Research in the focus

From fundamental virus research to food delivery

- a research report by Dr. Stephan Winter and Dr. Samar Sheat

One of the research places of the Department of Plant Viruses is the Democratic Republic of the Congo, actually to the island of Idwji and to the Plaine de la Ruzizi both located in South Kivu. It is there, the epicenter of the cassava virus diseases we work on, where we see the realities of the land, test our hypotheses and take the lessons learned from the farm back to the lab. We have not been visiting since early 2018 and there was no more time to wait in a virus epidemic situation rapidly expanding.  
Cassava (Manihot esculenta Crantz) is the crop we work on, it grows in tropical and sub-tropical regions of the world and in particular in Sub Sahara Africa. Indeed, Africa is the heart of cassava cultivation and the crop provides food for all and is the main source of living for smallholder farmers and the poor. Cassava is a super crop as it is drought resistant and grows even in very poor soils and without much input to produce a tuber that can be harvested and eaten. Unfortunately, cassava in Africa and thus the livelihood of people is threatened by virus diseases and this is where our research is anchored. The danger comes from viruses causing cassava mosaic disease (CMD) occurring wherever cassava is grown on the continent and from viruses causing even more serious disease, the cassava brown streak disease (CBSD). The latter is destroying the tuberous roots and the very recent outbreaks are now rapidly expanding from coastal zones of Kenya and Tanzania to threaten the entire cassava belt of Sub-Saharan Africa, from Mozambique to Nigeria.

Virus diseases of plants cannot be cured and thus, disease prevention and control are about the only means to manage virus diseases. It is already a complex task in industrialized modern agriculture, to comply with seed certification, phytosanitation and vector control, but almost impossible to implement in Africa, where agriculture often is done at the subsistence level and with little or no input.  

From the field to the lab

We try to find answers to the virus diseases of cassava. Running a reference virus collection, we started a long time ago to assess and maintain the global diversity of cassava viruses. Some of them, like frogskin disease in South America, are not yet clearly associated with viruses and thus we are on virus discovery missions. We use all weapons we have at hand and if not, we make new. For unknowns, we use high throughput sequencing. To reconstruct viruses, we assemble genomes and shoot them with a handheld gene gun into cassava. We remove virus genes, modify them and get them back to infect cassava, feed them to insects and see whether they are still transmitted. Our virology is not at all virtual; we maintain the cassava viruses in a live collection, ready at hand for reference diagnostics and virus experiments with hundreds of plants that have to be grown from tissue culture. At the cassava virus beginnings in 1996/97 we used the diverse cassava mosaic virus species we had collected to screen for virus resistance in cassava lines from the international Institute of Tropical Agriculture (IITA), in Nigeria. Our collaboration was super critical because the diverse virus species could not be screened in the field because of strict quarantine. Using specific ballistic delivery with cloned virus genomes to infect cassava and screening hundreds of cassava lines in Braunschweig, we eventually identified broad spectrum virus resistance against cassava mosaic viruses. This is now introgressed in almost all cassava varieties in Africa.    

At this time, the brown streak disease was not on stage yet and cassava virus diseases were no more an issue. Cassava brown streak virus was unintentionally collected in 1999 during virus surveys on the distribution of cassava mosaic viruses in Kenya. We kept the isolates on a side as we did not consider important what 7 years later became a major epidemic disaster in the region. All African cassava lines can be infected with either of the two viruses causing the disease, the Cassava brown streak virus (CBSV) and the Ugandan cassava brown streak virus (UCBSV) [1] and in particular vulnerable; the varieties with CMD resistance. Finding CBSD resistance is a daunting task and only incremental progress was made despite enormous investments and international research collaborations [2]. In 2014 we were asked by the Bill and Melinda Gate Foundation (BMGF) to help in the search for New sources of virus resistance. And because there was no resistance in Africa, we looked in the diversity of cassava genotypes in South America which is maintained in the germplasm collection of CIAT in Cali, Columbia. And finally, after infecting and testing hundreds of cassava lines, we found resistance to both viruses and their isolates in our laboratory using a very powerful and effective screening approach [3]. We found that in some cassava lines, virus replication is completely inhibited and in few others virus replication occurs but the virus remains restricted to the phloem cells only and cannot escape the companion cells. This “absolute” resistance character remains even with stress from other viruses or environmental disturbance and thus is a breath-taking finding that is now the main topic of our research; to elucidate the mechanism of this interaction leading to virus arrest.

However, finding sources of resistance in the laboratory is nice but by far not as exciting than bringing the plants to the field. We have many partners around the world to work with us and our partners of the NextGen Cassava Network were highly interested in our materials. And we have access to our own fields in South Kivu belonging to NGO associations of young woman and men who farm for a living. This part is less scientific but more of a logistic challenge to bring our materials, tissue cultures and well protected and pampered plantlets into the fields. How to grow, which medium to use, sterile or unsterile soils, how to protect the cuttings and what materials to use? Lots of questions to consider even before real field trials can start. And of course, we cannot take care of the little plants ourselves so we have to train our people how to do, what we may not know better.

