina, causing important losses in maize crops (Ornaghi et al. 2011 ). Congeners of L. maculigera include the species Leptodelphax dymas , reported in Africa as a vector of Napier grass stunt (NGS), a highly agressive phytoplasm belonging to the genus Candidatus (Obura et al. 2010 ). The wide range of hosts used by Delphachidae leafhoppers suggest that L. maculigera could be feeding and reproducing on other plants besides maize in Brazil, increasing the risk of population outbreaks and making the associated disease very hard to prevent. The confirmation of infectiveness by stunt pathogens in L. maculigera will aid in the outlining of managment strategies for this novel insect vector, and should be continued alongside the D. maidis ’ population surveys already conducted by the Cooperative Technical Network in Rio Grande do Sul. Further studies should explore the population patterns of L. maculigera in different maize producing regions of Brazil and detail its capacity to acquire and transmit stunt pathogens, as well as its potential adaptiveness to alternate plant hosts. Correct identification of specimens and thorough monitoring of field populations are recommended in order to attest L. maculigera’ s adaptiveness to Brazilian maize growing conditions and achieve an efficient, integrated management program for both leafhopper species. Declarations Conflicts of interest The authors have no relevant financial or non-financial interests to disclose. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Availability of data and material Not applicable. Code availability Not applicable. Authors contributions G.R.S. and C.W.G. conceived this research, designed the experiments and collected field data. C.C.L.A., T.M., Y.C. and I.F. carried out molecular analyses and procedures. H.P. assisted with writing and data discussion. All authors read and commented on the information provided in the manuscript and approved the final draft. References Bortolotto OC, Pazini JB, Molina RO, Garcia MH, Andrade CCL, Mituti T (2023) Vírus da-risca tem novo transmissor. Cultivar 292:30–35 Canale MC, Andrade MVS, Castilhos RV (2023) Cigarrinha-africana Leptodelphax maculigera Stal, 1859 (Hemiptera: Delphacidae) em Santa Catarina. Nota Técnica Epagri. https://publicacoes.epagri.sc.gov.br/nt/article/view/1763 . Accessed 07 May 2024 Canale MC, Manica MAP, Andrade MVS, Castilhos RV (2024) Leptodelphax maculigera (Hemiptera: Delphacidae) harbors the corn stunt complex pathogens. Plant Disease. Preprint. https://doi.org/10.1094/PDIS-01-24-0142-SC . Accessed 07 May 2024 Doyle J (1991) DNA Protocols for Plants. In: Hewitt GM, Johnston AWB, Young JPW (eds) Molecular Techniques in Taxonomy. Springer, Berlin, Heidelberg, pp 283–293 Ferreira KR, Barlett CR, Ache M, Silva LRS, Magalhães VS, Gondinho KCA (2023) First record of the African species Leptodelphax maculigera Stal, 1859 (Hemiptera: Delphacidae) in Brazil. Neotrop Entomol. Preprint. https://doi.org/10.21203/rs.3.rs-2818951/v . Accessed 07 May 2024 Giménez-Pecci MP, Oliveira E, Resende RO, Laguna IG, Conci LR, Avila A, Herrera P, Galdeano E, Virla E, Nome CF (2002) Ocorrência de doenças causadas por molicutes e por vírus nas Províncias de Tucumán e de Córdoba na Argentina. Fitopatologia Brasileira 27:403–407 Obura E, Midega C, Masiga D, Pickett J, Hassan M, Koji S, Khan Z (2009) Recilia banda Kramer (Hemiptera: Cicadellidae), a vector of Napier stunt phytoplasma in Kenya. Naturwissenschaften 96:1169–1176 Obura E, Midega C, Masiga D (2010) Napier stunt disease is transmitted by a leafhopper vector Maiestas (= Recilia ) banda in Western Kenya. In: Renard G (ed) ASARECA/ILRI Workshop on Mitigating the Impact of Napier Grass Smut and Stunt Diseases, Addis Ababa, Nairobi, Kenya Oliveira CM, Oliveira E, Souza RP, Alves E, Dolezal W, Paradell S, Lenicov AMRF, Frizzas MR (2013) Abundance and species richness of leafhoppers and planthoppers (Hemiptera: Cicadellidae and Delphacidae) in Brazilian maize crops. Fla Entomol 96:1470–1481 Oliveira CM, Frizzas MR (2022) Eight decades of Dalbulus maidis (DeLong & Wolcott) (Hemiptera, Cicadellidae) in Brazil: what we know and what we need to know. Neotrop Entomol 57:1–17 Ornaghi JA, March GJ, Moschini RC, Martínez MI, Boito GT (2011) Predicting population level of Delphacodes kuscheli , vector of Mal de Río Cuarto virus, and climate risk in the Argentine Pampas using meteorological models. Trop Plant Pathol 36:160–168 Pozebon H, Stürmer GR, Arnemann JA (2022) Corn stunt pathosystem and its