Viral diseases constitute obstacles to pepper production in the world. In Nigeria, pepper plants are primarily affected by pepper veinal mottle virus (PVMV), Cucumber mosaic virus (CMV), Pepper leaf curl Virus (TLCV), Tobacco mosaic virus (TMV), Pepper mottle virus (PMV) and a host of other viruses. The experiment was carried out with a diagnostic survey on the experimental field of the National Horticultural Research Institute, Ibadan, Nigeria and on pepper farms in six local government areas within Ibadan Oyo State, Nigeria, forty samples were collected from each of the farms. Diseased samples were obtained from the field and taken to the laboratory for indexing. In ELISA test some of the samples from the pepper farms showed positive reaction to single infection with PVMV (36.79%), CMV (22.14%) while some others showed positive reaction to mixed infection of the two viruses (10%) but some also negative reaction to PVMV and CMV antisera (31.07)

A Crescenzy

AA Brunt

AA Fajinmi

BD Harrison

C Huguenot

ECK Igwegbe

GI Atiri

JL Ladipo

Kehinde Titilope Kareem

MD Alegbejo

O Arogundade

Olawale Arogundade

Olusegun Samuel Balogun

P Palukaitis

R Nagaraju

Rao Prasada

REF Matthews

SB Lal

VB Vance

Y Otsuki

English

PubMed

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RESEARCH Open AccessOccurrence and distribution of pepper veinalmottle virus and cucumber mosaic virus inpepper in Ibadan, NigeriaOlawale Arogundade1*, Olusegun Samuel Balogun2 and Kehinde Titilope Kareem1AbstractViral diseases constitute obstacles to pepper production in the world. In Nigeria, pepper plants are primarilyaffected by pepper veinal mottle virus (PVMV), Cucumber mosaic virus (CMV), Pepper leaf curl Virus (TLCV), Tobaccomosaic virus (TMV), Pepper mottle virus (PMV) and a host of other viruses. The experiment was carried out with adiagnostic survey on the experimental field of the National Horticultural Research Institute, Ibadan, Nigeria and onpepper farms in six local government areas within Ibadan Oyo State, Nigeria, forty samples were collected fromeach of the farms. Diseased samples were obtained from the field and taken to the laboratory for indexing. InELISA test some of the samples from the pepper farms showed positive reaction to single infection with PVMV(36.79%), CMV (22.14%) while some others showed positive reaction to mixed infection of the two viruses (10%)but some also negative reaction to PVMV and CMV antisera (31.07).Keywords: Antisera, CMV, Diagnostic survey, ELISA, PVMV, NigeriaIntroductionPlant virus diseases cause major losses to agriculturalcrops around the world. Chemical agents similar to fun-gicides and bactericides are not effective in controllingvirus diseases. Strategies for virus management in plantare mostly aimed at eradicating the source of infectionto prevent it from reaching the crop and interferingwith the movement of vectors in order to prevent thespread of the disease. However, the most effectivemeans of controlling virus diseases is through cultivatingthe virus-resistant varieties.Generally, viral infection causes visible symptoms suchas various forms of mosaic and distortions in plants withconsequent reductions in crop growth and yield. Whilereduction in plant size is the most general symptominduced by virus infection, there is probably some stunt-ing of growth even with ‘masked’ or ‘latent’ infections,where the systemically infected plant shows no obvioussign of disease [1]. In nature, higher plants are commonlyco-infected with multiple viruses and a number of diseasesyndromes are caused by interaction of two independentviruses. The accumulation dynamics of the interactingviruses in such mixed infection often change drastically[2]. Besides, mixed infections with two unrelated viruses,which are common in field plants, especially in tropicalareas, often produce a more severe disease than thatcaused by either virus alone. For instance, tobacco oninfection with potato virus × (PVX) and potato virus Ynormally develop a more severe disease than thatinduced by either virus alone [3].Pepper veinal mottle virus (PVMV) was first recognizedas a distinct member of a group of viruses which was ori-ginally designated the Potato virus Y group but was laterrenamed the Potyvirus group [4]. PVMV occurs mainly inAfrica, although it affects Capsicum annuum L. crops inAfghanistan [5] and India [6]. PVMV also occurs in Capsi-cum spp. in Sierra Leone and Zaire, [7]. PVMV has beenreported in several West African countries, and in someparts of Nigeria [8,9]. There was a report that a strain ofPVMV occurs naturally in Telfairea occidentalis (Cucurbi-taceae) in Nigeria [10]. Strains of the virus are also experi-mentally transmissible to at least 35 species of theSolanaceae and to nine species of five other families(Aizoaceae, Amaranthaceae, Apocynaceae, Chenopodiaceae* Correspondence: arogundade_olawale@yahoo.co.uk1National Horticultural Research Institute, Idi Ishin, Jericho Reservation Area,P.M.B 5432, Dugbe, Ibadan, Oyo State, NigeriaFull list of author information is available at the end of the articleArogundade et al. Virology Journal 2012, 9:79http://www.virologyj.com/content/9/1/79© 2012 Arogundade et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.and Rutaceae) [11-14]. Symptoms expressed by the leavesof PVMV-infected plants include chlorosis of the veins, fol-lowed by systemic interveinal chlorosis, mottle, and smalldistortion of leaves and at times leaf abscission and fruitdistortion