​​Screening Cowpea Genotypes for Resistance to Cowpea Aphid Borne Mosaic Virus (CABMV) and Cowpea Severe Mosaic Virus (CPSMV) in Paraguay

Cowpea [Vigna unguiculata (L.) Walp.] seeds pose tremendous nutritional value for its high protein and carbohydrate content (Lambot, 2002). It is of extreme importance for Paraguayan rural families and is cultivated throughout the country on approximately 72,000 ha annually, involving about 230,000 small growers (Mag/dcea 2017).

Diseases and pests affect cowpea production resulting in severe yield losses. In Latin America, the main viruses that infect cowpea crops are cucumber mosaic virus (CMV) (genus Cucumovirus, family Bromoviridae); cowpea aphid-borne mosaic virus (CABMV) (genus Potyviridae, family Potyvirus); cowpea severe mosaic virus (CPSMV) (genus Comovirus, family Comoviridae) and cowpea golden mosaic virus (CPGMV) (genus Begomovirus, family Geminiviridae) (Lima et al., 2005; Pio-Ribeiro and Assis-Filho, 1997; Barreto, 1999). In Paraguay, only CABMV and CPSMV are present in cowpea crops (Gonzalez-Segnana et al., 2013; Delgado et al., 2014). 

CABMV was first described in Paraguay infecting sesame plants (Sesamum indicum) (Gonzalez-Segnana et al., 2011). In addition to cowpea and sesame, weeds and cover crops have been found infected with CABMV and its presence has been confirmed in at least three departments of the country (Gonzalez-Segnana et al., 2013). Natural transmission occurs in a nonpersistent manner by various aphid species (Bashir et al., 2002). Aphid crassivora proved to be vectors of CABMV in Paraguay (Natalia de Jesus et al., 2013).

CPSMV was first detected in Paraguay in seed-borne infected cowpea seedling from field symptomatic cowpea plants (Delgado et al., 2014). Natural transmission occurs in a semi-persistent manner by some Chrysomelidae beetles. Cerotoma arcuata was reported as the most important vector in Brazil (Costa et al., 1978; Lima et al., 2005).

Planting resistant cultivars is the most economical, effective and practicable method to minimize yield losses caused by CABMV and CPSMV (Taiwo, 2003). Plant resistance against viral diseases begun with the identification of an appropriate resistance source. Since biological properties of CABMV and CPSMV may differ among isolates worldwide (Bashir et al., 2002; Hampton et al., 1997) it becomes imperative to identify resistance sources against the local prevalent viral isolates.

This work aimed to evaluate the response of some local and introduced cowpea cultivars/genotypes to infection with local isolates of CABMV and CPSMV to identify resistant material to be used in further breeding programs.

Virus isolates
 
CABMV and CPSMV isolates collected from field-infected cowpea plants in the region of Choré, San Pedro Department, Paraguay, were used for the experiments. Virus isolates were separately maintained on cowpea plants through the periodical mechanical transmission. Plants were kept in a greenhouse at the Dept. of Biology FCA/UNA, San Lorenzo, Central department, Paraguay. Virus presence in source plants was confirmed by partial nucleotide sequencing of each virus target gene amplified by RT-PCR. Total RNA was extracted from cowpea sources plants using Trizol and analyzed by RT-PCR using potyvirus universal primers (forward: 5' -GATTTAGGTGACACTATAGT17-3', reverse: 5' -ATGGTTTGGTGYATYGARAAT-3') (Mackenzie et al., 1998; Maciel et al., 2011) and comovirus universal primers (forward: 5'-GCATGGTCCACWCAGGT-3', reverse: 5'-YTCRAAWCCVYTRTTKGGMCCACA-3') (Beserra et al., 2011). Obtained amplicons were purified with Wizard® SV Gel and PCR Clean-Up System (Promega) and sent for direct nucleotide sequencing at Macrogen (Seoul, South Korea). Nucleotide sequence of local CABMV isolate shared 95.56%-95.70% identity with CABMV isolates available in the GenBank (accession no. MK472693- KM597165). CPSMV isolate from Paraguay was partially sequenced for the first time and two nucleotide sequences were deposited in the GenBank (accessions no. KF793279- KF793280). They shared 93.77%-99.64% identity with corresponding nucleotide sequences of CPSMV isolates from Brazil (accessions no. GQ229416- AF263549). Molecular detection was carried out at ESALQ/USP.

Samples of CPSMV source plants were analyzed by transmission electron microscopy (TEM) to complement the identification of CPSMV, since the only available evidence of its presence in the country was done by serologic detection (Delgado et al., 2014). Examinations were made in a Zeiss EM 900 or JEOL 1011 transmission electron microscopes at ESALQ/USP and images were recorded digitally. TEM analysis of thin sections of symptomatic cowpea leaf tissue confirmed the presence of a high concentration of CPSMV isometric virus particles ca. 25 nm in diameter, often forming a crystalline array (Fig 1).



Evaluation of cowpea resistance to CABMV and CPSMV infection
 
For evaluation of the response of cowpea cultivars/genotypes to infection with each virus, independent experiments were conducted in 2018 and 2019, respectively. The seed of the following cowpea cultivars and/or genotypes were separately sown in 1.5 L plastic pots containing substrate; local cultivars: Arroz Rojo (Vigna angularis), Arroz Negro, Blanco Ojo Negro, Choré, Crema IAN, Ñu, Pyta, San Francisco; introduced genotypes: CNCX-698-128F TVu-379, TVu-382, TVu-3961, TE94-256-2E, TE94-200-49F, TE97-309G-9 and TE97-309G-3. After germination, seedlings were transplanted to 1.5 L plastic pots containing a mixture of soil, sand and sterile organic matter.

