Quantification of Gammacarmovirus glycinis (Soybean yellow mottle mosaic virus) to evaluate mungbean (Vigna radiata) and soybean (Glycine max) genotypes for resistance to seed transmission.

Quantification of Gammacarmovirus glycinis (Soybean yellow mottle mosaic virus) to evaluate mungbean (Vigna radiata) and soybean (Glycine max) genotypes for resistance to seed transmission - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice 3 Biotech . 2025 May 16;15(6):174. doi: 10.1007/s13205-025-04347-w Quantification of Gammacarmovirus glycinis ( Soybean yellow mottle mosaic virus ) to evaluate mungbean ( Vigna radiata ) and soybean ( Glycine max ) genotypes for resistance to seed transmission Nagamani Sandra 1, ✉ , Ankita Tripathi 1 , Sanjay Kumar Lal 2 , Harsh Kumar Dikshit 2 Author information Article notes Copyright and License information 1 Seed Pathology Laboratory, Division of Seed Science and Technology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012 India 2 Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi, 110012 India ✉ Corresponding author. Received 2025 Feb 21; Accepted 2025 May 4; Issue date 2025 Jun. © King Abdulaziz City for Science and Technology 2025. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. PMC Copyright notice PMCID: PMC12084199  PMID: 40386632 Abstract Soybean yellow mottle mosaic virus (SYMMV) is a newly emerging seed borne plant virus responsible for mosaic and mottling disease in mungbean and soybean. The present study aimed to evaluate the response of nine widely cultivated mungbean and 15 newly developed soybean genotypes to SYMMV-Mb infection under controlled environment conditions using agro-inoculation, followed by serological and molecular detection. The genotypes exhibited differential local symptoms at 13–17 dpi and systemic symptoms at 21–29 days post-inoculation (dpi), ranging from mild chlorosis to severe necrotic local spots with a PDI of 60–95% in mungbean and 30–50% in soybean genotypes. Among the mungbean genotypes, Sona Moong was found to be moderately susceptible (MS), six were susceptible and Pusa Vishal and SML-668 were highly susceptible. Among the soybean genotypes, nine were found to be immune, four were moderately resistant and two were MS. DAC-ELISA and RT-PCR confirmed the presence of SYMMV-Mb in all infected genotypes with higher absorbance values and amplification of the 1064 bp coat protein (CP) region. RT-qPCR analysis of whole seeds harvested from agro-inoculated plants revealed virus accumulation only in mungbean genotypes ranging from 6.33 × 10 1 to 9.00 × 10 5 but not in soybean genotypes. Progeny assays confirmed seed transmissibility of SYMMV-Mb at a rate of 20–50% in mungbean genotypes, and 0% transmission in soybean genotypes. Further studies are required to elucidate the plant defense mechanisms and viral factors involved in suppressing host resistance during seed embryo development. Supplementary Information The online version contains supplementary material available at 10.1007/s13205-025-04347-w. Keywords: Mungbean, Soybean, Soybean yellow mottle mosaic virus, DAC-ELISA, PCR Introduction Mungbean ( Vigna radiata L.) is an important grain legume crop, rich in essential amino acids, vitamins, and minerals. It is a short-duration Kharif crop, cultivated extensively in major tropical and subtropical regions of the world, especially in South Asian countries (Nair et al. 2019 ). Soybean ( Glycine max L.) is another precious legume crop, grown worldwide for its high-quality protein and oil content (Fried et al. 2018 ). In recent years, changes in global climate scenario has contributed to the emergence of new viral and the re-emergence of previously reported viral diseases affecting both of these crops. Among these, soybean yellow mottle mosaic virus ( Gammacarmovirus glycinis ; SYMMV) is a newly emerging virus infecting mungbean (Mb) and soybean and is responsible for mosaic and mottling symptoms (Nam et al. 2009 ; Sandra et al. 2015 , 2021 ; Wei et al. 2020 ). Soybean yellow mottle mosaic virus belongs to the genus Gammacarmovirus within the family Tombusviridae (Zerbini et al. 2023 ). The SYMMV consist of 28–30-nm isometric virion particles, monopartite with positive sense ssRNA genome of 3974 nucleotides encoding six