Serological detection of yam viruses in farmers' fields in Ogun State,Nigeria

A field survey was conducted in eight local government areas (LGA) of Ogun state, Nigeria to assess the incidence of viral diseases of yams in the areas. Leaf samples were collected from 90 yam plants which were either symptomatic or asymptomatic. These were bulked into 45 during serological tests and the viruses indexed include yam mosaic virus (YMV); Dioscorea alata bacilliform virus (DaBV) and cucumber mosaic virus (CMV). DaBV was the most prevalent virus on the field with incidence of 48.9% (22/45) followed by YMV which occurred in 42.2% (19/45). CMV had the lowest percentage of incidence; 2.2% (1/45). Of all the LGAs visited, Abeokuta north and Abeokuta south had the highest incidence of YMV and DaBV, respectively. Mixed virus infections were also detected.     

Strategies to combat the problem of yam anthracnose disease: Status and prospects

Yam (Dioscorea spp.) anthracnose, caused by Colletotrichum alatae, is the most devastating fungal disease of yam in West Africa, leading to 50%–90% of tuber yield losses in severe cases. In some instances, plants die without producing any tubers or each shoot may produce several small tubers before it dies if the disease strikes early. C. alatae affects all parts of the yam plant at all stages of development, including leaves, stems, tubers, and seeds of yams, and it is highly prevalent in the yam belt region and other yam-producing countries in the world. Traditional methods adopted by farmers to control the disease have not been very successful. Fungicides have also failed to provide long-lasting control. Although conventional breeding and genomics-assisted breeding have been used to develop some level of resistance to anthracnose in Dioscorea alata, the appearance of new and more virulent strains makes the development of improved varieties with broad-spectrum and durable resistance critical. These shortcomings, coupled with interspecific incompatibility, dioecy, polyploidy, poor flowering, and the long breeding cycle of the crop, have prompted researchers to explore biotechnological techniques to complement conventional breeding to speed up crop improvement. Modern biotechnological tools have the potential of producing fungus-resistant cultivars, thereby bypassing the natural bottlenecks of traditional breeding. This article reviews the existing biotechnological strategies and proposes several approaches that could be adopted to develop anthracnose-resistant yam varieties for improved food security in West Africa.     

A Turnip crinkle virus Isolate Lacking the CP Counter‐Defence Protein Gene Providing Protection Against the Wild‐Type Strain is Associated with Highly Localized RNA Silencing

Cross‐protection has been used successfully and commercially to control a range of virus diseases for which the selection of suitable mild strains of plant viruses is necessary. Turnip crinkle virus (TCV) is highly pathogenic on Arabidopsis plants and its silencing suppressor‐defective mutant, TCVΔCP, can induce highly localized RNA silencing which is differs from that of other protective strains. We found that TCVΔCP provides some protection against wild‐type TCV but lacks complete protection, and the relative locations of the protective virus and challenge virus affect the degree of cross‐protection. However, similar cross‐protection afforded by TCVΔCP is not observed in Nicotiana benthamiana plants. As expected, TCVΔCP pre‐infected Arabidopsis plants fail to protect against infection with the unrelated Cucumber mosaic virus, strain Fhy. It appears that cross‐protection afforded by TCVΔCP requires that the challenge virus be very similar in sequence, which is a characteristic of RNA silencing. In order to investigate whether the protection is associated with the highly localized RNA silencing, mutant plants involved in key silencing pathway genes of RNA silencing machinery, including dcl2, dcl4 and triple dcl2/dcl3/dcl4 mutants were used. The results demonstrate that cross‐protection afforded by TCVΔCP is dependent on host RNA silencing, and both DCL2 and DCL4 play important roles in this process.     

Infection of yam tubers by Aspergillus niger in relation to isolate, yam species, and temperatures

Infection of yam (Dioscorea spp.) tubers by a range of geographical isolates of Aspergillus niger from yam and other hosts was tested on tubers imported into the UK, using a standard inoculation procedure. A yam isolate of A. niger from Nigeria was generally more aggressive than one from Barbados or from isolates from different hosts. There were inconsistent differences in infection between different portions of the tubers. Infection and weight loss of yam tubers were evaluated at temperatures ranging from 18 to 36°C, using the Nigerian and Barbados isolates. Mean percentage infection increased across the temperature range for the Nigerian isolate; for the Barbados isolate, infection increased with temperatures only from 18 to 30°C. Comparison of different yam species in relation to A. niger infection is difficult due to lack of information on cultivar and provenance.     

