Cowpea Aphid-borne Mosaic Virus Isolates Research Articles

Interaction between Cowpea Aphid-Borne Mosaic Virus Isolates and Its Effect on Passion Fruit Woodiness Disease on Passiflora edulis Sims and Passiflora ligularis Juss

In East Africa, passion fruit woodiness disease is caused by potyviruses, among which are Cowpea Aphid-Borne Mosaic Virus (CABMV) and Uganda Passiflora Virus (UPV). Previous studies suggest that synergistic interaction of viruses causes mild or severe outcomes of the disease. However, mixed infections of these viruses have not been documented. The aim of this study was to determine the effects of the interaction between two CABMV isolates causing passion fruit woodiness disease in Kenya. Healthy plants were mechanically inoculated at six-leaf stage. Four treatments were used: CABMV isolate 1, CABMV isolate 2, mixture of the two isolates, and noninoculated control. The test plants were maintained in a screen house, and data on symptom severity and rate of disease development were recorded. ELISA test was conducted to confirm virus presence and distribution. The results indicate that the CABMV isolates used are systemic. There were various differences in disease progression, which was faster in the sweet passion variety than in the purple variety. However, the latter had a higher disease severity. Coinfected plants expressed severe symptoms compared to the singly infected ones, indicating synergistic interaction between the viral isolates. This implies that coinfection has adverse impact resulting in higher economic losses.

A survey of RNA genome viruses in Lima bean crops of Northeastern Brazil

ABSTRACT Lima bean plants showing mosaic and leaf distortion symptoms, similar to those induced by viruses, were observed in several production areas in the states of Ceara and Piaui, Northeastern Brazil. The aim of this study was to identify RNA genome viruses that infect lima bean. Field research was conducted from 2017 to 2018. Fifty-five symptomatic samples were randomly collected from seven properties in five municipalities and tested by RT-PCR and DNA sequencing with specific or universal primers for two viruses and two virus groups. Four virus species were identified: cucumber mosaic virus (CMV), cowpea mild mottle virus (CPMMV), cowpea aphid-borne mosaic virus (CABMV) and cowpea severe mosaic virus (CPSMV), which had infection levels of 21.8, 52.7, 47.2, and 1.8%, respectively, as well as double and triple infections. The CMV isolates belonged to subgroup IA. The CPMMV isolates had high nucleotide identity with CPMMV isolates from Brazil, USA and Mexico. The CABMV isolates showed moderate nucleotide identity with Brazilian isolates. Only one sample was infected with CPSMV. This is the first record of CPMMV and CPSMV naturally infecting lima bean. Approaches to virus control are discussed.

English

Bambara groundnut (Vigna subterranea) is an African legume with a great nutritional, economic and social potential. However, one of the main constraints to this crop is viral diseases which reduced yields. Indeed, approximately 12 viruses have been reported to infect bambara groundnut. Among these, only four were reported from Burkina Faso, namely, cowpea aphid-borne mosaic virus (CABMV), bean common mosaic virus strain-blackeye cowpea mosaic (BCMV-BlCM), peanut mottle virus (PeMoV), and cowpea mottle virus (CPMoV). This study was carried out in order to identify and characterize the main viruses occurring in bambara groundnut from Burkina Faso using serological and molecular tests. 140 plants were sampled in the three agro-climatic zones of Burkina Faso. Both Potyvirus CABMV and BCMV-BlCM were identified in 8.57% of the samples when double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test was used and 14.29% of the samples when reverse transcription-polymerase chain reaction (RT-PCR) test was used. Phylogenetic tree based on 476 nt in coat protein showed that all Burkina Faso CABMV isolates clustered together with Uganda isolate (KT726938). However, two groups were distinguished within these isolates. Burkina Faso BCMV-BlCM isolates are strengthly clustered with BCMV and BlCMV group. This study reports the first molecular characterization of CABMV and BCMV-BlCM infecting Bambara groundnut in Burkina Faso. Key words: Bambara groundnut, cowpea aphid-borne mosaic virus (CABMV), bean common mosaic virus strain-blackeye cowpea mosaic (BCMV-BlCM), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), reverse transcription-polymerase chain reaction (RT-PCR).

