Development of efficient RNAi methods in the corn leafhopper Dalbulus maidis, a promising application for pest control.

BACKGROUND: The corn leafhopper Dalbulus maidis is the main vector of important stunting pathogens that affect maize production. Currently, there are no effective methods available to manage this pest without adverse impact on the environment. In this context, genomic-based technologies such as RNA interference (RNAi) provide a more environmentally friendly pest control strategy. Therefore, we aimed to assess the application of RNAi in D. maidis and determine the function of a candidate gene related to insect reproduction and propagation. RESULTS: We have characterized the core RNAi genes and evaluated the functionality of the RNAi machinery. We assessed the potential of RNAi technology in D. maidis via injection or ingestion of double-stranded RNA (dsRNA) to adult females. We chose Bicaudal C (BicC) as a target gene due to its important role during insect oogenesis. Administration of dsRNABicC caused significant reductions in the transcript levels (fold changes up to 170 times) and ovipositions. Phenotypic analysis of the ovaries revealed alterations in oocyte development, providing additional confirmation for our results and supporting the idea that Dmai-BicC is a key player of D. maidis oogenesis. CONCLUSION: This is, to our knowledge, the first report of efficient RNAi in D. maidis. We believe our findings provide a starting point for future control strategies against one of the most important maize pests in the Americas. © 2022 Society of Chemical Industry.

Eight Decades of Dalbulus maidis (DeLong & Wolcott) (Hemiptera, Cicadellidae) in Brazil: What We Know and What We Need to Know.

The corn leafhopper Dalbulus maidis (DeLong & Wolcott) is one of the most important maize (Zea mays L.) pests in Latin America because of its ability to efficiently transmit pathogens [maize bushy stunt phytoplasma (MBSP) and corn stunt spiroplasma-Spiroplasma kunkelli Whitcomb et al. (CSS)] associated with corn stunt disease complex and maize rayado fino virus (MRFV). This leafhopper species, considered a secondary pest until a few years ago, was first reported in Brazil in 1938. Since 2015, corn stunt diseases have been the main phytosanitary threat to corn production in Brazil, and D. maidis has assumed the status of a key pest of the crop. In this study, we gathered pertinent information about the corn leafhopper, from the time it was first recorded in Brazil. Aspects such as origin, association with maize, bioecology, geographical distribution in the Americas, and its congeners are addressed. We present a history of studies performed with this species in the country, its importance as a pest, host plants, and survival strategies during the maize off-season. Based on the available scientific knowledge, the main management strategies for insect vectors and diseases are discussed. Finally, the main knowledge gaps for this insect vector and the prospects for future studies and actions to mitigate the damage caused by insect vectors in maize crops in Brazil are presented and discussed.

Maize Bushy Stunt Phytoplasma Favors Its Spread by Changing Host Preference of the Insect Vector.

Plant pathogenic bacteria may influence vector behavior by inducing physiological changes in host plants, with implications for their spread. Here, we studied the effects of maize bushy stunt phytoplasma (MBSP) on the host selection behavior of the leafhopper vector, Dalbulus maidis (DeLong and Wolcott). Choice assays contrasting leaves of healthy (mock-inoculated) vs. infected maize (Zea mays L.) were conducted during the asymptomatic and symptomatic phases of plant infection, with leafhopper males or females previously exposed to infected plants (bacteriliferous insects) or not. In each assay, 40 adults were released in choice arenas where only the leaves of two plants from each treatment were offered and visible, and the insects landed on the leaves were counted 1, 2, 3, 5, 7, 9, 11 and 23 h after release. During the asymptomatic phase of plant infection, an effect was observed only on bacteriliferous females, who preferred leaves of healthy plants 5 h after release or later. The symptomatic phase triggered a pull-push effect on non-bacteriliferous females, who were first attracted to symptomatic leaves but hours later moved to healthy leaves. Non-bacteriliferous males initially preferred symptomatic leaves (up to 5 h after release) and later became equally distributed between treatments. Bacteriliferous males and females initially did not discriminate between healthy and symptomatic leaves, but only the females tended to move to healthy leaves 9 h after release. Oviposition was drastically reduced on symptomatic leaves. The changes in vector behavior induced by MBSP favor its primary spread, since bacteriliferous females prefer healthy leaves at early (asymptomatic) stages of the crop. At later stages, secondary spread may be favored because non-bacteriliferous females are initially attracted to infected (symptomatic) leaves, allowing pathogen acquisition and subsequent transmission as they move to healthy plants.