Isolation, characterisation and identification of a potyvirus from Dioscorea alata L. (water yam) in Nigeria

A yam potyvirus was isolated from Dioscorea alata samples collected in Nigeria. The virus was not transmissible mechanically but was transmitted by Aphis craccivora to four cowpea cultivars (Ife Brown, IT84S-2114, IT82E-10 and TVu2657), and from which it could be mechanically transmitted between the cowpea cultivars. In infectivity- tests using cowpea extracts, the virus had a dilution end point of 10^-4, a thermal inactivation point of 60–65°C and longevity in vitro of 2 days at room temperature. The virus coat protein had an estimated molecular weight of 32 100 daltons. The virus was identified as an isolate of Dioscorea alata virus (DAV; syn. yam virus 1) due to its biological characteristics and its serological reaction with antiserum raised against DAV. The virus is not related to yam mosaic virus, but distantly related to blackeye cowpea mosaic virus and cowpea aphid-borne mosaic virus.     

Pathogenic and genetic variability among Colletotrichum gloeosporioides isolates from different yam hosts in the agroecological zones in Nigeria

Anthracnose, caused by Colletotrichum gloeosporioides Penz., is the most severe foliar disease of water yam (Dioscorea alata) worldwide. Population genetic analyses can yield useful insights into the evolutionary potential of C. gloeosporioides and thus lead to the development of appropriate disease management strategies. The genetic structure of C. gloeosporioides populations from yam and non‐yam hosts in three agroecological zones of Nigeria was investigated. Microsatellite‐primed polymerase chain reaction (MP‐PCR), virulence phenotyping using five putative D. alata differentials, cross‐inoculation tests, and the presence/absence of a Glomerella teleomorph in yam fields were used to infer the evolutionary potential of C. gloeosporioides on yam. We observed high genotypic diversity (GD = 0.99 to 1.00) for populations from all hosts and agroecological zones, with multiple pathogen genotypes in individual anthracnose lesions. Genetic differentiation was low among pathogen populations from different hosts (G_ST = 0.10, θ = 0.034), and agroecological zones (G_ST = 0.04, θ = 0.018), indicating limited host differentiation and significant gene flow. No evidence was found for the existence of C. gloeosporioides f. sp. alatae reported in previous studies. The fungus was recovered from several non‐yam host species commonly found in yam fields but non‐yam isolates caused only mild to moderate symptoms on yam. Eighteen C. gloeosporioides virulence phenotypes were identified among 217 isolates but there was a weak correlation (r = 0.02, P = 0.40) between virulence phenotype and MP‐PCR haplotype. Consistent with the above findings, we observed for the first time the Glomerella teleomorph on anthracnose‐infected yam plants in Nigeria, indicating that sexual recombination might play an important role in anthracnose epidemics on yam. The implications of these findings for C. gloeosporioides evolutionary potential and anthracnose resistance breeding are discussed.     

Severity of spoilage storage rots of white yam (Dioscorea rotundata Poir.)

Severity of storage rots in different sections of white yam tubers (Dioscorea rotundata Poir.) was investigated. Yam samples with rots were collected from a yam barn and from selected markets in Accra, Ghana, to identify the most predominant pathogens associated with the rots. Nine fungal spoilage microorganisms, including Aspergillus flavus, Aspergillus niger, Botryodiplodia theobromae, Fusarium culmorum, Fusarium oxysporium, Fusarium sp., Penicillium brevi‐compactum, Penicillium sp. and Rhizopus stolonifer and a bacterium species Erwinia carotovora were identified. The mean incidence of occurrence of the organisms on rotten tissues ranged from 1.2% to 28.5%. Of the 10 microorganisms isolated, B. theobromae, F. oxysporium and R. stolonifer were the most frequently encountered spoilage microorganisms in the markets. E. carotovora, Fusarium solani and Penicillium sp. were relatively sparse (incidence not exceeding 3%) compared to the other yam spoilage microorganisms. The surface area and weight of necrotic tissue induced by the spoilage fungi in the various zones of the tubers over a 28‐day storage period were assessed. All the spoilage microorganisms produced rots in the yams, although to different degrees. The severity of the rots increased in weight and area over the period when the tubers were in store but were normally not significantly different in the zones of tubers. There was, however, a linear progression of rots in the various zones of the yam tubers. Although there was generally no significant (P ≥ 0.05) difference in the severity of rots induced by the different microorganisms in the tubers, R. stolonifer commonly induced more rot in the zones of the tubers compared to B. theobromae and F. oxysporium.     

