Towards a Variety Resistant to Coffee Wilt Disease (CWD): a Case for Robusta Coffee (Coffea canephora) in Uganda

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1 Index Table of contents Towards a Variety Resistant to Coffee Wilt Disease (CWD): a Case for Robusta Coffee (Coffea canephora) in Uganda P.C. MUSOLI 1, A. KANGIRE 1, T. LEROY 2, A. NABAGGALA 1, S. NAKENDO 1, S. OLAL 1, J. OCHUGO 1, C. KABOLE 1, J. PANDE 1, C. CILAS 3, A. CHARRIER 4, D. BIEYSSE 5, J.A. OGWANG 1 AND D.T. KYETERE 1 1 Coffee Research Centre, P.O. Box 185, Mukono 2 Cirad, UMR PIA, Montpellier, F France 3 Cirad-CP, UPR Bioagresseurs de pérennes, Avenue Agropolis, TA80/ Montpellier Cedex 5, France 4 ENSAM, Place Viala, Montpellier, F France 5 Cirad UMR BGPI, Campus International Baillarguet, TA41/K, Montpellier Cedex 5, France SUMMARY Coffee wilt disease (CWD), which is caused by Fusarium xylaroides Steyaert, the conidial stage of Gibberella xylarioides Hem. & Saccas, is the most serious problem of Robusta coffee (Coffea canephora) production in Uganda, the Democratic Republic of Congo (DRC) and Tanzania. It is also affects Arabica coffee (Coffea arabica) production in Ethiopia. CWD spreads quickly and widely, thus it is a threat to coffee production in all continents. It can be controlled effectively by planting resistant varieties. In Uganda CWD was first observed on C. canephora in 1993, in areas bordering the DRC and by 2002 it had spread to many parts of the country, where it had destroyed 44.5% of the crop (Oduor, 2005). This reduced Uganda s coffee exports by nearly 50% and led to economic loss of US dollars million annually between 1996 and Consequently, a search for CWD resistant C. canephora varieties was initiated at the Coffee Research Centre (COREC) in This involved screening C. canephora germplasm in naturally infected fields and in artificial inoculations carried out in the screen house for resistance. The C. canephora genotypes both in field and screen house studies, responded quantitatively to the disease, implying that CWD resistance in C. canephora is controlled by many genes completely resistant genotypes were identified through large scale germplasm screening in artificial inoculations at COREC and these were planted in mother gardens and thereafter they were cloned and planted in field evaluation trials. 167 of the clones underwent preliminary evaluation and four (4) were selected for being resistant to leaf rust and red blister disease and having good cup and bean qualities. The four selected clones were planted in on-farm multi location evaluation trials, en route to being recommended for commercial cultivation. Similarly, four (4) CWD resistant clones were selected from among many genotypes screened in naturally infected fields at COREC and were also planted on-farm multi location trials, en route for recommendation for farmer cultivation. INTRODUCTION Coffee wilt disease (CWD), which is caused by Fusarium xylaroides Steyaert, the conidial stage of Gibberella xylarioides Hem. & Saccas, was first reported in 1927 on C. liberica spp. in the Central African Republic (CAR) (Figueres, 1940). The disease subsequently destroyed this crop during 1930s to 1950s within CAR and Cameroon (Mulller, 1997). During the same period, it destroyed C. canephora in Ivory Coast (Delassus, 1954). Fraselle (1950) reported 1472

