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African Crop Science Journal
African Crop Science Society
ISSN: 1021-9730 EISSN: 2072-6589
Vol. 6, Num. 1, 1998, pp. 69-78
African Crop Science Journal,Vol. 6. No. 1, pp. 69-78, 1998

OCCURRENCE OF TELFAIRIA MOSAIC VIRUS (TEMV) IN FLUTED PUMPKIN IN NIGERIA

S.A. Shoyinka and G. Thottappilly^1

Institute of Agricultural Research & Training, Obafemi Awolowo University, Moor Plantation, PMB 5029, Ibadan, Nigeria
^1 International Institute of Tropical Agriculture (IITA), Oyo Road, PMB 5320, Ibadan, Nigeria

(Received 3 February, 1997; accepted 29 October, 1997)

Code Number:CS98008
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Abstract

Fluted pumpkin (Telfaira occidentalis, Hook F.) fields, experimental plots and private gardens in 12 major pumpkin producing states in southern Nigeria were surveyed between 1987 and 1989 for relative incidences, distribution and prevalence of three viruses, namely, Telfairia mosaic potyvirus (TeMV), pepper veinal mottle potyvirus (PVMV) and Y-strain of cucumber mosaic cucumovirus (CMV). Using electron microscopy, host-plant reactions and enzyme-linked immunosorbent assay (ELISA) either alone or in combination, a total of 584 leaf samples from 85 sites was tested for the presence of the three viruses reported to infect the crop. Out of a total of 584 samples tested serologically, 498 (representing 85%) reacted positively to TeMV. However, no positive reaction was obtained when samples were tested against antisera to CMV or PVMV. TeMV infection was widely distributed in the area surveyed and its incidence varied considerably reaching 100% in some areas. Neither PVMV nor CMV infected fluted pumpkin in greenhouse tests but the two viruses were detected in their respective plant-hosts, pepper and cowpeas, growing with fluted pumpkin in the same or adjacent locations. No evidence of mixed infections with the three viruses was obtained from studies by host range, electron microscopy, or serological assay. The high incidences and wide distribution of TeMV recorded in this survey indicates that the virus may be of economic importance in the production of this vegetable in Nigeria.

Key Words: Cucumber mosaic cucumovirus, electron microscopy, greenhouse bioassay, pepper veinal mottle potyvirus, serological detection, Telfairia mosaic potyvirus

RESUME

Les champs de la citrouille (potrion) a cannelures, les parcelles experimentales et les jardins privees dans 12 etats producteurs de la citrouille au sud du Nigeria ont ete etudiees entre 1987 et 1989 en ce qui concerne les incidences relatives, la distribution et la prevalance des trois virus, notamment le potyvirus de la mosaique Telfairia (TeMV), le potyvirus de la teche verinee du poivre et la race - Y de la mosaique cucumovirus du concombre. Avec l'aide du microscope electronique, les reactions de la plante hote et l'essai de l'enzyme immunosorbante (ELISA) seul ou en combinaison, un total de 584 echantillons de feuilles provenant de 85 sites a ete teste pour evaluer la presence des trois virus susceptibles d'infecter la culture. Sur un total de 584 echantillons testes serologiquement, 498 (soit 85%) ont reagi positivement au TeMV. Toutefois, aucune reaction n'a ete obtenue lorsque les echantillons etaient testes contre l'antisera au CMV ou au PVMV. L'infestion TeMV a ete largement repandue sur la surface etudiee et son incidence variait considerablement jusqu' a 100% sur certaines surfaces. Ni PVMV ni CMV n'ont infecte la citrouille a cannelures dans les tests en serre mais les deux virus ont ete detectes dans leurs plantes-hotes respectives, le poivre et le cerfeuil, croissant ensemble avec la citrouille a cannelures aux memes endroits ou presque. Aucune evidence des infections mixtes avec les trois virus n'a ete obtenue des etudes sur ou par de nombreux hotes, a la aide d'un microscope electronique ou d'un essai serologique. Les grandes incidences et la large distribution de TeMV enregistres dans cette etude indiquent que le virus peut avoir une grande importance economique dans la production de ce legume au Nigeria.

