Arthropod-Plant Interactions (2020) 14:811–823 https://doi.org/10.1007/s11829-020-09783-4

ORIGINAL PAPER

Host plant selection and transmission by maidis are conditioned by infection in bicolor

Peter Klein1 · C. Michael Smith1

Received: 7 January 2020 / Accepted: 12 September 2020 / Published online: 26 September 2020 © The Author(s) 2020

Abstract Many plant are signifcant pathogens that are able to utilize vectors to infect a vast range of host plants, resulting in serious economic damage to world food . One such is Sorghum bicolor, grain sorghum, which is the ffth most important global cereal crop, it is grown for human consumption, feed, and biofuel. In this study, the Poty- viruses Johnsongrass (JGMV), dwarf mosaic virus (MDMV), mosaic virus (SCMV), and (SRMV) were tested for their rates of transmission into tissues of S. bicolor by the corn leaf , . In addition, virus infected and non-infected S. bicolor plants were assessed for their efects on R. maidis host plant selection behavior. Further, the propagation of each virus (viral ssRNA copy number) in infected plants was determined using qPCR amplifcation of viral coating protein gene fragments. The mean rate of JGMV transmission into S. bicolor plants by R. maidis was signifcantly lower than transmission of MDMV, SCMV, and/or SRMV. Sorghum bicolor plants infected with MDMV, SCMV or SRMV also attract signifcantly more R. maidis than non-infected plants. JGMV-infected plants do not efect a similar change in R. maidis plant choice preference. The preference of non-viruliferous R. maidis toward S. bicolor plants infected with MDMV, SCMV or SRMV, and lack of such attraction by JGMV-infected plants may play a role in virus transmission strategy and efciency by the vector.

Keywords Rhopalosiphum maidis · Potyvirus · Sorghum bicolor · Johnsongrass mosaic virus (JGMV) · (MDMV) · (SCMV) · Sorghum mosaic virus · Two-choice test · Volatile attraction

Introduction agriculture are estimated at $4.49 billion annually in the U.S. alone (Martin et al. 2015). Aphid feeding on phloem The earliest fossilized evidence of stylet feeding arthro- sap reduces chlorophyll and carotenoid, resulting in sig- pods was produced roughly 300 million years ago during nifcantly reduced plant biomass (Riedell and Kieckhefer the Carboniferous period (Labandeira 2013). In the ensu- 1995; Diaz-montano et al. 2007; Macedo et al. 2009; Ni and ing millenia, intense plant–arthropod interactions have Quisenberry 2006). resulted in the evolution of more than 4000 species of Allelochemical and biophysical traits introgressed into phloem-feeding () (Jaouannet et al. 2014), many crop plants have reduced aphid damage (Smith and consisting of ~ 100 economically relevant species (Adams Chuang 2014). Nonetheless, aphid-related yield losses et al. 2005) present on the vast majority of the global land- in U.S. grain sorghum continue to range between 10 and mass (Macfadyen and Kriticos 2012). Due to their global 50% in infested felds (Bowling et al. 2016). The ability of distribution on ~ 25% of all plants and immense reproductive aphids to transmit at least 275 plant viruses further magni- rates (Dedryver et al. 2010), aphid-related losses to global fes their detrimental efects on U.S. Agricultural Produc- tivity (1997). Although virus transmission strategies are highly diverse (Dietzgen et al. 2016). Around 75% of all Handling Editor: Yulin Gao and Heikki Hokkanen. known plant viruses are transmitted via a non-persistent * Peter Klein mode of transmission. In this process the virus acquired by [email protected] feeding on an infected host plant retains in the stylet of the vector without entering other tissues or propagation. The 1 Department of Entomology, Kansas State University, 124 W. vector remains viruliferous temporarily without horizontal Waters Hall, Manhattan, KS, USA

