Comparative analysis of genetic variation among Xanthomonas axonopodis pv manihotis isolated from the western states of Nigeria using RAPD and AFLP.

Xanthomonas axonopodis pv manihotis is the causal agent of cassava bacterial blight (CBB) worldwide. CBB disease is a major constraint to cassava cultivation, and losses can be extremely severe in regions where highly susceptible cultivars are grown. To develop an efficient disease management policy, the genetic diversity of the pathogens population must be known. There is dearth of information on the genetic diversity of X. axonopodis pv manihotis population in Nigeria. We used RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), a PCR-based technique, to characterize the X. axonopodis pv manihotis isolates from the western States of Nigeria. Thirteen strains Xam and 2 reference strains were tested with eight primers combination of AFLP and 4 RAPD primers. RAPD amplified DNA fragment data showed four major clusters at 80 % similarity coefficient level and two strains were not clustered by this analysis. Strains Kwa76A and Ond48A were also separated in the principal component analysis of the same data. Numerical analysis differentiated the AFLP patterns into four distinct clusters and grouped two strains separately at 66 % similarity. PCA assembly grouped the bacterial strains into 4 and one of the strains was singled out from the others. The two DNA analyses techniques seem to be complimentary to one another and informative on the genomic structure of Xam population in Western Nigeria. The genetic analysis presented here contributes to understanding of the Xam population structure in Western Nigeria.

Characterization of an 18,166 EST dataset for cassava (Manihot esculenta Crantz) enriched for drought-responsive genes.

Cassava (Manihot esculenta Crantz) is a staple food for over 600 million people in the tropics and subtropics and is increasingly used as an industrial crop for starch production. Cassava has a high growth rate under optimal conditions but also performs well in drought-prone areas and on marginal soils. To increase the tools for understanding and manipulating drought tolerance in cassava, we generated expressed sequence tags (ESTs) from normalized cDNA libraries prepared from dehydration-stressed and control well-watered tissues. Analysis of a total of 18,166 ESTs resulted in the identification of 8,577 unique gene clusters (5,383 singletons and 3,194 clusters). Functional categories could be assigned to 63% of the unigenes, while another approximately 11% were homologous to hypothetical genes with unclear functions. The remaining approximately 26% were not significantly homologous to sequences in public databases suggesting that some may be novel and putatively specific to cassava. The dehydration-stressed library uncovered numerous ESTs with recognized roles in drought-responses, including those that encode late-embryogenesis-abundant proteins thought to confer osmoprotective functions during water stress, transcription factors, heat-shock proteins as well as proteins involved in signal transduction and oxidative stress. The unigene clusters were screened for short tandem repeats for further development as microsatellite markers. A total of 592 clusters contained 646 repeats, representing 3.3% of the ESTs queried. The ESTs presented here are the first dehydration stress transcriptome of cassava and can be utilized for the development of microarrays and gene-derived molecular markers to further dissect the molecular basis of drought tolerance in cassava.

Effect of soil moisture stress on growth and yield of cassava in Nigeria.

Nine cassava genotypes were evaluated for their growth responses and adaptability to soil moisture stress on the field and in the screenhouse in Nigeria. Genotypes were evaluated in three savanna agroecologies in a randomized complete block design with three replicates. Screenhouse evaluation was conducted using three moisture regimes of 75, 50 and 25% Field Capacity (FC) in a two-factor factorial experiment in CRD with three replicates. Morphological and yield data were collected on the field and in the screenhouse. Results showed significant (p < 0.05) difference among genotypes on the field and in the screenhouse. Field moisture stress led to a decline in plant height by 47%, stem girth by 15%, number of tubers by 95% and tuber yield by 87%. Screenhouse moisture condition of 25% FC led to a reduction in plant height by 12.6 and 21.2%, stem girth by 16.3 and 21.7%, number of roots by 94.5 and 88.7% and root weight by 93.3 and 94.9%, respectively at 16 and 30 WAP. Moisture stress therefore resulted into considerable reduction in both vegetative growth and yield of cassava genotypes. Therefore, a concerted effort in breeding cassava for drought tolerance is needed as cassava cultivation is expanding into nontraditional semiarid regions of sub-Saharan Africa. Germplasm introduced from Latin America (especially north-eastern Brazil) is providing a unique source of variability to further broaden the genetic base for drought tolerance in cassava.

The use of biolistic inoculation of cassava mosaic begomoviruses in screening cassava for resistance to cassava mosaic disease.