Mots Cles: Mosaique cucumovirus du concombre, microscope electronique, Telfairia occidentalis, Hook F., serre, potyvirus de la teche veinee du poivre, detection serologique, mosaique potyvirus de Telfairia

Introduction

The production of fluted pumpkin (Telfairia occidentalis Hook F. Cucurbitaceae), a highly nutritious leafy vegetable of tropical West Africa, is reported to be mainly limited by a number of diseases, the most important of which is viral in nature (Anno-Nyako, 1988). Three viruses, inducing mosaic-type symptoms, have been reported on fluted pumpkin in Nigeria. These include Telfairia mosaic potyvirus, TeMV (Nwauzo and Brown, 1975; Shoyinka et al., 1987) later reported to be seed-transmitted (Anno-Nyako, 1988); a Y-Strain of cucumber mosaic cucumovirus (CMV-Y), which infects cowpeas (Atiri, 1985); and a strain of pepper veinal mottle potyvirus (PVMV-TYVC), which induces a yellow vein-clearing symptom in pepper (Capsicum sp.), its primary host (Atiri, 1986).

TeMV and CMV-Y are transmitted non-persistently by among others, the aphid-vector, Aphis spiraecola, and by sap-inoculation under experimental conditions (Atiri, 1985; Shoyinka et al., 1987), while PVMV-TYVC is transmitted non-persistently by A. craccivora (Atiri, 1986) and is also sap-transmissible. These vectors are however, not specific for the viruses (Shoyinka et al., 1987). Besides serological unrelatedness, the three viruses also differ substantially in their host ranges, and in in vitro and biophysical properties (Nwauzo and Brown, 1975; Atiri, 1986; Shoyinka et al., 1987). However, while TeMV was reported to be extensively widespread, accounting for about 84% of the 16 farms and private gardens surveyed in southwestern Nigeria (Shoyinka et al., 1987), the discovery of both CMV-Y and PVMV-TYVC suggested the possible existence of other viruses inducing mosaic disease in fluted pumpkin.

This study was, therefore, conducted to test samples from many fields throughout the main fluted-pumpkin producing areas of southwestern, southeastern and central regions of Nigeria, to determine the relative incidence, distribution and prevalence of the three viruses reported to infect T. occidentalis, and to examine whether there are any other virus(es) yet unidentified in the crop.

Materials and Methods

Survey procedures. Surveys were conducted between March 1987 and September 1989. Experimental plots, farmer's fields, and private gardens were surveyed in the Southwest, Southeast, middle belt, and Guinea savanna agroecologies of Nigeria. The fields to be sampled were chosen systematically, by making a stop every 50 km along the itinerary and then examining the nearest two fields. Field sizes varied between 0.1 ha and 8 ha. Twenty-five sites were selected and surveyed in 1987, 42 in 1988, and 18 in 1989 to cover a wide range of cultivars and soil types in the major agroecologies where fluted pumpkin is grown in Nigeria. Random samples were collected from survey sites, plants being selected irrespective of symptoms observed (Barnett, 1986). The survey covered the full cycle of the crop from nursery to harvest. In research plots and farmers' fields, sampling was done in plant population enclosed per m^2 at the four corners, midway along each edge, and at the centre.

The number of farms selected was proportional to the intensity of Telfairia cultivation in each geographic location. In home gardens where a systematic survey was not possible, individual plants showing virus-like symptoms were selected and assayed independently, and results of such assays were included in incidence ratings.

Field observations and sample collection. In each field, experimental plot, and home garden where samples were collected, the different symptoms suggestive of virus infection in the entire field were described. A representative sample of each symptom was collected and stored on ice for assay. The incidence of plants showing symptoms was assessed by counting the number of plants showing that symptom in the total area surveyed within the farm or field. In the laboratory, representative samples of each symptom were cut into 2mm pieces, dried over calcium chloride, and stored at 4 C for bioassay. Such materials were revived through sap-inoculation to young Telfairia seedlings before being assayed.

Virus identification. Identification of viruses was done by electron microscopy, biological indexing to selected indicator plants, and serology.

Electron microscopy. Representative samples (about 5%) were examined on a Philips TEM 201 using the leaf dip technique (Brandes, 1957). Samples were chopped in 0.2% sodium sulphite, stained in 2% uranyl acetate and the grids drained with filter paper and air-dried before examination.

Serology. Antisera against TeMV (Shoyinka et al., 1987) and CMV (G. Thottappilly, unpublished) were prepared while the commercial universal potyvirus (PTY) monoclonal antibodies were purchased from AGDIA Inc., U.S.A. The antiserum to PVMV was the one used in a previous investigation (Lana et al., 1975). The TeMV had titre of 1:4096 in tube precipitin test, the CMV had a titre of 1:512 in agar gel diffusion test while the universal potyvirus monoclonal antibody Pty had a titre of 1:20,000 in ELISA test. Enzyme-linked immunosorbent assay (ELISA) was used for detecting TeMV, CMV-Y strain, PVMV-TYVC, and other members of the potyvirus group that may occur in the samples collected. Procedures and chemical preparations were according to Clark and Adams (1977). PVMV-infected pepper and CMV-infected cowpea were used as positive controls for PVMV and CMV, respectively. Two microtitre plates were used to test each sample, thus giving two replicates for each sample.