Vol.:(0123456789)1 3 812 P. Klein, C. M. Smith or vertical virus transmission (Powell 2005). Among these aphid vector (Aphis gossypii) towards exposed plant leaves viruses, the aphid-vectored , is the largest plant infected with the virus in two-choice experiments (Sal- virus family, harboring at least 187 members (Adams et al. vaudon et al. 2013). Those results support the hypothesis 2005). have on average a 9.7 kb positive-sense that the altered volatile composition of virus-infected host single-stranded RNA genome encoding 10 mature proteins plants is able to manipulates the behavior of aphid vectors in a single large open-reading frame (Delmas et al. 2019). (Bosque-Perez and Eigenbrode 2011). Molecular studies demonstrated the involvement of these Grain sorghum, Sorghum bicolor, is the ffth most impor- proteins in virus transmission, membrane targeting (includ- tant cereal crop in the world and is grown in more than 100 ing virus movement within plants), viral RNA replication, countries to form the basic food staple for more than 500 and virion assembly (Peng et al. 1998; Cronin et al. 1995; million people (Luo et al. 2016a). Nevertheless, relatively Ivanov et al. 2014; Gallo et al. 2018). little is known about the dispersion dynamics and transmis- Sugarcane mosaic virus (SCMV), Sorghum mosaic virus sion efciencies of aphids transmitting JGMV, MDMV, (SRMV), Maize dwarf mosaic virus (MDMV) and Johnson- SCMV, and SRMV. The goal of this study was to determine grass mosaic virus (JGMV) are members of Potyviridae. All the efciency of R. maidis in transmitting each of the four are closely related and arranged in the same phylogenetic viruses, the efect of S. bicolor virus infection on R. maidis clade based on genomic RNA sequence identity, with the host plant choice, and viral RNA propagation in S. bicolor. exception of JGMV, which is localized in a neighboring Our study resulted in a clear attraction of R. maidis towards branch (Berger et al. 1997; Ward et al. 1992). However, addi- MDMV, SCMV and SRMV. However, no signifcant attrac- tional phylogenetic analysis on MDMV, SRMV, and SCMV tion towards JGMV infected sorghum plants was detected. isolates demonstrated a further segregation of these viruses Similarly, high transmission rates were observed for MDMV, into two groups. On the one hand, a closer related MDMV SCMV, and SRMV into sorghum plants accompanied by and SRMV emerged clustering the SCMV virus in a separate higher absolute viral CP gene accumulation in sorghum clade (Moradi et al. 2017a). These viruses have an overlap- plants infected with MDMV, SCMV, and SRMV. ping range of host plants that includes sorghum, sugarcane, maize, and Johnsongrass (Seifers et al. 2000; Kannan et al. 2018; Zhang et al. 2016). Plants infected by any of the four viruses develop yellowing leaves with mosaic-like infec- Material and methods tion patterns (Kannan et al. 2018; Xia et al. 2016; (Grisham and Pan 2007). Potyvirus infections are considered to be Maintenance and extraction of Potyviruses the most devastating viral diseases of sugarcane, sorghum, and maize (Moradi et al. 2017b). In addition, maize plants Sugarcane mosaic virus (SCMV), Sorghum mosaic virus infected with MDMV and SCMV have been shown to sufer (SRMV), Maize dwarf mosaic virus (MDMV) and John- reductions in height, weight, and cob weight of 16%, 37%, songrass mosaic virus (JGMV) Potyviruses were obtained and 27% respectively (1995). in dried and frozen plant tissues from the Kansas State Uni- Recent studies have shown that plant volatile factors play versity Agricultural Research Center in Hays, KS, USA. The an important role in vector-mediated virus dispersal under SRMV isolate was from an unknown location in Kansas. natural conditions (Dader et al. 2017). plants infected MDMV, JGMV and SCMV were isolated from tissue of with Rice Ragged Stunt Virus not only upregulate the plants collected near Hays, KS. Crude plant extracts of each expression of genes involved in defense response, but also virus were obtained by homogenizing infected plant material upregulate those genes involved in volatile-biosynthesis, in Phosphate bufered saline bufer (PBS: 1.54 mM NaCl, resulting in plants with increased attraction to vectors, which 5.6 mM ­Na2HPO4, 1.1 mM ­KH2PO4, pH 7.4) and centri- exponentially promotes their spread of Rice Ragged Stunt fuging in 1.5 ml reaction tubes for 10 min at 13,000 rpm in Virus (Lu et al. 2016). Similar results were obtained from a Thermo Scientifc/Legend Micro 21R centrifuge (Fisher studies of , a member of the Bro- Scientifc, Pittsburgh, PA, USA). To generate infected plants moviridae virus family, which induce a clear temporal pref- material for R. maidis virus acquisition the clear superna- erence of vectors to volatiles emitted from infected plants tants containing plant crude extracts, including the indi- (Mauck et al. 2010). Other vectors such as the bird-cherry vidual viruses, were used to brush-inoculate healthy 21- to aphid, , and the green peach aphid, 28 d-old S. bicolor seedlings in the two- to three-leaf stage, Myzus persicae, exhibit preferential responses to luteovirus- each planted in a separate pot. To ensure infection, virus infected and potato plants compared to uninfected applications were performed twice on 2 consecutive days. plants. Watermelon mosaic virus (WMV) and zucchini yel- Infected plants were kept in 24 °C day:20 °C night and a low mosaic virus (ZYMV), both Potyviruses, infected plants 14:10 [L:D] h photoperiod illuminated by 32 W fuorescent have been demonstrated to induce the immigration of it’s light source.