leafhopper vector in Brazil. J Econ Entomol 115:1817–1833 Ritchie SW, Hanway JJ (1989) How a corn plant develops. Iowa State University, Ames, USA. https://publications.iowa.gov/18027/1/How%20a%20corn%20plant%20develops001.pdf . Accessed 07 May 2024 Sabato O, Landau EC, Barros BA, Oliveira CM (2020) Differential transmission of phytoplasma and spiroplasma to maize caused by variation in the environmental temperature in Brazil. Eur J Plant Pathol 157:163–171 Wilson SW (2005) Keys to the families of Fulgoromorpha with emphasis on planthoppers of potential economic importance in the southeastern United States (Hemiptera: Auchenorrhyncha). Fla Entomol 88:464–481 Cite Share Download PDF Status: Published Journal Publication published 20 Aug, 2024 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted Editorial decision: Major revisions 02 Jul, 2024 Reviewers agreed at journal 31 May, 2024 Reviewers invited by journal 24 May, 2024 Editor assigned by journal 23 May, 2024 First submitted to journal 21 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4455424","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":306396083,"identity":"3e379f86-ea9e-4684-a1cc-460edfe23941","order_by":0,"name":"Glauber Renato Stürmer","email":"","orcid":"","institution":"CCGL-Tec","correspondingAuthor":false,"prefix":"","firstName":"Glauber","middleName":"Renato","lastName":"Stürmer","suffix":""},{"id":306396084,"identity":"d749feef-1167-49f7-b96c-76b15223cd34","order_by":1,"name":"Caroline Wesp Guterres","email":"","orcid":"","institution":"UFRGS: Universidade Federal do Rio Grande do 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3","display":"","copyAsset":false,"role":"figure","size":174655,"visible":true,"origin":"","legend":"\u003cp\u003ePresence of \u003cem\u003eLeptodelphax maculigera \u003c/em\u003eand infectiveness by corn stunt pathogens in Rio Grande do Sul state, Brazil, in the 2023/24 crop season.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4455424/v1/bfbe4f36e23a1e4615b059ef.png"},{"id":57827674,"identity":"796a8183-74a0-49df-81dd-1a7bdb8610ed","added_by":"auto","created_at":"2024-06-06 07:24:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":541666,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological differences between \u003cem\u003eDalbulus maidis\u003c/em\u003e and \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e. Photos and drawings by Glauber R. Stürmer\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4455424/v1/ee483477bc5d44d0ffd44cc1.png"},{"id":57827679,"identity":"4a8fc10f-f5b9-42e1-87f4-85b5ea0f2091","added_by":"auto","created_at":"2024-06-06 07:24:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105814,"visible":true,"origin":"","legend":"\u003cp\u003ePopulation level of \u003cem\u003eDalbulus maidis\u003c/em\u003e and \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e during 2023/24 crop season in Cruz Alta, RS\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4455424/v1/5b92387aea3f70313df2183b.png"},{"id":57828503,"identity":"241a4d32-330e-48ac-91be-f9326717c52c","added_by":"auto","created_at":"2024-06-06 07:32:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":22567,"visible":true,"origin":"","legend":"\u003cp\u003ePercentual amount of \u003cem\u003eDalbulus maidis\u003c/em\u003e and \u003cem\u003eLeptodelphax maculigera \u003c/em\u003esamples detected with each corn stunt infection (MRFV, MBSP and CSS) during 2023/24 crop season, in Cruz Alta, RS\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4455424/v1/d110644f9447e8e64f7620c0.png"},{"id":63300390,"identity":"4668288a-d41a-4e3a-9c28-dfc1570ca5e1","added_by":"auto","created_at":"2024-08-26 16:14:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4779566,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4455424/v1/904b76f5-53d1-4d58-a45a-c47ec1c950bd.pdf"}],"financialInterests":"","formattedTitle":"Simultaneous infection of Leptodelphax maculigera (Hemiptera: Delphacidae) by Maize rayado fino virus (MRFV) and Maize bushy stunt phytoplasma (MPSP) detected in Rio Grande do Sul, Brazil.