DETACHED BERRIES INOCULATION FOR CHARACTERIZATION OF COFFEE RESISTANCE TO COFFEE BERRY DISEASE

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1 Journal of Plant Pathology (2012), 94 (3), Edizioni ETS Pisa, DETACHED BERRIES INOCULATION FOR CHARACTERIZATION OF COFFEE RESISTANCE TO COFFEE BERRY DISEASE F. Pinard 1, C.O. Omondi 2 and C. Cilas 3 1 CIRAD-icipe, PO Box 30777, Nairobi, Kenya 2 Coffe Research Foundation, PO Box 4, 00232, Ruiru, Kenya 3 CIRAD, UR 106, Avenue Agropolis, TA A31/02, Montpellier Cedex 5, France SUMMARY Introgression of resistance to coffee berry disease (CBD), caused by Colletotrichum kahawae, is one of the best control methods for this disease. In a set of three successive experiments, we assessed the efficiency of detached berries inoculation to characterize coffee resistance to CBD. Cvs Ruiru 11 and SL 28 were inoculated with a suspension containing from 10 3 to 10 6 fungal spores ml -1. The number of infected berries and their degree of infection were assessed and statistically analyzed. Results indicated that berry resistance could be separated into two components: one against the pathogen penetration and the other against its growth in the berries. The existence of a third dynamic component of resistance is suggested: the continued evolution and eventual decrease of resistance during berry expansion and maturation stages. Resistance to C. kahawae penetration was higher in cv. Ruiru 11 than in cv. SL 28, but was bypassed at the highest inoculum concentration tested (10 6 spores ml -1 ). The resistance of the inner berry tissues to pathogen growth appeared similar in both cultivars, but was broken in cv. SL 28 at the highest inoculum concentration. It is assumed that both forms of resistance could be of quantitative nature because they were modulated by the inoculum concentration. As the varieties were ranked in the same order in the three experiments, it was concluded that the technique is reliable for a relative ranking of the tested cultivars. Key words: coffee berry disease, Colletotrichum kahawae, host pathogen interaction, resistance component. INTRODUCTION Arabica coffee is one of the main cash crops in East Africa contributing to the livelihood of a large part of the rural populations of Ethiopia, Kenya, Uganda, Corresponding author: C. Cilas Fax: christian.cilas@cirad.fr Rwanda, Burundi, Tanzania and Democratic Republic of Congo. Diseases remain the major constraint to Arabica coffee production in the region, coffee berry disease (CBD) being one of the most important (Firman, 1977). CBD, caused by Colletotrichum kahawae Waller and Bridge (Waller et al., 1993) is, to a large extent, responsible for reduced productivity and increased cost of production, thereby reducing the competitiveness of the region s coffee. The disease, which is confined to the African continent, attacks all stages of the developing crop including flowers and occasionally leaves (Mulinge, 1970). Symptoms on green berries appear as small dark sunken lesions typical of anthracnose which may spread to cover the whole berry. Under wet and cold conditions, the fungus readily sporulates forming a mass of pink conidia and penetrates the interior of the berries destroying the beans. The resulting dry, black mummified berries have no commercial value and may be shed off the tree or remain attached. The disease severity is higher on coffee trees exposed to sunlight than on those located under the shade (Mouen et al., 2007, 2008). Host resistance to CBD has been the subject of intense studies in the East African region (Firman, 1977; Van der Vossen and Walyaro, 1981; Van Der Graaff, 1992). It is widely accepted that resistant varieties provide an opportunity for the farmers to produce coffee in a manner that is sustainable, competitive and environmentally benign. Some countries in the region including Ethiopia, Kenya and Tanzania have released resistant varieties for commercial production but the challenges of long breeding cycle associated with long juvenile period have slowed down the pace of further varietal improvement. Screening varieties under development for CBD resistance in the field is difficult, as it depends on the occurrence of suitable climatic conditions for infection and disease expression. Therefore, the screening procedures still largely rely on artificial inoculations. Pre-selection on 6-week-old seedlings has been widely used to overcome the long juvenile period (Van der Vossen et al., 1976; Van der Graaff, 1978; Vander der Vossen and Walyaro, 1980; Omondi et al., 2000, 2001), but it must be combined with field evaluation to confirm mature plant resistance (Van der Graaff, 1978). The ino-