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Immunological Potyvirus identifcation h photoperiod. Each of the three replicates consisted of 10 two- or three-leaf stage plants being infected with each virus To identify and verify S. bicotor plants infections with by transferring 5 viruliferous adult R. maidis and allowed MDMV, JGMV, SCMV or SCMV ELISA assays were to feed for 1 h subsequently infecting the plants with one of performed 21 d-post-inoculation. In total, 0.05 g leaf tis- the tested Potyviruses (JGMV, MDMV, SCMV, and SrMV). sue from each plant tested plant was homogenized in 200 µl After the feeding period R. maidis were removed manually. 0.02 M PBST bufer and tested for the presence of each virus To avoid plant resistance efects induced by aphid feeding with Potyvirus ELISA kits specifc for each virus (Agdia on the host plant during the choice assay mock-inoculated Inc., Elkhart, IN, USA). All healthy S. bicolor plants were control plants treated with 5 adult non-viruliferous R. maidis also confrmed negative by ELISA assay. For storage pur- for 1 h were simultaneously generated. Infected and mock- poses, tissue from positively tested plants was homogenized innoculated plants were incubated in separately for 21 d at in 5 ml PBS 10% glycerol bufer, centrifuged at 4000 rpm, 24 °C:20 °C in a 14 h:10 h light/dark photoperiod illumi- and the supernatant stored for later use at − 80 °C. nated by 32 W fuorescent light source. A two-choice bioassay was conducted with 21 d post- Rhopalosiphum maidis virus transmission efciency inoculation plants after confrming healthy and infected plants by ELISA as described above. A 15 cm diameter To initiate virus acquisition, approximately 50 adult R. plastic petri dish with two 1.5 cm diameter glass tubes on maidis obtained from virus-free S. bicolor plants were opposite sites served as an arena to assess R. maidis food starved for 1 h, placed on leaves of S. bicolor infected with source preference. Infected and mock-inoculated control one of the tested Potyviruses (JGMV, MDMV, SCMV, and plant leaves were inserted into opposite glass tubes facing SrMV) and allowed to feed for 30 min, to acquire the virus. each other. The outer end of each tube was plugged with For each virus tested, a single viruliferous R. maidis was cotton to prevent R. maidis escape (Fig. 1). For each pref- then transferred to 30 non-infected two leaf-stage S. bicolor erence assay 20 adult and non-viruliferous R. maidis were plants using a wet fne brush under a 20 × Stereoscope. starved for 1 h in a 10 cm Petri dish. Starved aphids were These aphids were removed manually from exposed plants placed by a fne brush in the middle of each arena ofering R. at 1 h post-transfer and all plants were incubated for 21 d maidis a choice either between a healthy Sorghum plant or at 24 °C day:20 °C night and a 14:10 [L:D] h photoperiod a Sorghum plant infected with one of the four tested Potyvi- illuminated by 32 W fuorescent light source. ruses. A constant 32 W fuorescent light source maintained To identify the infection rate a crude cell extract sample a stable illumination across all choice test experiments obtained from 0.05 g tissue of each plant was subjected to eliminating a variance in visual cues for R. maidis across ELISA assay to determine virus transmission rates for each all experiments. R. maidis were allowed to move across the tested Potyvirus according to manufacturer’s instructions entire arena freely and probe on ofered healthy and virus- (Agdia Inc., Elkhart, IN, USA). Each of the four ELISA infected Sorghum plant. After 2 h unrestricted movement assays included a negative control obtained from healthy S. the number of individual R. maidis located on healthy and bicolor plant tissue extract and a commercial positive con- virus infected Sorghum plant leaf was counted. The fnal R. trol. The hydrolysis of the substrate p-nitrophenyl phosphate maidis counts for settling preference after 2 h was obtained by alkaline phosphatase is catalyzed in the presence of Poty- virus particles turning the testing solution yellowish that can be recorded at 405 nm with a Vmax Kinetic microplate reader (Molecular Devices, San Francisco, CA, USA) after 2 h incubation in darkness at room temperature. Absorption values of n = 30 independent transmission experiment for each tested Potyvirus were subjected to one-way ANOVA (GraphPad Prism V. 8.20) to determine diferences in trans- mission efciency of each virus.

Efect of virus infection on R. maidis plant preference Fig. 1 Schematic representation of two-choice bioassay system. For each virus tested, 30 S. bicolor plants, each in a separate Leaves of mock-inoculated (a) and potyvirus infected (b) S. bicolor 10 cm diam × 9 cm high pot containing Metro-Mix® 360 plants were inserted into glass tubes on the opposite sites of the arena (c). The arena rests on a cardboard platform (d). For each bioassay 20 soil mix (Sun Gro Horticulture Inc, Pine Bluf, AR, USA), adult R. maidis starved for 1 h were placed in the middle of the arena were grown under a 24 °C day:20 °C night and a 14:10 [L:D] and their location recorded after unrestricted movement for 2 h