Inoculation of cassava with infectious clones of cassava mosaic geminiviruses (Geminiviridae: Begomovirus) and total DNA extracts from plants infected with well-characterised viruses was evaluated using the Bio-Rad Helios Gene Gun System. Total DNA extracts from infected plants and cloned viruses were produced for coating gold particles and bombardment onto new cassava genotypes, 96/1089A, 96/1039, 96/0160, 96/0304 and three local landraces TME 117, TME 3 and TME 4. Cloned DNA of a Kenyan isolate of the recombinant variant of East African cassava mosaic virus (EACMV-UG2-[Ka]), was only infectious to TME 117 (7/10 plants), 3 weeks post-inoculation with mild infection symptoms in the newly developing leaves. Biolistic inoculation with a chimeric pseudorecombinant virus between DNA A and B components from EACMV-[Ke-Kilifi] and EACMV-UG2-[Ka], respectively, was infectious to TME 117, 96/1039 and 96/0304 and developed very severe and persistent symptoms. TME 3 and TME 4 also developed symptoms, 12 days post-inoculation (d.p.i.). Total DNA extracts of ACMV and EACMV-[Ke-Kilifi] resulted in serious infections with symptoms already evident, 10d.p.i. In general, biolistic inoculation trials with total DNA extracts resulted in a higher number of infected plants expressing symptoms at a much earlier stage (10-12d.p.i.) compared with trials inoculated with virus clones.

Status of Cassava Begomoviruses and Their New Natural Hosts in Nigeria.

A diagnostic survey was conducted in 2002-03 to determine the status of cassava mosaic begomoviruses in Nigeria and to ascertain if the virulent Ugandan variant of East African cassava mosaic virus (EACMV-Ug2) was present. Of the 418 farms visited, 48% had cassava with moderately severe or severe symptoms, whereas 52% had cassava with mild symptoms. These distributions were at random. Of the 1,397 cassava leaf samples examined, 1,106 had symptoms. In polymerase chain reaction tests, 74.1% of the symptom-bearing samples tested positive for African cassava mosaic virus (ACMV) alone, 0.3% for EACMV alone, 24.4% for mixed infections by the two viruses, and 1.2% did not react with any of the primers used. The two viruses also were detected in 32% of the 291 symptomless plants and in the whitefly vector samples. EACMV-Ug2, Indian cassava mosaic virus, and South African cassava mosaic virus were not detected in any of the whitefly or leaf samples. Most farms had ACMV in single infection as well as in mixed infections with EACMV. Most doubly infected plants showed severe symptoms. Two biological variants of ACMV were identified based on symptom expression on cassava in the field. ACMV and EACMV were detected in the leguminous plant Senna occidentalis (L.) Link and the weed Combretum confertum Lams.; these are new natural hosts of the viruses. Although the virulent EACMV-Ug2 was not detected, the occurrence of variants of ACMV and a high proportion of mixed infections by ACMV and EACMV, which could result in recombination events such as the one that produced EACMV-Ug2, demands appropriate measures to safeguard cassava production in Nigeria.

Whitefly Bemisia tabaci (Homoptera: Aleyrodidae) infestation on cassava genotypes grown at different ecozones in Nigeria.

Large-scale screening of cassava, Manihot esculenta Crantz, genotypes for resistance to infestation by whitefly Bemisia tabaci Gennadius, the vector of cassava mosaic geminiviruses, is limited. A range of new cassava elite clones were therefore assessed for the whitefly infestation in the 1999/2000 and 2000/2001 cropping seasons in experimental fields of International Institute of Tropical Agriculture, Ibadan, Nigeria. On each scoring day, between 0600 and 0800 hours when the whiteflies were relatively immobile, adult whitefly populations on the five topmost expanded leaves of cassava cultivars were counted. All through the 6-mo scoring period, there was a highly significant difference in whitefly infestation among the new cassava elite clones. Vector population buildup was observed in Ibadan (forest-savanna transition zone) and Onne (humid forest), 2 mo after planting (MAP). Mean infestation across cassava genotypes was significantly highest (16.6 whiteflies per plant) in Ibadan and lowest in Zaria (0.2). Generally, whitefly infestation was very low in all locations at 5 and 6 MAP. During this period, cassava genotypes 96/1439 and 91/02324 significantly supported higher infestations than other genotypes. Plants of 96/1089A and TMS 30572 supported the lowest whitefly infestation across cassava genotypes in all locations. The preferential whitefly visitation, the differences between locations in relation to whitefly population, cassava mosaic disease, and the fresh root yield of cassava genotypes are discussed.

Assessment of laboratory methods for evaluating cassava genotypes for resistance to root rot disease.