ORFs. Host range studies through mechanical sap transmission and agro-inoculation have shown that SYMMV primarily restricted to Leguminosae members , causing symptoms such as mosaic, mottling, and necrosis. Recently, the SYMMV Mb isolate has been reported to be seed transmitted in both its natural host, Mb and an experimental host French bean ( Phaseolus vulgaris ) (Sandra et al. 2017 , 2020 ). The development of resistant cultivars against newly emerging plant viruses that can spread quickly and impact crop fields is a crucial aspect of modern plant disease management. Hence, identification of resistant germplasm through phenotyping is a first step in breeding programs targeted at developing virus-resistant cultivars (Singh et al. 2019 ). However, natural field screening has significant challenges like mixed viral infections, which can influence symptom expression. Therefore, artificial screening approach such as agro-inoculation is essential for accurate identification of genotypes with true resistance to specific viruses. Seed transmission is a key factor in the survival and dissemination of plant viruses, posing a serious threat to seed producers, as seeds infected with viruses can facilitate long-distance dispersal across regions (Johansen et al. 1994 ; Ali and Kobayashi 2010 ; Sastry 2013 ; Dombrovsky and Smith 2017 ). Beyond initial spread, early stage viral infections during embryo development can cause genetic and morphologic abnormalities, reducing crop vigor and yield, ultimately resulting in financial losses (Escalante et al. 2024 ). In the present study, attempts were made to identify resistant genotypes to SYMMV-Mb infection and its seed transmission by screening widely cultivated Mb and newly developed soybean genotypes through agro-inoculation. This approach will also provide valuable insights into which genotypes might be used in breeding programs to develop more resilient varieties. Further, understanding the mechanisms underlying seed transmission will allow more targeted and effective strategies to limit virus spread and ensure crop security across new geographic regions. Materials and methods Seed material and virus source The present study involves the evaluation of nine popularly cultivated Mb and 15 newly developed soybean genotypes currently under initial varietal trial phase (IVT) to soybean yellow mottle mosaic virus mungbean isolate (SYMMV-Mb) infection (Table 1 ). Seed material was obtained from the Division of Genetics, ICAR-Indian Agricultural Research Institute (IARI), New Delhi, India. For inoculation, a full-length infectious clone of SYMMV-Mb present in the pORE O4 binary vector (p35S-SYMMV-OCS) available at Seed Pathology Laboratory, ICAR-IARI was used (Sandra et al. 2017 ). Table 1. Response of mungbean and soybean genotypes following agro-inoculation with SYMMV full-length infectious clone S.No Name of the Cultivar No. of symptomatic plants Symptoms b Confirmation of leaf samples Confirmation of seed samples Rep I Rep II PDI% Local DPI a Systemic DPI DAC-ELISA c RT-PCR d DAC-ELISA c RT-PCR d RT-qPCR e Mungbean ( Vigna radiata ) 1 IPM-02–3 7 6 65 NS − Nls 22–24 + + + + + 2 Pusa 1371 8 7 75 NlS 15–17 Vn, Nls 23–25 + + + + + 3 Pusa 1413 8 8 80 NS − Nls 22–25 + + + + + 4 Pusa-9531 7 6 65 Vn 14–16 Vn, Nls 21–24 + + + + + 5 Pusa Ratna 6 8 70 Cls 13–15 Cls, Clb 20–23 + + + + + 6 Pusa Vishal 9 9 90 NlS 13–17 Nls, Mo 20–23 + + + + + 7 MH-421 8 6 70 NS − MCls 21–25 + + + + + 8 SML-668 10 9 95 NS − Nls 21–24 + + + + + 9 Sona Moong 6 6 60 Vn 14–16 Vn, Nls 20–24 + + − − − Soybean ( Glycine max ) 1 DS-3109 − − 0 − − − − + + − − − 2 NRC-146 4 6 50 − − VMm 26–28 + + − − − 3 PS-1634 − − 0 − − − − + + − − − 4 JS-21–71 − − 0 − − − − + + − − − 5 MACS-1566 5 4 45 − − MMo 26–28 + + − − − 6 SL-1191 − − 0 − − − − + + − − − 7 Himso-1688 − − 0 − − − − + + − − − 8 PS-24 − − 0 − − − − + + − − − 9 RSC-11–17 − − 0 − − − − + + − − − 10 MAUS-734 4 3 35 Cls 14–17 Cls 25–27 + + − − − 11 DSB-33 4 4 40 Mo 15–18 Mo, Cls 26–28 + + − − − 12 NRC-138 − − 0 − − − − + + − − − 13 JS-21–72 − − 0 − − − − + + − − − 14 PS-1637 3 3 30 NS − Mmo 24–27 + + − − − 15 AUKS-176 3 4 35 NS − Vmm 27–29 + + − − − Positive 9 8 85 Mo 5–8 Clb, Mo, LR 15–17 + + + + + Healthy − − − − − − − − − − − − Open in a new tab a Days after