Mathew M. Abang

Differential selection on Rhynchosporium secalis during parasitic and saprophytic phases in the barley scald disease cycle

ABSTRACT Competition among eight Rhynchosporium secalis isolates was assessed during parasitic and saprophytic phases of the disease cycle in field experiments conducted at two locations and over two growing seasons. The eight isolates were inoculated onto six barley populations exhibiting varying degrees of resistance. Microsatellite analysis of 2,866 isolates recovered from the field experiments showed significant, and sometimes opposite, changes in the frequencies of R. secalis genotypes during the growing season (parasitic phase) and between growing seasons (saprophytic phase). Isolates that showed the most complex virulence in greenhouse seedling assays had the lowest fitness in the field experiment. Significant differences in isolate fitness were found on different host populations and in different environments. Selection coefficients were large, indicating that evolution can occur rapidly in field populations. Although inoculated isolates had the lowest overall fitness on the moderately resistant landrace cv. Arabi Aswad, some isolates were more virulent and consistently increased in frequency on this landrace, suggesting a risk of directional selection and possible erosion of the resistance following its widespread deployment in monoculture. These results provide the first direct evidence that R. secalis pathogen genotypes differ in their saprophytic ability and parasitic fitness under field conditions.

Molecular taxonomic, epidemiological and population genetic approaches to understanding yam anthracnose disease

Water yam (Dioscorea alata L.) is the most widely cultivated yam species globally. The major limitation to the profitable and sustainable production of D. alata is its susceptibility to anthracnose disease. The availability of resistant varieties could potentially form the cornerstone of an integrated management strategy for yam anthracnose; however, anthracnose resistance breeding is hampered by the dearth of knowledge on pathogen identity and diversity. Four forms of Colletotrichum are now known to be associated with foliar anthracnose of yam: the slow-growing grey (SGG), the fast-growing salmon (FGS), the fast-growing olive (FGO), and the fast-growing grey (FGG) forms. The close phylogenetic relationship of the first three forms to reference isolates of Colletotrichum gloeosporioides, and the fact that only strains of these forms have been observed to induce typical anthracnose symptoms on D. alata, recently confirmed that C. gloeosporioides is the causal agent of yam anthracnose disease. The FGG form possibly represents a distinct, endophytic, species as indicated by morphological, biological and molecular criteria. Previous research emphasized epidemiology and control but limited progress was made in understanding yam anthracnose disease based on this classical approach. Molecular approaches have started to unravel the systematics and ecology of Colletotrichum strains associated with yam anthracnose, as well the population biology of C. gloeosporioides on yam. Sexual recombination is a likely mechanism contributing to the high genetic diversity of C. gloeosporioides in yam-based cropping systems. Studies have been initiated to understand the mechanisms that generate genetic variation in C. gloeosporioides, and to gain some insight into the biochemistry of the interactions between the pathogen and yam. Our thesis in this article is that integrating traditional and molecular approaches to understanding C. gloeosporioides systematics, epidemiology and population genetics will lead to a much better understanding of yam anthracnose disease, and thus to the development of effective and sustainable control measures. Research successes and challenges are discussed, as well as their implications for future studies on pathogen evolutionary potential, anthracnose resistance breeding, and the deployment of resistance genes. Key words : Anthracnose, Colletotrichum gloeosporioides, Dioscorea spp., molecular markers, molecular systematics, population biology, resistance breeding, yam. African Journal of Biotechnology Vol. 2 (12), pp. 486-496, December 2003

Harnessing modern biotechnology for tropical tuber crop improvement: Yam ( Dioscorea spp.) molecular breeding

Yams (Dioscorea spp.) constitute a staple food crop for over 100 million people in the humid and subhumid tropics. They are polyploid and vegetatively propagated. The Guinea yams, Dioscorea rotundata and D. cayenensis, are the most important yams in West and Central Africa where they are indigenous, while D. alata (referred to as water yam) is the most widely distributed species globally. The genetics of yams is least understood among the major staple food crops due to several biological constraints and research neglect. Research to unravel the apparent complexity of the yam genome will have far-reaching implications for genetic improvement of this important tuber crop. Some progress has been made in recent years in germplasm characterization and the development of molecular markers for genome analysis. A genetic linkage map based on amplified fragment length polymorphism (AFLP) markers has been constructed for Guinea and water yams. These linkage maps were used to scan the genome for quantitative trait loci (QTL) associated with genes conferring resistance to Yam Mosaic Virus (YMV) in D. rotundata and anthracnose (Colletotrichum gloeosporioides) in D. alata. In addition, candidate random amplified polymorphic DNA (RAPD) markers associated with major genes controlling resistance to YMV and anthracnose have been identified that could be used for selection and pyramiding of YMV and anthracnose resistance genes in yam improvement. Also, molecular markers such as RAPDs, AFLPs, and microsatellites or simple sequence repeats (SSRs) have been developed for yam genome analysis. An initial c-DNA library has been constructed in order to develop expressed sequence tags (ESTs) for gene discovery and as a source of additional molecular markers. This paper will review the advances made, discuss the implications for yam genetic improvement and germplasm conservation, and outline the direction for future research.

