Evaluation of in situ and ex situ forage germplasm collections reveals the first occurrence and seed-transmission of alfalfa mosaic virus and southern bean mosaic virus in Urochloa species

Grasses of the genus Urochloa (Urochloa species syn. Brachiaria species) are among the most important tropical forage grasses of African origin. The International Livestock Research Institute (ILRI) Forage Genebank maintains 671 Urochloa grass accessions, belonging to 28 species, of which 261 (39%) accessions are Urochloa brizantha (A.Rich.) R.D.Webster. The susceptibility of Urochloa grasses to disease and insect pests is a key challenge to the sustainable production of these grasses in Africa. In this study, using a dot blot assay and RT-PCR, we report for the first time the association of Alfalfa Mosaic Virus (AMV) and Southern Bean Mosaic Virus (SBMV) with 92% of the tested accessions conserved in situ and ex situ in the ILRI forage genebank. In addition, virus transmission from seed to plant and from legume to grass was confirmed through the bioassay test. The detection of these two viruses in Urochloa species presents a new challenge to germplasm conservation, distribution, and their sustainable production in the region, and demands immediate attention towards developing an effective method of cleaning viruses from the conserved germplasm. Furthermore, there is an urgent need to assess the economic importance of diseases in the grasses, and for the selection of germplasm that shows resistance to diseases.


Evaluation of in situ and ex situ forage germplasm collections reveals the first occurrence and seed-transmission of alfalfa mosaic virus and southern bean mosaic virus in Urochloa species
Currently, the ILRI forage Genebank maintains 671 Urochloa grass accessions, containing 28 species, of which 261 (39%) accessions are Urochloa brizantha.The susceptibility of Urochloa species to diseases and insect pests is a key challenge to the sustainable production of these grasses in Africa.Worldwide, tropical forage grasses are known to be infected by at least 21 viruses of different genera (Jones 2013).In Ethiopia, potyviruses such as elephant grass mosaic virus (EGMV), johnsongrass mosaic virus (JGMV), maize dwarf mosaic virus (MDMV), and sugarcane mosaic virus (SCMV) are reported as the major challenges for the forage grasses (Mih and Hanson 2004).The improved cultivars of Urochloa have also been reported to be infected by fungi causing diseases such as leaf rust, leaf spot, leaf blight, and smut in Kenya (Nzioki et al. 2016), and leaf blight, rust, leaf spot and ergot in Rwanda (Uzayisenga 2021).
Seeds have been identified as a means of long distance spread of seed-borne viruses (Hampton et al. 1982;Bos and Makkouk 1993).To ensure that only seeds of satisfactory phytosanitary status are distributed to recipients in geographically diverse areas, the ILRI genebank routinely monitors seed-borne diseases during seed multiplication and in the field genebank.Among the targeted quarantine pathogens, seedborne fungi such as Alternaria, Bipolaris, Fusarium, Macrophomina and Phoma, and viruses of the potyvirus group are frequently detected in seeds of Urochloa species (unpublished: personal communication with Yesuf Eshete, Mycologist at Germplasm Health Unit, ILRI, December 2022).In 2022, regular seed sample health testing found alfalfa mosaic virus (AMV) and southern bean mosaic virus (SBMV) for the first time in Urochloa grasses.Alfalfa mosaic virus is one of the most economically significant seedborne plant pathogens, occurring worldwide and known to naturally infect 698 species of 167 genera in 71 families (Jaspars andBos 1980 Edwardson andChristie 1997;Fletcher 2001;Xu and Nie 2006).The disease results in yield losses of up to 41% for shoot dry weight and 45% for seeds (Latham et al. 2004).Southern bean mosaic virus (SBMV) is a seedborne pathogen.The genus Sobemovirus, named after the type member SBMV, comprises 11-14 virus species that infect both monocots and dicots.The species SBMV has a very narrow host range with various species of Leguminosae known to be susceptible to infection (Hull 2004).Initial infections in new areas are usually due to transmission of the virus from seeds, with subsequent spread by beetles (Kopek and Scott, 1983).The virus can reduce bean yields by 83-94%, affecting both the amount and weight of seed produced by infected plants (Morales and Castaño 1985).These two viruses have not previously been detected in forage grasses such as Urochloa, thus, this study reports for the first time the association of AMV and SBMV with Urochloa grasses, conserved in situ and ex situ in the ILRI forage genebank, and also confirms their seed-borne nature these grass species.