post-inoculation b Key to symptoms: Clb: chlorotic blotches; Cls: Chlorotic spots; MCls: Mild chlorotic spots; Mo: Mottle; Mmo: Mild mottle; NS: No symptoms; Nls: Necrotic local spots; LR: Leaf rolling; Vmm: Veinal mild mottling; Vn: Veinal necrosis c Absorbance value at 405 nm (1 h reading): 0.84–2.05; Healthy plant sample value: 0.30; + : positive results; -: negative results d RT-PCR was carried out with SYMMV Coat protein-specific primers e RT-qPCR was carried out with the SYMMV partial coat protein-specific primers that amplify 178 bp Agro-inoculation and plant growth conditions For agro-inoculation, the full-length SYMMV-Mb infectious clone was introduced into Agrobacterium tumefaciens strain GV3101 competent cells via transformation. The transformed Agrobacterium cultures harboring the SYMMV-Mb clone were prepared and processed for inoculation following the previously described protocol (Sandra et al. 2017 ). Mungbean and soybean seedlings were agro-inoculated at the two leaf stage using pin pricking method where 20 µl of Agrobacterium culture was applied at the site of inoculation. Each cultivar was tested in two replications. Plants inoculated with Agrobacterium cells carrying the empty pORE O4 vector served as negative controls, while French bean plants mechanically inoculated with native SYMMV-Mb sap were used as positive controls. All the plants were maintained under controlled environmental conditions at National Phytotron Facility (NPF), IARI with a temperature of 26 ± 2 °C, 16/8-h photoperiod and 70% relative humidity. Plants were regularly monitored for symptom development. Upon maturation, total seed was harvested from the agro-inoculated plants and stored for further studies. Disease incidence percentage of mungbean and soybean cultivars Percent disease incidence (PDI) was calculated by recording the number of symptomatic plants out of ten plants inoculated plants per genotype assessed over two replications and expressed as a percentage. A disease rating scale of 0–5 was used to evaluate symptom severity in both mungbean and soybean genotypes (modified from Bachkar et al. 2019 ). The genotypes were classified as immune (I), resistant (R), moderately resistant (MR), susceptible (S) and Highly Susceptible (HS) based on their responses to SYMMV-Mb infection. Serological and molecular detection of SYMMV from leaf and seed samples Systemic trifoliate leaf samples collected from all inoculated and control plants were assayed for SYMMV-Mb infection through direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) using in-house generated SYMMV polyclonal antibodies (Sandra et al. 2015 ). DAC-ELISA was also carried out on seed harvested from agro-inoculated mungbean and soybean genotypes. Initially, the seeds were surface sterilized with 1% sodium hypochlorite for 1 min followed by rinsing with sterile distilled water and allowed to imbibe water for overnight (Sauer and Burroughs 1986 ). Groups of three randomly selected seeds were ground together in coating buffer containing 2% polyvinylpyrrolidone to prepare the seed extract. The ELISA procedure was carried out at 37 °C following the method described by Sandra et al. ( 2020 ). The presence of SYMMV was determined by measuring absorbance values (OD 405 ) using an ELISA plate reader 60 min after substrate addition (Edwards and Cooper 1985 . A sample was considered positive for SYMMV-Mb if its OD₄₀₅ value was at least twice that of the healthy control. Total RNA was extracted from Mb and soybean leaf samples collected 25 dpi from both agro-inoculated and mock-inoculated plants. For RNA isolation from seeds, an initial starch removal step was performed using starch removal extraction buffer (Li and Trick 2005 ). Subsequent RNA extraction was carried out from the aqueous phase using the SV total RNA isolation system (Promega, Madison, USA) as per manufacturer’s protocol. The quality and quantity of isolated RNA were analyzed using a Nanodrop spectrophotometer and RNA integrity was confirmed via agarose gel electrophoresis. First-strand cDNA synthesis was performed using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, India) with the reverse primer (5′-caaagcttagtgtgtgggttttaactggcgttgtta - 3′). Subsequently, polymerase chain reaction (PCR) was