1 3 814 P. Klein, C. M. Smith from 30 independent choice tests. To identify statistically phylogenetic clade were extracted, realigned, and the primer signifcant diferences in R. maidis settling behavior between binding sites were determined as previously described for healthy and infected plants the one-way ANOVA (GraphPad the most conserved gene coding region. The designed degen- Prism V. 8.20) test was performed. erated JGMV primer set (5′ primer AAGAAR​ GAR​ TAY​ GAY​ ​ GTT​RATGA, 3′ primer: TAY​GCT​TCWGCKGCR​TCA​ Viral RNA extraction and cDNA synthesis CTRAA), spans a genomic region of 218 bp, whereas the common degenerated primer pair for MDMV, SCMV, and The abundance of Potyvirus RNA representing the virion SRMV (5′ primer: CAY​TTY​AGT​GAT​GCA​GCT​GAAGC, particle quantity in plant tissue was quantifed with reverse 3′ primer: TAY​GCT​TCWGCKGCR​TCA​CTRAA) covers a transcription-quantitative PCR (RT-qPCR). For each virus distance of 242 bp (Y = C or T, R = A or G, W = A or T, 10 randomly chosen S. bicolor plants infected with one of K = G or T). the four tested viruses were used. To obtain total RNA from each plant a Qiagen RNeasy Mini Kit was used following Reverse transcription‑quantitative PCR manufacturer’s instructions (Qiagen Inc., Germantown, MD, and standard curve generation USA). Total RNA was extracted from 0.3 g tissue from each plant. Reverse transcription reactions were performed with The RT-qPCR analysis was carried out using the CFX 3 µg total RNA as template using the SuperScript III First- Connect Real-Time System and reagents from BIO-RAD, Strand Synthesis System (Fisher Scientifc, Pittsburgh, PA, Hercules, CA, USA. Each 20-µl reaction contained 10 µl USA) and 0.1 µM random hexamers (IDT), according to iTaq Universal SYBR Green Supermix, 1 µl forward and manufacturer’s instructions. The obtained complementary reverse primer, 1 µl cDNA template and 7 µl ­H2O. To gener- DNA (cDNA) was diluted 1:2, starting from 64-fold dilu- ate standard curves for each viral CP gene, PCR-amplifed tion, for further processing. coat protein (CP) templates were generated by PCR. The qPCR cycling program was run with the following settings: Primer design for reverse transcription‑quantitative 3 min initial denaturation at 95 °C and 40 cycles alternating PCR between 30 s at 95 °C, 35 s at 51 °C and 50 s at 72 °C. The amplicons were purifed using the Wizard SV Gel and PCR Primers suitable to detect MDMV, SCMV, SRMV, and Clean-Up System (Promega) and the DNA concentration for JGMV cDNA were generated from full and partially avail- each sample was determined by NanoDrop 2000C (Thermo able viral genomes from NCBI (Table 1). The coat protein Scientifc) and the DNA copy number calculated as follow- (CP) coding regions of each virus were extracted and phy- ing: DNA copy number = (DNA ng 6.022 × 1023)/(length logenetic analyses were performed utilizing MEGA inte- 1 × 109 650) (Liu et al. 2012). Each sample was diluted grated MUSCLE alignment (Edgar 2004)] and Neighbour- in twofold linear fashion. The standard curves for JGMV, Joining Tree phylogenetic software (Saitou and Nei 1987)] MDMV, SCMV, and SRMV CP gene were generated with with 500 Bootstrap replications (data not shown). The 21 1 µl CP gene fragment dilutions as template starting from clustering MDMV, SCMV, and SRMV CP coding regions 1/128 to 1/4096. For each virus and dilution, resulted Cq were realigned and the most highly conserved regions were values were plotted against the log template copy number. selected as common primer binding sites for those viruses. The efciency of the amplifcation reactions was determined All 9 JGMV CP coding regions clustering in a separate by the linear regression slope of the linear equation and the

Table 1 Potyvirus accessions Sugarcane mosaic virus Maize dwarf mosaic Sorghum mosaic virus Johnsongrass used for coat protein coding virus mosaic virus region extraction and comparative analysis to generate AY042184 AJ001691 AJ310198 KY952243 suitable qPCR primer sets AY569692 NC_003377 AJ310196 KY952242 AF494510 AM110758 SMU07219 KY952241 AY149118 FM883211 KY659307 EU091075 JQ403608 KU746868 GU474635 JQ403609 AY387826 NC_003398 JX185302 KR911666 AM110759 JQ280313 JMU07218 AJ278405 Z26920 AJ297628

1 3 Host plant selection and virus transmission by Rhopalosiphum maidis are conditioned by… 815

R2 15 regression coefcient ( ) for each viral CP gene fragment. infected c c For CP gene copy number assessment, 10 cDNA samples per c mock-innoculated tested virus were obtained from infected plants and analyzed R. maidis a ab 10 b for the abundance of viral coat protein cDNA. The reac- b b tions were loaded into Hard-Shell PCR Plates (BIO-RAD, catalog# HSP9645) and sealed with Microseal ‘B’ (BIO- 5 RAD catalog # MSB1001). The qPCR cycling program was run with the following settings: 3 min initial denaturation at