2 observation of CWD on C. canephora at Yangambi in the DRC in 1948, which later became a serious problem in many parts of the country. Lejeune (1958) reported similar disease symptoms on Arabica coffee (C. arabica) in Ethiopia and later Kranz and Mogk (1973) confirmed that the disease on C. arabica was also caused by F. xylarioides. Later studies revealed that F. xylariodes strains causing CWD on C. canephora and C. arabica are different. By 1970 s, the CWD had been effectively controlled in the Central and West African countries, by combined use of resistant varieties and cultural practices (Muller, 1997). However, in the 1980s, new outbreaks were reported on C. canephora in the DRC (Flood and Brayford, 1997), from where it spread to Uganda in 1993 and subsequently to Tanzania in 1997, affecting the same species. In Uganda coffee is the most important cash crop and a major source of foreign currency into the country. Over 8 million Ugandans derive their livelihood directly from coffee (Uganda Bureau of Statistics-UBS, 2007). The crop contributes 18-21% of Uganda s foreign currency earnings and about 64% of the earnings from traditional export crops alone (Mugambe, 2007). Coffee has remained the lead foreign currency earner despite government s efforts to diversify to other non-traditional exports (Masiga and Ruhweza, 2007). C. canephora contributes 70-90% of the coffee production in Uganda and C. arabica contributes 10-30%. The recent devastation of C. canephora by CWD has significantly dented Uganda s coffee exports and thus the national economy and social welfare of people involved in its supply chain has been disrupted. Surveys conducted in 2002 found the disease in all the C. canephora growing areas of Uganda and on over 90% of the farms, where it had destroyed over 44% of the crop nationwide (Oduor et al., 2005). The overall effect of CWD was a significant reduction in production and export volumes, from 4.2 million 60 Kg bags of green beans exported in 1996/97 to 2.0 million bags in 2005/06, which translated into annual monitory loss of US $ million (Uganda Coffee Trade Federation-UCTF, 2007). In 1997 scientists at COREC initiated a breeding programme to develop CWD resistant varieties of acceptable farm and market traits. The objective of the programme was to develop C. canephora varieties, which are resistant to CWD and other major coffee diseases; are high yielding and possess good bean and cup qualities. MATERIALS AND METHODS C. canephora germplasm was screened for CWD resistance at Kituza (Headquarters of COREC) in Mukono district, which is one of the locations where CWD was first observed in Uganda. The screening was carried out in naturally infected fields and in artificial inoculations in a screen house; followed by planting resistant genotypes in mother gardens and cloning and re-evaluating the resistant clones in the field trials. Evaluation of C. canephora germplasm for CWD resistance in naturally infected field 20 C. canephora clones raised from rooted nodal stem cuttings and planted in the field in 1997 were carefully evaluated (figure 1). The clones included four commercial varieties (1s/2, 1s/3, 223/32 and 257s/53) and 16 hybrids selected for high yielding, good bean quality and resistance to leaf rust and red blister disease, from among various progenies of specific crosses involving various parental whose CWD resistance was not known by then. The 20 clones were planted in a randomized complete block design trial with four replications and each replicate consisting of 20 plots, each plot constituted by six coffee trees arranged in continuous patterns of 3 rows by 2 columns, spaced at 3 x 3 m. Clones Q/6/1 and Q/1/1 were only planted in replicates 1 & 2, due to insufficient planting materials. Clones H/4/1 and R/14 were only planted in replicates 1, 2 and 3, also due to insufficient planting materials. The field was maintained following routine procedure of maintaining C. canephora gardens. 1473

3 Although CWD started affecting coffee trees in this trial in 1999, systematic assessment started in April Prior to April 2001 infected trees were uprooted and burnt on observation of first CWD symptoms as a way trying to minimizing and / or eradicate the disease from the Centre. During systematic assessment, each tree was scored for CWD severity by assessing percentage defoliation caused by the disease. The scoring was done twice in a month using a symptom severity scale of 1 to 5, where 1 = no disease, 2 = 1-25% defoliation, 3 = 26-50% defoliation, 4 = 51-75% defoliation, 5 = % defoliation. Trees in level 5 were considered dead. Percent mortality of trees was computed for each clone. Disease progress curves of each of the clones were plotted from the percentage mortality data and the genotypes were arbitrary classified into resistance classes using data of the last scoring (31 March 2006). Genotypes in resistant and moderately classes were cloned and planted in multi-location on-farm trials. Screening germplasm through artificial inoculation This involved inoculation of tens of thousands of open pollinated seedlings and rooted nodal cuttings of various parent genotypes obtained from germplasm plots at COREC, survivors in heavily devastated farms and wild and feral population. The plant materials were raised in the coffee nursery at Kituza following routine procedures (MAAIF) and were inoculated with a suspension of F. xylarioides conidia in water at concentrations of 1.3x10 6 using root dip method (Musoli et al., 2001), when at 6-9 months old. Inoculated plants were incubated in the screen house at room temperature and watered regularly as they were monitored for CWD symptoms for at least 8-12 months. Plants that survived this long were re-inoculated using the same procedure. The re-inoculated plants were also watered and monitored for mortality for at least 8 months. All plants that survived in the second inoculation after this long, were considered resistant and were planted in mother gardens for vegetative multiplication, en route to planting in field trials for further evaluations. Evaluation of CWD resistant clones in field trials All CWD resistant genotypes identified in field and artificial studies were planted in mother gardens, as they became available. All the genotypes in the mother gardens were cloned and planted in on-station (Kituza) field trials. These trials were established in gardens heavily infected with CWD. The gardens were previously planted with other coffee lines, which were continuously destroyed by CWD, therefore assumed to have high concentrations of F. xylarioides inoculum. Each of the clones was planted in one plot constituted of a single row of 6 trees spaced at 3 m by 3 m. Besides CWD incidence, these clones were assessed for yield, incidence and severity of coffee leaf rust and red blister disease. Leaf rust and red blister diseases were scored on a scale of 1-5, where 1 = no disease and 5 is when most of the plant organs (leaf for leaf rust and fruit for red blister disease) were destroyed. Dry samples of coffee beans at 11-13% moisture content, were analyzed at the Uganda Coffee Development Authority laboratories for physical and cup qualities. Cup quality was analyzed using sensory taste by a panel of experts. RESULTS Evaluating C. canephora clones in a naturally infected field Curves showing mortality of trees or the 20 clones evaluated for resistance against CWD in a naturally infected field are given in Figure 1. It is notable that tree mortality among the clones was at different levels when the evaluation started in April 2001 and it progressed at varying rates to varying levels by March Clone J/1/1 did not succumb to CWD throughout the assessment period. Mortality increased rapidly among clones P/3/6, H/4/1, E/3/2, C/1/7 and 1474