2 518 Coffee resistance to coffee berry disease Journal of Plant Pathology (2012), 94 (3), culation of detached berries under artificial conditions offers the advantage of a direct assessment of C. arabica/c. kahawae interaction at the fruit level (Bock, 1956), making possible the characterization of the resistant component operating under field conditions. Earlier workers (Bock, 1956; Firman, 1964; Nutman and Roberts, 1960; Griffiths et al., 1972) claimed the great potential of this technique, which was successfully used in some selection process (Van der Graaff, 1978), but others (Van der Vossen et al., 1976) questioned the ability of the technique to correctly reveal at the laboratory level the coffee field resistance to CBD. According to Van der Graaff (1992) this conflicting situation was mainly due to the fact that the conditions of the protocol to be used were never precisely established, leading to difficult implementation or misinterpretation of the results. The objective of this study was therefore to confirm the potential of the detached berry inoculation technique to correctly assess CBD resistance of coffee genotypes and to determine the experimental conditions for a possible routine screening procedure. In particular, this paper investigates the effect of inoculum concentration and cultivar as two of the main variables controlling disease expression. MATERIALS AND METHODS Plant material. All material was collected from the experimental plots of the Coffee Research Foundation (CRF) in Ruiru, Kenya. In a set of three successive experiments, expanding berries of 14, 15 and 16 weeks of age were collected during the last two weeks of March This stage of maturation is the most susceptible to CBD before the hardening stage, when berries exhibit a higher level of resistance (Muller, 1980; Mulinge, 1970; Nutman and Roberts, 1960). All berries originated from single mature trees of the susceptible cv. SL 28 and the resistant cv. Ruiru 11 (Van der Vossen and Walyaro, 1980). The wounded stalk end of the berries was removed with a sterile scalpel to avoid contamination and their surface was cleaned with liquid soap (0.01%), rinsed and dried. Berries were then aligned on a wet tissue paper supported by a metallic grid and placed in a plastic box partially filled with water. The box was sealed to provide the saturated humid conditions necessary for disease development. Inoculations. A spore suspension was prepared from a 7-day-old culture of a single spore colony of C. kahawae strain KW 33 isolated by and maintained at CRF. This strain was selected for its medium aggressiveness in the Ruiru environment. Berries were inoculated with a 20 µl suspension drop, then kept in a refrigerated room at C. After 24 h, the remnants of the inoculum were removed with a dry tissue paper. The symptoms were regularly scored at 21 to 25 days post inoculation (dpi), a period during which the berries remained free of contamination other than CBD. The infection potential of several inoculum concentrations, from 2x10 3 to 2x10 6 spores ml -1 was investigated and compared with a control treatment inoculated with sterile water. Three independent experiments were conducted: in experiment 1 the protocol included 3 replications of 50 berries each; in experiment 2, 2 replications of 40 berries each; and in experiment 3, 1 replication of 30 berries. Disease assessment. CBD development was assessed using a visual scale from 0% to 100% of the total berry surface affected. In particular, disease incidence was expressed as the percentage of infected berries, whereas disease severity was determined on each berry as the percentage of necrotic area. The mean incubation periods (IP) were estimated as the weighed mean of the IP evaluated for each berry: IP = Σ i (n i /n)d i where i is the number of scoring dates, n i the number of newly infected berry at the i date, n the total number of infected berry for the corresponding treatment, and d i the number of days corresponding to the i th observation Statistical analysis. Curves for symptoms development were drawn for the infection rate and for the necrotic area on the infected fruits. These curves helped defining the dates for the statistical comparison between treatments (inoculum concentration, cultivars) and interaction between these main factors. At these dates, the variable severity was transformed using Bliss transfor- Table 1. Inoculum concentration effect on symptoms expression by cvs Ruiru 11 and SL 28 varieties in three successive experiments. GLM (χ 2 ) and ANOVA (F) analysis conducted at the date indicated between brackets. (dpi, days post inoculation). Infection rate Necrosis development Ruiru 11 SL 28 Ruiru SL 28 χ 2 P χ 2 P F P F P Experiment 1 (22 dpi) < < Experiment 2 (19 dpi) < < Experiment 3 (21 dpi) < <