Mean SE± number of 0 95 °C and 40 cycles alternating between 30 s at 95 °C, 35 s JGMV MDMV - SCMV SRMV at 51 °C and 50 s at 72 °C. Melt curves were generated on Potyvirus infecting S. bicolor plant PCR products from 65 to 95 °C in 0.5 °C and 5 s increments. The number of initial template CP copies for each virus was Fig. 2 R. maidis S. bicolor calculated from previously obtained standard curves. The Mean ± SE number of on leaves of plants C infected or mock-inoculated with Potyviruses Johnsongrass mosaic q values for each analyzed plant were collected. To iden- virus (JGMV), Maize dwarf mosaic virus (MDMV), Sugarcane tify whether there is a signifcant diferences in CP gene mosaic virus (SCMV), and Sorghum mosaic virus (SRMV) at 2 h abundance across plants infected with diferent Potyviruses after placement in a two-choice bioassay chamber. n = 30 infected and (JGMV, MDMV, SCMV, and SrMV) the one-way ANOVA 30 mock-inoculated pairs of plants for each virus. Means that do not share a letter difer signifcantly (P < 0.05) using Tukey test (GraphPad Prism V. 8.1.0) test was performed. The actual signifcant diferences in viral CP RNA abundance in plants infected with diferent viruses was determined by using number of R. maidis settling on mock-inoculated plants Tukey test (Lee and Lee 2018)] with P values < 0.05. across all conducted choice experiments. However, there is no diference between the mean number of R. maidis on Statistical analyses JGMV-infected plants and mock-inoculated control plants (P > 0.05) (Fig. 2). All datasets were subjected to a one-way ANOVA (Graph- Pad Prism V 8.1.0) to identify signifcant changes across Rhopalosiphum maidis virus transmission efciency datasets (P value ≤ 0.05), a Shapiro–Wilk test (Ghasemi and Zahediasl 2012)] for normality and Bartlett’s test (Struchalin The potential threat posed by the vector R. maidis to transmit et al. 2010)] for homogeneity of variation were performed. plant diseases was accessed by testing its ability to transmit The signifcance comparison of all possible pairs of means Potyviruses (JGMV, MDMV, SCMV, and SrMV) into a suit- was calculated by Tukey’s Honest Signifcant Diference able host plant such as S. bicolor. Therefore, the virus trans- test. Samples failing the normality test were subjected to mission rate by a single viruliferous R. maidis into healthy Kruskal–Wallis with subsequent Dunn’s test means sepa- Sorghum plant was measured. The mean virus transmis- ration test to assess the signifcance between experimental sion percentage obtained from 30 individual transmission groups. experiments per virus ranged between 70% for MDMV, 70% SCMV, and 73.33% for SRMV with P value > 0.05 not showing a signifcant diference between each other. This Results result suggests a similar R. maidis virus transmission poten- tial for those viruses into S. bicolor. However, JGMV trans- Efect of virus infection on R. maidis plant mission efciency by R. maidis is with 40% signifcantly preference lower if compared to those of MDMV, SCMV or SRMV (P < 0.05) indicating a reduced vector-virus or host-virus In general, R. maidis exhibited a signifcant preference for S. compatibility (Fig. 3). bicolor plants infected with MDMV, SCMV or SRMV com- pared to mock-inoculated control plants by the end of the ELISA verifcation of viral transmission bioassay period (Fig. 2). The mean numbers of R. maidis on plants infected with MDMV, SCMV or SRMV were signif- To identify JGMV, MDMV, SCMV and SrMV infected cantly (P < 0.05) greater than those exposed to mock-inoc- plants on the one hand and to determine the virion quan- ulated S. bicolor plants. Although, due to the experimental tity qualitatively on the other hand ELISA tests were per- setup, no direct comparison between R. maidis settlement formed. The one-way ANOVA test computing the ELISA on plants infected with MDMV, SCMV or SRMV is pos- 405 nm absorbance values for plants infected with each virus sible, a clearly similar preference towards infected plants resulted in P values of 0.4, 0.82, 0.81, and 0.88 for JGMV, is observable. A similar trend can be observed in the mean MDMV, SCMV, and SRMV, respectively. Absorbance

1 3 816 P. Klein, C. M. Smith

100 fragments are 90%, 90%, 105%, and 98%, respectively. The correlation coefcients (R2) of all linear regression slopes

ts were > 0.99, ranging from 0.9946 for JGMV to 0.9994 for b 80 b SRMV confrming a good confdence into the amplifcation

plan b efciency and subsequently in the absolute quantifcation of viral particles in plant tissue (Fig. 5). 60 fected a Viral CP gene copy number quantifcation in

)% 40 The viral ssRNA genome copy number of each virus was determined based on the Cq values for the viral coat pro-

(±SE tein (CP) gene in 10 cDNA samples from individual S. bicolor plants infected with one of the four tested viruses. 20 Mean ± SE Cq values were as follows: JGMV, 32.4 ± 0.3; Mean MDMV, 28.0 ± 0.1; SCMV, 26.8 ± 0.3; and SRMV, 27.3 ± 0.3 (Fig. 6a) passing the Shapiro–Wilk normality and Tukey test 0 with P values > 0.05 (0.59, 0.64, 0.4, and 0.16). The calcu- JGMV MDMV SCMV SRMV lated mean JGMV CP gene copy ± SE number of 439 ± 85.1 Potyvirus infecting S. bicolor plant was signifcantly (P < 0.001, P = 0.001 and P < 0.001) lower than MDMV, SCMV, and SRMV CP gene copy numbers of Fig. 3 R. maidis potential to transmit Potyviruses into Sorghum host plant. Mean (± SE) transmission efciency in percent for Johnson- grass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), and Sorghum mosaic virus (SRMV) by a single viruliferous R. maidis into S. bicolor plants. Potyvirus infecting S. bicolor plant n = 30 S. bicolor plants. Means that do not share a letter difer signif- cantly (P < 0.05) due to Dunn’s test 0.8 bbb

values from S. bicolor plants infected with MDMV, SCMV, m n and SRMV yielded mean ± SE ELISA 405 nm absorbance

405 0.6 values of 0.71 ± 0.04, 0.69 ± 0.03, and 0.71 ± 0.03, respec- t tively. Those absorbance values correlate to the virion ea

quantity in the tested plant extract. The absorbance data nc a obtained from plant material infected with MDMV, SCMV, and SRMV was not signifcantly diferent from one another. 0.4

In contrast, the mean ± SE ELISA absorbance value of bsorba plants infected with JGMV (0.44 ± 0.02) was signifcantly )a

(P < 0.05) less than values of plants infected with MDMV, SE ± SCMV or SRMV according to Tukey test indicating lower ( 0.2 JGMV virion count in host plant S. bicolor if compared to an other three tested Potyviruses (Fig. 4). Me