4 B/6/2 and by March 2006 and the final mortality of these clones ranged from 87.5% for clone E/3/2 to 95.8% for C/1/7. Mortality increased very slowly on clones Q/3/4, R/1/4 and 1s/3 and by March 2006 their mortality ranged from 4.2 for clone Q/3/4 to 33.3 for clones 1s/3 and R/1/4. Clone 1s/2 appeared to be resistant until April 2002, when its trees started dying in high numbers. By March 2006, 1s/2 was among clones with very high. Highly susceptible Moderately susceptible Moderately resistant Resistant Highly susceptible Moderately susceptible Moderately resistant Resistant Figure 1. Progression of tree mortality of 20 C. canephora clones evaluated for CWD resistance in a naturally infected field. Screening C. canephora germplasm in artificial inoculation Because tens of thousand of plants were inoculated, it is impossible to present results of all the genotypes tested. Figure 2 gives comparative responses of wild, cultivated and feral populations. There were variable responses to CWD infection among and between C. canephora populations studied in artificial inoculations. Figure 3 presents response of 24 open pollinated seedlings inoculated in the screen house. These results are typical to what was 1475

5 observed in other inoculations. Survivors of the 24 progenies are among the many that were planted in the mother gardens at Kituza for cloning en route to field evaluation. Figure 2. Response of open pollinated progenies of different C. canephora populations to CWD artificial inoculation with CWD pathogen. Field evaluation of CWD resistant clones None of the CWD resistant clones identified through artificial inoculations succumbed to CWD in the field, implying that the screening method was effective and thus the clones are effectively resistant to the disease. At the time of this report, only 167 clones had been evaluated for visual impression, quality and resistance to leaf rust and red blister diseases. Results of these assessment revealed that clones vary in their phenotypic impressions. Clone responses to leaf rust and red blister disease and their quality, is illustrated by 23 clones in Table 1. These results show that cup and bean qualities vary among the clones. Response to leaf rust and red blister diseases also varied. On the basis of the visual impression, cup and bean qualities and response to leaf rust and red blister diseases, 22 clones were selected from the on-station field trials and these together with 4 elite clones identified from the field trial, were planted in on-farm trials. Four (4) out of the 22 elite clones identified from on-station field trials plus the four (4) identified in the field trial, were proposed for limited cultivation by farmers, in the agro-ecological location, were Kituza is located. Assessment of yield of the elite CWD resistant clones commenced in 2005/06 and it will continue for at least 4-5 years. DISCUSSION Revelation of CWD resistance within Ugandan C. canephora natural populations gave ways towards the development of commercial varieties, which are resistant to the disease without involving many cycles of introgressing resistance from other species, particularly C. arabica. This coupled with availability of individuals with good market qualities, which are resistant to leaf rust and red blister disease, among the CWD resistant genotypes, will hasten availing of appropriate varieties for cost effective management of CWD.Revelation of the CWD resistance also removed the fear that C. canephora could be exterminated by CWD, although the genetic diversity is still threatened. It is feared that as farmers adopt new CWD resistant varieties, which are a product of a very narrow genetic base, they will destroy few individuals that survived CWD in farms, and as losing genes for other traits. 1476