Field conditions and layout
The ILRI forage genebank has maintained 658 accessions of Brachiaria grasses (Urochloa spp.) in-situ at its Zwai field site Ethiopia, over the past 30 years.Each Urochloa accession is planted in six rows, 3 m in length.The spacing between plants and rows is 50 cm each.The field site is located about 100 miles south of Addis Ababa, on the border between Oromia and the Southern Nations, Nationalities, and Peoples regions at 7.9000 N, 38.7348 E and altitude of 1650 m a.s.l.The average annual minimum and maximum temperatures are 13 and 27 °C, respectively and annual average rainfall is 600 mm (mostly occurring between July and September).Soil is loamy sand with 0.5 m depth and pH from 8.1 to 8.4.
In 2022, during the routine monitoring at Zwai field genebank, mosaic and yellowing symptoms were observed on some Urochloa species, particularly on U. brizantha (Figure 1).An initial investigation of symptomatic leaves collected from twelve accessions of U. brizantha revealed the association of AMV and SBMV with all the accessions.Further investigation was carried out on individual 136 accessions: 72 U. brizantha (53%), 27 U. decumbens (20%), 29 U. dictyoneura (21%) and 8 U. jubata Sosef (6%).During March 2022, leaf samples were collected from the two middle and the top two leaves from randomly selected ten plants from both symptomatic and asymptomatic accessions.All the leaf samples were combined for each accession separately.The samples were placed in a cool box, having ice packets around the samples to keep them cool during sample processing and subsequent transportation to ILRI-GHU laboratory, Addis Ababa.In the laboratory, samples were kept at −80 °C prior to freeze drying.Samples were dried in a freeze drier for three days at a vacuum temperature of −55 °C and sample temperature of −30 °C.

Leaf samples
During the growth period of the field trials in February-March 2022, mosaic and yellowing symptoms were observed on some Urochloa species, particularly on U. brizantha (Figure 1).An initial investigation of symptomatic leaves collected from twelve accessions of U. brizantha revealed the association of AMV and SBMV with all the accessions.Leaf samples were collected from the Zwai field site during March 2022 from 136 Urochloa accessions, 72 U. brizantha (53%), 27 U. decumbens (20%), 29 U. dictyoneura (21%) and 8 Urochloa jubata (6%).The two middle and the top two leaves were collected from 10 randomly selected plants from both symptomatic and asymptomatic accessions.All the leaf samples were combined for each accession separately.The samples were placed in a cool box, with ice packs around the samples to keep them cool during sample processing and subsequent transportation to ILRI-GHU laboratory, Addis Ababa.In the laboratory, samples were kept at -80 °C prior to freeze drying.Samples were dried in a freeze drier for three days at a vacuum temperature of −55 °C and sample temperature of −30 °C.

Seed samples
In addition to the leaf samples, seeds of the same 136 accessions were taken from the ILRI Genebank to be tested for the presence of AMV, SBMV and potyvirus.The selection of the accessions was made based on the availability of enough seeds for testing.The origins of the selected accessions were Burundi, Congo, Ethiopia, Kenya, Rwanda, Tanzania and Zambia (Table 1).For the dot-blot assay, 70 seeds per accession were surface-sterilised using 10% sodium hypochlorite (NaOCl) and placed onto three layers of moist sterile blotter paper in petri dishes (100 mm × 15 mm).The petri dishes were incubated at 23 ± 1 °C with alternate cycles of 12 hours of light and dark for two weeks.All the germinated seedlings per accession were then collected into a sample bag (Universal 12 × 15 cm Art.No. 430100, BIOREBA Switzerland) and crushed and ground thoroughly in the sample bag using a sample homogeniser (HOMEX 6, Art.No. 400005/400004, BIOREBA, Switzerland).The resulting extracts were used for the dot-blot assay.