occur [11]. There have been reports of one hun-dred percent losses of marketable pepper fruit due to infec-tion with pepper viruses causing whole field to beabandoned prior to harvest and in some areas making cul-tivation of pepper to be uneconomical in some parts ofNigeria [8].Cucumber mosaic virus (CMV) is worldwide in distribu-tion. The virus causing cucumber mosaic has perhaps awider range of host and attacks a greater variety of vegeta-bles, ornamentals, weeds, and other plants than otherviruses [15]. CMV is transmitted mainlyby the greenpeach aphid, Myzus persicae, and by Aphis gossypii, but itcan be transmittedby other species of aphids [16].In this paper we report the distribution of PVMV andCMV infection in pepper plants within Ibadan, Oyo state,Nigeria.Materials and methodsSurvey for Pepper veinal mottle virus, genus Potyvirus,and Cucumber mosaic virus, genus Cucumovirus wasconducted during the 2009 planting season in sevenlocations in Ibadan, Oyo State in the southwest agro-ecological zone of Nigeria.The locations were the experimental field of theNational Horticultural Research Institute (NIHORT),Lagelu Local government area, Akinyele Local govern-ment area, Egbeda Local government area, Ona AraLocal government area, Oluyole Local government areaand Ido Local government area.Collection of diseased leaf samplesDiseased Leaf Samples were randomly collected from theexperimental fields containing pepper in NIHORT whilein the Local government areas two cultivated pepperfarms were randomly surveyed per Local government.On each site 20 plants were randomly sampled from thepopulation of plants on the field. Forty plants weresampled per location from cultivated pepper plants show-ing symptoms of mosaic, chlorosis, yellowing, stuntingand mottle making a total of two hundred and eightysamples in all. The sampled leaves were then storedunder Calcium chloride and were placed in the refrigera-tor prior to indexing.Virus detectionInternational Iinstitute of Tropical Agriculture (IITA)Virology laboratory modified protocols for Direct anti-gen coating (DAC - ELISA) was used for the detectionof the presence of PVMV and CMV both from the fortyinfected pepper leaf samples collected per location. ThePVMV and CMV antibodies used were AAB 328 anti-serum diluted in the ratio 1:1000 and 1:3000 respectivelywith Phosphate Buffered Saline (PBS-T) (0.05% Tween20: pH 7.4: 8.0 g NaCl, 0.2 g KH2 PO4, 1.1 g Na2 HPO40.2 g KCl, 0.2 g NaNO3 in 1 l H2O + 0.5 ml Tween 20(0.05%)) from the Virology Laboratory of the Interna-tional Institute of Tropical Agriculture (IITA) Ibadan.Viral indexing protocolsOne hundred micro litre of antigen (e.g. sap) ground at 0.1g of leaf sample in 1 ml of coating buffer was dispensedinto each well of ELISA plate. The plate was then incu-bated at 37°C for 1 h and later washed three times withPBS-T after the incubation period. Cross adsorption wasmade by grinding 1 g of healthy pepper leaf in 20 mls ofconjugate buffer conjugate buffer (1/2 PBS + 0.05% Tween20 + 0.02% egg albumin + 0.2% PVP + 0.02 g NaN3).100 μl of PVMV and CMV polyclonal (AAB 328) antiseradiluted 1:1000 and 1:3000 in the adsorption solution wasadded to each of the ELISA plate and then incubated at37°C for 1 h. After incubation the ELISA plate was washedthree times with PBS-T. 100 μl of protein, A- alkalinephosphatase conjugate diluted in the ratio 1:15000 in con-jugate buffer (1/2 PBS + 0.05% Tween 20 + 0.02% eggalbumin + 0.2% PVP + 0.02 g NaN3) was added per welland the plate incubated at 37°C for 1 h. The plate waswashed three times with PBS-T. 100 μl of 0.001 g/ml of p-nitrophenyl phosphate substrate in substrate buffer (97 mldiethanolamine + 800 ml H2O + 0.2 g NaNO3 add HCl togive pH 9.8) was added per well and incubated at roomtemperature for one hour. For all incubations plates werecovered with ELISA cover plates to avoid edge effects andto maintain uniform temperature. Healthy pepper plants(Capsicum sp.) were used as negative control while PVMVand CMV infected Capsicum sp were used as positive con-trol. After one hour the absorbance was measured at405 nm using multiscan ELISA reader. The samples wereconsidered positive when the ELISA reading exceeded thatof the healthy control or was at least twice the reading forthe healthy control.Results and discussionAll the farms visited recorded the presence of PVMV andCMV as a single infection as well as mixed infection insome cases. Out of the two hundred and eighty leaf sam-ples suspected to be virus infected collected from thefarmers field in Ibadan, Oyo State of Nigeria 36.79%tested positive to PVMV, 22.14% tested positive to CMV,10% tested positive to both PVMV and CMV through theserological test used, while 31.07% tested negative to thetwo antisera (Figure 1). However, from Table 1, out ofthe 40 leaf samples collected from each of the surveypoint Lagelu have the highest single infection of PVMV[3] while Ido have the highest single infection of CMVArogundade et al. Virology Journal 2012, 9:79http://www.virologyj.com/content/9/1/79Page 2 of 4[12] while Egbeda have the highest incidence of mixedinfection of PVMV and CMV [9].All the pepper farms surveyed within Ibadan showeddifferences in PVMV and CMV as observed in the per-centage viral incidence (Figure 2). The percentage viralincidence of PVMV and CMV ranges between 55.0% inOna-ara to 87.50% in Ido.Pepper is highly susceptible to virus diseases in Iba-dan, Oyo state, Nigeria as all samples picked from allthe surveyed locations showed