conducted in 50 μl reaction volume using DreamTaq DNA polymerase with CP-specific primers (F: 5′-atgaattcatgaatggaacaatgctcaccgt-3′; R: 5′- caaagcttagtgtgtgggttttaactggcgttgtta-3′) at an annealing temperature of 62 °C. The amplified PCR products were analyzed in 1% agarose gel (Sandra et al. 2020 , 2021 ). Viral RNA quantification in infected seed samples through RT-qPCR Reverse transcription quantitative PCR (RT-qPCR) was employed to quantify viral RNA load in infected and control Mb and soybean seed samples. For absolute quantification of SYMMV-Mb , a standard curve was generated using tenfold serial dilutions of a TA-cloning vector harboring the SYMMV CP gene . Prior to qPCR, primer specificity was validated through conventional PCR. For RT-qPCR, 1 µL of cDNA (20 ng) synthesized from a group of three infected seeds was used as a template. Amplification targeted a partial region of the CP gene (F: 5′-atggctggcgctgtctctgg-3′; R: 5′-caccgagggcgaagggagtg-3′) using PowerUp™ SYBR® Green Master Mix (Applied Biosystems, Thermo Fischer Scientific, India). Each genotype was run in three technical replicates on an Mx3005P QPCR system (Agilent Technologies, India) with a thermal cycling program of 95 °C for 5 min and 30 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 5 s. The SYMMV viral copy number was estimated using the standard formula ( Supplementary material ). Seed transmission assay of SYMMV-Mb through grow-out test Seed transmission of SYMMV-Mb was evaluated in the progeny of agro-inoculated genotypes through grow-out test. For each genotype, 50 seeds from both symptomatic and control (mock-inoculated) plants were separately sown in pots and maintained under controlled environmental conditions with 26 ± 2 °C, 16/8-h photoperiod at NPF, IARI. The seedlings were monitored regularly for symptom development over a period of 30 days. At 15 days after sowing (DAS), randomly selected seedlings were subjected to DAC-ELISA to detect SYMMV-Mb infection. Seed transmission percentage was calculated as number of ELISA positive seedlings out of total number of seedlings tested. Results Phenotypic response of mungbean and soybean genotypes following agro-inoculation Agro-inoculated plants from all the genotypes displayed the varied local symptoms at 13–17 dpi and systemic symptoms at 21–29 dpi. Mb genotypes IPM-02–3, Pusa 1371, Pusa Vishal and SML-668 exhibited systemic necrotic spots by 20–25 dpi. Pusa-9531 and Sona Moong showed both local and systemic veinal necrosis around 20–24 dpi while the genotypes MH-421, Pusa 1413, and Pusa Ratna displayed mild chlorosis and chlorotic spots (Cls) by 20–23 dpi (Fig. 1 A; Table 1 ). In contrast, most soybean genotypes remained asymptomatic up to 30 dpi, with a few exceptions. The genotypes DSB-33 and MAUS-734 developed systemic mottling and Cls while PS-1637, MACS-1566, and AUKS-176 showed mild veinal mottling and Cls (Fig. 1 B; Table 1 ). Mechanically, sap-inoculated French bean cultivar showed the symptoms of systemic chlorotic blotches (Clb), mottling and leaf rolling (Table 1 ). Fig. 1. Open in a new tab Symptoms of soybean yellow mottle mosaic virus mungbean isolate (SYMMV-Mb) on mungbean ( A ) and soybean ( B ) genotypes following agro-inoculation. A Mungbean genotypes exhibited distinct symptoms: necrotic local spots on IPM-02–3, Pusa 1371, Pusa Vishal and SML-668; Veinal necrosis on Sona Moong and Pusa 9531; Mild chlorotic spots on genotypes Pusa Ratna and MH-421. B Soybean genotypes showed chlorotic spots in MAUS-734 and DSB-33, while veinal mild mottling symptoms were observed in PS-1637 and AUKS-176 Disease incidence percentage in mungbean and soybean genotypes The disease score and PDI were recorded for all agro-inoculated genotypes under controlled environmental conditions. The PDI among Mb genotypes ranged from 60–90% while in soybean genotypes ranged from 30–50%. Among the nine Mb genotypes evaluated, Sona Moong was found to be MS (60% PDI), 6 genotypes were classified as susceptible (60.1 to 80%) and two genotypes Pusa Vishal and SML-668 were found to be highly susceptible (HS) with 90 and 95% PDI, respectively. Of the 15 soybean genotypes tested against SYMMV-Mb, nine genotypes exhibited no visible symptoms and classified as immune; four genotypes