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe corn stunt disease, caused by Maize rayado fino virus (MRFV), Maize bushy stunt phytoplasma (MBSP) and \u003cem\u003eSpiroplasma kunkelii\u003c/em\u003e (Mycoplasmatales: Mycoplasmataceae) (CSS), is the main production drawback currently faced by maize (\u003cem\u003eZea mays\u003c/em\u003e L.) (Poales: Poaceae) growers in Brazil. Yield losses equal or higher than 70% have been reported in susceptible maize hybrids subjected to high disease pressure (Pozebon et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The corn stunt pathogens colonize the phloem tissues of maize plants and are transmitted in a persistent-propagative manner by \u003cem\u003eDalbulus maidis\u003c/em\u003e (DeLong \u0026amp; Wolcott) (Hemiptera: Cicadellidae), which was long considered the only vector for these pathogens in Brazil (Oliveira and Frizzas \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the African leafhopper \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e (Hemiptera: Delphacidae) was recently found in Brazil, feeding on maize, signalgrass (\u003cem\u003eBrachiaria\u003c/em\u003e sp.), Napier grass (\u003cem\u003eCenchrus purpureus\u003c/em\u003e) and common bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e) in the state of Goi\u0026aacute;s (Ferreira et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The species was later reported in the states of Paran\u0026aacute; (Bortolotto et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Santa Catarina (Canale et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with positive results for infectiveness by corn stunt pathogens (Canale et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, this information is still lacking for the state of Rio Grande do Sul, an important maize producing region. Therefore, the aim of this study was to assess infectiveness by corn stunt pathogens in \u003cem\u003eL. maculigera\u003c/em\u003e individuals captured in different sites of Rio Grande do Sul state, Brazil, during 2023/24 crop season. Infectiveness of \u003cem\u003eD. maidis\u003c/em\u003e by the same pathogens was also assessed and used for comparison.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eThe study was carried out as a cooperation among researchers from Cooperativa Central Ga\u0026uacute;cha LTDA (CCGL), Universidade Federal do Rio Grande do Sul (UFRGS) and Agron\u0026ocirc;mica Laboratory of Phytosanitary Diagnosis. Yellow sticky traps were set up in June, 2023 in maize and wheat fields, including areas with volunteer maize plants, in the municipalities of Passo Fundo, Santa Rosa and Cruz Alta (all located within Rio Grande do Sul state, Brazil). The traps were sampled weekly and taken to the CCGL Entomology Laboratory for counting and morphological identification of the collected specimens. Seven samples identified as \u003cem\u003eL. maculigera\u003c/em\u003e were sent to Agron\u0026ocirc;mica Laboratory to assess infectiveness by corn stunt pathogens using molecular tools.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;1\u003c/b\u003e Yellow sticky trap used to capture corn leafhoppers.\u003c/p\u003e \u003cp\u003eA 1,20 m tall light trap was also used in the experimental maize field of Cruz Alta (Fig.\u0026nbsp;2), which was sowed on 20/08/2023 and emerged on 07/09/2023 (maize hybrid AG9021 PRO3). The lamp was kept turned on once a week during night time, beginning on 15/09/2023. Sampling was carried out weekly from 15/10/2023 to 24/02/2024, followed by counting and morphological identification of the collected specimens in the CCGL Entomology Laboratory. The experimental maize field was harvested on 24/01/2024.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;2\u003c/b\u003e Light trap used to capture corn leafhoppers.\u003c/p\u003e \u003cp\u003eInfectiveness by stunt pathogens was assessed in both trials for 11 different sampling times, at Agron\u0026ocirc;mica Laboratory, Porto Alegre (Rio Grande do Sul state) using RT-qPCR (reverse transcription quantitative real-time PCR), which is 10.000x more sensitive to corn stunt pathogens compared to detection by conventional PCR. Acid nucleic was extracted following Doyle (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) adapted. Amplification of the pathogens DNA (MRFV, MBSP and CSS) by RT-qPCR multiplex was carried out using master mix GoTaq\u0026reg; Enviro RT-qPCR System (Promega, Madison) in a QuantStudio 5 Real-Time PCR System (Applied Biosystems) thermocycler, using specific primers for the three pathogens, according to method developed by Agron\u0026ocirc;mica Laboratory (2023) in the regions: coat protein, cpn60 and spiralin lipoprotein, respectively. The resulting data were analysed using QuantStudio\u0026trade; Design \u0026amp; Analysis Software (Applied Biosystems).