Viral CP gene standard curve generation 0.0 JGMV MDMV SCMV SRMV The standard curve qPCR slopes of virus specifc CP gene amplicons were determined as − 3.578 for JGMV, − 3.402 Fig. 4 Qualitative virion quantifcation by ELISA. Mean (± SE) for MDMV, − 3.2 SCMV, and − 3.359 for SrMV. Those val- 405 nm absorbance values of leaf extracts from S. bicolor plants ues are within the tolerance of − 3.1 and − 3.6 representing infected with Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), or Sor- acceptable amplifcation efciency between 90 and 110%. ghum mosaic virus (SRMV). Means that do not share a letter difer Based on the individual slopes the calculated amplifcation signifcantly (P < 0.05) using Tukey test efciencies for JGMV, MDMV, SCMV, and SRMV CP gene

1 3 Host plant selection and virus transmission by Rhopalosiphum maidis are conditioned by… 817

Fig. 5 Standard curves, PCR amplifcation plots, linear regression slopes and correlation coefcients of serially diluted coat protein gene frag- ments from Potyviruses Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), and Sor- ghum mosaic virus (SRMV)

1 3 818 P. Klein, C. M. Smith

A computing JGMV, MDMV, SCMV, and SRMV coat protein 40 amplicons/ ng total RNA template confrmed the normal distri- a bution with P values of 0.16, 0.36, 0.25, and 0.94. Amplicons b b b obtained from qPCR were separated on 2% agarose gel for 30 primer and product quality assessment. DNA bands from 10 plant samples infected with each virus exhibited the expected molecular size of 218 bp for the JGMV CP gene fragment and 242 bp for the closely related MDMV, SCMV, and SRMV CP 20 gene amplicons (Fig. 7). Cq mean (±SE) 10 Discussion Potyvirus transmission 0 Potyviruses such as JGMV, MDMV, SCMV, and SRMV JGMV MDMV SCMV SRMV cause significant global yield losses in a wide range of Potyvirus infecting S. bicolor plant crop plants. In this study, inoculation experiments with a single viruliferous R. maidis successfully determined B the transmission efficiency of each virus into sorghum 12000 seedlings. A 1 h R. maidis starvation period prior virus b acquisition proved to be suitable for maximum virus RNA b 10000 transmission (Powell et al. 1995). In our transmission tal b to efficiency experiments the MDMV, SCMV, and SRMV 8000 ng infection rate varied slightly between 70% –73% was confirmed immunologically by ELISA assay. On the ber/ 6000 other hand the measured JGMV transmission efficiency um rate of 43% was significantly lower. These results are the en 4000 first to determine JGMV, MDMV, SCMV, and SRMV en transmission efficiency by R. maidis into S. bicolor.

Pg 2000 1000 However, results of a previous related study (Lucio- )C 800 Zavaleta et al. 2001)] align with the high observed virus SE R. maidis (± 600 a transmission efficiencies. , which also trans-

an 400 mits yellow dwarf virus (BYDV), was shown to e

M 200 transmit different BYDV RMV strains at rates ranging 0 from 10 to 82%. Two MDMV haplotypes obtained from JGMV MDMV SCMV SRMV Johnsongrass showed a variable transmission rate into maize host plants depending on virus haplotype and Potyvirus infecting S. bicolor plant transmitting vector. In the same study, R. maidis was shown to be the most suitable vector of MDMV into

Fig. 6 Mean (± SE) fractional CR cycle (Cq) and mean (± SE) coat maize among four vectors tested, reaching transmission protein gene copy in 10 S. bicolor plants infected with Potyviruses efficiencies between 29 and 54%, depending on the hap- Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus lotype, indicating that the abundance of a specific vector (MDMV), Sugarcane mosaic virus (SCMV), and Sorghum mosaic population may shape the virus population in a given virus (SRMV). a Mean ± SE Cq value obtained from 10 S. bicolor plants infected with one of the four tested Potyviruses. b Calculated area (Achon and Serrano 2010). mean ± SE coat protein gene copy number per ng of total RNA in 10 Previously. R. maidis has been shown to transmit SCMV host plants infected with one of the tested plant viruses. Means that infections into maize seedlings at rates up to 92% (Sahi do not share a letter difer signifcantly (P < 0.05) using Tukey test. and Imanat 2003), and similar rates of transmission were observed for MDMV infections into sorghum (Gordon and 8864 ± 691.4; 6584 ± 1300; and 9065 ± 1500/ng total RNA. Thottappilly 2003). A related study with peach potato aphid, According to Tukey’s test multiple comparison test calculated M. persicae, demonstrated 40% and 85% MDMV transmis- P values ranged between 0.35 and 0.99 for MDMV, SCMV, sion into maize seedlings, respectively (Tu and Ford 1971). and SRMV group (Fig. 6b). The Shapiro–Wilk normality test High coinfection rates with multiple viruses such as SRMV

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Fig. 7 Agarose gel electrophoresis of Potyviruses Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), and Sorghum mosaic virus (SRMV). CP gene amplicons, 100 bp DNA marker and corresponding PCR product melt curves