different viral symptomsranging from different degree of mosaic, mottle, yellow-ing, stunting, puckering and in some cases reduction inleaf size which was similar to the earlier describedsymptoms of PVMV and CMV by Atiri [17] and Aro-gundade et al. [18]. It was however observed that fromall the six local governments used as study area in thissurvey and the experimental field of NIHORT, pepper ismostly infected with PVMV than CMV and the mixedinfection of PVMV and CMV, however the combineeffect of PVMV and CMV is more on pepper than allother vegetable viruses combine as observed during thesurvey (Figure 1).The high percentage incidence of PVMV and CMV inthe experimental field of NIHORT could be as a resultof numerous alternative host species surrounding thepepper field such as tomato, solanum, cucurbits and ahost of other vegetables. This corroborates the findingsof Alegbejo [19] who reported that the proximity ofpepper plants to certain important weed hosts also hascontributed greatly to the spread of viral diseases ofpepper. These weeds include Solanum nigrum, S. gracil,Physalis angulata, Vigna rosea, Vigna sinensis, Comme-lina nudiflora, Petunia hybrida, Physalis floridana, P.micrantha and Solanum incanum.Author details1National Horticultural Research Institute, Idi Ishin, Jericho Reservation Area,P.M.B 5432, Dugbe, Ibadan, Oyo State, Nigeria. 2University of Ilorin, Faculty ofAgriculture, Crop Protection Department, P.M.B 1515, Ilorin, Kwara State,Nigeria.Received: 13 July 2011 Accepted: 11 April 2012 Published: 11 April 2012References1. Matthews REF: Plant Virology. 3 edition. San Diego: Academic; 1991, 835.2. Otsuki Y, Takebe I: Double infection of isolated tobacco mesophyllprotoplasts by unrelated viruses. J Gen Virol 1976, 30:309-316.3. Vance VB: Replication of potato virus × is altered in co-infections withpotato virus Y. Virology 1991, 182:486-489.4. Harrison BD, Finch JT, Gibbs AJ, Hollings M, Shepherd RJ, Valenta V,Wetter C: Sixteen groups of plant viruses. Virology 1971, 45:356-363.5. Lal SB, Singh S: Identification of some virus diseases of vegetable cropsin Afghanistan. Plant Prot Bull (Faridabad) 1988, 36(2):83-89.6. Nagaraju R, Reddy HR: Occurrence and distribution of bell pepper virusesaround Bangalore. Current research, University of Agricultural Sciences,Bangalore; 1980:10(9/10):155; 6.Figure 1 Percentage of PVMV, CMV, PVMV + CMV and OtherVirus diseases of pepper in Ibada.Table 1 Distribution of PVMV and CMV in IbadanLocation Number of samples collected PVMV CMV PVMV + CMVNIHORT Experimental Field 40 17 10 6Lagelu Local Government 40 19 10 0Akinyele Local Government 40 14 7 4Egbeda Local Government 40 11 8 8Ona Ara Local Government 40 9 6 7Oluyole Local Government 40 15 4 5Ido Local Government 40 18 13 4Figure 2 Percentage Incidence of PVMV and CMV in NIHORTexperimental Station and Six other areas of Oyo State.Arogundade et al. Virology Journal 2012, 9:79http://www.virologyj.com/content/9/1/79Page 3 of 47. Huguenot C, Furneaux MT, Clare J, Hamilton RI: Sero-diagnosis of Pepperveinal mottle viru in West Africa using specific monoclonal antibodies inDAS-ELISA. J Phytopathol 1996, 144:29-32.8. Alegbejo MD, Uvah II: Effect of intercropping pepper with tall companioncrops on the incidence of Pepper veinal mottle viru on pepper. Nigerian JEntomology 1987, 7:82-87.9. Fajinmi AA: The incidence, spread and possible control strategies ofPepper veinal mottle Potyvirus (PVMV) disease on pepper (Capsicumannuum.L.) in Nigeria. Ph. D thesis University of Ibadan Oyo State Nigeria;2006.10. Atiri GI: A disease of fluted pumpkin (Telfairia occidentalis Hook.f.) causedby a yellow vein clearing strain of Pepper veinal mottle virus in Nigeria. JPlant Prot Tropics 1986, 3(2):105-110.11. Brunt AA, Kenten RH, Phillips S: Symptomatologically distinct strains ofPepper veinal mottle viru from four (4). West African Solanaceous crops.Ann App Biology 1978, 88:115-119.12. Igwegbe ECK, Waterworth HE: Properties and serology of the strain ofPepper veinal mottle virus isolated from eggplant (Solanum melongenaL.) in Nigeria. Phytopathologische Zeitschrift 1982, 103(1):9-12.13. Ladipo JL, Roberts IM: Pepper veinal mottle viru associated with a streakdisease of tomato in Nigeria. Ann Appl Biol 1977, 87:133-138.14. Prasada Rao, RDVJ Yaraguntaiah RC: The occurrence of Pepper veinalmottle virus on chilli in India. Mysore J Agric Sci 1979, 13(4):445-448; 6.15. Crescenzy A: Cucumber mosaic cucumovirus populations in Italy undernatural epidemic conditions and after a satellite - mediated protectiontest. Plant Dis 1993, 77:28-33.16. Palukaitis P, Roossinck MJ, Dietzgen RG, Francki RIB: Cucumber mosaicvirus.Adv Virus Res 1992, 41:281-349.17. Alegbejo MD: Identification of a weed host of Pepper veinal mottle virusin Northern Nigeria. Samaru J Agric Res 1987, 5(1 and 2):65-70.18. Atiri GI: Progress of Pepper veinal mottle viru disease in Capsicu peppers.Crop Prot 1992, 11(3):255-259.19. Arogundade O, Balogun OS, Shokalu O, Aliyu TH: Influence of CowpeaMottle Virus and Cucumber Mosaic Virus on the Growth and Yield of sixLines of Soybean (Glycine max L.). J Agric Sci 2010, 2(1):72-78.doi:10.1186/1743-422X-9-79Cite this article as: Arogundade et al.: Occurrence and distribution ofpepper veinal mottle virus and cucumber mosaic virus in pepper inIbadan, Nigeria. Virology Journal 2012 9:79.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitArogundade et al. Virology Journal 2012, 9:79http://www.virologyj.com/content/9/1/79Page 4 of 4

