ANALYSIS OF RESISTANCE TO YAM MOSAIC VIRUS , GENUS POTYVIRUS IN WHITE GUINEA YAM ( DIOSCOREA ROTUNDATA POIR . ) GENOTYPES

Resistance to Yam mosaic virus (YMV), genus Potyvirus was studied in 10 populations of selected white Guinea yam (Dioscorea rotundata). Plants of resistant genotypes: TDr 35, TDr 1621, TDr 93-1, TDr 93-32, TDr 95107, TDr 93-23, and susceptible ones: TDr 87/00211, TDr 87/00571 and TDr 95-127 were screened for their reaction to the pathogen by symptom severity scoring scale of 1-5, and by quantifying virus multiplication by triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). Controlled crosses were made among the genotypes within and between the groups according to reactions to the pathogen. The resultant F1 progenies were evaluated for the infection by disease symptom development and by TAS ELISA to detect a symptomless infection in an insect-proof screenhouse for the assessment of inheritance of resistance to YMV. A genetic analysis of the reactions of progenies derived from the D. rotundata genotypes to inoculation with YMV strongly suggests that resistance to the virus is a dominantly inherited trait. Segregation ratios obtained from the families indicate that at least two dominant genes are involved.


Introduction
Yam mosaic virus (YMV) genus Potyvirus, family Potyviridae is the most important virus infecting both cultivated and wild yams especially Dioscorea rotundata, D. alata, D. cayenensis and D. praehensilis in the yam-growing areas of the world (Thouvenel and Fauquet, 1979;Porth et al., 1987).YMV is reported in the growing regions of Nigeria, Ghana, Cameroon, Benin, Côte d'Ivoire, Burkina Faso and Togo in West Africa (Thouvenel and Fauquet, 1979;Porth et al., 1987;Thottappilly, 1992;Brunt et al., 1996) and also in Guadeloupe and Guyana (Brunt et al., 1996).YMV is the most widespread among all the viruses infecting yams in Nigeria (Hughes et al., 1997) and in Ghana (Olatunde, 1999).YMV causes several symptoms including mottling, leaf and vein chlorosis, leaf mosaic, leaf distortion and malformation, shoestringing of leaf as well as plant stunting.YMV is reported to cause about 40% of yield reduction in D. rotundata (IITA, 1981).Among all the viruses identified to infect yams, YMV is the causal agent of the most important virus disease (Thouvenel andDumont, 1988, 1990;Goudou-Urbino, 1995).
Protection of crops against pathogens may be realized by different means such as chemical application, phytosanitation, and use of biological control agents or resistant varieties.Breeding for resistance is usually preferred due to its costeffectiveness and the minimal impact on the environment (Hogenboom, 1993).This is because once resistance to the causal agent of the disease is established it can be transferred from generation to generation.
For a higher level of food production, greater income and improved nutritional status of the poor people in sub-Saharan Africa, resistant landraces grown in the region must be identified and hybridized with other genotypes having desired qualities to bring about high level resistant offspring with improved and reliable yield.
Several studies by various authors identified inheritance of resistance.Legnani et al. (1995) identified a peculiar type of resistance in F 2 progenies of Lycopersicon hirsutum cv.PI247087 (a resistant line) to infection with isolates of Potato virus Y (PVY) genus Potyvirus which allows the virus to multiply in the inoculated leaves but could not establish a systemic infection, thus suggesting a mechanism which interferes with the long distance migration of the virus in infected plants.Numerous studies have been conducted to elucidate the mode of inheritance of resistance of several cultivars of soybeans (Glycine max) to isolates of Soybean mosaic virus (SMV) genus Potyvirus (Kiihl and Hartwig, 1979;Buzzell and Tu, 1984;Lim, 1985;Buss et al., 1989;Bowers et al., 1992).Mignouna et al. (2001) reported that segregation into resistant and susceptible individuals in a three population's inheritance study indicated that resistance in D. rotundata is inherited differentially as a dominant and recessive character, thus suggesting that two major genes control resistance to Yam mosaic virus (YMV), genus Potyvirus in white guinea yams.
The objectives of this study were to contribute to the knowledge of genetic control of resistance to YMV, in D. rotundata using landraces with a range of resistance/susceptibility to YMV under vector-controlled conditions, and to study the F 1 hybrids produced.