were identified as moderately resistant (20.1 to 40% PDI) and 2 cultivars (NRC-146 and MACS-1566) were considered MS based on phenotypic response and PDI (Table 2 ). Table 2. Grouping of mungbean and soybean genotypes upon SYMMV infection as per disease rating scale Scale a Description Disease score No. of genotypes Genotypes 0 No symptoms on plants Immune (I) 9 DS 3109, PS 1634, JS 21–71, SL 1191, Himso 1688, PS-24, RSC 11–17, NRC 138, JS 21–72 1 0.1 to 20% of the plants exhibiting mosaic, mottling mild chlorotic blotches, chlorotic spots, necrotic spots, veinal necrosis and veinal mild mottle on leaves Resistant (R) 0 – 2 20.1 to 40% of the plants exhibiting mosaic, mottling, mild chlorotic blotches, chlorotic spots, necrotic spots, veinal necrosis and veinal mild mottle on leaves Moderately resistant (MR) 4 AUKS-176, DSB-33, MAUS-734, PS-1637 3 40.1 to 60% of the plants exhibiting mosaic, mottling, mild chlorotic blotches, chlorotic spots, necrotic spots, veinal necrosis and veinal mild mottle on leaves Moderately susceptible (MS) 2(Sb); 1(Mb) MACS-1566, NRC-146, Sona Moong 4 60.1 to 80% of the plants exhibiting mosaic, mottling, mild chlorotic blotches, chlorotic spots, necrotic spots, veinal necrosis and veinal mild mottle on leaves Susceptible (S) 6 IPM-02–3, Pusa 1371, Pusa-9531, Pusa 1413, MH-421, Pusa Ratna 5 80.1 to 100% of the plants exhibiting mosaic, mottling, mild chlorotic blotches chlorotic spots, necrotic spots, veinal necrosis and veinal mild mottle on leaves Highly susceptible (HS) 2 Pusa Vishal, SML-668 Open in a new tab a Key to disease score: 0: Immune(I); 1: Resistant (R); 2: Moderately resistant (MR); 3: Moderately Susceptible; 4: Susceptible; 5: Highly susceptible Confirmation of SYMMV-Mb from agro-inoculated plants and seeds SYMMV-Mb infection was confirmed in all agro-inoculated genotypes using DAC-ELISA and RT-PCR. In DAC-ELISA, the absorbance values at OD₄₀₅ ranged from 0.84 to 1.45 in Mb and from 0.41 to 0.92 in soybean leaf samples. In contrast, control plants agro-inoculated with an empty binary vector showed much lower absorbance values, ranging from 0.12 to 0.20 (Fig. 2 ). Fig. 2. Open in a new tab Estimation of viral titre in mungbean ( A ), soybean ( B ) leaves, and mungbean seed ( C ) using DAC-ELISA (OD A 405nm ) with SYMMV polyclonal antibodies (dilution 1:500). RT-PCR confirmation of SYMMV-Mb in infected seeds collected from agro-inoculated mungbean genotypes. (Lane details: M: GeneRuler 1 kb DNA ladder; + Ve: Positive control; -Ve: Negative control (healthy seed); Lanes 1–8: RT-PCR amplification from whole seeds of different mungbean genotypes) Among the Mb genotypes, IPM-02–03, Pusa 1413, Pusa-9531, Pusa Vishal, MH-421, SML-668 exhibited higher absorbance values (Fig. 2 A). Similarly, soybean genotypes DS 3109, SL 1191, and PS-24 recorded the highest absorbance values (0.79–0.91) among the tested soybean lines (Fig. 2 B). RT-PCR analysis using CP-specific primers resulted in the amplification of 1065 bp in all the tested Mb and soybean genotypes (Supplementary Figure F1 ). Interestingly, despite the presence of the viral CP in all soybean genotypes as confirmed by RT-PCR, none of the genotype exhibited any visible symptoms. DAC-ELISA testing of seed harvested from Mb genotypes revealed absorbance values ranging from 0.34 to 0.96, providing preliminary evidence for the presence of SYMMV-Mb in the seed material (Fig. 2 C). The genotypes Sona Moong exhibited good vegetative growth but failed to produce pods and was therefore not included in further seed testing. In contrast, seeds from all soybean genotypes tested negative in DAC-ELISA, with absorbance values ranging from 0.10 to 0.35, which were comparable to healthy controls. RT-PCR analysis of seed samples from eight Mb genotypes showed amplification of the 1065 bp fragment corresponding to the CP region in seven genotypes, with the exception of Pusa Ratna (Fig. 2 D). No amplification was observed in any of the soybean seed samples using SYMMV-CP-specific primers. Accumulation of SYMMV RNA in the seed material The accumulation of SYMMV-Mb RNA in seeds harvested from Mb and soybean genotypes was estimated using RT-qPCR. In Mb, viral RNA was detected in seeds from all genotypes although at low copy number. Notably, genotypes