Acknowledgements
10.3. Assessment of Resistance
identifying genuinely resistant genotypes. This will include having easy but effective protocols for testing and quantifying the resistance. The screening protocols involve assessing genotypes for resistance under natural and artifi- cial infections. There are almost no special manipulations involved when as- sessing coffee plants for resistance in naturally infected gardens, and limited variation in methodologies is anticipated, except perhaps in the quantifica- tion of resistance. There are more methodological variations when assessing genotypes for CWD resistance in artificial conditions, perhaps due to varia- tion of costs required by different methods and the facilities available for the studies. Thus, a greater part of this section deals with methods involved in artificial inoculation.
10.3.1. Inoculation methods
Different inoculation procedures have been used by different scientists for artificially inoculating coffee plants when testing them for CWD resistance (Meiffren, 1961; Van der Graaff and Pieters, 1978; Bieysse, 2005; Musoli et al., 2001; Musoli, 2005). In Uganda, a number of artificial inoculation meth- ods were evaluated to determine their efficacy when testing for CWD resis- tance in C. canephora (Hakiza et al., 2002). The inoculation methods evaluated include
• Root dip method: involved inoculation by dipping the entire root system of the plants into a standard conidia inoculum of G. xylarioides;
• Stem wounding: involved introducing a standard inoculum through ar- tificial wounds on the stems of the study plants;
• Soil infection: involved planting test plants in soil contaminated with the CWD pathogen;
• Soil drenching without root wounding: involved drenching the soils where test plants are growing with a standard inoculum;
• Soil drenching with root wounding: involved drenching the soils where the test plants are growing with a standard inoculum but after wound- ing the roots while the plants remain in situ; and
• Leaf wounding: involved applying the inoculum on to the leaves through artificially made wounds.
All the inoculations were carried out using inoculum derived from a sin- gle conidium obtained from the same host plant and standardized at 1.3 × 106 conidia per millilitre of water. The inoculum was applied on 6- to 8-month- old seedlings or cuttings of known C. canephora genotypes (Plate 25). Plants inoculated by root dipping developed the disease symptoms earlier than plants inoculated by other methods and had a higher incidence of diseased plants, and there was clear contrast between susceptible and resistant geno- types. This method was therefore adopted for large-scale germplasm screen- ing by scientists in Uganda and Tanzania. However, root dipping required a lot more inputs (polythene pots, soils and manpower) than the other methods and therefore is more costly. It was also suspected that some of the plants
infected by this method could have developed the disease because of extra stress resulting from root damage incurred when stripping off soils from roots in preparation for dipping. At CIRAD, where labour is costly, stem nicking was adopted. This method can also clearly enable differentiation of resistant and susceptible genotypes. Stem nicking was also adopted for germplasm screening in Ethiopia, as it is considered to be less expensive although the disease levels among plants inoculated by root dip were always higher.
10.3.2. Inoculum concentration
At COREC, it was thought that a high inoculum concentration such as the 1.3 × 106 used by scientists during the past epidemics in Central and West African countries for routine germplasm screening could kill plants with moderate resistance, leading to discarding of useful material. Conversely, lower inoculum concentrations may not be effective enough to select plants that are resistant enough to the disease pressure in fields over long peri- ods, as in the case where the variety is planted in heavily infested gardens.
To test the optimum concentration, 6- to 8-month-old C. canephora seedlings were artificially inoculated by dipping their entire root system into inocu- lum (derived from a single conidium) at concentrations of 1.3 × 101, 1.3 × 102, 1.3 × 103, 1.3 × 104, 1.3 × 105, 1.3 × 106, 1.95 × 106 and 2.6 × 106conidia per millilitre of water. Results of these tests revealed that infection occurs even at the lowest concentration (1.3 × 101), but this inoculum concentration was associated with long incubation periods (for symptom development) and the lowest disease incidence among the inoculated plants (Fig. 10.3). Increase in
0 10 20 30 40 50 60 70 80 90 100
40 50 60 70 80 90
% Wilt Incidence
Days after inoculation
1.3 x 101 1.3 x 102 1.3 x 103 1.3 x 104 1.3 x 105 1.3 x 106 1.95 x 106 2.6 x 106
Fig. 10.3. Effect of inoculum concentration on incidence of CWD on artificially inoculated open pollinated seedlings of C. canephora.
inoculum concentration resulted in reduced incubation times and increased wilt incidence. It was decided that concentrations of 1.3 × 103, 1.3 × 104 and 1.3 × 105 conidia per millilitre should be adopted for routinely screening for resistance against CWD.