Field evaluation of six cassava genotypes for resistance to root rot disease was compared with three rapid laboratory methods (whole root inoculation, root slice inoculation, and stem inoculation) for resistance screening. Both the field evaluation and the three laboratory methods separated the varieties into resistant and susceptible groups. Genotypes 30572 and 91/02324 were resistant while 92/0247, 92/0057 and TME-1 were susceptible. One genotype (30001) was not consistent in its reaction between field evaluation and laboratory assays. In the laboratory assays with three fungal pathogens, different pathogens varied in their levels of virulence on host genotypes. With the most virulent pathogen (Botryodiplodia theobromae), the majority of the genotypes reacted in the same way across trials with the root slice and whole root assays. Due to the good correlation between the whole root assay and the field results, we recommend this for the routine assessment of cassava resistance to root rot disease and for the analysis of virulence of pathogen isolates. However, because of the advantages in terms of economy of labour, space, time, quantity of root and inoculum required, the root slice assay could be used for the preliminary screening of large cassava accessions. The selected genotypes can then be further screened with the whole root inoculation method.

Effect of temperature on the survival and infectivity of Pseudotheraptus devastans vector.

The aim of this study was to investigate under a controlled environment, the effect of temperature on the survival and infectivity of Pseudotheraptus devastans Distant, a cassava anthracnose disease vector. The insect P. devastans was collected from young cassava (Manihot esculenta Crantz) field plots, at the International Institute of Tropical Agriculture, (IITA), Ibadan, Nigeria. A mixture of the different developmental stages of eggs, first to fifth instar nymphs, and adults, were incubated in controlled environment chambers, under various constant temperatures of: 15, 17, 22, 25, 27, 30, and 35 degrees C. Relative humidity at different temperature conditions were recorded and maintained at 90%, 85%, 80%, 75%, 70%, 65%, and 60%, respectively. A significant increase in insect survival was observed between 22 and 27 degrees C temperature conditions while a significant decrease in survival was observed at 15 degrees C and above 30 degrees C. Lesion number, lesion diameter and infectivity among the insect stages varied as a function of temperature and relative humidity. Infectivity was highest at 22-25 degrees C maintained at 75-80% RH and lowest at 15 degrees C and above 30 degrees C maintained respectively, at 65% RH and 90% RH. There was considerable low vector infectivity due to low survival of the insects at extreme temperatures.

Simple sequence repeat marker diversity in cassava landraces: genetic diversity and differentiation in an asexually propagated crop.

Cassava (Manihot esculenta) is an allogamous, vegetatively propagated, Neotropical crop that is also widely grown in tropical Africa and Southeast Asia. To elucidate genetic diversity and differentiation in the crop's primary and secondary centers of diversity, and the forces shaping them, SSR marker variation was assessed at 67 loci in 283 accessions of cassava landraces from Africa (Tanzania and Nigeria) and the Neotropics (Brazil, Colombia, Peru, Venezuela, Guatemala, Mexico and Argentina). Average gene diversity (i.e., genetic diversity) was high in all countries, with an average heterozygosity of 0.5358 +/- 0.1184. Although the highest was found in Brazilian and Colombian accessions, genetic diversity in Neotropical and African materials is comparable. Despite the low level of differentiation [F(st)(theta) = 0.091 +/- 0.005] found among country samples, sufficient genetic distance (1-proportion of shared alleles) existed between individual genotypes to separate African from Neotropical accessions and to reveal a more pronounced substructure in the African landraces. Forces shaping differences in allele frequency at SSR loci and possibly counterbalancing successive founder effects involve probably spontaneous recombination, as assessed by parent-offspring relationships, and farmer-selection for adaptation.

Variants of East African cassava mosaic virus and Its Distribution in Double Infections with African cassava mosaic virus in Nigeria.

In a survey for cassava mosaic begomoviruses conducted in 1997 and 1998 in Nigeria, East African cassava mosaic virus (EACMV) was detected by the polymerase chain reaction together with African cassava mosaic virus (ACMV) in 27 out of 290 cassava leaf samples of infected plants from 254 farmers' fields in five agroecological zones. One plant was infected with EACMV only. Five variant isolates of EACMV were observed based on their reactions to primers that could detect Cameroonian and East African strains of EACMV. Isolates of variants 1 and 3 occurred mostly in the derived or coastal and southern Guinea savannahs, while variants 4 and 5 predominated in the humid forest region. Isolates of variant 2 were widely distributed across the three agroecologies. EACMV was not detected in the northern Guinea savannah and arid and semiarid zones. Most doubly infected plants showed more severe symptoms than plants with single infection. Occurrence of EACMV variants together with ACMV detection and information about their distribution in Nigeria could be used for the selection of cassava clones in cassava mosaic disease resistance programs.

In vitro, greenhouse and field assessments of cassava lines for resistance to anthracnose disease caused by Colletotrichum gloeosporioides f.sp. manihotis.