Photoperiod affects the growth and development of yam plantlets obtained by in vitro propagation

The effects of photoperiod on the development of in vitro grown plantlets of yam (Dioscorea alata L.), were investigated. Plantlets were transplanted into pots, acclimatizated until they reached vegetative stages V₁ (3 leaves) or V₂ (8 leaves), and then grown under 12-h or 16-h photoperiod. The formation and development of underground tubers was only induced under 12-h photoperiod. Tuber initiation was not related to the initial vegetative stage of plants, and the tubers were visible at about 18 – 24 d. On the contrary, a 16-h photoperiod inhibited tuber formation and stimulated vine and leaf growth. The total dry matter production and the number of leaves per plant of V₁ stage plants were 50 and 30 % lower respectively, after 44 d under 12-h compared to 16-h photoperiod. These parameters were not influenced by photoperiod in V₂ stage plants. Consequently, the effect of 12-h photoperiod on dry matter of V₁ plants was attributed to a source limitation related to the early initiation of tuberization. The transfer of plants grown under 12-h to 16-h photoperiod stopped tuber growth and starch accumulation. On the other hand, it stimulated the shoots and the roots to grow.Abbreviations

Production and properties of monoclonal antibodies to Solanum nodiflorum mottle sobemovirus

Black raspberry necrosis virus (BRNV) reaches only very low concentrations in herbaceous plants and is difficult to maintain in culture. However, in a mixed culture with an unrelated virus, Solanum nodiflorum mottle (SNMV), in the genus Sobemovirus, the concentration of BRNV particles increases about 1000‐fold. In attempts to produce monoclonal antibodies (MAbs) to BRNV for diagnostic use, purified virus particles from the mixed virus culture were used as immunogen and the resultant antibodies screened against cultures of SNMV alone, BRNV+SNMV and healthy plant extracts. None of the virus‐specific MAbs obtained in this way was specific to BRNV but six were specific to SNMV. Although the original objective was not achieved, the SNMV MAbs were characterised and used to study serological properties of SNMV and other Sobemoviruses. Characterisation of the six SNMV MAbs showed that four were IgG3, one IgG1 and the other IgG2b. SNMV was detected by all six MAbs in ELISA, by five in Western blotting, by three in agarose gel double diffusion tests, but only one was suitable for trapping virus particles in immuno‐electron microscopy (IEM). In Western blotting using virus in sap extracts of Nicotiana clevelandii, each of the five MAbs detected a single major band of Mc. 31 000 in sap containing SNMV, and additional bands of lower mass attributed to degradation of coat protein. In various serological tests, no cross‐reactions were detected between SNMV and seven other viruses from the genus Sobemovirus. However, in IEM but not in Western blotting, significant cross‐reactions were observed between SNMV and Velvet tobacco mottle virus, another species from the genus Sobemovirus. The significance of these different findings is discussed.     

Mycoflora of tuber surface of white yam (Dioscorea rotundata poir) and postharvest control of pathogens with Bacillus subtilis

Bacillus subtilis (Enrenberg) Cohn was investigated for its antagonistic properties against surface mycoflora of yam (Dioscorea rotundata Poir) tubers in storage. Yam tubers inoculated with a spore suspension of B. subtilis in potato dextrose broth using a knapsack sprayer showed a drastic reduction in the range and number of mycoflora, including pathogens of the tuber surface in contrast to the control tubers, during the five-month storage period in a traditional yam barn. However, B. subtilis maintained a high frequency of occurrence during the same period. Botryodiploidia theobromae Pat, Fusarium moniliforme Wollen and Reink., Penicillium sclerotigenum Yamamoto, and Rhizoctonia sp. were displaced completely on the treated tubers. The antagonism of B. subtilis was so effective that the normal tuber surface mycoflora was greatly reduced throughout the storage period of five months by a simple initial application of the antagonist.This revised version was published online in October 2005 with corrections to the Cover Date.     