10.3.3. Duration of exposure to the inoculum for root dipping
To maximize the number of plants inoculated by root dipping, plants were assessed after various times of exposure to the inoculum standardized at 1.3 × 106 conidia per millilitre of water. The exposure times tested included immediate root dip (which involved withdrawal of the plants immediately after their roots had been dipped into the inoculum), 20 min of exposure (which involved withdrawal of plants after 20 min in the inoculum), 40 min of exposure, 60 min of exposure, 4 h of exposure, 8 h of exposure, 12 h of exposure and 24 h of exposure. C. canephora seedlings of 6–8 months were used in these tests, and the tests were all conducted twice. All inoculated seedlings developed CWD symptoms irrespective of duration of the expo- sure time. The differences in incidence of diseased plants, especially during the first 40 days after inoculation, were insignificant. These results showed no advantage in leaving plants in the inoculum for more than 20 min since shorter exposure periods allow more plants to be inoculated within a given period.
10.3.4. Quantifying CWD resistance in artificial inoculation
An effective and reliable method of quantifying resistance was necessary for comparison of results among experiments and among scientists, and it was necessary for the selection of genuinely resistant genotypes, irrespective of whether the evaluation was carried out on mature plants in the field or young plants in the screen house. The method adopted will depend on the purpose of the study. Where the study aims at determining relative resis- tance between progenies or clones, resistance can be assessed as numbers of infected plants compared to those uninfected. The numbers can be expressed as percentage infection. Relative resistance between progenies and/or clones can also be expressed on a disease symptom severity scale. In Uganda, the plants studied in artificial inoculations were commonly assessed on a scale of 1 to 5, where 1 = no disease, 2 = curling leaves and stunted growth, 3 = leaf wilting and yellowing, 4 = leaf necrosis, leaf wilting, and abscission and 5 = plants are dead. Mature plants studied in fields were also assessed on a scale of 1 to 5, but the quantification of the disease levels in the field was slightly different. In field assessment (mature coffee trees) 1 = no disease, 2 = 1%–25%
defoliation, 3 = 26%–50% defoliation, 4 = 51%–75% defoliation and 5 = 76%–
100% defoliation. Plants scored as level 5 are normally considered dead. The studies carried out at COREC (Uganda) and CIRAD showed that coffee trees, even of the same genotype and in heavily infected gardens, get affected at
different rates (Section 10.3.5), and that the time lapse between the first ob- served symptoms and the death of trees varies between genotypes. Thus, there was an attempt to quantify relative resistance among C. canephora clones using duration of the disease period (average time taken by plants from appearance of first symptoms to death). However, this quantification did not give consistent results because some moderately resistant clones had a shorter disease period than some highly susceptible clones and vice versa, and its cor- relations with other traits were not significant (Table 10.1).
Where the aim was to identify genotypes with total resistance, plants are classified as either diseased or not diseased. Since all plants that develop CWD symptoms eventually die, only plants without the symptoms after a long period of infection (6 months for plants in artificial inoculations and not less than 5 years for plants evaluated in heavily infested fields) were considered resistant. Where necessary, these plants can be re-inoculated and assessed again for another 6 months to ascertain their resistance. The plants that remained healthy after the re-inoculation were considered to have com- plete resistance, and such plants were planted in mother gardens for cloning and further assessments.
10.3.5. Evaluation for CWD resistance in the field
Studies carried out on mature trees of 20 C. canephora clones in a field at COREC revealed variable responses of the clones to CWD infection (Fig. 10.4).