6 Table 1. Quality and resistance to leaf rust and red blister disease of 21 CWD resistant and 6 commercial C. canephora clones. Source Variety Body Flavour Colour 100 bean weight Retained by 16/64 CWD resistance Resistance to rust Resistance to Red blister Identified through screen house tests Identified from field trials Previous commercial lines 1 NFCT3 Fair+ Fair Greenish brown JB5109.4/5/1 Fair+ Fair- -do Erecta unknown 20 Fair Fair-- -do Erecta unknown 14 Fair Fair+ -do s/21/1 Fair+ Fair+ -do /22/2 Light Fair -do J24/13/20/4 Fair++ Fair -do Erecta unknown 11 Fair- Fair-- -do MFCT2 Fair Fair -do J94/2/64/1 Fair+ Fair -do MFCT1 -do /32/2 Fair Fair-- -do J12.01/16/1 Fair Fair -do /1 Fair++ Fair- -do /2 Fair+ Fair+ -do /22/12 Fair+ Fair+ -do J1/14/21/1 Fair Fair+ -do /37/7 Fair+ Fair -do J/1/ Q/3/ R/1/ s/ s/ Quality analysis: ++ is very good; + is good; - is fair, - is poor and - - is poor Disease analysis: ++ is resistant; + is fairly resistant; - is susceptible and - - is very susceptible Yield

7 Figure 3. % survival among C. canephora artificially progenies inoculated with CWD pathogen in the screen house. Although 8 elite varieties identified in this study have the potential for farmer cultivation, it should be noted that data on yield and their interaction with the environments in different C. canephora farming regions is not yet known. But in the mean time they can be disseminated to farmers in the agro-geographical location where Kituza is located, awaiting validation of their performance in multi-location trials. These 8 varieties might not be the best in terms of yield and bean and cup qualities and it is unthinkable that only 8 clones will suffice to support the Robusta coffee growing in the whole country. Therefore there is still need for developing more varieties. This process is continuing at COREC through identification and evaluation of other CWD resistant clones plus generation, evaluation and selection of multi-trait hybrids. Currently there are 1519 CWD resistant clones planted in the mother garden at Kituza, out of which only 167 were evaluated. The other clones are being evaluated and good performing clones shall be advanced to on-farm trials en route to being released to farmers. Through this process, many more superior varieties will be identified and supplied into the farming system to reverse the devastating effects of the CWD menace. 1478

8 REFERENCES Delassus E La trachéomycose du caféier. Bulletin Scientifique du Ministère des Colonies, Section Agronomie Tropicale, 5: Delassus E La trachéomycose du caféier. Bulletin Scientifique du Ministère des Colonies, Section Agronomie Tropicale, 5: Flood J. and Brayford D The re-emergence of Fusarium wilt of coffee in Africa. In: ASIC 1997 Proceedings of the International 17 th Scientific Colloquium on Coffee Conference. ASIC, NAIROBI. Fraselle J Observations préliminaire sur une trachéomycose de Coffea robusta. Bulletin Agricicole, Congo Belge, XLI, Kranz J. and Mogk M Gibberella xylarioides Heim et Saccas on arabica coffee in Ethiopia. Phytopathology, 78: Lejeune J.B.H Rapport au Gouvernement Imperial d'ethiopie sur la production caféière. Rapport du la FAO, Rome FAO158/3/1881. Lejeune, J. B. H Rapport au Gouvernement Imperial d'ethiopie sur la production caféière. Rapport du la FAO, Rome FAO158/3/1881. MAAIF (Ministry of Agriculture Animal Industry and Fisheries) Clonal Robusta coffee hand book Part 2: Nursery management practices. Communication Centre, Entebbe. Muller R.A Some aspects of past studies conducted in Western and Central Francophone Africa on tracheomycosis (Cote d Ivoire, Cameroon and Central African Republic). In: Hakiza JG, Birikunzira B, Musoli P (eds.) Proceedings of the first regional workshop on coffee wilt disease (Tracheomycosis). (pp ) International Conference Centre, Kampala, Uganda. Masiga M., Ruhweza A. (2007). Commodity Revenue Management: Coffee and Cotton in Uganda. YOMA Consultants; International Institute for Sustainable Development. Winnipeg, Manitoba, pp. 18. Mugambe K. (2007). The Coffee Year Book, 2006/07. Uganda Coffee Trade Federation; pp Musoli P., Olal S., Nabaggala A. and Kabole C Screening Robusta coffee germplasm for resistance against coffee wilt disease. CORI progress report on coffee wilt disease research and development Musoli, P., Olal, S., Nabaggala, A. and Kabole, C Screening Robusta coffee germplasm for resistance against coffee wilt disease. CORI progress report on coffee wilt disease research and development Musoli P.C., Sources of Resistance against coffee wilt disease caused by Fusarium xylarioides in Uganda. Ph.D. Thesis of Montpellier II University. Oduor G., Phiri N., Hakiza G. J., Abebe M., Asiimwe T., Kilambo D. L., Kalonji Mbuyi A., Pinard F., Simons S., Nyasse S. and Kebe I Surveys to establish the spread of coffee wilt disease, Fusarium (Gibberella) xylarioides, in Africa (2004) In: ASIC 2004, Proceedings of 2O th International Conference on Coffee Science. (pp ), Bangalore, India. 1479