Pusa 1371, Pusa 1413, and MH-421 followed by Pusa Vishal exhibited low C t values (21.00 to 24.97) indicating relatively higher level of virus accumulation (Table 3 ). In contrast, none of the soybean genotypes showed detectable levels of SYMMV-Mb RNA in seeds collected from agro-inoculated plants, clearly indicating the absence of seed transmission of SYMMV-Mb in these soybean genotypes. Table 3. SYMMV viral RNA quantification from infected mungbean and soybean seed tissues through reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) S. No Mungbean genotypes Ct Value (Means ± Standard error) Copy Number S. No Soybean genotypes Ct Value (Means ± Standard error) Copy Number 1 IPM-02–3 28.72 ± 0.15 6.33 × 10 1 1 DS-3109 30.88 ± 0.22 4.22 × 10 0 2 Pusa 1371 22.81 ± 0.13 1.08 × 10 5 2 NRC-146 30.57 ± 0.07 6.17 × 10 0 3 Pusa 1413 22.00 ± 0.08 2.83 × 10 5 3 PS-1634 30.54 ± 0.07 6.41 × 10 0 4 Pusa 9531 28.42 ± 0.16 9.14 × 10 1 4 JS-21–71 30.77 ± 0.13 4.84 × 10 0 5 Pusa Ratna 29.11 ± 0.09 3.87 × 10 1 5 MACS-1566 30.61 ± 0.11 5.92 × 10 0 6 Pusa Vishal 24.97 ± 0.63 6.93 × 10 3 6 SL-1191 30.88 ± 0.23 4.20 × 10 0 7 MH-421 21.08 ± 0.31 9.00 × 10 5 7 Himso-1688 30.73 ± 0.12 5.07 × 10 0 8 SML-668 29.12 ± 0.01 3.81 × 10 1 8 PS-24 31.00 ± 0.19 3.63 × 10 0 9 Sona Moong – – 9 RSC 11–17 30.38 ± 0.16 7.89 × 10 0 Positive 8.23 ± 0.24 4.354 × 10 12 10 MAUS-734 30.75 ± 0.11 4.92 × 10 0 Negative – – 11 DSB-33 31.01 ± 0.30 3.59 × 10 0 12 NRC-138 30.20 ± 0.25 9.89 × 10 0 13 JS 21–72 30.33 ± 0.25 8.40 × 10 0 14 PS-1637 29.43 ± 0.11 2.58 × 10 1 15 AUKS-176 30.55 ± 0.36 6.35 × 10 0 Open in a new tab Efficiency of SYMMV seed transmission to the next generations Germination percentage and seedling emergence were adversely affected by SYMMV-Mb infection in both the Mb and soybean genotypes. In Mb, germination ranged from 40 to 76% while soybean genotypes showed 60 to 80% germination, both accompanied by delayed seedling emergence. In contrast, seeds harvested from uninoculated plants showed the 85–95% germination in Mb and 81–90% germination in soybean genotypes. In the grow-out test, germinated seedlings of Mb genotypes Pusa 1371, Pusa 1413, Pusa Vishal, MH-421, and SML-668 exhibited mild chlorotic and necrotic local spots, although at a low infection rate. Other Mb genotypes i.e., IPM-02–3, Pusa-9531 and Pusa Ratna did not show any distinctive symptoms. All soybean seedlings remained symptomless throughout the grow-out period, regardless of genotype. DAC-ELISA testing of ten randomly selected Mb seedlings per genotype revealed that two to five seedlings tested positive for SYMMV-Mb using polyclonal antibodies, with absorbance values ranging from 0.20 to 0.80 (Table 4 ). These results clearly indicated seed transmissibility of SYMMV-Mb in Mb, with a transmission rate ranging from 20 to 50% across cultivars (Table 4 ). In contrast, none of the soybean seedlings tested positive in DAC-ELISA, showing absorbance values between 0.15 and 0.30, confirming the absence of seed transmission of SYMMV-Mb in the evaluated soybean genotypes. Table 4. Effect of SYMMV on seed transmission efficiency in mungbean and soybean through grow-out method Plant species Germination% (No. of seeds germinated/Total No. of seed sown) Seedling infection % (No. of seedlings positive/No of seedlings tested) Symptoms a Confirmation through ELISA Mungbean ( Vigna radiata ) genotypes IPM-02–3 60 (30/50) 20 (3/10) NS 0.29–0.80 Pusa 1371 66 (33/50) 40 (4/10) Nls 0.28–0.73 Pusa 1413 56 (28/50) 30 (2/10) Nls 0.20–0.71 Pusa 9531 70 (35/50) 20 (2/10) NS 0.25–0.60 Pusa Ratna 76 (38/50) 40 (4/10) NS 0.29–0.74 Pusa Vishal 40 (20/50) 40 (4/10) Ns, Vn 0.30–0.65 MH-421 56 (28/50) 50 (5/10) Cls 0.26–0.54 SML-668 50 (25/50) 20 (2/10) Nls 0.29–0.60 Sona Moong – – – – Healthy cultivars 85–95 Soybean ( Glycine max ) genotypes No symptoms were observed for any of the cultivars in grow-out test 0.15–0.30 Diseased genotypes 60–80 Healthy genotypes 81–90 Positive (French bean cv. Arka Sharat) 80 (40/50) 1.70 Healthy 90(45/50) – – 0.15 Open in a new tab Discussion The evaluation of popular and newly developed genotypes against emerging plant viruses is a critical step in identifying resistant sources, which is essential for designing long-term, sustainable, and eco-friendly