Pathotype IV, a New and Highly Virulent Pathotype of Didymella rabiei, Causing Ascochyta Blight in Chickpea in Syria

The causal agent of Ascochyta blight disease of chickpea (Cicer arietinum L.) is highly variable because of the presence of a sexual phase (Didymella rabiei). There is also selection pressure on the pathogen due to wide adoption of improved resistant chickpea cultivars in some countries. The pathogen is able to produce pathotypes with specific virulence on particular cultivars. Three pathotypes, I, II, and III, have been reported (3). In this study, we confirmed the presence of a new and highly virulent pathotype that we designate as pathotype IV. To test the pathogenicity of the isolates collected and maintained at ICARDA, 10 isolates representing a wide spectrum of pathogenic variation, including those classified by S. M. Udupa et al. (3) and a putatively identified more virulent type, which was collected from a chickpea production field in the Kaljebrine area, Syria, were inoculated onto a set of differential chickpea genotypes. The differential genotypes, ILC 1929, ILC 482, ILC 3279, and ICC 12004, were sown in individual 10-cm-diameter pots containing potting mix and arranged in a randomized block design with three replications in a plastic house maintained at 18 to 20°C. Each differential genotype was inoculated individually with the 10 isolates following the methodology of S. M. Udupa et al. (3). DNA was extracted from single-spored isolates to compare the genotypes of the isolates using three simple sequence repeat (SSR) markers (ArA03T, ArH05T, and ArH06T) (2) and to determine the frequency of mating types (MAT) through the use of MAT-specific PCR primers for MAT1-1 and MAT1-2 (1). Host genotype reactions were measured on a 1 to 9 rating scale (1 = resistant and 9 = plant death). On the basis of the pathogenicity tests, the isolates were classified into four pathotypes: I (least virulent, killed ILC 1929 but not ILC 482, ILC 3279, or ICC12004); II (virulent, killed ILC 1929 and ILC 482 but not ILC 3279 or ICC12004); III (more virulent, killed ILC 1929, ILC 482, and ILC 3279 but not ICC12004); and IV (highly virulent, killed all four host differentials). Of 10 single-spore isolates tested, four showed similar disease reactions unique to pathotype I, four revealed pathotype II reactions, and one isolate each behaved like pathotype III or pathotype IV. SSR fingerprinting of these isolates provided evidence for genetic diversity since SSR ArH05T was highly polymorphic and amplified five bands, including pathotypes III- and IV-specific bands, which need further investigation to discern if this locus has any role to play in the virulence. MAT-type analysis showed that seven isolates were MAT1-1 while the remaining three isolates were MAT1-2. Only pathotype I showed the profile of MAT1-2 and the other three pathotypes were MAT1-1. Initially, a number of chickpea wild relatives were screened to identify sources of resistance to pathotype IV, but none of the accessions tested showed resistance. However, efforts are underway to combine minor and major gene(s) available in the breeding program in addition to a further search of the wild gene pools to control pathotype IV. References: (1) M. P. Barve et al. Fungal Genet. Biol. 39:151, 2003. (2) J. Geistlinger et al. Mol. Ecol. 9:1939, 2000. (3) S.M. Udupa et al. Theor. Appl. Genet. 97:299, 1998.

Genomics of Yams, a Common Source of Food and Medicine in the Tropics

Yams (Dioscorea spp., Dioscoreaceae), grown either for their starchy tubers or medicinal properties, are important crops in the tropics and subtropics. Yams broaden the food base and provide food security and income to over 300 million people. They are vegetatively propagated and comprise both diploid and polyploid species. Despite their economic and socio-cultural importance, very little is known about the genetics and genomics of yams due to research neglect and several biological constraints. Consequently, conventional breeding efforts have been severely hampered. Research to unravel the apparent complexity of the yam genome will have far-reaching implications for genetic improvement of this important tuber crop. Nevertheless, progress has been made recently towards understanding Dioscorea phylogeny and phylogenetic relationships within the genus. Also, improved molecular technologies have been developed for genome analysis, including germplasm characterization, cytogenetics, genetic mapping and tagging, and functional genomics. Genetic linkage maps have been constructed for D. rotundata and D. alata, and quantitative trait loci associated with resistance to Yam mosaic virus in D. rotundata and anthracnose (Colletotrichum gloeosporioides) in D. alata have been identified. In addition, candidate random amplified polymorphic DNA markers associated with major genes controlling resistance to Yam mosaic virus and anthracnose have been identified. These markers could be converted to sequencecharacterized amplified regions and used formarker-assisted selection for resistance to diseases. An initial cDNA library has been constructed to develop expressed sequence tags for gene discovery and as a source of additional molecular markers. Genetic engineering offers a powerful tool, complementing conventional breeding approaches, for yam improvement. Methods for yam transformation, including in vitro plant regeneration, gene delivery, selection of transformed tissues, and recovery of transgenic plants have been developed but still need improvements. This chapter reviews advances made in yam molecular marker development for genome analysis, phylogeny, molecular cytogenetics, characterization of genetic diversity, genetic mapping and tagging, and progress in functional genomics.