3 Journal of Plant Pathology (2012), 94 (3), Pinard et al. 519 Fig. 1. Disease incidence on cvs Ruiru 11 (a-c) and SL 28 (d-f) inoculated with various concentration of CBD inoculum. o: 10 6 spores ml -1 ; : spores ml -1 ; : 10 5 spores ml -1 ; *: spores ml -1 ; : 10 4 spores ml -1 ; : 10 3 spores ml -1. DAI: days after inoculation. Experiment 1: a, d; experiment 2: b, e; experiment 3: c, f. mation (arsin ) in order to homogenize variance, then analysed by a classical ANOVA followed by a Newman and Keuls multiple test comparison. At the same dates, disease incidence was analysed by a generalized linear model (Nelder and Wedderburn, 1972) because of the binomial distribution (0/1) with the logit linked function. RESULTS In all experiments, controls remained symptomless, proving the initial healthy condition of the tested berries. Disease incidence. CBD occurrence in the three experiments is shown in Fig. 1. Disease incidence evolved as follows: cv. SL 28 was more infected in experiments 2 and 3 than in experiment 1. The same trend was observed on cv. Ruiru 11 but later during the evaluation period for the incidence was higher in experiment 3 than in 1 and 2. In all 3 experiments, the concentration effect was highly significant (Table 1), the highest inoculum concentration leading to the highest level of infection. Ruiru 11 was never infected with the 10 3 spores ml -1 inoculum and was infected only once with the 10 4 spores ml -1 one (Fig. 1c). On the contrary, cv. SL28 was always infected, even though at a low rate, at the lowest concentration of 10 3 spores ml -1. Table 3 shows that the inoculum concentration effect was associated to an increase of IP, which is obvious on cv. Ruiru 11 and weaker on cv. SL 28 in experiment 3 (Fig. 1f).

4 520 Coffee resistance to coffee berry disease Journal of Plant Pathology (2012), 94 (3), The cultivar effect on CBD incidence was significant for all the inoculum concentrations (except for 10 4 spores ml -1 ) in experiment 2 (Table 2), where cv. Ruiru 11 showed fewer attacks than cv. SL 28. In experiment 1 (Fig. 1 a, d) and 3 (Fig. 1 c, f), the cultivar effect was also evident but only at concentrations lower than 10 6 spores ml -1 ; with the exception of the 10 4 spores ml -1 suspension, in experiment 3, where the infection of cv. SL 28 appeared quite low and similar to that of cv. Ruiru 11. Disease severity. The inoculum concentration had no impact on disease severity on cv. Ruiru 11 in the 3 experiments (Table 1). In experiment 2 (Fig. 2 b, e), the suspension of 10 5 spores ml -1 led to too few infected berry to impact the statistical analysis. One can note that with this treatment, necrosis was delayed (4.4 days, Table 3), progressed during the first 19 days at the same speed than the other inoculum concentrations tested (Fig. 2 b), then showed an quicker growth rate so as to elicit a necrotic area of the same size as that generated by higher inoculum concentration. A similar pattern was observed on cv. SL 28 at the concentration of 10 3 and 10 4 spores ml -1 in experiment 2 (Fig. 2 e) and at the concentration of 10 4 spores ml -1 in experiment 3 (Fig. 2 f). On cv. SL 28, the concentration effect was weak, and detected only in experiment 2 (Table 1) for, at 19 dpi, the 10 6 spores ml -1 inoculum had a more serious effect than the 10 4 spores ml -1 and 10 3 spores ml -1 treatments. By 21 dpi this difference decreased; only the 10 4 spores ml -1 treatment showed significantly smaller necroses compared to the other treatments. Fig. 2. Disease severity on varieties Ruiru 11 (a-c) and SL 28 (d-f) inoculated with various concentration of CBD inoculum. o: 10 6 spores ml -1 ; : spores ml -1 ; : 10 5 spores ml -1 ; *: spores ml -1 ; : 10 4 spores.ml -1 ; : 10 3 spores ml -1. DAI: days after inoculation. Experiment 1: a, d; experiment 2: b, e; experiment 3: c, f.

