“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.

Developing dual-resistance cassava

Cassava root rot caused by CBSD. Photo by IITA.
Cassava root rot caused by CBSD. Photo by IITA.

This year, we moved closer to developing North Face Sale a few too mild properly and so forth!
cassava with dual resistance to Cassava Mosaic Disease (CMD) and Cassava Brown Streak Disease (CBSD) – the most devastating diseases of the crop in Eastern and Central Africa and the greatest threats to the food security and livelihoods of over 200 million people.

In Uganda, we selected eight clones with resistance to CMD and CBSD and other farmer-preferred traits. These clones, which are the first ones with dual resistance suitable to the mid-altitude areas of the Great Lakes regions, were sent to the Kenya Plant Health Inspectorate Services for cleaning and multiplication in preparation for regional distribution to national partners. An additional 41 yellow-fleshed clones, also with dual resistance to CMD and CBSD, are undergoing advanced evaluation.

This is the fourth year of trials for dual-resistance cassava for mid-altitudes in Uganda. The trials are being conducted in Mukono and Namulonge, considered hot spots of CBSD and CMD in the country. The breeding work started with over 5000 true seeds of parents with tolerance to CBSD from Tanzania that were sent to Uganda for crossing with IITA varieties that are resistant to CMD.

Cassava grown from the Tanzanian seeds were repeatedly subjected to high disease pressure along with susceptible varieties for comparison. From each growing season, only 10 percent of the crop was selected for the next stage. After four growing seasons, the field has been narrowed down to eight very promising varieties.

Similar dual-resistance evaluation was carried out in Tanzania. Eight clones that have resistance to both CMD andeducate North Face Jacket Sale yourself on the way th much CBSD were deliberately subjected to the diseases by grafting them with infected plants. Five of these clones are being evaluated on-farm, while 11 clones with dual resistance and high starch content – a preferred trait by farmers – are also being evaluated.

Cassava that survives these tests, thereby producing a true dual-resistant variety, can then be used for further disease-resistance breeding in other countries in the Great Lakes region such as Rwanda, Kenya, and DR Congo. Throughout the selection process, farmers were actively engaged to ensure that the varieties meet their preferences such as cooking taste, texture, and yield.