True Yams (Dioscorea): A Biological and Evolutionary Link between Eudicots and Grasses

Dioscorea (true yams) is a large genus that contains species important as food (with edible tubers) or as sources of bioactive substances used in a range of applications. Dioscorea is a major staple food in many parts of the world, especially in West Africa and the Pacific islands, and serves as a famine food in many regions. It is a critically important but neglected crop, which is likely to increase in importance as climate change leads to necessary changes in global food systems. It is a herbaceous, climbing, tropical monocot that looks rather like a dicot, and is part of a lineage that is relatively closely related to the phylogenetically derived group containing the grasses. Therefore, it represents an important biological link between the eudicots and grasses--groups that contain all the model flowering plant species--and has the potential to fill gaps in our knowledge of plant biology and evolution. Yams also offer us the possibility to gain new insights into processes such as tuberization and sex determination, which cannot be studied in current model organisms. This combination of rising importance due to its socioeconomic significance and interesting biology and evolutionary position justify its potential as a model organism. This potential remains to be harnessed, and much of the current work on yam is directed toward its role as a food crop. This aspect will remain important, but its potential for answering questions of basic biological interest will be a major motivation for researchers interested in this organism.

Colletotrichum circinans and Colletotrichum coccodes can be distinguished by DGGE analysis of PCR-amplified 18S rDNA fragments

The rDNA 18S region of Colletotrichum circinans and C. coccodes was amplified by PCR to evaluate this DNA region as a tool for species delineation. PCR amplification of the 18S of both species produced 1.65 Kb long fragments that covered most of the entire 18S rDNA molecule. DGGE analysis of the amplified fragments distinguished C. circinans from C. coccodes isolates. This result provides molecular evidence that supports the current treatment of C. circinans as a species distinct from C. coccodes , in spite of the failure of previous attempts at genetic differentiation of the two species based on RFLP analysis of the rDNA ITS region. Key Words: DGGE; Colletotrichum circinans; Colletotrichum coccodes; molecular differentiation; species delineation. African Jnl Biotechnology Vol.3(3) 2004: 195-198

Development, characterization and linkage analysis of microsatellite loci for the Ascochyta blight pathogen of faba bean, Didymella fabae.

Eighteen microsatellite markers were developed for Didymella fabae, seventeen of which were highly polymorphic among a sample of D. fabae isolates from Syria. Genetic linkage analysis assigned the markers to eight linkage groups. These markers will facilitate population and evolutionary studies of D. fabae and related species.

Culturing meristematic tissue and node cuttings from yams (Dioscorea).

This protocol describes how to produce whole yam (Dioscorea) plantlets in vitro through yam meristem culture and from yam node cuttings.

Seed-borne pathogens detected in consignments of cereal seeds received by the International Center for Agricultural Research in the Dry Areas (ICARDA), Syria.

During 1995–2004, the Seed Health Laboratory at the International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria, tested all incoming cereal seed from 41 countries (251 seed lots comprising 91,993 samples) for the presence of seed-borne pathogens. Pest detection methods used included direct visual inspection, wash-filter, freezing-blotter, embryo, seed-gall nematode and growing-on tests. Analysis of seed lots with ≥30 seeds (53,566 seeds in total) revealed that 22.03% (11,797) of the seeds, predominantly of barley and wheat, were infected. Of these, 20.02% were infected with Tilletia caries and/or T. foetida, followed by T. controversa (0.99%), Ustilago tritici (0.30%), T. indica (0.27%), Fusarium spp. (0.25%), Helminthosporium spp. (0.09%), Ustilago spp. (0.03%), Urocystis agropyri (0.02%), Anguina tritici (0.02%) and Ustilago hordei (0.01%). The frequency of Tilletia indica-infection in seed samples was: Ethiopia 13.31%, Azerbaijan 10.20%, Tajikistan 0.64%, and Turkey 0.22%. T. indica and T. controversa do not occur in Syria and are considered as quarantine pests with zero tolerance. The implications of these findings for the safe movement of cereal germplasm are discussed.