5 Journal of Plant Pathology (2012), 94 (3), Pinard et al. 521 The cultivar effect on the CBD severity was quite evident only at the concentration of 10 6 spores ml -1 in experiment 1 and 2 (Table 2). In these cases, cv. SL 28 was more damaged than cv. Ruiru 11. In experiment 3, the disease severity on cv. Ruiru 11 was higher than in experiments 1 and 2, reducing the differences between the two cultivars. This is in agreement with our observation on infection rates: more disease was expressed in experiment 3 than in experiment 1 and 2. DISCUSSION During these experiments, the controls remained healthy, suggesting the absence of latent infections which are quite frequent with anthracnose diseases of other fruit tree species (Prusky and Plumbley, 1992), and is suspected with CBD (Mulinge, 1970; Firman and Waller, 1977). Only the typical black sunken CBD lesions were observed on inoculated berries and, unlike Firman (1964), we were unable to detect the inactive scab lesion of CBD. This could be explained by the very humid conditions of our experiments which are highly favourable to the development of the active lesion type (Nutman and Roberts, 1960). It could also be due to the fact that the samples berries were healthy and had not been exposed earlier to C. kahawae, thus they were unable to react to the fungus attack by developing the scab interaction (Griffiths et al., 1971; Firman and Waller, 1977). Our observations confirmed and documented what was first suggested by Firman (1964) on the possible existence of two resistance components of coffee to CBD: one against the penetration and the installment of the pathogen, which can be assessed analysing disease incidence, and the other against the extension phase of the fungus colony, assessed by the evolution of the severity index. Both were considered as partial as they did not prevent the pathogen infection and colonization. Our results indicated that cv. Ruiru 11 expressed a greater resistance to penetration of C. kahawae than cv. SL 28. One of the mechanisms of this resistance appeared to be of quantitative nature as it could be modulated by inoculum concentration. As a consequence, the cultivar effect on the resistance to penetration varied with the inoculum concentration. The differences of resistance expressed by both cultivars were clear-cut at 5x10 5, 10 5 and spores ml -1. The cultivar effect was also significant at 10 3 spores ml -1, because of the complete re- Table 2. Variety effect on symptoms expression by cvs Ruiru 11 and SL 28 inoculated with a range of CBD inoculum concentration (spores ml -1 ) in three successive experiments. GLM (χ 2 ) and ANOVA (F) analysis conducted at the date indicated between brackets. (dai, days post inoculation). Infection rate Necrosis development Treatment Inoculum concentration χ 2 P F P Experiment 1 (22 dpi) < compared to Experiment 2 (19 dpi) < < < Experiment 3 (21 dpi) Table 3. Average incubation period (IP) of CBD on cvs Ruiru 11 and SL 28 inoculated with a range of inoculum concentration (spores ml -1 ) in 3 successive experiments (ni: no inoculation; nc: no contamination; *: inoculated with spores ml -1 inoculum). Average IP (days) Ruiru 11 SL28 inoculum concentration Exp 1 Exp 2 Exp 3 Exp 1 Exp 2 Exp ni ni * nc nc 15.6 ni nc nc nc

6 522 Coffee resistance to coffee berry disease Journal of Plant Pathology (2012), 94 (3), sistance of cv. Ruiru 11 at this inoculum concentration. The suspension of 10 4 spores ml -1 led to non-significant results because of the too small number of successful infections on both cultivars and its variation between experiments 1, 2 and 3. Interestingly, it was observed that the pathogen, at the concentration of 10 6 spores ml -1, was able to break the resistance to penetration of cv. Ruiru 11, inducing a susceptible pattern, similar to that of cv. SL 28. The resistance component against C. kahawae colony extension was seen only in experiment 1 and 2, at the concentration of 10 6 spores ml -1 : in these cases, cv. Ruiru 11 was again more resistant than cv. SL 28. This observation may be indicative of a possible breakdown of cv. SL 28 resistance at the highest inoculum