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe presence of \u003cem\u003eL. maculigera\u003c/em\u003e was confirmed in the municipalities of Cruz Alta, Passo Fundo and Santa Rosa from samples collected in 03/08/2023, 12/09/2023 and 01/10/2023, respectively. However, only the specimens captured in Cruz Alta were infected by corn stunt pathogens, showing simultaneous infection by MRFV and MBSP (Fig.\u0026nbsp;3). This is the first report of infection of \u003cem\u003eL. maculigera\u003c/em\u003e by both pathogens in the state of Rio Grande do Sul, diverging from the results obtained by Obura et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), who found the species incapable of acquiring and transmiting MBSP to Napier grass in Kenya.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;3\u003c/b\u003e Presence of \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e and infectiveness by corn stunt pathogens in Rio Grande do Sul state, Brazil, in the 2023/24 crop season.\u003c/p\u003e \u003cp\u003eThe confirmation of simultaneous MRFV and MBSP infection in \u003cem\u003eL. maculigera\u003c/em\u003e raises an alert for the potential threat that this novel species represents to maize production in the Rio Grande do Sul state, and in Brazil as a whole. The morphological closeness between \u003cem\u003eL. maculigera\u003c/em\u003e and the more common \u003cem\u003eD. maidis\u003c/em\u003e is also of concern, as field samples could be easily misidentified and lead to underrated population estimates. Certain morphological traits are unique to \u003cem\u003eL. maculigera\u003c/em\u003e, however, and can be used to correctly identify the species. \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e is straw colored, hyaline winged and black eyed, and is about 0,4 cm smaller than \u003cem\u003eD. maidis.\u003c/em\u003e It also displays a single dark spot in the clypeus (i.e. lower face), whereas \u003cem\u003eD. maidis\u003c/em\u003e has two characteristic dark spots in the upper face (Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eThe antennae of \u003cem\u003eL. maculigera\u003c/em\u003e are also distinct, with a visible dilated pedicel near its insertion in the insect\u0026rsquo;s head, whereas \u003cem\u003eD. maidis\u0026rsquo;\u003c/em\u003e pedicels are not dilated. \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e and other Delpachidae leafhoppers also present tibiae with movable apical spurs in the third pair of legs, which are absent in \u003cem\u003eD. maidis\u003c/em\u003e (Fig.\u0026nbsp;4). The correct identification of corn leafhoppers is the first step in establishing adequate management measures for each detected species, ensuring that infesting populations are controlled before reaching economic injury level in maize and other crops.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;4\u003c/b\u003e Morphological differences between \u003cem\u003eDalbulus maidis\u003c/em\u003e and \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e. Photos and drawings by Glauber R. St\u0026uuml;rmer\u003c/p\u003e \u003cp\u003eThe population density of Cicadellidae and Delphacidae leafhoppers was previously assessed in two Brazilian regions during three crop seasons by Oliveira et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), who found that \u003cem\u003eD. maidis\u003c/em\u003e made up 90.1% of the collected samples. The authors reported nine species of Delphacidae among the samples, three of them for the first time in Brazil, and raised concern about the possibility of these species acting as potential vectors for viruses and mollicutes in maize. In our study, the highest population of leafhoppers (653 individuals) was captured in the lamp trap between 26/11/2023 and 20/01/2024, with \u003cem\u003eD. maidis\u003c/em\u003e representing 87% of the population (569 individuals). During this period the corn plants progressed from growth stage V4 (four leaves) to R5 (dent) (Ritchie and Hanway \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Only 2.3% of the samples collected in the same period were identified as \u003cem\u003eL. maculigera\u003c/em\u003e (15 individuals), most of them captured between 14/01/2024 and 20/01/2024 (10 individuals), when maize plants were at growth stage R5 (Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e Population level of \u003cem\u003eDalbulus maidis\u003c/em\u003e and \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e during 2023/24 crop season in Cruz Alta, RS\u003c/p\u003e \u003cp\u003eAssessment of infectiveness by stunt pathogens showed positive results for 72.7% of the \u003cem\u003eD. maidis\u003c/em\u003e samples and 50.0% of the \u003cem\u003eL. maculigera\u003c/em\u003e samples. The positive \u003cem\u003eD. maidis\u003c/em\u003e samples showed 2.8% of single MBSP infection, 16.8% of single MRFV infection, 76.7% of simultaneous MRFV\u0026thinsp;+\u0026thinsp;MBSP infection and 3.7% of simultaneous MRFV\u0026thinsp;+\u0026thinsp;MBSP\u0026thinsp;+\u0026thinsp;CSS infection. As for the positive \u003cem\u003eL. maculigera\u003c/em\u003e samples, 23.1% were of single MRFV infection and 76.9% of simultaneous MRFV\u0026thinsp;+\u0026thinsp;MBSP infection (Fig.