Selection of parental lines and hybridization
Following visual scoring of disease symptoms as described by Mignouna et al. (2001) and Odu et al. (2004) on a scale of 1-5, serological evaluation, and observation of flowering pattern to determine the sex, 10 D. rotundata genotypes were selected for use as parental lines from the 24 evaluated genotypes in four agro-ecological zones in Nigeria (Table 1 The genotypes were planted at a density of one plant per square meter and each experimental plot (6 m x 5 m) was planted with 30 sets of each genotype and replicated four times.The experimental design was a randomized complete block (RCBD).Individual staking of each of the plant stands was done at emergence to increase the area of leaf exposure to sunlight and to facilitate controlled crossing of individual plants.

Planting and germination of yam seeds
A layer of coco-peat was placed at the bottom of a 0.9 x 0.6 m metal tray and Jiffy 7 peat pellets (Forestry Supplies Inc. U.S.A.) were arranged side by side on the coco-peat with each tray having 240 pellets.The pellets were then sprayed with water to the saturation point.After about 3-5 minutes, more water was added and this was repeated until the pellets were fully expanded.Each of the pellets at this time was about 1 5 / 8 inches high.
The expanded pellets were then sprayed with fungicide (5 g litre -1 of benlate in water containing 50% benomyl (w/w)), water-soaked and drilled in the middle.The seeds were planted in the holes with one seed per pellet, labeled with plastic pegs to differentiate between the crosses and watered when necessary to ensure the germination of the seeds and uptake of nutrients from the pellets and the coconut peat.Germination counts were done at 2-day interval for each of the crosses, using pushpins of assorted colors (one color per day) to distinguish already counted seedlings.
Germinated seedlings were transplanted into 7 or 8-inch Stewart's pots which were previously filled with a mixture of heat sterilized top-soil and coconut peat (1:3).Individual plants from each of the crosses were labeled and randomized within the screenhouse.There were 15 blocks of parent plants and progenies arranged in 20 rows of 6 plants per block.
YMV detection in F 1 and their parents Three leaf samples were collected from all plants (both F 1 and parents) in the screenhouse and indexed serologically to detect YMV infection prior to mechanical inoculation.Triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) described by Thottappilly et al. (1998) was used for the detection of YMV in leaf samples collected from F 1 plants and the parents as reported by Odu et al. (2004).The monoclonal antibody used was YMV-M24, produced against YMV at the IITA, which was found to detect YMV isolates from other countries including Cameroon, Nigeria, Benin, Togo, Côte d'Ivoire, Burkina Faso and Guinea (Njukeng, 1998).

Mechanical inoculation of progenies (F 1 population) and their parents with YMV
Plants of Nicotiana benthamiana infected with standard isolate of YMV (Mignouna et al., 2001) were used as source of inoculums 10 days after inoculation with the virus antigen.All F 1 progenies and parents planted in the screenhouse were mechanically inoculated with the inoculum after being dusted with carborundum (600 mesh).Inoculation buffer was 0.03M sodium phosphate buffer pH 8.3, containing 0.2% DIECA, while sterilized mortars and pestles were used and disposable gloves were worn during inoculation.All plants were inoculated twice, with the second inoculation taking place three weeks after the first.Ten plants of each genotype were inoculated with the buffer as healthy controls.