Dot blot assay
The dot blot assay was carried out using the method described by Cardosa et al. (2002).The crude extracts (6 μl) from homogenised leaves extracted in carbonate buffer comprising 2% polyvinyl pyrrolidine (PVP, MW 40 000) were spotted onto a pre-washed nitrocellulose membrane (NCM), soaked in 1x phosphate buffer saline (PBS), pH 7.2.The NCM was air-dried, and the free binding sites were blocked by soaking the membrane in 5% non-fat skimmed milk prepared in 1 x PBS for 30 minutes.All the membranes coated with the samples were then washed five times at 10 minute intervals with 1 x PBS, pH 7.2.The membranes coated with the sample extracts were then soaked in specific antiserum (DSMZ, Germany) diluted (1:1 000 dilution) in PBS-TPO (PBS with 0.05% Tween 20, 2% PVP and 0.2% ovalbumin) and incubated at 37°C for 1 hour.They were then incubated for another 1 hour at 37°C in a 1:3 000 dilution of goat anti-rabbit IgG-Alkaline phosphatase conjugate (Sigma Aldrich, St. Louis, USA) prepared in PBS-TPO.Subsequently, the membranes were incubated in freshly prepared substrate solution of 5-Bromo-4-chloro-3-indolyl phosphate (BCIP)/ 4-nitro blue tetrazolium chloride (NBT) in dark conditions, achieved by covering the tray with an aluminium sheet, at 37°C for 15-20 mins or until the development of a purple-blue colour.All the membranes were washed with 1 x PBS 5 times at 10 minute intervals after each step of the dot-blot assay.

RNA extraction, Reverse transcriptase PCR (RT-PCR) and sequencing
Total RNA was extracted from freeze dried leaf samples of all 136 Urochloa accessions using TRI Reagent ® and following the protocol of Chomczynski and Sacchi (2006).As a reference, RNA was also extracted from the freeze-dried inoculums of AMV and SBMV, obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Germany.Approximately 50 mg of freeze-dried leaf material was homogenised using a mortar and pestle and mixed with 1 ml of Trizol reagent (Thermo Fisher Scientific, USA) followed by phase separation using extraction with an equal volume of chloroform and centrifugation (12 000 × g for 15 mins at 4°C).The aqueous phase was pipetted into a clean centrifuge tube, the RNA was then precipitated using isopropyl alcohol,  washed with 75% ethanol, and re-dissolved in RNase-free water.The quality and quantity of RNA was determined using a nanodrop spectrophotometer (DeNovix DS-11, Thermo Fisher Scientific).
A pair of virus-specific primers, each for AMV (GBV53F/ GBV97R) and SBMV (SBMVF1/SBMVR1) was developed in this study from the coat protein (CP) region and CP and 3' UTR regions respectively for the specific detection of each virus.Potyvirus genus-specific primers were used to determine the status of potyvirus infection in the Urochloa grasses (Table 2).The virus was detected by RT-PCR in a two-step process in which the first strand cDNA was synthesised in a 20 μl reaction mix, where 2 μl of RNA template (̴ 500 ng) was mixed with 1 µl each of Oligo dT primer and 10 mM dNTP mix with the volume adjusted to 15 μl using nuclease-free water.The mixture was heated to 65 °C in a thermocycler for 5 minutes followed by the addition of 4 μl of 5 x RT buffer and 1 μl Maxima H Minas Enzyme mix (ThermoFischer, Scientific, USA).The first strand cDNA synthesis was conducted by heating the mixture to 50 °C for 30 minutes followed by heating at 85 °C for 5 minutes.After this, PCR was carried out in a 25 μl reaction volume containing 2 μl of cDNA, 12 μl DreamTaq Master Mix (Thermo Fisher, USA), 1 μl of 10 μM each of forward and reverse primer, and 9 μl of nuclease-free water.The thermal cycling conditions were an initial denaturation step at 98 °C for 30 seconds followed by 35 cycles of 98 °C for 10 seconds, annealing temperatures of 54 °C for AMV, 53 °C for SBMV and 45 °C for Potyvirus for 1 minute.This was followed by exposure to 72 °C for 1 minute with a final extension at 72 °C for 10 minutes.The amplified products were resolved by electrophoresis on 1% agarose gel and visualised under a UV gel documentation system (Intas, Germany).As the specificity of the primers for AMV (GBV53F/GBV97R) was confirmed earlier by RT-PCR using RNA extracted from the pure isolate of AMV maintained on Chenopodium amaranticolor (data not shown), only PCR products of SBMV were sequenced using the commercial facility at Macrogen, Republic of Korea, for sequencebased confirmation of association of SBMV with Urochloa accessions.

Bioassay
The test was carried out to confirm the seed transmission of AMV and SBMV as well as transmission of the viruses from legumes to grasses.

