Races of Hemileia vastatrix and Variation in Pathogenicity of Colletotrichum kahawae Isolates to Compact Coffee Genotypes in Tanzania

Eighteen clones of coffee rust differentials were used to characterize rust races prevailing in the coffee growing areas in Arusha, Kilimanjaro, Mara, Bukoba, Kigoma, Mbeya and Ruvuma regions. The results showed the presence of seven new Hemileia vastatrix races causative agent for coffee leaf rust (CLR) in the surveyed coffee growing areas. The races were: XLI (v2, 5, 8), XLII (v2, 5, 7, 8 or v2, 5, 7, 8, 9), XV (v4, 5), XXX (v5, 8), XXXIII (v5, 7 or v5, 7, 9), XXXIV (v2, 5, 7 or v2, 5, 7, 9), XXXIX (v2, 4, 5, 6, 7, 8, 9). Four strains of Colletotrichum kahawae originating from 4 coffee ecosystems were used to test their level of pathogenicity on 16 compact coffee genotypes, Ctr088 and commercial variety N39. The four C. kahawae strains were less pathogenic to six compact coffee genotypes; CVT2, CVT4, CVT5, CVT7, CVT8 and CVT13, completely nil pathogenic to Ctr088, but significantly highly (P < 0.05) pathogenic to N39. Coffee genotype Ctr088 was confirmed to be a potential donor of developing coffee berry disease (CBD) varieties with durable resistance. Differential interaction between the compact genotypes and the four C. kahawae strains needs further investigation.


Introduction
Coffee berry disease (CBD) caused by Colletotrichum kahawae Waller and Bridge sp.nov.(Waller et al., 1993), and coffee leaf rust disease caused by Hemileia vastatrix Berk et Br., causal agent of orange rust on coffee plants are the main cause of yield loss of Arabica coffee in Tanzania.Colletotrichum kahawae isolates from coffee plants including the one causing CBD were previously referred to as Colletotrichum coffeanum Noack, but recently it was recognized that there is a clear distinction between pathogenic form named as Colletotrichum kahawae Waller and Bridge Sp.Nov., and saprophytic form (Waller et al., 1993).However, there are pathogenic variations within C. kahawae strains.Studies by Rodrigues Jr. et al. (1992) found out that C. kahawae strains from Angola and Malawi exhibited slightly higher level of virulence than Kenyan strains.Similarly difference in the level of pathogenicity was found among C. kahawae strains in Tanzania (Kilambo et al., 2008).In the same study, C. kahawae strain from Cameroon was found to have higher level of pathogenicity than those from Tanzania, Kenya and Zimbabwe.Inspite of the differences in aggressiveness of C. kahawae strains, differential interactions between host and pathogen were seldom quantified (Kilambo et al., 2008;van der Graaf, 1981).There is therefore a need to update the characteristics of existing C. kahawae isolates established from coffee growing areas in order to obtain detail information on the variability of the CBD pathogen in Tanzania.
Worldwide, there are around 40 known physiological races of H. vastatrix causative agent for coffee leaf rust (CLR); of which seven were recorded to exist in Tanzania (Rodrigues Jr. et al., 1975).These were races I, II, III, XVII, XXIV, XI and XX.Coffee rust disease surveys carried out from 2006 to 2007 recorded new rust pathogen races XXII and XXXIV (CIFC, 2007).Two years later, five new coffee leaf rust pathogen races were recorded (TaCRI, 2009).These were races XXIII, XXIV, XXV, XXVIII and XXXI.Due to this observation a need to update information of coffee leaf rust pathogen races in coffee growing areas is important.Therefore, according to Flor (1971) varieties used for the identification of coffee leaf rust pathogen races called differential coffee leaf rust pathogen races were used to study the current pathogen variation in Tanzania.
The objective of this study was to characterize races of Hemileia vastatrix and variation in pathogenicity of Colletotrichum kahawae strains in the key coffee growing areas of Tanzania.

Description of the Study Area
Coffee leaf samples infected with Hemileia vastatrix and Colletotrichum kahawae isolates were collected from the sites as shown in Table 1.The study areas are marked with red dot (Figure 1).