Symptom evaluation and serological virus detection for YMV
All the inoculated plants were scored for the presence/absence of virus symptoms and severity 5, and 10 weeks after the second inoculation as previously reported by Mignouna et al. (2001) and Odu et al. (2004).Plants with score of 1 (no symptom) were considered as resistant while those scoring between 2 and 5 (2: moderate or mild symptoms, 3: severe symptoms, 4: very severe symptoms, 5: distortion, malformation of leaf or stem) were evaluated as susceptible.
Leaf samples were collected from all inoculated F 1 progenies and parent plants in the screenhouse, and tested for the presence of YMV by TAS-ELISA.Plants having leaf samples with mean absorbance value twice or more than that of the healthy sample were considered susceptible.A DYNEX MRX ELISA plate reader (DYNATECH, USA) was used to determine the absorbance value for each of the samples.

Analysis of inheritance
The Chi-square statistical test (Little and Hills, 1978) was performed to determine if the observed ratios of resistant to susceptible plants deviated from expected ratios.The observed ratio was considered to fit the expected ratio if the appropriate chi-square (χ 2 ) value had a corresponding probability of >0.05 (Gomez and Gomez, 1984).
Analysis of inheritance of resistance to YMV was carried out using the data from the virus symptom severity rating of the hybrids and their parents, as well as from the data from the serological indexing.

Results and Discussion
Pre-inoculation indexing of the F 1 progenies by TAS-ELISA did not detect any YMV infection.Although seed transmission of many viruses has been reported in several studies (Sekar and Sulochana, 1988;Thottappilly, 1992;Ndiaye et al., 1993;Brunt et al., 1996), it was not detected among the 1,564 seedlings screened during the course of this study.This included the use of TAS-ELISA serological detection method to detect any masked/latent infection by YMV in the D. rotundata progenies.This result concurs with the findings of Thouvenel and Fauquet (1979), when they screened progenies of crosses among several D. cayenensis genotypes for YMV infection by symptomatology alone.
Inoculation of transplanted seedlings growing in the screenhouse with YMV induced various YMV-associated symptoms such as mosaic, green vein-banding and chlorosis in some of the inoculated plants.Symptoms appeared on newly formed leaves 3 weeks after the first inoculation.
Of all the 1,564 F 1 plants that were established in the screenhouse for resistance study, a total of 380 seedlings showed symptoms while 460 tested positive for the presence of YMV by TAS-ELISA (Table 2), suggesting that 80 plants were also infected but symptomless.These symptomless infections could result from a tolerance mechanism operating in the particular genotypes.This confirms the findings of Mignouna et al. (2001), when they observed asymptomatic F 1 progenies which were ELISA-positive.The cross between TDr 35 and TDr 87/00211 had the highest number of plants that expressed symptoms and also those that were positive for YMV by TAS-ELISA.Resistance to viruses is often evaluated by the expression of disease symptoms without necessarily quantifying virus multiplication (Fraser, 1990).Plants can be classified as resistant when no symptoms or necrotic lesions develop and as susceptible when symptoms appear (Scholten et al., 1996).Observation of symptom expression complemented with the use of ELISA serological technique for the detection of virus infection in this study has shown that TAS-ELISA is both sensitive and consistent.TAS-ELISA was able to detect symptomless infection in genotypes that would have been taken as resistant if symptom observation had been employed as the sole method of indexing.
Although the variations in symptom severity may be due to differences in virus concentration, some genotypes could express symptoms independently of the rate of virus concentrations (Kuhn et al., 1981).