The tree mortality for different clones was at varying levels when assess- ment started, and it progressed at varying rates to varying final levels. This showed that the clones had variable levels of resistance and the disease re- sistance is most likely imparted by many genes, which are variably avail- able in the clones. Clone J/1/1, which did not succumb to CWD throughout the assessment period, and clones Q/3/4, R/1/4 and 1s/3, whose disease levels were comparatively very low, were considered resistant. Clone 1s/2 appeared to be resistant until April 2002, when its trees started dying in high numbers. This shows that some genotypes require higher inoculum concen-
Table 10.1. Correlation of field resistance of C. canephora clones to CWD measured by different traits.
Disease severity
(1–5 scale) Disease period Sqr. AUDPC
% plant mortality 0.997 (<0.0001) -0.446 (0.064) 0.935 (<0.0001) Disease severity
(1–5 scale)
-0.454 (0.059) 0.931 (<0.0001)
Disease period -0.281 (0.274)
Sqr. AUDPC is coefficient of correlations performed on the square root of AUDPC. Figures in parentheses are probability values. AUDPC = area un- der disease progress curve.
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0
% mortality
Time in months
H/4/1 C/1/7 Q/1/1 257/53 C/6/1
P/5/1 1S/3 Q/3/4 R/1/4 B/2/1
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0
% mortality
Time in months
E/3/2 223/32 1S/2 B/1/1 J/1/1
G/3/7 Q/6/1 P/3/6 L/2/7 B/6/2
Highly susceptible Moderately susceptible
Moderately resistant Resistant
Highly susceptible Moderately susceptible
Moderately resistant Resistant
Fig. 10.4. Tree mortality of 20 C. canephora clones evaluated for CWD resistance in a field at Kituza.
trations for infection, and thus, there is need to assess the plants for many years, to allow the inoculum build up, when screening them for resistance in the field. Analysis of variance revealed significant genetic differences be- tweenC. canephora clones for resistance to CWD in the field.
10.3.6. Comparison of resistance in the field and in artificial inoculations
Rooted cuttings and open pollinated seedlings of some of the clones studied for field resistance were studied for resistance in artificial inoculations in a screen house at COREC, and their final per cent tree mortality were correlated with the final per cent tree mortality in the field. It was observed that even clone J/1/1, which had no diseased trees in the field, had 15% of its cuttings killed in artificial inoculation, but it was among the least affected (Table 10.2).
Clone B/2/1 had 54.2% mortality in the field but did not have any dead cuttings after artificial inoculation. This clone could be having differential reactions to infection under the different conditions. However, there was an overall significant correlation (P = 0.002) between mortality in the field and mortality of cuttings in the screen house (Table 10.3). This indicates that re- sistance detected in artificial inoculation is depicted in the field, and there- fore, the artificial inoculation is a good protocol for screening germplasm for CWD resistance. Where correlations between mortality among open pol- linated progenies in the screen house and mortality of their parents in the field and in the artificial inoculations (rooted cuttings) were not significant,
Table 10.2. Mortality of C. canephora clones in the field and their rooted cuttings and progenies in artificial inoculation.
Clone Field mortality Rooted cuttings
Open pollinated progenies
J/1/1 0.0a 15.0b
Q/3/4 4.2b 20.0b
1S/3 33.3c 35.0abcd
R/1/4 33.3c 44.4bc 35.0abcd
C/6/1 50.0cd 65.0def
Q/6/1 50.0cd 80.0fg
B/2/1 54.2cd 0.0a 10.0a
223/32 58.3cde 90.0d 65.0def
L/2/7 62.5def 50.0bcd 25.0abc
Q/1/1 66.7defg 53.0cde
B/1/1 75.0defgh 85.0fg
257/53 83.3efgh 25.0b 40.0bcde
G/3/7 83.33efgh 80.0cd 50.0cde
P/5/1 87.5fgh
E/3/2 87.5fgh 25.0abc
1S/2 87.5fgh 100e 20.0ab
P/3/6 91.7gh 69.0efg
B/6/2 91.7gh 68.0efg
H/4/1 94.4gh 70.0cd 60.0def
C/1/7 95.8h 63.6cd 95.0g
Means separated by Student Newman–Keuls mean separation test. Figures represented by different letters are significantly variable.
then the response of open pollinated seedlings does not effectively represent CWD resistance of their parents.