Modern Maize Hybrids Have Lost Volatile Bottom-Up and Top-Down Control of Dalbulus maidis, a Specialist Herbivore.

Following damage by herbivores, many plants release volatiles that dissuade future conspecifics from feeding. In many crop plants however, induced volatiles mediating this kind of interactions among plants, herbivores and also their natural enemies have been altered through the process of domestication. The selection of crops for increased yield may have gone at a cost of defense, possibly including defense-related volatiles. Dalbulus maidis (Hemiptera: Cicadellidae), a specialist leafhopper that only feeds on Zea spp., is a vector of Corn Stunt Spiroplasma, a serious maize disease. Here, we compared the volatiles released following D. maidis attack by a maize landrace and two maize hybrids of temperate and tropical background. Also, we performed behavioral assays with the leafhopper contrasting healthy non-attacked maize seedlings versus attacked seedlings. The maize landrace produced more than 6-fold larger quantities of induced volatiles compared to the maize hybrids after herbivory. Corn leafhopper females were able to detect and significantly preferred the odors of healthy seedlings over the attacked ones only in the landrace. They did not discriminate between the attacked and non-attacked hybrids. Additionally, we found that the attraction of the parasitoid wasp Anagrus virlai (Hymenoptera: Mymaridae) to its host was diminished in the tested hybrids. The parasitoid was able to detect the odors of the attacked landrace, however it was unable to discriminate between healthy and attacked maize hybrid plants. These results suggest that those more domesticated germplasms may have lost the ability not only to release volatiles that avoid colonization of future herbivores, but also to attract their natural enemies in a tritrophic system.

Bacteriomes of the corn leafhopper, Dalbulus maidis (DeLong & Wolcott, 1923) (Insecta, Hemiptera, Cicadellidae: Deltocephalinae) harbor Sulcia symbiont: molecular characterization, ultrastructure, and transovarial transmission.

In this study, we surveyed the bacteriome-associated microbiota of the corn leafhopper Dalbulus maidis by means of histological, ultrastructural, and molecular analyses. Amplification and sequencing of 16S rDNA genes revealed that the endosymbiont "Candidatus Sulcia muelleri" (Phylum Bacteroidetes) resides in bacteriomes of D. maidis. Phylogenetic analysis showed that the sequence was closely allied to others found in representatives of the subfamily Deltocephalinae. We failed to amplify other sequences as "Candidatus Nasuia deltocephalinicola," a co-primary symbiont frequently associated to deltocephaline leafhoppers. In addition, a metagenetic analysis carried out in order to investigate the presence of other bacteriome-associated bacteria of D. maidis showed that the sequence of Sulcia accounted for 98.56 % of all the sequences. Histological and ultrastructural observations showed that microorganisms harbored in bacteriomes (central syncytium and cytoplasm of uninucleate bacteriocytes) look like others Sulcia described in hemipteran species and they were transovarially transmitted from mother to offspring which is typical of obligate endosymbionts. The only presence of Sulcia in the bacteriomes of D. maidis was discussed.

Molecular confirmation of Maize rayado fino virus as the Brazilian corn streak virus

Maize rayado fino virus (MRFV), present in various countries in Latin America, has shown similarities to corn streak virus that occurs in Brazil, regarding pathogenic, serological and histological characteristics. In the current report both virus were molecularly compared to confirm the similarities between them. MRFV was identified by nucleic acid hybridization in samples of maize tissues exhibiting symptoms of "corn stunt" disease, collected from two Brazilian States - São Paulo and Minas Gerais. The coat protein gene and 3'non-translated region of MRFV were amplified from infected tissues by reverse transcription-polymerase chain reaction (RT-PCR) using MRFV-specific primers, and were characterized by nucleotide sequence and phylogenetic analysis of the cloned PCR products. Phylogenetic analysis of the relationships between the Brazilian isolates and isolates obtained from Latin America and the United States reveals a close relationship to isolates from Brazil, Peru and Bolivia. Results support the proposal that the Brazilian corn streak virus be regarded as an isolate of MRFV and provide evidence for the presence of MRFV in "corn stunt' disease in Brazil.