English

Woodiness disease is the most important disorder of passion fruit worldwide. The causal agent in Brazil is the Cowpea aphid-borne mosaic virus (CABMV), and despite the economic relevance of passion fruit for agriculture there have been recently very few studies about this virus in Brazil and worldwide. This work reveals the phylogenetic relationships of 10 newly identified CABMV isolates from Bahia State, the region where CABMV was first identified (in that time reported as PWV) in South America before its outbreak. The coat protein of 10 CABMV isolates (CABMV-Lns1 - CABMV-Lns10) from Livramento de Nossa Senhora Country, Bahia State, were sequenced and presented very close identity between themselves (nucleotide: 97 to 99%, amino acid: 95 to 100%). They are phylogenetically closely related to Brazilian CABMV, however forming an isolated cluster within the Brazilian clade. According to previous evidences, our data demonstrate that CABMV-Lns are more closely related to isolates from Southern rather than from Northern Africa. Other two isolates from Bahia State clustered separately from CABMV-Lns, but together with isolates from other Brazilian regions thus suggesting that CABMV-Lns are a strain likely restricted to Bahia. The characterization of new populations of CABMV enables greater resolution of the evolution of viruses causing woodiness disease in passion fruit vines. Our data shed light on an as yet unexplored population of CABMV in Brazil and contributes to the understanding of its evolutionary history.   Key words: Passion fruit, Livramento de Nossa Senhora, Bahia, phylogenetics, woodiness disease.

Phylogeny and recombination analysis of Brazilian yellow passion fruit isolates of Cowpea aphid-borne mosaic virus: origin and relationship with hosts

The Cowpea aphid-borne mosaic virus (CABMV) causes the most important disease in passion fruit (Passiflora spp.) in Brazil. The genetic diversity of Brazilian CABMV isolates infecting yellow passion fruit was evaluated. Leaves presenting symptoms of mosaic and blister were collected in orchards in four Brazilian states. Samples were submitted to RNA extraction, RT-PCR, sequencing and phylogenetic analyses of two regions of the CABMV genome: complete coat protein (CP) and partial cytoplasmic cylindrical inclusion (CI), representing together about 20 % of the genome. Potential alternative hosts to CABMV in areas with intensive passion fruit farming in the state of Sao Paulo were analyzed. Isolates did not infect cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris). Only Macroptilium atropurpureum behaved as a differential host to an isolate from the southernmost region of the state of Sao Paulo. In spite of the little variation in biological properties, CABMV isolates from Sao Paulo presented moderate genetic diversity between one another, especially between isolates from the other states, with nucleotide identity between 88 and 99 % (84 to 100 % amino acid identity) for CP and 85 % and 99 % (93 to 100 % amino acid identity) for CI. Isolates did not group consistently by origin or hosts in the maximum likelihood tree. The parent strains of CABMV isolates in Sao Paulo occupied a more basal position in the phylogenetic tree, suggesting the possible origin of Brazilian isolates. Recombination events were observed only for CP. Isolates from Sao Paulo were the major parents of CABMV recombinant strains of cowpea, passion fruit and peanut from other Brazilian states. These results agree with the expansion pattern of passion fruit cultures in the country, and suggest that the Sao Paulo region is the origin of CABMV in Brazil, from which isolates spread to other regions and evolved.

Obtenção de plantas de feijão-caupi resistentes ao Cowpea severe mosaic virus e ao Cowpea aphid-borne mosaic virus

Dentre os vírus que infectam o feijão-caupi (Vigna unguiculata L. Walp.) destacam-se, respectivamente, pela severidade e ampla ocorrência o Cowpea severe mosaic virus (CPSMV) e o Cowpea aphid-borne mosaic virus (CABMV). Portanto, objetivaram-se, no presente trabalho, obter e avaliar plantas de feijão-caupi com resistência ao CPSMV e ao CABMV, visando ao desenvolvimento de cultivares essencialmente derivadas e novas cultivares. Realizaram-se oito cruzamentos seguidos de retrocruzamentos, utilizando a linhagem TE 97-309G-9 e a cultivar Patativa como genitores resistentes, e as cultivares BR3-Tracuateua, BRS-Urubuquara, BRS-Novaera, BRS-Guariba e Pretinho como genitores suscetíveis. As gerações F2 e F2RC1 foram desafiadas quanto à resistência por meio de inoculação mecânica com isolados do CPSMV e do CABMV. Nas gerações F2RC1, além da resistência foram avaliados os caracteres: número de dias para o início da floração, comprimento das vagens, número de grãos. vagem-1, peso de cem grãos e produção de grãos.planta-1. Todos os indivíduos F2 e F2RC1 foram analisados pelo teste χ² e se ajustaram à frequência esperada de 15 plantas suscetíveis 1 planta resistente a ambos os vírus. As médias das plantas F2RC1 resistentes, de cada retrocruzamento, foram comparadas com a média do seu respectivo genitor recorrente pelo teste 't' e as médias dos retrocruzamentos foram comparadas pelo teste de Scott-Knott. Foi detectada variabilidade genética entre os retrocruzamentos para todos os caracteres. Todos os retrocruzamentos foram considerados promissores para produção de cultivares essencialmente derivadas resistentes ao CPSMV e ao CABMV e as plantas selecionadas possuem características que possibilitam a seleção de linhagens com grãos de bom padrão comercial e altamente produtivas.