Inhibited Long-Distance Movement of Potato Leafroll Virus to Tubers in Potato Genotypes Expressing Combined Resistance to Infection, Virus Multiplication and Accumulation

Plants of two potato clones which, in preliminary greenhouse assessments, showed resistance to multiplication and accumulation of potato leafroll virus (PLRV) were graft or aphid inoculated with the virus and grown in the greenhouse; plants of a moderately susceptible cultivar were used for comparison in all experiments. A high concentration of aphid‐borne inoculum was used to ensure strong infection pressure. Clone M62759 appeared to be highly resistant to PLRV infection, whereas clone PS1706 was more susceptible. Both clones expressed a high level of resistance to virus multiplication, when primary or secondary infection was assayed by enzyme‐linked immunosorbent assay. Moreover, PLRV was detected in only few or none of the progeny plants of clone M62759, which thus strongly inhibited virus transport to tubers. The study on PLRV translocation from aphid‐inoculated shoots to uninoculated shoots sprouted from the same tubers showed that no specific mechanisms are likely to impair PLRV movement through the tubers of the resistant genotypes. These results indicate that three valuable components of the resistance to PLRV are probably closely linked in the genotype, a combination that seems to occur rather rarely in potato clones. Nevertheless, selecting potato genotypes for the complex resistance to PLRV may prove to be a worthwhile part of breeding programmes, provided that the genetic mechanisms governing particular types of resistance are better recognized.     

Strong resistance to potato tuber necrotic ringspot disease in potato induced by transformation with coat protein gene sequences from an NTN isolate of Potato virus Y

The potato cv. Igor is susceptible to infection with Potato virus Y (PVY) and in Slovenia it has been so severely affected with NTN isolates of PVY causing potato tuber necrotic ringspot disease (PTNRD) that its cultivation has ceased. Plants of cv. Igor were transformed with two transgenes that contained coat protein gene sequence of PVY^NTN. Both transgenes used PVY sequence in a sense (+) orientation, one in native translational context (N‐CP), and one with a frame‐shift mutation (FS‐CP). Although most transgenic lines were susceptible to infection with PVY^NTN and PVY^O, several lines showed resistance that could be classified into two types. Following manual or graft inoculation, plants of partially resistant lines developed some symptoms in foliage and tubers, and virus titre in the foliage, estimated by ELISA, was low or undetectable. In highly resistant (R) lines, symptoms did not develop in foliage and on tubers, and virus could not be detected in foliage by ELISA or infectivity assay. Four lines from 34 tested (two N‐CP and two FS‐CP) were R to PVY^NTN and PVY^O and one additional line was R to PVY^O. When cv. Spey was transformed with the same constructs, they did not confer strong resistance to PVY^O.     

Incidence of potato virus Y infection in seed and ware tubers of the potato cv. Record

The incidence of potato virus Y (PVY) infection was assessed in samples of potato tubers, cv. Record, taken from Scottish seed stocks and English ware crops grown from some of these seed stocks. PVY was readily detected by ELISA of tuber sprouts. PVY-infected tubers were found in 10 seed stocks of 84 tested. The mean level of virus infection was 0.23%, 0.76% and 0.56% in Super Elite, Elite and AA stocks respectively. In 46 commercial ware crops grown from some of these seed stocks, a substantial proportion of the harvested tubers in all but one of the crops were infected with PVY, the mean percentage of infected tubers was 58.5%. Ware crops grown from seven seed stocks in which PVY had been detected (mean 6.2% infection in seed) contained a mean of 70% infected tubers, compared with 56% infection in crops grown from 39 stocks in which PVY was not detected in the seed tubers. The predominant PVY strain detected in the ware crops was the veinal necrosis strain (PVY^vn).     

Delimitation of physiological regions in yam tubers (Dioscorea sp.) and distribution pattern of saccharide degrading enzymes,cell sap pH and protein in these regions

Physiological regions of yam tubers were morphologically defined in different specie into ‘Head’, ‘Middle’ and ‘Tail’, while the limits of these regions were studied using phosphorylase activity. Variation in enzyme activity, pH and protein concentration was found in different regions of the tubers. Old yam tubers had significantly higher activities of saccharide degrading enzymes, hexokinase, phosphorylase, glucose-6-phosphate dehydrogenase, phosphofructokinase and pyruvate kinase, than the new tubers. However, activity of phosphofructokinase in newD. rotundata was higher than that of old tuber. The high activity of phosphorylase in different regions of all the yam tubers examined indicates a very important role of this enzyme in starch degradation inDioscorea species. The measured pH and protein concentration were also higher in old yam tubers. Except for phosphorylase, these enzymes had alkaline pH optima.     

Rapid assessment of resistance of tissue-cultured water yam (Dioscorea alata) and white guinea yam (Dioscorea rotundata) to anthracnose (Colletotrichum gloeosporioides Penz.)