concentration, cv. Ruiru 11 being less or not at all affected by this treatment. Such situation would be in favour of a quantitative nature of the resistance to the fungal growth in the inner berry tissue. If confirmed, it could also explain the high but late speed of necrosis development seen in experiment 1 and 2, once the fungal colony reached a critical size. In this work, an increase on both cultivars of the global level of the disease was observed from experiment 1 to 3, this being evident earlier on cv. SL 28 than on cv. Ruiru 11. Assuming that the experimental conditions and the level of aggressiveness of the used C. kahawae strain remained stable during the trial period, the change of susceptibility of the berries could be explained by an evolution of their physiology. It is well known that the resistance of coffee berries to CBD varies with their stage of development (Rayner, 1952), but the chronology and the magnitude of the successive events, from the supposed resistance of the young berries (Mulinge, 1970), the susceptibility of expanding berries and the resistance of the mature green berries (Muller, 1980; Mulinge, 1970; Nutman and Roberts, 1960), to the final susceptibility of the ripening berry (Nutman and Roberts, 1960), is not well understood. Our results may suggest the existence of a dynamic aspect of the evolution of susceptibility during the expanding stage (from 4-6 weeks to weeks after flowering), which is slower on cv. Ruiru 11 than on cv. SL 28. More work, however, is necessary for confirming this hypothetic dynamic process that could be cultivar-dependent (Gichuru, 2007). In this case, experiments on detached berry could no longer be indicative, as it is difficult to precisely control the physiological stages of the berry in the field and to synchronize the berry development of various cultivars when comparing them in one experiment. Nevertheless, bearing in mind the resistance evolution of coffee berry to CBD, our experiments showed the possible use of the detached berry technique for a relative ranking of cultivar resistance at different dates after flowering, which is still useful for a differential interaction analysis and varietal characterization. In Kenya s plantations, cv. Ruiru 11 is always ranked as resistant, compared to the susceptible cv. SL 28. In this cultivar CBD damages are rare, of little consequence, and generally associated with exceptional humid and cold weather conditions. In our experimental conditions, this type of resistance was seen only at inoculum concentration around or below 10 5 spores ml -1, while susceptibility was induced by suspension at 10 6 spores ml -1. Such observation reveals the high level of inoculum pressure induced by the detached berry inoculation technique, as seen also from the data by Firman (1964) who was able to break down the high level of cv. Ruiru 11 field resistance. Bock (1956) recommended the use of inoculum suspension of 10 6 spores ml -1 for optimal results. According to our data, this concentration is too high and should be reduced to at least 10 5 spores ml -1. Under these specific conditions, it is then expected that the detached berry inoculation test will compare with field observation and could be integrated in a selection index for C. arabica breeding (Cilas et al., 1998). These conditions should constitute the basis for further studies to confirm the ability of the detached berry technique to mimic field interaction and therefore its potential to contribute to a better understanding of the CBD epidemiology. An important diversity exists in C. kahawae (Derso and Waller, 2003), and this technique could also be used to compare the aggressiveness of several strains of this pathogen. ACKNOWLEDGEMENTS The authors are thankful to J.M. Ithiru and S.K. Kiarie for their technical support. This work received the financial support from the European Union through an INCO-DEV grant: CBDRESIST. Part of these results were communicated at the ASIC Conference held on September 2006 at Montpellier, France. REFERENCES Bock K.R., Investigations of coffee berry disease laboratory studies. 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