Simple and multiple resistances to viruses in cowpea genotypes

The objective of this work was to identify new sources of simple and multiple resistances to Cowpea severe mosaic virus (CPSMV), Cowpea aphid-borne mosaic virus (CABMV) and Cucumber mosaic virus (CMV) isolates in cowpea (Vigna unguiculata). Thirty-three genotypes from the germplasm bank of Universidade Federal do Ceará were tested as to their resistance to four CPSMV isolates, two CABMV isolates and one CMV isolate. Twenty-five days after the first virus inoculations, all inoculated plants, including the asymptomatic ones, were tested by serology. Genotypes were classified as: immune, plants without symptoms and negative serology; resistant, plants with mild mosaic and positive serology; susceptible, plants with mosaic and positive serology; and highly susceptible, plants with severe mosaic, other systemic symptoms, including systemic necrosis, and positive serology. Simple and multiple resistances to viruses were identified among the evaluated genotypes, but none of them showed multiple immunities to all isolates. Four genotypes showed immunity to all CPSMV isolates, two were immune to CABMV and two showed immunity to CMV. Eleven genotypes showed multiple resistances to two viruses, allowing for the development of new cultivars with more stable and broader resistance. Genotypes Purple Knuckle Hull-55, MNC-03-731C-21 and CNCx284-66E show resistance to CABMV, even when inoculated with CMV.

Indigenous and introduced potyviruses of legumes and Passiflora spp. from Australia: biological properties and comparison of coat protein nucleotide sequences

Five Australian potyviruses, passion fruit woodiness virus (PWV), passiflora mosaic virus (PaMV), passiflora virus Y, clitoria chlorosis virus (ClCV) and hardenbergia mosaic virus (HarMV), and two introduced potyviruses, bean common mosaic virus (BCMV) and cowpea aphid-borne mosaic virus (CAbMV), were detected in nine wild or cultivated Passiflora and legume species growing in tropical, subtropical or Mediterranean climatic regions of Western Australia. When ClCV (1), PaMV (1), PaVY (8) and PWV (5) isolates were inoculated to 15 plant species, PWV and two PaVY P. foetida isolates infected P. edulis and P. caerulea readily but legumes only occasionally. Another PaVY P. foetida isolate resembled five PaVY legume isolates in infecting legumes readily but not infecting P. edulis. PaMV resembled PaVY legume isolates in legumes but also infected P. edulis. ClCV did not infect P. edulis or P. caerulea and behaved differently from PaVY legume isolates and PaMV when inoculated to two legume species. When complete coat protein (CP) nucleotide (nt) sequences of 33 new isolates were compared with 41 others, PWV (8), HarMV (4), PaMV (1) and ClCV (1) were within a large group of Australian isolates, while PaVY (14), CAbMV (1) and BCMV (3) isolates were in three other groups. Variation among PWV and PaVY isolates was sufficient for division into four clades each (I-IV). A variable block of 56 amino acid residues at the N-terminal region of the CPs of PaMV and ClCV distinguished them from PWV. Comparison of PWV, PaMV and ClCV CP sequences showed that nt identities were both above and below the 76-77% potyvirus species threshold level. This research gives insights into invasion of new hosts by potyviruses at the natural vegetation and cultivated area interface, and illustrates the potential of indigenous viruses to emerge to infect introduced plants.

First Report of Cowpea aphid-borne mosaic virus on Sesame in Paraguay.