There was an approximately 50% correlation between the average symptom severity score of the crossed parents and the average severity ratings of the F 1 population (Figure 1).This shows that there is the relationship between the crossed parents and the resultant offspring with relation to symptom expression.Correlation analysis was carried out between ELISA reading (A 405 ) and the symptom severity scores in the F 1 generation of the hybrids for YMV infection.There was a highly significant positive correlation (p<0.0001) in the correlation matrix between symptom score and the ELISA reading of all plants.Classification of plants in the segregating families into susceptible or resistant genotypes was possible due to the presence of symptoms and also due to the reliability of TAS-ELISA for the detection of YMV in infected symptomless F 1 plants.The segregation for resistance to YMV based on the virus symptom severity scores shows that most of the crosses conform to ratio 3:1 proposed for the ratio of the resistant to susceptible in the populations derived in the inheritance studies (Table 3).On the other hand, considering the serological indexing (Table 4), most of the crosses conform to the ratios 3:1 (assuming random chromosome assortment) and 2.48:1 (assuming random chromatid assortment) proposed for the ratio of the resistant to susceptible plants in the populations derived in the inheritance studies (Mignouna et al., 2001).Table 3. Segregation for resistance to Yam mosaic virus (YMV), genus Potyvirus based on symptom severity scores of 20 F 1 families in a nursery obtained by crossing resistant and susceptible Dioscorea rotundata genotypes.The segregation ratio obtained in the cross between TDr 1621, a resistant genotype and TDr 87/00211, a susceptible genotype, fits both the 3:1 (χ 2 = 0.004; P = 0.951) and 2.48:1 (χ 2 = 0.505; P=0.477).A better fit is obtained with a 3:1 ratio, which is expected in a cross between two different heterozygotes with two dominant genes in a simplex condition.The same segregation ratio is obtained in a cross between TDr 35, a resistant genotype and TDr 87/00211, a susceptible line, where a better fit is obtained with a 3:1 (χ 2 = 0.544; P = 0.461).
Segregation ratios of progenies from crosses involving TDr 35, TDr 32, and TDr 93-1 were more complicated.Progenies from their crosses to TDr 87/00211 fitted the 3:1 ratio as for the previous genotypes.Segregation of progenies from the crosses of TDr 35 to TDr 95-115 and TDr 95-127 fitted 1:1 and 2.48:1 ratios respectively although the latter combination could also fit a 3:1 ratio.Progenies from crosses of TDr 93-32 and TDr 93-1 to TDr 95-127 segregated in a 1:1 ratio.These segregation ratios are more consistent with the actions of a single dominant gene in each situation.Mignouna et al. (2001) attributed this apparent complexity to the possibility of genotypic mixtures among the parental lines.
The cross between TDr 93-32, a moderately resistant genotype and TDr 95-127, a susceptible genotype produced a ratio that had tendency to fit to a ratio of 1:1.The same goes for the cross between TDr 93-1, a moderately resistant genotype and TDr 95-127.It should be noted that all genotypes designated moderately resistant were actually infected, as they permit the multiplication and spread of YMV, but they only had mild symptoms arising from infection.Mignouna et al. (2001) reported that ELISA detected the presence of YMV in parental resistant genotypes, though at a level far below those obtained in susceptible genotypes.The segregation ratios obtained in the cross between TDr 93-1 and TDr 87/00211 on one hand, and TDr 93-1 and TDr 95-127 on the other hand corroborated the findings of resistance in TDr 93-1 by Mignouna et al. (2001).This may also be true for some of the other genotypes designated as moderately resistant (MR).
Regression of average YMV symptom severity ratings of F 1 population on mid-parent values was significant, and the heritability estimate calculated was 0.23 (Figure 1).

Conclusion
On the basis of the results obtained in this study, tetrasomic inheritance of resistance to YMV is implicated in D. rotundata genotypes.It is also strongly suggested that resistance is dominantly inherited.Segregation of the progenies derived from some resistant genotypes and the susceptible genotype TDr 87/00211 showed that resistance is controlled by two dominant genes in a simplex condition assuming random chromosome assortment.

Figure 1 .
Figure 1.Relationship between parents and F 1 population in virus symptom severity score.

Table 1 .
). Accession numbers, names, sources and sexes of Dioscorea rotundata genotypes used in the resistance studies.

Table 2 .
Dioscorea rotundata parental genotypes and F 1 populations showing numbers of plants exhibiting symptoms of infection and numbers of plants that tested positive for Yam mosaic virus genus Potyvirus infection by TAS-ELISA after mechanical inoculation.
a Enzyme-linked immunosorbent assay.

Table 4 .
Segregation ratios of F 1 populations obtained in crosses between resistant and susceptible Dioscorea rotundata genotypes based on serological indexing to detect Yam mosaic virus (YMV), genus Potyvirus after mechanical inoculation with the virus.