IgGs to TeMV, CMV, and PVMV at concentrations of 1ug/ml were found appropriate for coating plates. Dilutions of enzyme-labelled IgGs were standardised at 1/250 (V/V) for TeMV, 1/250 for PVMV-TYVC, and 1/500 for CMV. The end-point of virus-infected leaf extracts was 1/1000 (W/V) for TeMV.

In the case of universal potyvirus monoclonal antibodies, plate trapped (antigen coated) indirect ELISA was used. Plant extracts were prepared in coating buffer (1.59 g Na2CO3, 2.93 g NaHCO3, and 0.2 g NaN3 per litre), to which 2% of polyvinyl pyrrolidone (PVP) was added. The plates were kept at 37 C for 2 hr. After washing, universal potyvirus antibodies at a dilution of 1/1000 were added. Each step was followed by thorough washing with PBS-Tween buffer. Goat antimouse IgG_alkaline phosphatase conjugate (Agdia Incorporated) at a dilution of 1/2000 was applied. Finally the enzyme substrate, p-nitrophenyl phosphate, was added for colour development.

Enzyme substrate reactions were measured at 405 nm. Samples were considered positive when ELISA readings exceeded that of a healthy control by a factor of two. The OD values for the healthy samples were less than 0.1. A value of 0.2 - 0.3 was considered a weak reaction denoted by "+", while ++ means an OD value of 0.3 - 0.6. Any OD value above 0.6 was designated by +++. Each sample-test was replicated. Glasshouse-grown healthy or virus-infected fluted pumpkin plants were used as negative and positive controls, respectively. To prevent false-negative reactions, samples revealing values around threshold were subjected again to ELISA. All samples that tested negative in ELISA were examined in the EM and by inoculation to test plants.

Screenhouse bioassay. One virus-infected sample each from Ibadan, Ilishan, one home-garden crop, and one sample from a commercial farm each from Mgbator and Itu were selected for bioassay in 1988. Each sample was macerated in 0.1M phosphate buffer, pH 7.4, containing 0.1% 2-mercaptoethanol and inoculated on to indicator host plants, raised under natural light in the screenhouse at 24-31 C, with cotton swabs that were pre-dusted with 600-mesh carborundum.

Results

A total 498 of the 584 samples collected (85%) were virus positive (Table 1, Part 1, Part 2, Part 3), but only TeMV was present in all regions sampled. In some cases, TeMV incidence reached virtually 100% in experimental and commercial fields in Ibadan, Ilishan, Asaba, and Itu. All fields surveyed were found to contain only TeMV. Neither PVMV-TYVC, nor any other potyvirus or CMV-Y, was detected in samples from any location. Numbers and percentage incidences of TeMV-infected plants varied from one location to another, especially in the southwestern and southeastern states where fluted pumpkin cultivation predominates. About 80% of the Telfairia planting was in either home gardens or small holdings; large-scale commercial farms covering over 1ha were few (Table 1 and Fig. 1).

Field symptoms. Symptoms exhibited by field-grown TeMV-infected fluted pumpkin were variable, depending on age of plant at time of survey and probably genetic variability of the cultivars grown. In general, symptoms observed closely resembled those previously described in the literature (Nwauzo and Brown, 1975; Atiri, 1986; Shoyinka et al., 1987). These include severe mosaic, vein-clearing, vein-banding, leaf-distortion, and generally stunted growth.

Electron microscopy. Electron microscopic examinations of all selected 30 samples exhibiting various types of symptoms showed only flexuous, rod-shaped particles, characteristic of potyviruses. The modal particle-length was ca. 800nm. No spherical or other shape particles were seen under the electron miscroscope.

Serology. All TeMV isolates detected appeared to be serologically indistinguishable; the uniformity of absorbance readings thus suggests that only one TeMV strain exists throughout the survey area. Universal potyvirus antibodies also detected the same 498 samples which gave positive results to TeMV antibodies. No field-infected fluted pumpkin isolate reacted positively to either CMV or PVMV antiserum used. However, strong positive reactions were obtained to glasshouse-grown cowpea and pepper plants infected with CMV and PVMV, which were included as positive controls in the studies. Also, the positive controls to PVMV reacted positively with universal potyvirus antibodies.