Yam anthracnose is caused by the pathogen Colletotrichum gloeosporioides Penz. and has been identified as the most important biotic constraint to yam production worldwide. Rapid assessment of the disease is vital to its effective diagnosis and management. In this study, tissue-cultured yam plantlets of five lines of Dioscorea alata and nine of D. rotundata were rapidly assessed for their reactions to two isolates of yam anthracnose. The plantlets, obtained from meristem of the nodal cuttings, were grown for 8?weeks on Murashige and Skoog (MS) basal medium, acclimatised for 3?weeks, hardened for an additional 3?weeks, arranged in screen house in completely randomised design and sprayed with spore inocula prepared from 7?day-old culture of the two strains of Colletotrichum gloeosporioidies Penz. The relative resistance of the different Dioscorea spp. was evaluated using three disease indices – severity at seventh day after inoculation, SD7; area under disease progress curve, AUDPC; and disease severity rate, Rd. A modified rank-sum classification method put TDa 1425 and TDr 2040, with rank sum of 2.0 each, as resistant. TDr 2121, TDr 2287 and TDr 2048 were susceptible with rank sum of 27.50, 25.50 and 24.50, respectively. Dioscorea alata TDa 1425 and Dioscorea rotundata TDr 2040 were recommended in areas endemic with yam anthracnose, and also as parent lines while breeding for resistance to anthracnose.     

Studies on Biological Control of Postharvest Rot in Yams (Dioscorea spp.) using Trichoderma viride

From in vitro and in vivo screening tests for antagonism by isolates of Trichoderma against postharvest pathogens of yams (Dioscorea spp.), an isolate of Trichoderma viride Pers. ex S.F. Gray was selected as the most promising candidate for the biocontrol of postharvest rot of yams. Inoculation of white yam (Dioscorea rotundata Poir.) with conidiaspores of T. viride and subsequent storage of the tubers under the ambient environment conditions of a traditional yam barn, resulted in a drastic reduction in the frequency of occurrence of the normal tuber surface mycoflora over a 4‐month (December‐April) storage period. Trichoderma viride on the other hand, maintained a high frequency of occurrence during the same period. Furthermore, whereas up to 52.0%) rot was found among groups of tubers that were artificially inoculated with the postharvest pathogens of yams, Aspergillus niger Van Tiegh., Botryodiplodia theobromae Pat. or Penicillium oxalicum Currie and Thom, and also the group that was not inoculated with any organism (control), among groups of tubers that were inoculated with T. viride the rot was either totally suppressed or only a low percentage of rot occurred. The significance of the findings is discussed in relation to yam storage especially by farmers with limited resources.     

Inheritance of resistance to Yam mosaic virus, genus Potyvirus, in white yam (Dioscorea rotundata)

Yam mosaic virus (YMV) causes the most-widespread and economically important viral disease affecting white yam (Dioscorea rotundata) in West Africa. The genetic basis of resistance in white yam to a Nigerian isolate of YMV was investigated in three tetraploid D. rotundata genotypes: TDr 93–1, TDr 93–2 and TDr 89/01444. F₁ progeny were produced using TDr 87/00571 and TDr 87/00211 as the susceptible parents. Segregation ratios indicated that a single dominant gene in a simplex condition governs the resistance in TDr 89/01444, while the resistance in TDr 93–2 is associated with the presence of a major recessive gene in duplex configuration. Segregation of progeny of the cross TDr 93–1×TDr 87/00211 fitted a genetic ratio of 2.48:1 resistant:susceptible, which can be expected when two simplex heterozygotes are crossed, indicating the possible modifying effect of the susceptible parent. A triple antibody immunosorbent assay (TAS-ELISA) was used for virus detection in inoculated plants. Slight mosaic symptoms appeared on most resistant individuals, while asymptomatic resistant genotypes with high ELISA (A₄₀₅) values were observed in all crosses. Such a heterogeneous response suggests the influence of additional modifier genes that segregate in the progeny. The finding that resistance can be inherited as a dominant or recessive character has important implications for YMV resistance breeding. Received: 15 August 2000 / Accepted: 12 April 2001     

Detection of measles,mumps and rubella viruses by immuno‐colorimetric assay and its application in focus reduction neutralization tests

Measles, mumps and rubella are vaccine‐preventable diseases; however limited epidemiological data are available from low‐income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture‐based rapid and reliable immuno‐colorimetric assay (ICA) was established and its utility studied. Twenty‐three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT‐PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post‐infection in Vero or Vero‐human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post‐infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero‐epidemiological, cross‐neutralization and pre/post‐vaccine studies.