viral disease management strategies (Gomez et al. 2009 ). In a previous study aimed at identifying resistance to the SYMMV-Mb isolate in widely cultivated soybean genotypes, we observed successful virus replication and systemic movement, with mild or no visible symptom expression (Srikant et al. 2025 ). As a further study, the present investigation involved the screening of nine popular Mb genotypes and 15 recently developed soybean genotypes through agro-inoculation. The study further evaluated the potential for SYMMV-Mb seed transmission under controlled glasshouse conditions. The findings of the present study showed that agro-inoculation delayed symptom expression across all tested genotypes, consistent with previous study (Sandra et al. 2017 ). Most of the agro-inoculated soybean genotypes did not display distinct viral symptoms, except for a few, whereas the Mb genotypes showed severe phenotypic responses, including necrotic local spots and veinal necrosis. This phenotypic response variation may be due to several factors including host genotype, virus strain, plant age and environmental conditions (Eid et al. 2023 ). Similar type of symptom variation was also reported during the evaluation of soybean cultivars against SMV infection, where some cultivars exhibited complete resistance while others were HS (Bachkar et al. 2019 ; Eid et al. 2023 ). Among the nine Mb genotypes evaluated in the present study, six were categorized as susceptible, two as HS, and one as MS. In contrast, out of 15 soybean genotypes, nine were classified as immune, four as moderately resistant, and two as MS based on a 0–5 disease severity scale. The differential sensitivity reaction between Mb and soybean genotypes may be due to the inability of the SYMMV-Mb isolate to induce visible symptoms in the newly developed soybean genotypes. A similar disease scoring approach of 0–5 and 0–9 scale has been employed in previous studies to screen soybean and Mb germplasm against MYMIV under field and net house conditions (Akhtar et al. 2011 ; Ramesh et al. 2019 ; Rahman et al. 2023 ). The DAC-ELISA technique was used for the initial detection of SYMMV-Mb in agro-inoculated genotypes. Among the tested genotypes, the highest virus titres were observed in Mb genotypes IPM-02–3, Pusa 1413, Pusa-9531 and Pusa Vishal followed by soybean genotypes DS-3109 and SL-1191. This elevated titre may be attributed to rapid viral replication and systemic movement of SYMMV-Mb from the inoculation site in these genotypes compared to others. Further confirmation of systemic leaves from agro-inoculated plants through RT-PCR consistently detected the CP gene, with a 1064 bp amplicon, indicating efficient sgRNA production and successful viral replication. The results of DAC-ELISA and RT-PCR in soybean genotypes suggested that there is not necessarily a direct correlation between virus accumulation and visible symptom expression. Similar results have been reported for other plant viruses viz . , SMV and tomato spotted wilt virus (Cho and Goodman 1979 ; Glasa et al. 2003 ). Seed transmission of plant viruses typically require embryo infection which may occur either directly after fertilization or indirectly through gamete infection before fertilization (McDonald and Hamilton 1972 ; Sastry 2013 ; Sandra and Mandal 2024 ). In the present study, the detection of SYMMV-Mb in Mb seeds using DAC-ELISA and RT-PCR revealed high abundance of the virus in seed tissues. However, SYMMV-Mb was not detected in the seeds of soybean genotypes as no amplification was observed via RT-PCR. This could be due to the presence of virus at very low titres in seed tissues (Pagan et al. 2014 ). Even though SYMMV-Mb was detected in Mb seed, the exact mechanism by which seed transmission is prevented in newly developed soybean genotypes remains unclear. Moreover, there is scanty information on the host genes involved in either preventing or facilitating the transmission of plant viruses through seed (Domier et al. 2011 ; Montes et al. 2021 ). Therefore, seed samples were further analyzed through RT-qPCR to estimate viral copy numbers. Real-time PCR utility for quantifying the plant pathogenic viruses has been well- documented because of its enhanced sensitivity compared to the nucleic acid