Screenhouse bioassay. When virus susceptibility and symptom expression on assay (test) plants inoculated with selected TeMV isolates were assessed along with the confirmatory serological reactions (Table 2), only TeMV was detected serologically in infected plants. None of the infected test plants resulted in positive serological reaction to either CMV-Y or PYMV-TYVC antisera, whereas CMV was detected in diseased cowpea plants, and PVMV was detected in pepper plants with polyclonal antisera.

Discussion

The results of this survey indicate that only TeMV was present in fluted pumpkin in all the locations sampled. Both CMV-Y and PVMV, although present in their natural hosts, were not detected in field-grown fluted pumpkin and could not infect Telfairia occidentalis under experimental conditions. These findings confirm the earlier observation of Shoyinka et al. (1987) that TeMV was endemic in T. occidentalis grown in southwestern Nigeria.

The wide distribution of TeMV may be partly due to its being seed-transmitted in some varieties (Anno-Nyako, 1988). The generally lower incidence of TeMV infections in some areas of main cultivation in eastern Nigeria may be due to continuous conscious selection of fruits from virus-free plants by farmers. Such a method has been shown to produce virus-resistant T. occidentalis (Atiri and Varma 1983).

In some locations, notably at Ibadan, Ikenne, and Ilishan, fluted pumpkin, cowpea, and pepper were being cultivated in adjacent fields. Since the aphid vectors of the three non-persistently transmitted viruses under investigation were present at these locations, we expected to detect mixed infections, either through host range tests, serology or electron microscopy. Infected pepper plants reacted only with the PVMV and with universal PTY antiserum, while infected fluted pumpkin reacted only with the TeMV antiserum and universal PTY monoclonal antibodies. PVMV has been shown to be serologically unrelated to TeMV (Shoyinka et al., 1987). The detection of TeMV in apparently healthy fluted pumpkin indicated the efficiency of the ELISA technique developed for both diagnostic and epidemiological studies.

Infection by TeMV was reported to cause extreme reduction in fruit size (Nwauzo and Brown, 1975). This, coupled with the high incidence recorded in many fields and its widespread distribution over a large geographical area during this survey, indicates that TeMV may be economically important in vegetable production in Nigeria.

REFERENCES

Atiri, G.I. 1985. An isolate of cucumber mosaic virus from field-infected fluted pumpkin in Nigeria-Phytopath Zeischrift 114:268-273.

Atiri, G.I. 1986. A disease of fluted pumpkin (Telfairia occidentalis Hook. F.) caused by a yellow vein clearing strain of pepper veinal mottle virus in Nigeria. Journal of Plant Protection in the Tropics 3:105-110.

Atiri, G.I. and Varma, A. 1983. Development of improved lines of Telfairia occidentalis, Hook. f.) resistant to mosaic disease. Tropical Agriculture (Trinidad) 60:95-96.

Anno-Nyako, F.O. 1988. Seed transmission of Telfairia mosaic virus in fluted pumpkin (Telfairia occidentalis Hook f.) in Nigeria. Journal of Phytopathology 121:85-87.

Barnett, O.W. 1986. Surveying for Plant Viruses: design and consideration. In: Plant Virus Epidemics: Monitoring, Modelling and Predicting outbreaks. Mclean, G.D., Gamet, R.G. and Ruesnit, W.G. (Eds.), pp147 - 166 - Sydney, Austrtalia, Academic Press.

Brandes, J. 1957. Eine elektronenmikrosp-kopische Schnellmethode zum Nachweis faden-und stŠbchenfšrmigen Viren, insbesondere in Kartoffeldunkel Keimen. - Nachrichtenbl. dt Pflschuntzdients. 9:151 - 152.

Clark, M.F. and Adams, A.N. 1977. Characteristic of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34:475-483.

Lana, A.O., Gilmer, R.M., Wilson, G.F. and Shoyinka, S.A. 1975. Identification and characterization of a severe strain of pepper veinal mottle virus isolated from pepper (Capsicum annum and C. rutescens) in Nigeria. Nigeria Journal of Plant Protection 1:76 - 81.

Nwauzo, E.E. & Brown, W.M. 1975. Telfairia (Cucurbitaceae) mosaic virus (TeMV) in Nigeria. Plant Disease Reporter 59:430 - 432.

Shoyinka, S.A., Brunt, A.A., Lesemann, D., Thottappilly, G. and Lastra, R.J. 1987. The occurrence, properties and affinities of Telfairia mosaic virus, a poty-virus prevalent in Telfairia occidentalis (Cucurbitaceae) in South Western Nigeria. Journal of Phytopathology 119:13-27.

Copyright 1998, African Crop Science Society


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