Our most promising CBSD resistant cassava lines from South America were brought to Africa …. and died! Here it is the proof of a hypothesis. We had brought the best of all resistances against the brown streak viruses and this was still holding but 17 of the 18 lines died from severe infection and hypersensitivity against the mosaic viruses in Africa. Now, what to do, how to find a location where CMD is not present and our partners can cross lines with both resistances? Actually, how to cross cassava when it is not flowering or the flowering is not synchronised and one line flowers 3 months after the other? We found this location in Columbia and brought the cassava there to be crossed by our CIAT partners. Last June, during the shutdown, we got the seeds back, 250 pieces from 18 families. These viruses of cassava do not occur in South America, so it was again on us to do the virus infections and screening. We learned that cassava seeds only germinate after a long time and forest fires, so we simulated fire by boiling the seeds; got little seedlings which we watched growing in our kindergarden.

Now, our excitement rose to its maximum. Can we see something after the first crosses already or do we need to backcross and test the next generation? And how is the heritability, dominant recessive… questions a virologist never has to ask, not to mention, understand. We changed our workflow to increase speed and still maintain the precision of testing. Only 9 months later, we got the results; some lines were infected and some lines not, others with two viruses and few with none! Dual resistance against both cassava mosaic and cassava brown streak viruses. Evident in the first days of January 2021! And, we could not wait longer to bring this into the African field. No way of sending the material without us; the flight from Frankfurt to Goma with the first group of plantlets and Stephan Winter was on the 4th of February and on 14th of February Samar Sheat brought the next batch for planting.

From the lab to field

We carried materials that we deemed important to build plastic houses and we dried substrate from coco husks to grow our tissue cultures in small plastic containers used in the canteen for a fresh salad. Stephan Winter explored the sites and got his gang together to built the first plastic house on the island of Idjwi, we now designated as our virus-free zone. 

The cassava plantlets and cuttings and tissue cultures were transferred and the houses closed for establishment of the cultures.  And we moved to our next site, the epicenter of the brown streak disease on the Plaine de la Ruzizi, between the Lake Kivu and Lake Tanganyika. Compared with Idjwi, this is a rough place that lets you freeze in humility on how much people can take, not only because of plant diseases. Cassava tubers are in high demand as the market is empty and the prices in the sky because of cassava brown streak disease that essentially wiped out cassava. Resistant cassava planting materials is on top of all farmers wishes and thus we are welcomed with raised eyebrows. And more plastic houses were built and plants planted and transplanted directly to the field.

Field testing

We jumped on the motorbikes to conduct field surveys in the region to identify sites with high infection pressure where all viruses meet and cassava is suffering seriously. We collected more virus specimens for lab identification and we choose sites as our sentinel plots, where we plant our lines directly into the inferno. The farmers, almost all women, were very happy to be part of this effort and prepare the land to receive our material in June/ July when we are ready. Now we have lots of eye (brows) on us…

Working together

Many aspects of our research and development are now in the hands of our partners and this is the last and most critical step in our workflow. It requires lots of talking, showing, reflecting and listening. We learned that we have to bring all on board and if it has to be discussed hundreds of times, then this is what is necessary. We spoke with everyone in French, Lingala, Suaheli, hands, heads and feet showed how symptoms are identified and learned what is better not to do and about the enormous impact when wrong materials are used. Workshops followed to introduce our graft-infection technique, how and which samples to collect for laboratory testing and what parameters to use to assess growth performance photo 8. We are on for success but we also know that this is only a beginning. Aside from virus resistance, there is a lot of uncertainty. Will our crosses from Africa/South America develop well in this environment and produce sizable tubers that taste well, have high dry matter and fit into the food habits of the people. We are ready to explore this as we will not stop to bring more lines from our research into the field.  
It is about the coolest thing to follow a beginning to a likely end. We find it highly rewarding that we are given the opportunity above publication of our findings to bring a product of our research to the field and beyond speculation, to see for ourselves whether we were right. We also learned how wonderful it is to work together with people that acknowledge our efforts and are on with it. This is what we carry back and unpack this sentiment for hard laboratory days when clouds hang deep because nothing goes back and forth.
 
References
1.    Winter, S.; Koerbler, M.; Stein, B.; Pietruszka, A.; Paape, M.; Butgereitt, A., Analysis of cassava brown streak viruses reveals the presence of distinct virus species causing cassava brown streak disease in East Africa. J Gen Virol 2010, 91, 1365-1372.
2.    Kawuki, R. S.; Kaweesi, T.; Esuma, W.; Pariyo, A.; Kayondo, I. S.; Ozimati, A.; Kyaligonza, V.; Abaca, A.; Orone, J.; Tumuhimbise, R.; Nuwamanya, E.; Abidrabo, P.; Amuge, T.; Ogwok, E.; Okao, G.; Wagaba, H.; Adiga, G.; Alicai, T.; Omongo, C.; Bua, A.; Ferguson, M.; Kanju, E.; Baguma, Y., Eleven years of breeding efforts to combat cassava brown streak disease. Breeding Sci 2016, 66, (4), 560-571.
3.    Sheat, S.; Fuerholzner, B.; Stein, B.; Winter, S., Resistance Against Cassava Brown Streak Viruses From Africa in Cassava Germplasm From South America. Frontiers in plant science 2019, 10.