hybridization and serological methods (Korimbocus et al. 2002 ; Mason et al. 2008 ; Ramesh et al. 2019 ). RT-qPCR assay used in the present study for detecting SYMMV-Mb RNA in seed tissues provided strong evidence of SYMMV-Mb seed transmission in various Mb genotypes by revealing the viral copy number. Complementing the DAC-ELISA and RT-PCR, RT-qPCR based viral titre estimation has helped further in confirming the absence of SYMMV-Mb seed transmission in newly developed soybean genotypes. This may be attributed to the genotype’s inherent ability to resist virus multiplication and the presence of seed infection resistance mechanisms that prevent viral entry into the seed (Morales 1986 ). In addition, a good correlation was also observed between the viral copy number measured by RT-qPCR and the seed transmission percentage in Mb genotypes. It was reported that virus infection had a detrimental effect on the overall plant development and the seed quality (Walkey et al. 1985 ). In the present study, seed collected from agro-inoculated Mb and soybean genotypes exhibited poor quality, with a reduced germination percentage compared to seeds from healthy plants. Furthermore, the absence of pod production despite good vegetative growth in Mb cv. Sona Moong definitely indicates the potential economic losses due to SYMMV-Mb infection. Similar detrimental effects of plant viral infections on crop productivity have been reported in other crops as well (Camara et al. 2013 ). Grow-out tests conducted in the present study revealed a seed-to-seedling transmission rate of 20–50% for SYMMV in Mb cultivars, which corresponded with higher absorbance values, while no transmission (0%) was observed in soybean genotypes. The seed transmissibility of SYMMV in Mb may be attributed to the virus entering the embryo during seed development. In contrast, the absence of seed transmission in newly developed soybean genotypes could be due to physical or molecular barriers that prevent the virus from reaching the embryo, or due to the inherent inability of SYMMV-Mb to invade embryonic tissues. Progeny testing is considered a more specific method for assessing vertical transmission, as virus titres tend to be higher in progeny seedlings than in the seeds themselves. Similar variation in seed transmission has been reported for black gram mottle virus, with transmission rates ranging from 1.3% to 15.9% across different blackgram ( Vigna mungo ) cultivars (Varma et al. 1992 ). In conclusion, the SYMMV-Mb isolate was found to be seed-transmissible in all tested Mb genotypes but not in the newly developed soybean genotypes. Ongoing experiments aim to elucidate the mechanisms of host–virus interactions that may prevent viral entry into the seeds of these soybean genotypes. The combination of agro-inoculation and RT-qPCR-based quantification of seed viral load offers a rapid and reliable approach for virus detection in seeds. Further investigation of SYMMV-Mb across diverse leguminous cultivars is essential to identify resistance sources and to better understand the mechanisms underlying seed transmission of this virus. Supplementary Information Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 41 KB) (41.1KB, docx) Acknowledgements We thank National Phytotron Facility (NPF) for providing controlled environmental conditions to carry out the experiments. Funding This work was carried out under the financial support provided by Science and Engineering Research Board (DST-SERB) through Early Career Research Award Grant ECR/2016/001016. Data availability All data generated during the study is included in the manuscript and its supplementary files. Declarations Conflict of interest The authors declare that they have no competing interests. Consent for publication All the authors of this article agree to the publication. Human and animal participants This study does not involve any human participants and animals. References Akhtar KP, Sarwar G, Abbas G et al (2011) Screening of mungbean germplasm against mungbean yellow mosaic India virus and its vector Bemisia tabaci . Crop Prot 30(9):1202–1209 [ Google Scholar ] Ali A, Kobayashi M (2010) Seed transmission of cucumber mosaic virus in pepper. 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