1 3 820 P. Klein, C. M. Smith and SCMV have been also reported. In Tucumán, Mexico, of tested aphids moved towards infected plants in a dual SCMV and SRMV was detected in 70% and 94% of sampled choice assay and remained arrested over a 1 h testing period sugarcane with a coinfection rate of 64% (Perera et al. 2012). (Eigenbrode et al. 2002). Analog observations of SRMV and MDMV occurrences Increased volatile production in wheat infected with reaching 75% in Chinese sugarcane-growing provinces sug- BYDV attracts also signifcantly higher numbers of R. padi gest that this Potyvirus maintains a high rate of transmission when plants are in close proximity to one another. Further, and co-infection with other Potyviruses (Luo et al. 2016b). headspace volatile analyses of infected plants identifed 20 The pairwise analysis of the JGMV CP nucleotide cod- compounds, with (Z)-3-hexenyl acetate being signifcantly ing sequence to the nucleotide coding sequences of MDMV, overproduced, suggesting it as a potential R. padi arrestant SCMV, and SRMV resulted in a slightly greater than 70% or attractant (Jiménez-Martínez et al. 2004). Similar to identity (de Souza et al. 2017). Thus, considering the essen- those observations, the results of the current study also tial role of the CP and helper component proteinase (HC- show the clear appeal of MDMV-, SCMV-, and SRMV- pro) proteins during virus acquisition by R. maidis and a infected plants to R. maidis. In contrast, JGMV-infected higher genetic diversity if compared to other tested viruses plants exhibit no such efects on R. maidis, and no signif- might be a factor for the reduced JGMV transmission ef- cant diferences in R. maidis choice of host plant after 2 h ciency by R. maidis. Studies on M. persicae transmission roaming period. Similarly, the majority of alate R. maidis of the potyvirus zucchini yellow mosaic virus suggest the has shown a tendency to arrest on infected plants soybean involvement and necessity of the viral HC-Pro (Peng et al. mosaic virus (SMV/Potyvirus) infected soybean plants for 1998)], as HC-Pro mutations can result in total loss of infec- more than 1 hour after landing before transitioning to a tion. Coat protein sequence diversity may also alter the healthy plant (Fereres et al. 1999). The green peach aphid capacity of the vector to acquire the viral helper component (M. persicae) prefers virus infected Jalapeño pepper plants (HC-pro), and afect the binding afnity of the CP to the for longer period of time before transitioning to healthy helper protein in the vector stylets (Ng and Perry 2004). plants (Safari et al. 2019). Studies on host plant quality On the vector side, CP afnity is determined by HC-pro showed a signifcant change in soluble metabolites such as recognition in the stylet (1987). However, mutations in the sugar and free amino acid content between Papaya rings- HC-pro protein can also afect virus transmission, suggest- pot virus (Potyvirus) infected and healthy plants and may ing a continuous co-evolution and a distinct range of viruses play a role in aphid host plant choice, growth, and settling transmitted by the vector with variable efciencies (Ng and behavior (Gadhave et al. 2019). The preferred settlement Perry 2004). and feeding of nonviruliferous vectors on virus infected host plants is a consistently reported observation through- Efect of virus infection on R. maidis plant out scientifc literature (Mauck et al. 2018). preference

Vectors of plant viruses are attracted to infected host plants Viral RNA quantifcation by visual and olfactory cues that enhance vector attraction, as demonstrated by studies on BYDV- and Cucumber mosaic The usage of highly specific degenerated primer sets virus (CMV)-infected plants that induce a clear vector pref- designed based on the viral HC-Pro sequence analysis, erence compared to uninfected control plants (Ingwell et al. including JGMV, for diagnostic purposes has been already 2012; Mauck et al. 2014). Viral infections cause changes reported (Ha et al. 2008). However, no qPCR efciency for in host plant physiology, that in turn alters the host plant JGMV, MDMV, SCMV, and SRMV CP based degenerated volatile profle. However, to our knowledge, little is known primer sets is reported. Using the designed primer sets for about plant volatile compound content in response to viral this study the PCR efciency stretches from 90.2 to 102% infections. Studies of Cucurbita pepo plants infected with with R2 values between 0.9946 and 0.9994. Considering CMV have shown infected plants to be inferior hosts for the fact that MDMV, SCMV, and SRMV standard curve the vectors M. persicae and Aphis gossypii while attracting PCR reactions were performed with the same primer sets signifcantly more vectors by emitting an increased volatile leads to the conclusion that same the primer pair can be level without signifcant changes in the overall blend com- used for diagnostic and quantifcation purposes for those position causing a winged vector migration to healthy plants viruses. after a short probing period. However, in the same study As previously mentioned, the Cq value in real-time PCR unwinged aphids have shown a signifcant attraction towards analysis resonates the initial template quantity present in the virus infected plants over long period of time (Mauck et al. sample. Therefore, a standard curve based absolute quanti- 2010). Experiments on M. persicae attraction towards virus fcation of viral ssRNA in plants infected with each of the infected potato plants showed a similar efect. Roughly 70% tested Potyviruses is realizable. The CP copy numbers in