Mechanical transmissions were performed using extracts from each viral source plants prepared by grinding symptomatic leaves in a mortar in the presence of 0.01 M phosphate buffer (pH 7.0) with 0.1% sodium sulfite, diluted 1:5 (w/v). The extracts were then applied to 10 plants of each cultivar, at 4-6 leaf stage. The inoculum was manually rubbed on the surface of two leaves previously dusted with carborundum. Ten plants per cultivar were mock inoculated as controls. Plants were kept in the greenhouse of the Dept. of Biology FCA/UNA, for further development. Eight weeks later, all plants were analyzed by symptoms expression and PTA-ELISA for virus detection. The experiment was repeated twice.
 
Virus detection
 
PTA-ELISA was performed to detect each virus in newly emerged leaves of mechanically inoculated test plants as described by Mowat and Dawson (1987). CABMV and CPSMV antisera were diluted 1:1000. CABMV antiserum was produced by one of the authors (JAMR, unpublished data), whereas the other was produced by Dr. M.T. Lin, Universidade de Brasília, DF, Brazil. Alkaline phosphatase conjugated anti-rabbit (Sigma) diluted at the ratio of 1:7000 was used. The reaction was considered positive when the OD405nm reading was at least three times that of the healthy control. A Perlong model DNM 9602 ELISA reader was used. The immunoassays were carried out at FCA/UNA.

The reaction of plants of eight local and eight introduced cowpea cultivar/genotypes to infection with CABMV and CPSMV throughout mechanical inoculation is shown in Table 1.



Plants of the introduced cowpea genotypes TVu 379, TVu 382, TE94-256-2E and TE97-309G-9 were resistant to infection with CABMV, whereas CNCX-698-128F, TVu 379, TVu 382, TVu-3961, TE97-309G-9 and TE97-309G-3 were resistant to CPSMV.

CABMV and CPSMV have been accounted as the most widespread viruses on cowpea crops worldwide (Emechebe and Lagoke, 2002; Bashir et al., 2002). Several studies for identifying CABMV and CPSMV resistant cowpea genotypes were made and used in cowpea breeding programs (Lima et al., 1986; Dhanasekar and Reddy, 2015; Leao et al., 2016; Assuncao et al., 2005). In Brazil, Lima et al., (1986) identified four genotypes resistant to both viruses (TVu 379, TVu 382, TVu 966 and TVu 3961), two of those genotypes (TVu 379, TVu 382) evaluated in the present study were resistant to local isolates of CABMV and CPSMV. However, TVu 379, TVu 382 are considered outside the commercial standard, needing a pre-breeding before being included in a breeding program (Oliveira, 2012; Nogueira et al., 2011).

Cultivar TVu 382 was used as a donor parent of CPSMV resistance gene in a few works. Crosses segregate in the proportion of 3 susceptible plants: 1 resistant plant and was considered promising for obtaining resistant cowpea cultivars (Lima, 2019; Lima et al., 1986; Assuncao et al., 2005). Similarly, cultivar TE97-309G-9 was also found resistant to each local virus isolates and used as donor parent to identify cowpea-resistant plants by retro-crossing. Segregation of the F2 plants in all crosses was homogeneous, in the proportion of 15 susceptible: 1 resistant (Barros et al., 2013).

Plants of CNCx698-128F were susceptible to CABMV in the present study; however, they were resistant to this potyvirus in the study made by Nogueira et al., (2011) in Brazil. This difference can be attributed to virus isolates differences since distinct CABMV serotypes/isolates have been reported worldwide and biological properties of CABMV may differ among isolates (Bashir et al., 2002; Huguenot et al., 1993, Ndiaye et al., 1993; Barros et al., 2013). The same has been reported for CPSMV, as nine serotypes have already been detected worldwide (Hampton et al., 1997). Four serological groups were identified (I, II, III and IV) in Brazil (Lin et al., 1981; Lin et al., 1984). In this context, genotype TE94-256-2E was susceptible to the local isolate of CPSMV and the serotype I of a Brazilian isolate, as reported by Oliveira et al., (2012). However, plants of this genotype were resistant to the Brazilian serotype I of CPSMV (Oliveira et al., 2012).  The same authors showed that cultivar TE97-309G-9 was susceptible to infection with serotype I. Nevertheless, in the present study, plants of TE97-309G-9 were resistant to infection with the local isolate of CPSMV. No serotype characterization is available for the Paraguayan isolates of CABMV and CPSMV. These results can serve as biological evidence for future serotype characterization.

Local cultivars (Arroz Negro, Blanco Ojo Negro, Choré, Crema IAN, Ñu, Pyta and San Francisco) were highly susceptible to infection with both virus and developed variable symptoms. Plants of cultivar Arroz rojo (V. angularis) were resistant to infection with both viruses, as none of the inoculated plants developed symptoms; neither PTA-ELISA detected the viruses in the inoculated plants.

The control of viral diseases on cowpea crops worldwide has been achieved using resistant cultivars and hybrids. Since simultaneous infection with both viruses is expected, selecting resistant cowpea cultivars for controlling this double infection is important (Lima et al., 1986). In the present work, cowpea genotypes TVu 379, TVu 382 and TE97-309G-9 were resistant to infection with local isolates of CABMV and CPSMV. These genotypes presented asymptomatic plants under mechanical experimental inoculation with both viruses and tested negative for virus detection in PTA-ELISA. Other studies had shown their potential for obtaining new cultivars with multiple resistance to CPSMV and CABMV (Barros et al., 2013; Lima et al., 2019).