Sesame (Sesamum indicum L.) is cultivated mainly in the central region of the Departamento de San Pedro in Paraguay from October to February and the seed are exported to Asia. The crop is grown on 100,000 ha annually and Escoba blanca is the most common cultivar. The crop plays an important socioeconomical role since it is cultivated mostly by small growers. A disease characterized by yellowing and curling down leaves and shortening of the internodes has been observed in almost all sesame-growing areas. It is referred to locally as "ka'are" because the affected sesame plant resembles Chenopodium ambrosioides L. This disease occurred occasionally and was of marginal importance prior to 2005, but during the last five growing seasons the disease incidence has increased substantially, with some growers losing the entire crop. To determine the causal agent, symptomatic leaf samples were collected from five commercial fields near Colonia San Pedro and Choré, Departamento San Pedro in December 2009. Preliminary transmission electron microscopy (TEM; Zeiss EM900) of extracts from symptomatic leaves revealed the presence of elongated flexible particles resembling a potyvirus. Mechanical transmission assays resulted in chlorotic local lesions on C. quinoa and C. amaranticolor, mosaic on Vigna unguiculata and Nicotiana benthamiana, and symptoms on sesame that are similar to those observed in the field. The disease could also be reproduced in sesame by aphid (Myzus persicae) transmission in a nonpersistent manner. TEM examination of leaf sections of these naturally or experimentally infected plants showed the presence of the type I cylindrical inclusions and masses of filamentous particles. Leaf extracts of naturally or experimentally infected sesame and test plants were positive for Cowpea aphid-borne mosaic virus (CABMV) on the basis of plate-trapped antigen (PTA)-ELISA. CABMV as the causal agent of "ka'are" disease of sesame in Paraguay was further confirmed by analyzing part of the nucleotide sequence of CABMV coat protein and 3' nontranslated region that were obtained directly from reverse transcription-PCR product amplified with PV1-antisense primer (5'-gatttaggtgacactatagt17-3') and WCIEN-sense primer (5'-atggtttggtgyatygaraat-3') (1,2). Comparisons of the 676-bp nucleotide sequence of two sesame virus isolates (GenBank Accession Nos. HQ336402 and HQ336403) revealed 92% identity with the corresponding nucleotide sequence of CABMV available in the GenBank (Accession No. AF348210). Thus, all the assays indicated that the "ka'are" disease of sesame in Paraguay is caused by an isolate of CABMV. Several cowpea fields, nearby sesame diseased crops, also contained plants exhibiting mosaic symptoms. Transmission assays, electron microscopy, PTA-ELISA, and nucleotide sequence analysis indicated that they were also infected by CABMV and may play an important role in the epidemiology of this disease on sesame. CABMV isolates from passion fruit and cowpea from Brazil were mechanically transmitted to sesame but induced milder symptoms. CABMV-infected sesame was described in the United States (3), but to our knowledge, this is the first report of a severe disease on sesame caused by this virus in Paraguay.

Cowpea aphid-borne mosaic virus (CABMV) is widespread in passionfruit in Brazil and causes passionfruit woodiness disease

Leaf samples of yellow passionfruit (Passiflora edulis f. flavicarpa) displaying fruit woodiness symptoms were collected in seven Brazilian states and the Federal District. Viral infection was confirmed by host range and ELISA, and fourteen viral isolates were obtained. All isolates were capable of infecting several leguminous host species, although differences in symptom severity were noticeable. Woodiness symptoms were reproduced in yellow passionfruit, and mosaic symptoms were induced in common bean. All isolates infected cowpea, reported as a non-host of passion fruit woodiness virus (PWV). Indirect ELISA demonstrated that all isolates were serologically related to each other and also to cowpea aphid-borne mosaic virus (CABMV). The complete sequence of the capsid protein was determined for all isolates. Comparison of these sequences with those of other potyviruses indicated the highest identity with CABMV isolates (85 to 94%). Identity with PWV isolates ranged from 54 to 70%. Phylogenetic analysis grouped all of the Brazilian isolates in a monophyletic cluster with the CABMV isolates, clearly distinct from the PWV isolates. Furthermore, this analysis demonstrated that a group of previously characterized isolates from Brazil that had been designated as PWV should be reclassified as CABMV. Together, these results provide unequivocal evidence that, in Brazil, passionfruit woodiness disease is primarily caused by CABMV. The presence of PWV in Brazil has yet to be confirmed.

The potyvirus associated with the dappled fruit of Passiflora edulis in Kagoshima prefecture, Japan is the third strain of the proposed new species East Asian Passiflora virus (EAPV) phylogenetically distinguished from strains of Passion fruit woodiness virus

A potyvirus (isolate IB) causing dappled or faded fruits and foliar mosaic symptoms of purple passionfruit, was found in the botanical garden of Kagoshima University, Japan. This isolate--differed in host range from isolates of Passion fruit woodiness virus (PWV)-AO, previously reported to cause "woodiness" in Japan. Isolates IB and AO had 83% amino acid identity in their coat proteins (CPs). In phylogenetic analysis, East Asian isolates IB, AO, and PWV-Taiwan clustered together, and were distinguishable from Australian PWV and Brazilian Cowpea aphid-borne mosaic virus isolates, which also cause "woodiness" in passionfruit. We propose the name "East Asian Passiflora virus (EAPV)" for the new potyvirus species.