Fifty-three cassava lines were selected from breeding populations at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria and screened in vitro for resistance to cassava anthracnose disease (CAD). The in vitro inoculation of stem cuttings with the fungus Colletotrichum gloeosporioides f.sp. manihotis showed significant differences (p +/- 0.05) in acervuli production and in the sensitivity of the cassava lines to the fungal infection after 7 days of incubation at 25 degrees C. Cassava lines 88/01084, 91/00595, 91/00475, 91/00344, 91/00684, 91/00313, 91/00422, and 91/00344 were highly resistant, with necrotic lesion sizes less than 7 mm. In contrast pedigree lines 88/02549, 89/0008, 91/00390 and 91/00402 were highly susceptible with the largest necrotic lesion size being greater than 20 mm. Ten cassava lines from the in vitro screening that showed varying levels of resistance to CAD were selected, based on their flowering abilities for diallel hydridization trials, and were further screened in greenhouse and field trials for CAD resistance. The greenhouse and field screening showed significant varietal differences (p +/- 0.05) in sensitivity to the fungus. In all cases, the progeny lines showed correlated levels of resistance irrespective of the type of screening or assessments. Correlation analysis of the in vitro, greenhouse and field assessments showed that there was a good correspondence among all three methods of evaluating for CAD.

First Report of East African Cassava Mosaic Begomovirus in Nigeria.

Cassava (Manihot esculenta Crantz) is an important food crop in sub-Saharan Africa. One of the major production constraints is cassava mosaic disease caused by African cassava mosaic (ACMV) and East African cassava mosaic (EACMV) begomoviruses. ACMV is widespread in its distribution, occurring throughout West and Central Africa and in some eastern and southern African countries. In contrast, EACMV has been reported to occur mainly in more easterly areas, particularly in coastal Kenya and Tanzania, Malawi, and Madagascar. In 1997, a survey was conducted in Nigeria to determine the distribution of ACMV and its strains. Samples from 225 cassava plants showing mosaic symptoms were tested with ACMV monoclonal antibodies (MAbs) in triple antibody sandwich enzyme-linked immunosorbent assay (1). Three samples reacted strongly with MAbs that could detect both ACMV and EACMV. One of them did not react with ACMV-specific MAbs while the other two reacted weakly with such MAbs. With polymerase chain reaction (2), the presence of EACMV and a mixture of EACMV and ACMV in the respective samples was confirmed. These samples were collected from two villages: Ogbena in Kwara State and Akamkpa in Cross River State. Co-infection of some cassava varieties with ACMV and EACMV leads to severe symptoms. More importantly, a strain of mosaic geminivirus known as Uganda variant arose from recombination between the two viruses (2). This report provides evidence for the presence of EACMV in West Africa. References: (1) J. E. Thomas et al. J. Gen. Virol. 67:2739, 1986. (2) X. Zhou et al. J. Gen. Virol. 78:2101, 1997.

First Report of Colletotrichum gloeosporioides f. sp. manihotis, Cause of Cassava Anthracnose Disease, Being Seed-borne and Seed-Transmitted in Cassava.

Cassava anthracnose disease is a major economic disease of cassava in the tropics (2). Infection can lead to a significant loss in planting materials and total field crop failure. The disease has been reported to be transmitted mainly by a bug (Pseudotheraptus devastans Dist) (1). Open pollinated seeds from 13 cassava (Manihot esculenta Crantz) genotypes, stored for 10 months after harvest in 1994, were used to investigate the presence of the cassava anthracnose disease fungus. Seeds, 200 from each genotype, were surface sterilized, cultured on potato dextrose agar (PDA), and incubated for 8 days, at 25 ± 2°C. Microscopic examination indicated that Colletotrichum gloeosporioides was one of the seed-borne fungi, with up to 40% incidence recorded in some genotypes. Seeds from five susceptible genotypes selected for seed transmission studies were planted in fine, steam-sterilized soils in jiffy pots and watered daily for seedling emergence. At a height of 10 to 15 cm, the seedlings were transferred to plastic pots (10.5 cm in diameter) filled with sterilized mixture of soil and sand (2:2, vol/vol). Pots were placed close to each other to obtain a thick plant canopy. Temperature of 25 to 32°C and humidity of 80 to 98% were maintained. After 45 days, some plants had cassava anthracnose symptoms, including defoliation, wilt, and necrotic lesions. Stems, leaves, and roots of infected plants were washed, surface sterilized, and plated on PDA for 5 to 7 days. Microscopic observation of the fungus showed conidia of C. gloeosporioides. The rest of the plants were monitored for 3 months under vector-free conditions for typical anthracnose symptoms. Mean maximum wilt and defoliation of 35 to 38% was recorded in some genotypes. Conidial suspensions of C. gloeosporioides were used in stem-puncture inoculations of young, healthy cassava plants. The typical anthracnose symptoms of stem necrosis were observed 2 weeks after inoculation, confirming isolates as C. gloeosporioides f. sp. manihotis. This is the first report of C. gloeosporioides f. sp. manihotis being seed-borne and seed-transmitted in cassava. References: (1) B. Boher et al. Agronomie 3:989, 1983. (2) J. C. Lozano. PANS 20:30, 1974.