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1 ng total RNA from 10 infected JGMV, MDMV, SCMV or Open Access This article is licensed under a Creative Commons SRMV S. bicolor plants was determined as following 439.7 Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, for JGMV and 8,886, 6,584, and 9,065 for MDMV, SCMV, as long as you give appropriate credit to the original author(s) and the and SRMV respectively, confrming the pathological poten- source, provide a link to the Creative Commons licence, and indicate tial towards S. bicolor. Nonetheless, the virus propagation if changes were made. The images or other third party material in this rate of JGMV based on CP gene copy number is signifcantly article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not lower compared to other three tested Potyviruses. Potyvi- included in the article’s Creative Commons licence and your intended ruses belong to secondary movement type viruses recruiting use is not permitted by statutory regulation or exceeds the permitted the viral CP protein in addition to other proteins for cell to use, you will need to obtain permission directly from the copyright cell movement (Otulak and Garbaczewska 2011). Decreased holder. To view a copy of this licence, visit http://creat​iveco​mmons​ .org/licen​ses/by/4.0/. pathogen-host plant compatibility might be a factor for the low JGMV CP copy number in S. bicolor and afect viral spread throughout plant tissues. Further, evidence for a higher degree of recombination within the Potyviridae group References has been found reducing the host specifcity and broaden the host plant ranges (Kehoe et al. 2014). Therefore, a thorough Achon MAADN, Serrano L (2010) Maize dwarf mosaic virus diversity bioinformatic recombination event analyses of the JGMV, in the Johnsongrass native reservoir and in maize. Plant Pathol MDMV, SCMV, and SRMV genomes could provide addi- 60:8 tional clues about rates of divergent infection and shed light Adams MJ, Antoniw JF, Fauquet CM (2005) Molecular criteria for genus and species discrimination within the family Potyviri- on virus evolution and future pathogenic potential of those dae. Arch Virol 150:459–479. https​://doi.org/10.1007/s0070​ viruses. 5-004-0440-6 Berger PH, Wyatt SD, Shiel PJ, Silbernagel MJ, Drufel K, Mink GI (1997) Phylogenetic analysis of the Potyviridae with emphasis on Conclusions legume-infecting potyviruses. Arch Virol 142:1979–1999 Bosque-Perez NA, Eigenbrode SD (2011) The infuence of virus- induced changes in plants on aphid vectors: insights from The results of this study demonstrate that R. maidis is an ef- luteoviruspathosystems. Virus Res 159:201–205. https​://doi. cient vector of JGMV, MDMV, SCMV, and SRMV into S. org/10.1016/j.virus​res.2011.04.020 bicolor Bowling RD et al (2016) Sugarcane aphid (: Aphididae): a able to transmit all four tested Potyviruses. However, new on sorghum in North America. J Integr Pest Manag. https​ its transmission efciency capacity for JGMV in S. bicolor is ://doi.org/10.1093/jipm/pmw01​1 signifcantly lower than that for MDMV, SCMV or SRMV. Cronin S, Verchot J, Haldeman-Cahill R, Schaad MC, Carrington JC In two-choice bioassays S. bicolor infection with MDMV, (1995) Long-distance movement factor: a transport function of the potyvirus helper component proteinase. Plant Cell 7:549–559. SCMV, and SRMV manifest in a behavioral manipulation https​://doi.org/10.1105/tpc.7.5.549 of R. maidis, which shows a signifcant R. maidis preference Dader B, Then C, Berthelot E, Ducousso M, Ng JCK, Drucker M for S. bicolor plants infected with MDMV, SCMV, SRMV (2017) transmission of plant viruses: multilayered interac- compared to non-infected or JGMV infected plants. The low tions optimize viral propagation. Insect Sci 24:929–946. https​:// S. bicolor doi.org/10.1111/1744-7917.12470​ propagation of JGMV virus within tissue com- Dedryver CA, Le Ralec A, Fabre F (2010) The conficting rela- pared to MDMV, SCMV, and SrMV was demonstrated by tionships between aphids and men: a review of aphid dam- absolute virion quantifcation using the qPCR method. The age and control strategies. C R Biol 333:539–553. https​://doi. viral CP gene primers designed in this study exhibit a high org/10.1016/j.crvi.2010.03.009 Delmas B, Attoui H, Ghosh S, Malik YS, Mundt E, Vakharia VN, target specifcity and can be used for virus identifcation Ictv Report C (2019) ICTV virus profle: Picobir- or early diagnostics of JGMV, MDMV, SCMV and SrMV naviridae. J Gen Virol 100:133–134. https​://doi.org/10.1099/ virion RNA along with subsequent quantifcation of virion jgv.0.00118​6 particles in plant tissue. de Souza IRP, Silva XA, Medeiros Carvalho SG, Oliveira SE, Gon- çalves IAM, Noda RW, Santos RJ (2017) Johnsongrass mosaic However, the reason behind the inferior JGMV transmis- virus infecting sorghum in Brazil. Int J Curr Res 9:7 sion efciency and propagation in S. bicolor is still unclear, Diaz-montano JRJC, Schapaugh WT, Campbell LR (2007) Chlorophyll along with the physiological mechanisms involved in the loss caused by (Hemiptera: Aphididae) feeding on R maidis attraction towards Potyvirus infected S. bicolor soybean. J Econ Entomol 100:5 Dietzgen RG, Mann KS, Johnson KN (2016) Plant virus-insect vec- plants. tor interactions: current and potential future research directions. Viruses. https​://doi.org/10.3390/v8110​303 Acknowledgements This is Contribution No. 20-057-J of the Kansas Edgar RC (2004) MUSCLE: multiple sequence alignment with high Agricultural Experiment Station. accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https​://doi.org/10.1093/nar/gkh34​0

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