O vírus do rayado fino do milho (MRFV), presente em vários países da América Latina, tem mostrado semelhança ao vírus da risca do milho que ocorre no Brasil, em relação a características patogênicas, sorológicas e histológicas. No presente trabalho, ambos os vírus foram comparados molecularmente, visando confirmar a similaridade entre os mesmos. O MRFV foi identificado por hibridização de ácido nucléico em amostras de tecido que apresentavam sintomas de enfezamento, coletadas nos estados de São Paulo e Minas Gerais. O gene da capa protéica e a região 3' não traduzida do MRFV foram amplificados, a partir de tecidos infectados, através da transcrição reversa por reação em cadeia da polimerase (RT-PCR), usando-se os iniciadores específicos para o vírus, obtendo-se a seqüência nucleotídica e a análise filogenética dos produtos de PCR clonados. A análise filogenética relacionando isolados brasileiros, da América Latina e dos Estados Unidos revela a estreita relação entre isolados do Brasil, do Peru e da Bolívia. Os resultados demonstraram que o vírus da risca do milho, presente no Brasil, pode ser considerado como um isolado do MRFV e também evidenciaram a presença do MRFV associado à doença conhecida como enfezamento do milho que ocorre no Brasil.

Molecular confirmation of Maize rayado fino virus as the Brazilian corn streak virus

Maize rayado fino virus (MRFV), present in various countries in Latin America, has shown similarities to corn streak virus that occurs in Brazil, regarding pathogenic, serological and histological characteristics. In the current report both virus were molecularly compared to confirm the similarities between them. MRFV was identified by nucleic acid hybridization in samples of maize tissues exhibiting symptoms of "corn stunt" disease, collected from two Brazilian States - São Paulo and Minas Gerais. The coat protein gene and 3'non-translated region of MRFV were amplified from infected tissues by reverse transcription-polymerase chain reaction (RT-PCR) using MRFV-specific primers, and were characterized by nucleotide sequence and phylogenetic analysis of the cloned PCR products. Phylogenetic analysis of the relationships between the Brazilian isolates and isolates obtained from Latin America and the United States reveals a close relationship to isolates from Brazil, Peru and Bolivia. Results support the proposal that the Brazilian corn streak virus be regarded as an isolate of MRFV and provide evidence for the presence of MRFV in "corn stunt' disease in Brazil.

O vírus do rayado fino do milho (MRFV), presente em vários países da América Latina, tem mostrado semelhança ao vírus da risca do milho que ocorre no Brasil, em relação a características patogênicas, sorológicas e histológicas. No presente trabalho, ambos os vírus foram comparados molecularmente, visando confirmar a similaridade entre os mesmos. O MRFV foi identificado por hibridização de ácido nucléico em amostras de tecido que apresentavam sintomas de enfezamento, coletadas nos estados de São Paulo e Minas Gerais. O gene da capa protéica e a região 3' não traduzida do MRFV foram amplificados, a partir de tecidos infectados, através da transcrição reversa por reação em cadeia da polimerase (RT-PCR), usando-se os iniciadores específicos para o vírus, obtendo-se a seqüência nucleotídica e a análise filogenética dos produtos de PCR clonados. A análise filogenética relacionando isolados brasileiros, da América Latina e dos Estados Unidos revela a estreita relação entre isolados do Brasil, do Peru e da Bolívia. Os resultados demonstraram que o vírus da risca do milho, presente no Brasil, pode ser considerado como um isolado do MRFV e também evidenciaram a presença do MRFV associado à doença conhecida como enfezamento do milho que ocorre no Brasil.