“DNA Barcoding”: new initiative in determining the diversity and checking the spread of pathogens

Diagnostic tools is vital to disease surveillance, development of host plant resistance, quarantine monitoring, and supporting safe conservation and exchange of germplasm. Good knowledge on pathogen population structure and genetic diversity is a pre-requisite to developing unambiguous diagnostic tools and is critical in establishing disease management tactics. Increasingly, modern diagnostic tools are being based on the DNA characteristics of the pathogen.

Clustering of 25 yam isolates based on rDNA sequences. Image by Lava Kumar, IITA.
Clustering of 25 yam isolates based on rDNA sequences. Image by Lava Kumar, IITA.

We undertook a new initiative to genetically characterize pathogen populations and recognize unique stretches of sequences. Called ‘DNA Barcodes’, they can be used as markers for diagnosing pathogens and pests affecting African food crops.

For instance, we conducted molecular characterization of fungal pathogen(s) causing anthracnose – the most destructive disease of yam and cassava in West Africa. In yams, anthracnose appears as leaf spot that spreads rapidly, killing the leaves, shoots, and entire plant. In cassava, it appears as cankers on stems at the base of leaf petioles, and also kills the leaves, shoots, and whole plant. The disease causes severe yield losses in both crops.

The causal fungus, Colletotrichum gloeosporioides Penz., is widespread in West Africa. We identified various isolates of this fungi differing in morphology, growth characters, and pathogenicity, and investigated their genetic relatedness and diversity through molecular analysis using a set of 25 reference isolates (17 from yam and 8 from cassava). Based on the symptoms they induce, they were grouped into spot (S) and blight (B) isolates. Both isolates infect yam, but only B isolates infect cassava. We assessed the genetic diversity in these isolates by nucleotide sequencing and cluster analysis of the ~540 base pair (bp) nuclear ribosomal internal transcribed spacer region (ITS1, ITS2 and the 5.8S gene) and partial gene sequences of actin (~240 bp) and histone (~370 bp).

Phylogenetic cluster analysis grouped the 25 isolates into two major clades and two sub-clades within the major clades. Both the S and B isolates were distributed between the two clades (see figure). All the isolates in clade 1 were unique to yam. Seven of these isolates (YA08-1, YA08-2, YA08-3, YA08-4, YA08-7, Y-83, Y-84) formed a genetically-distinct lineage indicating that they could be new strains unique to yam. Isolates in clade 2 infect both cassava and yam suggesting their capability to infect wide range of plants. Clade 2 isolates could be the most frequently occurring on yam and cassava because of their ability to survive on weeds and other crops. We recognized unique sequence motifs and designed diagnostic PCR primers for specific amplification of C. gloeosporioides infecting yam and cassava directly from infected plant tissues.

Using a similar approach, we characterized the fungal agent associated with grey leaf spot (GLS), the most destructive disease of maize. We found that GLS in Nigeria is caused by a distinct species of Cercospora, but not C. zeae-maydis. This work, in addition to confirming the GLS etiology, allowed us to establish a unique set of primers for specific identification of GLS pathogen prevalent in Nigeria.

Through comparative genomics, we identified common genome regions in cassava mosaic begomoviruses occurring in sub-Saharan Africa. We developed a simple multiplex PCR assay that can detect all the major viruses in cassava mosaic disease etiology. This test has been institutionalized for virus indexing of cassava propagated in vitro.

To aid in diagnostics research, we developed a simple and cost-effective procedure suitable for extraction of DNA from seeds, leaves, stems, tubers, and even roots. The resultant DNA is suitable for PCR-based diagnoses of fungi, bacteria, and viruses in the infected tissues in a wide range of plant species, and is handy for quarantine monitoring of germplasm. We are establishing a repository of diagnostic protocols in an approach we call “Diagnostic Basket” and make it available to users.

Fighting a two-pronged attack on African bananas

Flesh of banana fruit infected by BXW. Photo by Piet van Asten.
Flesh of banana fruit infected by BXW. Photo by Piet van Asten.

Among the many diseases that affect bananas and p exciting new major f use of Ralph Lauren Outlet Australia product ion lantains in Africa, the two greatest threats are Banana Xanthomonas Wilt (BXW) and Banana Bunchy Top Disease (BBTD). Combined, these diseases have the potential to wipe out these economically- and food security-vital crops from the continent. Their rapid spread in recent years has alarm bells ringing in banana-producing countries across Africa Hüpfburgen. BXW, which in the past had only been prevalent in Ethiopia, has been highly active in East Africa this past decade. On the other hand, BBTD, which was first reported in the 1920s and then the 1960s in Egypt and DR Congo, respectively, has been spreading rapidly in the East African highlands and in Central and Southern Africa. In the face of this two-pronged threat, we have been actively engaged in a number of complementary disease-management research. These include developing specific diagnostic assays, initiating regional surveillance to map current disease distribution and future spread, developing management tools to minimize establishment, spread, and impact, and working on host-plant resistance through germplasm screening and biotechnology approaches.

Banana plants showing classic symptoms of BBTD. Photo by Fen Beed.
Banana plants showing classic symptoms of BBTD. Photo by Fen Beed.

As the BXW pathogen is closely related to other Xanthomonas pathogens that affect maize, sorghum, and sugarcane, we, together with advanced laboratories in the USA, developed a highly specific assay to identify the banana variant using genomic tools. We also developed a sensitive assay for the banana bunchy top virus (BBTV), the causal agent of BBTD. These two assays are now being used for verification of suspected BXW and BBTD cases in East and southern Africa in cooperation with national and private sector partners. The development of sensitive diagnostics for BXW and BBTD permits epidemiological studies in plants that do not show symptoms during the latent period, rapid deployment of control measures, and effective detection of the pathogens by quarantine officers along borders. We also conducted surveys with national partners in southern and Central Africa to map the extent of spread of BBTV, and to determine the abundance and distribution of the banana aphid, the only known insect vector of BBTV.

Map showing distribution of BBTD and BXW in Africa. Image provided by Fen Beed, IITA.
Map showing distribution of BBTD and BXW in Africa. Image provided by Fen Beed, IITA.

We recognize that insects play an important role in the spread of banana diseases, so we initiated studies on host-plant resistance to the banana aphid that spreads BBTV. We also plan to explore for natural enemies of the banana aphid in its putative area of origin in 2010. In Malawi, we established a field trial of various banana cultivars to study host reaction to BBTV and assess virus concentration. Many insects have been implicated in the medium-distance, farm-to-farm spread of BXW. To help us develop management guidelines, we have been conducting studies in  th Ralph Lauren Mens Polo Shirts Australia do you an isolated and controlled site in a forest reserve to further understand how insects spread the BXW pathogen. Together with the Southern Africa Development Community,  the Association for Strengthening Agricultural Research in Eastern and Central Africa, FAO, Bioversity International, and other partners, we co-organized an international workshop in Arusha, Tanzania in August to integrate recent information on these diseases and develop control strategies. The workshop recommended measures to slow the spread of these diseases into new regions and offset their impact in already-affected areas. These included large-scale awareness and surveillance campaigns, community-level cooperative actions, establishment of reporting, communication, and monitoring systems, improved “seed” systems, development of national contingency plans, and long-term programs for eradication and/or management of BXW and BBTD. A follow-up meeting in 2010 is being planned to establish the framework of a region-wide disease-management and production strategy.