\u0026nbsp;6). Transmission efficiency of MBSP and CSS is dependent on temperature, with MBSP being more tolerant to lower temperatures (Sabato et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this experiment, triple infection (MRFV\u0026thinsp;+\u0026thinsp;MBSP\u0026thinsp;+\u0026thinsp;CSS) was only detected in the samples collected between 31/12/2023 and 06/01/24, whereas single infection by MBSP was detected since the second sampling week (22/10/2023 to 28/10/2023). Vector competence of Cicadellidae and Delphacidae leafhoppers to transmit MBSP has been documented by Obura et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;6\u003c/b\u003e Percentual amount of \u003cem\u003eDalbulus maidis\u003c/em\u003e and \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e samples detected with each corn stunt infection (MRFV, MBSP and CSS) during 2023/24 crop season, in Cruz Alta, RS\u003c/p\u003e \u003cp\u003eThe Delphacidae family includes several leafhoppers of economic importance, which can transmit up to 25 different viruses to maize and other grasses, such as sugarcane (\u003cem\u003eSaccharum officinarum\u003c/em\u003e), millet (\u003cem\u003ePanicum miliaceum\u003c/em\u003e), sorghum (\u003cem\u003eSorghum bicolor\u003c/em\u003e), rice (\u003cem\u003eOryza sativa\u003c/em\u003e), wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e), oat (\u003cem\u003eAvena sativa\u003c/em\u003e) and Bermuda grass (\u003cem\u003eCynodon dactylon\u003c/em\u003e) (Wilson \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The species \u003cem\u003eDelphacodes kuscheli\u003c/em\u003e, for instance, is the main vector of MRCV (Mal de R\u0026iacute;o Cuarto virus) in Argentina, causing important losses in maize crops (Ornaghi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Congeners of \u003cem\u003eL. maculigera\u003c/em\u003e include the species \u003cem\u003eLeptodelphax dymas\u003c/em\u003e, reported in Africa as a vector of Napier grass stunt (NGS), a highly agressive phytoplasm belonging to the genus \u003cem\u003eCandidatus\u003c/em\u003e (Obura et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The wide range of hosts used by Delphachidae leafhoppers suggest that \u003cem\u003eL. maculigera\u003c/em\u003e could be feeding and reproducing on other plants besides maize in Brazil, increasing the risk of population outbreaks and making the associated disease very hard to prevent.\u003c/p\u003e \u003cp\u003eThe confirmation of infectiveness by stunt pathogens in \u003cem\u003eL. maculigera\u003c/em\u003e will aid in the outlining of managment strategies for this novel insect vector, and should be continued alongside the \u003cem\u003eD. maidis\u003c/em\u003e\u0026rsquo; population surveys already conducted by the Cooperative Technical Network in Rio Grande do Sul. Further studies should explore the population patterns of \u003cem\u003eL. maculigera\u003c/em\u003e in different maize producing regions of Brazil and detail its capacity to acquire and transmit stunt pathogens, as well as its potential adaptiveness to alternate plant hosts. Correct identification of specimens and thorough monitoring of field populations are recommended in order to attest \u003cem\u003eL. maculigera\u0026rsquo;\u003c/em\u003es adaptiveness to Brazilian maize growing conditions and achieve an efficient, integrated management program for both leafhopper species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cbr /\u003e The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.R.S. and C.W.G. conceived this research, designed the experiments and collected field data. C.C.L.A., T.M., Y.C. and I.F. carried out molecular analyses and procedures. H.P. assisted with writing and data discussion. All authors read and commented on the information provided in the manuscript and approved the final draft.