Seed transmission study
Seedlings of five accessions of U. brizantha (13467.3, 13584.2, 13352.2, 13368.2 and 13373.2),which were pregerminated on moist blotting paper for approximately two weeks, were transplanted into pots containing a soil mixture of sand, compost, and clay in equal ratios.To avoid infection of plants from external sources, all plants were kept in isolated/ insect proof mesh cages.When the plants reached the three-leaf stage (after two months), all plants were pooled into nine groups, in which each pool consisted of five plants.Initially, the composite samples from each group were tested for AMV and SBMV infection using RT-PCR following the procedure described above, followed by testing the individual plants of those groups which tested positive by RT-PCR.The percentage seed infection was calculated by dividing the number of positive plants by the total number of plants ( 45) tested and multiplied by 100.

Mechanical transmission study
The two upper leaves of two-month-old seedlings of ten (five for each virus) healthy U. brizantha plants (tested negative by RT-PCR) were mechanically inoculated with inocula of the two viruses which had been collected from legumes, and purchased from DSMZ, Germany.For inoculation, dry plant material (inoculum) was placed into a pre-cooled mortar and pestle, reconstituted in 4 ml of inoculation buffer (0.05 M sodium/potassium phosphate buffer, pH 7.0, 1 mM EDTA, 5 mM DIECA, 5 mM thioglycolic acid) and thoroughly ground until a green paste was obtained.The composition of inoculation buffer used in the experiment was suggested by DSMZ, Germany, from where the inocula were purchased.The virus extract was gently rubbed onto the leaves of Urochloa plants which had been dusted with a sterile carborundum powder (Size ca.24 grit, Thermo Fisher Scientific).After inoculation, the plants were rinsed/ sprayed with water to remove residual inoculum and to prevent excessive evaporation.Gloves were changed between each plant to avoid carryover of infectious material.After four weeks of inoculation, leaf samples were collected, and the associations of the viruses were detected by RT-PCR using specific primers.

RT-PCR detection of AMV, SBMV and Potyvirus in leaf samples collected from the field
Leaf samples of all the 136 Urochloa accessions (Table 1) collected from the ILRI Zwai field genebank were tested for target viruses in a two-step RT-PCR reaction using virus/ genus specific primers (Table 2).Regardless of the species, the test results revealed that 95 (70%) and 108 (79%) of the accessions tested positive for AMV and SBMV respectively, with the expected product size of 632 bp (AMV) and 231 bp (SBMV).One hundred and twenty-five (92%) of the accessions were positive for AMV and/or SBMV viruses and AMV and SBMV were detected on all the four species.The highest percent infection was detected in U. jubata which was 8 (100%) and 7 (88%) for AMV and SBMV, respectively, followed by U. decumbens with 25 (93%) for AMV and 22 (81%) for SBMV.In the case of U. brizantha, AMV and SBMV were detected in 36 (50%) and 57 (79%) of the accessions, respectively.However, the infection for potyvirus was recorded only in 19 (14%) Urochloa accessions (Table 3).Further, BLASTn analysis of the sequences obtained from the PCR products also confirms the association of SBMV with the Urochloa grass (Supplementary Table S1).

Serological detection of AMV, SBMV and Potyvirus in seed samples
The dot-blot assay performed on the seeds of the 136 accessions of Urochloa species using the virus-and genusspecific antisera revealed that 83 (61%) of the accessions exhibited a positive reaction with the AMV antiserum, followed by the SBMV 82 (60%) and potyvirus 80 (59%) antisera (Table 3).Similar to the test result of leaf samples, SBMV and potyvirus were detected in all the four species, whereas AMV was detected in U. brizantha, U. decumbens, and U. dictyoneura but not in U. jubata (Table 4).

Seed transmission of AMV and SBMV
Of the nine groups formed in the process, each containing a pool of five plants, the expected 632 bp (AMV) and 231 bp (SBMV) fragments extracted by RT-PCR were obtained only for three groups.Of the fifteen individual plants tested from these three groups, 8 and 10 of the plants were found to be positive for AMV and SBMV, respectively, indicating seed transmission (seed-borne) of the two viruses with transmission rates of 18% (AMV) and 22% (SBMV).

Mechanical transmission of AMV and SBMV
The sap transmission of virus inocula from legumes to the healthy U. brizantha plants produced mosaic and chlorotic lesion symptoms in the plants at 30 days post inoculation (Figures 2a and 2b).Viral infection in the inoculated U. brizantha was confirmed by RT-PCR (Figure 2c), which confirmed the association of AMV and SBMV in all (100%) of the inoculated plants.This indicates that both legumes and Urochloa grasses could be infected by the same strains of AMV and SBMV.