Identification of Coffee Leaf Rust Pathogen Races
Field sampling of coffee leaf of genotype N39 infected with Hemileia vastatrix (coffee genotype N39 is known to be attacked with available possible CLR pathogen races in specific area) was done in 2010 and 2011.The infected leaves were dried while pressed between newspapers, in this way rust lesions would be kept afresh.The uredospores from the lesions were then scraped into conical flask contained sterilized dH 2 O.In the solution a drop of tween 80 was added to allow uniform dispersion of uredospores.The uredospores concentration was calibrated at 1 x 10 6 /ml.Inoculation was done by dipping camels brush into uredospores concentration then rub on the underside of twelve ( 12) leaves of each of the 18 CLR differentials (Eskes, 1982).The inoculated leaves of the CLR differentials were placed in a labeled plastic box of 30 cm length x 15 cm width x 10 cm height, covered with black polythene.Dark conditions stimulate formation of the germ tube and subsequently followed by appressorium, which in turn, initiate of the infection process.Two un-inoculated healthy leaves were included as control.Reactions were recorded 40 days after inoculation.Information on the available coffee leaf rust races were determined according to Rodrigues et al. (1975) and Varzea and Marques (2005).They collected samples of the coffee leaves infected by H. vastatrix from different coffee growing areas, and then artificially inoculated the leaves of the coffee rust differentials to establish physiological rust races.

Variation in Pathogenicity of Colletotrichum kahawae Isolates to Compact Genotypes
Four (4) Colletotrichum kahawae strains [2006/14 (Kibosho-Kombo Moshi), 2010/2 (Lunji Mbeya), 2010/1 (Kigoma), and 2006/21 (Mbinga)] were used to test the pathogenicity of the compact coffee genotypes.Purity of the strains was done by single spore isolation.F 1 hybrid seed produced through artificial pollination between male parents (listed in Appendix 1) and selected compact coffee lines Ctr086, Ctr088 and Ctr127 each year in the seed garden were used as a host in determining the pathogenicity of the C. kahawae strains.F 1 of each of the 16 hybrids represented by 40 hypocotyls were raised in a plastic box at a spacing of 5 cm x 5 cm containing sterilized sand.The experiment was arranged in the laboratory in a completely randomized design (CRD) with three replications.Hypocotyls at 5 to 6 weeks old or 5 to 6 cm tall were sprayed and inoculated with suspension of C. kahawae strains at a concentration of 2.0 x 10 6 spores/ml twice at 48 hr intervals using a method described by van der Vossen et al. (1976).The inoculum was prepared from 17-23 old days colonies of the four C. kahawae strains grown in 3.4% malt extract agar containing 0.04% streptomycin.Conidia were dislodged by pipetting 4-6 ml of distilled water onto cultures and then rubbing gently the surface of the mycelia mat with a spatula.The suspension was filtered and the concentration calibrated using haemocytometer to obtain the spore concentration of 2.0 x 10 6 spores /ml.Three weeks after the date of first inoculation, coffee seedlings were individually scored for CBD symptoms developed on the hypocotyls using a rating scale from 0 to 4 developed by van der Graff (1982), where 0 is nil and 4 intense CBD infections.
For each genotype disease intensity reaction (DIR) for sixteen F 1 hybrids was determined to define the pathogenicity as follows: where, i is the disease class, ni is the number of seedlings in class i and n is the total number of seedlings scored.In determining the DIR of a coffee genotype it requires: assessing the symptoms developed on the hypocotyls using the scale of 0-4, determining the number of hypocotyls infected per each rating scale, followed by determining the percent infection for each rating scale which then is multiplied by susceptibility of coefficient (0-4).Summation of the products is divided by 4 categories of resistance: Resistance, 0-25; Moderately Resistance, 26-50; Moderately Susceptible; and Susceptible, 76-100).
The level of pathogenicity of the four C. kahawae strains was determined on hypocotyls and green berries of N39, a susceptible variety at an expansion stage.Means of DIR were separated using Tukey's separation test.Data for CBD and CLR were not transformed as the raw data were symmetrically distributed; between the 16 compact coffee genotypes and N39.The data was subjected to analysis of variance (ANOVA) using Genstat software and effects declared significant at 5% level.

Identification of Hemileia vastatrix Races
The results on the reaction of rust races on leaves of differentials are as indicated in Table 3 and Figure 2. Differentials 849/1, 63/1x, 32/1, H 420/2, and 1396 had the highest frequency of rust reaction.In this study, the identified coffee rust races are presented in Table 5 whereby seven new coffee rust races were recorded.The new coffee leaf rust races were: XLII, XLI, XXX, XXXVIII, XV, XXXIV, and XXXIX (Table 4)., 3, 5, 7 or v2, 3, 5, 7, 9 NB: * Further research work required to establish a type of the race.