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBortolotto OC, Pazini JB, Molina RO, Garcia MH, Andrade CCL, Mituti T (2023) V\u0026iacute;rus da-risca tem novo transmissor. Cultivar 292:30\u0026ndash;35\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanale MC, Andrade MVS, Castilhos RV (2023) Cigarrinha-africana \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e Stal, 1859 (Hemiptera: Delphacidae) em Santa Catarina. 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J Econ Entomol 115:1817\u0026ndash;1833\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitchie SW, Hanway JJ (1989) How a corn plant develops. Iowa State University, Ames, USA. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://publications.iowa.gov/18027/1/How%20a%20corn%20plant%20develops001.pdf\u003c/span\u003e\u003cspan address=\"https://publications.iowa.gov/18027/1/How%20a%20corn%20plant%20develops001.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 07 May 2024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSabato O, Landau EC, Barros BA, Oliveira CM (2020) Differential transmission of phytoplasma and spiroplasma to maize caused by variation in the environmental temperature in Brazil. Eur J Plant Pathol 157:163\u0026ndash;171\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson SW (2005) Keys to the families of Fulgoromorpha with emphasis on planthoppers of potential economic importance in the southeastern United States (Hemiptera: Auchenorrhyncha). Fla Entomol 88:464\u0026ndash;481\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"corn stunt disease, phytoplasma, spiroplasma, Zea mays","lastPublishedDoi":"10.21203/rs.3.rs-4455424/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4455424/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe corn stunt disease is a major threat to maize production in Brazil. In addition to the more common insect vector \u003cem\u003eDalbulus maidis\u003c/em\u003e (Hemiptera: Cicadellidae), the African species \u003cem\u003eLeptodelphax maculigera\u003c/em\u003e (Hemiptera: Delphacidae) was recently found in the Brazilian states of Goi\u0026aacute;s, Paran\u0026aacute; and Santa Catarina, raising concern regarding its potential ability to transmit stunt pathogens. We confirmed the presence of \u003cem\u003eL. maculigera\u003c/em\u003e in the state of Rio Grande do Sul by conducting a population survey with yellow sticky traps and light traps in three different sites, although it corresponded to only 2.3% of the \u003cem\u003eD. maidis\u003c/em\u003e population captured during the same period. Molecular analysis via RT-qPCR multiplex confirmed the simultaneous infection of \u003cem\u003eL. maculigera\u003c/em\u003e by two corn stunt pathogens \u0026ndash; Maize rayado fino virus (MRFV) and Maize bushy stunt phytoplasma (MBSP) \u0026ndash; in 76.9% of the samples, while the remaining 23.1% showed single infection by MRFV. The confirmation of infectiveness by two stunt pathogens in \u003cem\u003eL. maculigera\u003c/em\u003e will aid in the outlining of managment strategies for this insect vector.\u003c/p\u003e","manuscriptTitle":"Simultaneous infection of Leptodelphax maculigera (Hemiptera: Delphacidae) by Maize rayado fino virus (MRFV) and Maize bushy stunt phytoplasma (MPSP) detected in Rio Grande do Sul, Brazil.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-06 07:24:39","doi":"10.21203/rs.3.rs-4455424/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-07-02T05:13:56+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-05-31T05:29:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-24T13:33:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-23T07:20:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2024-05-21T10:04:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c61c5c96-5fef-4de1-8bcc-7e0f1b8bf4ae","owner":[],"postedDate":"June 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:04:46+00:00","versionOfRecord":{"articleIdentity":"rs-4455424","link":"https://doi.org/10.1007/s41348-024-00988-w","journal":{"identity":"journal-of-plant-diseases-and-protection","isVorOnly":false,"title":"Journal of Plant Diseases and Protection"},"publishedOn":"2024-08-20 15:57:47","publishedOnDateReadable":"August 20th, 2024"},"versionCreatedAt":"2024-06-06 07:24:39","video":"","vorDoi":"10.1007/s41348-024-00988-w","vorDoiUrl":"https://doi.org/10.1007/s41348-024-00988-w","workflowStages":[]},"version":"v1","identity":"rs-4455424","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4455424","identity":"rs-4455424","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}