Discussion and conclusion
Evaluation of in situ and ex situ Urochloa germplasm revealed the first evidence of the occurrence and seed-transmission of AMV and SBMV in several Urochloa species.Alfalfa mosaic virus causes widespread disease resulting in yield losses of up to 41% for shoot dry weight and 45% for seeds (Latham et al. 2004).Alfalfa mosaic virus was first isolated from alfalfa (Medicago sativa L.), which is the main natural host, and the pathogen infects over 165 species, mainly in the Fabaceae and Solanaceae families (Edwardson and Christie 1997).The virus is predominantly seed-borne but can also be transmitted by several aphids and mechanical inoculation (McLaughlin 1991;Valkonen et al. 1992;He et al. 2010).Southern Bean Mosaic Virus, of the genus Sobemovirus, has a very narrow host range; previously only species of Leguminosae have been shown to be susceptible to infection (Hull 2004).It can be transmitted by a vector (insect), sap, mechanical inoculation, grafting and seeds.In the present study, these two viruses were detected in 92% of the tested Urochloa accessions conserved in situ and ex situ at the ILRI forage genebank, which is the first recorded report known to these authors This implies that both cultivated and wild forages  could act as reservoirs for important plant pathogens with a great risk of cross-border spreading to new areas.Jaspars and Bos (1980) and Hajimorad and Francki (1988) reported that AMV develops distinct strains and variants which help the virus in the expansion of it's host range and increases it's standing as being one of the most economically important diseases worldwide (Trucco et al. 2021;Topkaya 2022).
The tested leaf and seed samples represented accessions from four species of Urochloa: U. brizantha, U. decumbens, U. dictyoneura, and U. jubata.Regardless of the discrepancy in the number of samples tested in each species, AMV and SBMV were detected in all the tested species.All the germplasm used in the present study originated from Africa (Burundi, Congo, Ethiopia, Kenya, Rwanda, Tanzania, and Zambia), and was from three of the four Urochloa species of African origin (Keller-Grein et al. 1996); that is, excepting U. jubata.The centre of crop origin is seen as the centre of variability of pathogens and pests (Jennings and Cock 1977), which might be the possible reason for the infection of Urochloa by these two uncommon viruses in grasses.Mih and Hanson 2004 reported that potyviruses, which include elephant grass mosaic virus (EGMV), johnsongrass mosaic virus (JGMV), maize dwarf mosaic virus (MDMV), and sugarcane mosaic virus (SCMV), are the most common viruses affecting grasses in Ethiopia.In the present study the high percent of potyvirus infection exhibited in 59% of seeds and 14% of leaf samples supports the previous report of Mih and Hanson (2004).Similarly, the high levels of AMV and SBMV infection on leaf and seed samples indicate that these two viruses are equally important for Urochloa species.
Seeds have been identified as a cause of long-distance spread of seed-borne viruses such as AMV and SBMV (Hampton et al. 1982;Bos and Makkouk 1993).The present study supports the seed transmission of the two viruses in Urochloa grasses, and possible infection in legumes (beans) and Urochloa grasses by the same strains of AMV and SBMV.In addition to the previously reported hosts, these two viruses could be spread to new areas through the exchange of infected seed of Urochloa grass.Therefore, it is important to make sure that only seeds of satisfactory phytosanitary status for AMV and SBMV are distributed.The detection of these two viruses in Urochloa species presents a new challenge to germplasm conservation, distribution, and its sustainable production in the region and demands immediate attention towards developing an effective virus-cleaning method of the conserved germplasm.Furthermore, there is also an urgent need to assess the economic importance of the diseases on the grass and selection of germplasm that has greater resistance to the diseases.

Figure 1 :
Figure 1: Leaves of Urochloa brizantha showing (a) symptoms of viral infection and (b) healthy leaves

Figure 2 :
Figure 2: Leaf damage caused by viruses after sap inoculation of (a) AMV and (b) SBMV on Urochloa brizantha plants.(c) RT-PCR confirmation of viruses in U. brizantha plants inoculated with AMV and SBMV extracts as follows: Lane 1-5: Amplification of AMV from sap inoculated individual plant numbers 1 to 5 Lane 6: Positive control for AMV Lane 7-11: Amplification of SBMV from sap inoculated individual plant numbers 1 to 5 Lane 12: Positive control for SBMV; M = 1 Kb plus ladder

Table 1 :
List of Urochloa species, origin and number of accessions tested in the study

Table 2 :
Primer sequences, annealing temperature and expected fragment size

Table 3 :
RT-PCR detection of viruses in Urochloa species collected from Zwai field genebank site in 2022