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Occurrence and distribution of pepper veinal mottle virus and cucumber mosaic virus in pepper in Ibadan, Nigeria

Abstract Viral diseases constitute obstacles to pepper production in the world. In Nigeria, pepper plants are primarily affected by pepper veinal mottle virus (PVMV), Cucumber mosaic virus (CMV), Pepper leaf curl Virus (TLCV), Tobacco mosaic virus (TMV), Pepper mottle virus (PMV) and a host of other viruses. The experiment was carried out with a diagnostic survey on the experimental field of the National Horticultural Research Institute, Ibadan, Nigeria and on pepper farms in six local government areas within Ibadan Oyo State, Nigeria, forty samples were collected from each of the farms. Diseased samples were obtained from the field and taken to the laboratory for indexing. In ELISA test some of the samples from the pepper farms showed positive reaction to single infection with PVMV (36.79%), CMV (22.14%) while some others showed positive reaction to mixed infection of the two viruses (10%) but some also negative reaction to PVMV and CMV antisera (31.07).</p

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C: Sixteen groups of plant viruses. Virology

Reddy HR: Occurrence and distribution of bell pepper viruses around Bangalore. Current research,

Replication of potato virus × is altered in co-infections with potato virus Y. Virology

Singh S: Identification of some virus diseases of vegetable crops in Afghanistan. Plant Prot Bull (Faridabad)

Takebe I: Double infection of isolated tobacco mesophyll protoplasts by unrelated viruses.

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Occurrence and distribution of pepper veinal mottle virus and cucumber mosaic virus in pepper in Ibadan, Nigeria

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