Occurrence of Cowpea aphid-borne mosaic virus in Peanut in Brazil

Surveys of peanut crops in northeastern Brazil since 1995 showed the occurrence of a hitherto unreported virus disease. Characteristic leaf symptoms were ring spots and blotches. The virus was seed transmitted in peanut (1/610) and cowpea (47/796). Local and systemic symptoms were observed in cowpea (cv. TVu 3433) known to be susceptible to most Cowpea aphid-borne mosaic virus (CABMV) isolates. The virus was transmitted by aphids Toxoptera citricidus and Aphis gossypii. Using degenerate primers, the 3' terminal region of the viral genome was cloned and sequenced. Sequence analyses of the coat protein and the 3' untranslated region indicated that the potyvirus was most closely related to CABMV isolates from South Africa, Zimbabwe, and the United States. On the basis of genome analysis, the virus was identified as CABMV. The natural occurrence of CABMV on peanut has so far not been reported. The significance of this finding especially for germ plasm exchange is discussed.

Reaction of cowpea (Vigna unguiculata L. Walp.) to six isolates of blackeye cowpea mosaic virus (BlCMV) and cowpea aphid-borne mosaic virus (CAMV), two potyviruses infecting cowpea in Nigeria

Five potyvirus isolates from various cowpea-producing areas (Guinea and Sudan savannas and forest zones) in Nigeria were identified by serology and infectivity tests as BlCMV (Isolates IT15, IT16 and WC) or as CAMV (isolates IT11 and ZARIA) and compared with the standard isolate of blackeye cowpea mosaic virus (BlCMV) (isolate ONNE) used at the International Institute of Tropical Agriculture (IITA) for screening purposes. According to the type and severity of symptoms on Nicotiana benthamiana and on selected susceptible cowpea cultivars, the BlCMV and CAMV isolates studied were grouped into three categories. Members of the first group (isolates IT15 and IT16) induced mild-to-severe bright yellow mosaic on selected susceptible cowpea cultivars and could not easily infect Nicotiana benthamiana, but the other two groups of isolates readily infected this plant. In the second group of isolates, ONNE and WC induced mosaic with variable severity in dark green and yellow patches. The third group (isolates IT11 and ZARIA) induced mild green mosaic and veinbanding symptoms on different cowpea lines. An evaluation of cowpea germplasm accessions and breeding lines using these BlCMV and CAMV isolates has shown that germplasm accessions TVu 401, TVu 1453 and TVu 1948 and breeding lines IT82D-885, IT82D-889 and IT82E-60 possess resistance genes to all six potyvirus isolates. These lines could be used in breeding programs at IITA to develop new cowpea varieties.

Blackeye cowpea mosaic virus from asparagus bean (Vigna sesquipedalis) in Thailand and Malaysia, and their relationships to a Japanese isolate.

A sap-transmissible virus isolated from infected asparagus bean (Vigna sesquipedalis) in Thailand and Malaysia had relatively wide host range, but the plants showed systemic symptom were limited to Leguminosae. In asparagus bean juice, infectivity was lost by heating at 55-65C for 10 minutes, by diluting at 10-4-10-5, and by aging at 20C for 1-3 days. The virus was flexuous filaments of about 750nm in length, and was transmitted by aphids in a non-persistent manner. The isolate from Thaliand was seed-borne in cowpea but not in asparagus bean. In double-diffusion tests in agar gel plates containing lithium 3, 5-diiodosalicylate, the virus reacted strongly with antiserum to the Florida isolate of blackeye cowpea mosaic virus (B1CMV) and bean common mosaic virus (BCMV), but not with antiserum to the Morocco isolate of cowpea aphid-borne mosaic virus (CAMV). From these results, this virus was identified as B1CMV. In double diffusion test, the isolate of CAMV from Tokyo (Tsuchizaki et al., 1970) reacted strongly with antisera to the Florida isolate of B1CMV, the Thailand isolate of B1CMV, but not with antiserum to the Morocco isolate of CAMV. Based on the results of serological tests, the Tokyo isolate previously designated as CAMV was considered an isolate of B1CMV.

Detection of cowpea aphid-borne mosaic virus in Queensland

Cowpea aphid-borne mosaic virus (CAMV) is shown to cause a mosaic disease of cowpea (Vigna unguiculata) in north Queensland. The virus was identified on the basis of its host range and symptoms, particle morphology, transmission by sap, aphids, and in seed, and its serological reaction with a known antiserum to an East African isolate of CAMV. No resistance was found in 14 cowpea cultivars. The potential of CAMV as a cause of disease in beans grown in the Burdekin bean seed production area was investigated but most bean cultivars tested were resistant. Only the cultivars Black Turtle Soup, Climbing Blue Lake, Limelight, Pinto and the navy bean lines Actolac, Gallaroy and Kerman were susceptible and produced severe sumptoms.