Variation in Pathogenicity of Colletotrichum kahawae Isolates to Compact Coffee Genotypes
The results in Table 5 show the pathogenicity of C. kahawae strains on hypocotyls of the compact coffee genotypes.There was a significant (P < 0.05) variation in the pathogenicity of Colletotrichum kahawae strains to compact coffee genotypes.The four C. kahawae strains were less pathogenic to the coffee genotypes CVT2 (Ctr088 x (SL34 x HdT) x Kent x Rume Sudan), CVT4 (Ctr088 x (SL34 x HdT) x Rume Sudan), CVT5 (Ctr 088 x (Rume Sudan x Catuai), CVT7 (Ctr088 x (HdT x N39) x SL28) x (N39 x Rume Sudan), CVT8 (Ctr088 x (N39 x HdT) x (N39 x HdT) x Rume Sudan) and CVT13 (Ctr127 x (Blue Mountain Jamaica x Cioccie) x Rume Sudan).The pathogenic level of the four C. kahawae strains was highly expressed to the check variety N39; (DIR = 100), compared to Ctr088 a pure line of compact coffee genotype with zero score of DIR (Table 5).The levels of pathogenicity of the four C. kahawae strains were determined on green berries of N39 at an expansion stage.The differences of the lesions were as shown in Figure 5. Green berries infected with C. kahawae strain 2006/14 had wider black lesions of 4-5 mm than the rest of the strains with 1-3 mm recorded seven days after artificial inoculation (Figure 5).

Discussion
Updating of information of available coffee rust races forms an important activity in producing coffee varieties with durable resistance to Hemileia vastatrix.In Tanzania the first information on the existence of coffee leaf rust races was done in 1975.Rodrigues et al. (1975) reported the existence of seven rust races collected in different coffee growing ecosystems in Tanzania.The races recorded included race I, II, III, XVII, XXIV, XI, and XX.This information was updated 22 years later whereby CIFC (2007) recorded the existence of two new rust races XXXIV andXXII, andin 2009 races XXIII, XXIV, XXV, XXVIII andXXVI (TaCRI, 2009).In this study seven new races have been identified within a period of two years; 2010 and 2011.The coffee leaf rust races identified were; XLII, XLI, XXX, XXXIII, XV, XXXIV, and XXXIX.This information is very useful in formulating strong breeding programmes.Van der Vossen (2005) reported that resistance genes S H I-S H 9 have all been successively defeated in C. arabica by emerging new physiologic races of the CLR pathogen H. vastatrix in some coffee growing areas in the world.It is reported that mutation is the likely mechanism for new race formation as no alternative host to H. vastatrix has been found (Varzea & Marques, 2005).Catimors including genotypes Ctr086, PNI088 and Ctr127 still provides full resistance to all types of coffee rust races ( Van der Vossen, 2005).As these races were identified from areas where the 16 compact genotypes were exposed, implies that there are genotypes with complete resistance to CLR.
The results on DIR of C. kahawae strains found on coffee genotypes CVT2, CVT4, CVT5, CVT7, CVT8 and CVT13; showed that there were potential compact genotypes with high levels of resistance to CBD.These can be used to improve coffee breeding programme in Tanzania.Some of these genotypes like CVT2 were found to produce economical yields and cup taste classified for export value.Colletotrichum kahawae strain 2006/14 showed a high level of pathogenicity.The strain demonstrated to be pathogenic on hypocotyls and green berries of N39.Studies done by Kilambo (2008) identified this strain as one of the most aggressive C. kahawae strains in the country.The bio-chemical analysis using iso-enzymes applied to 25 C. kahawae strains collected from coffee growing areas in Tanzania confirmed that strain 2006/14 formed a peculiar identity compared to other strains (Kilambo et al., 2008).The findings confirm that this strain can be used to screen for genotypes with resistance to CBD. High levels of CBD shown by Ctr088 confirms studies done by Gichuru et al. (2008) when studying the F 2 populations of Ctr088 x SL28 to map the Ck-1 gene by molecular markers.The genotype can be used as a donor parent for genes of CBD resistance to breed coffee varieties with resistance to C. kahawae.
In the light with the occurrence on variability of H. vastatrx and C. kahawae strains in Tanzania, TaCRI will quite often be faced with the challenges of finding the information on the CLR and CBD populations and their evolution.This will assist in improving coffee genotypes found to have good agronomic characteristics which have also high spectra of resistance.Recently, additional physiological races of H. vastatrix were identified in Kenya (Gichimu et al., 2012) revealling a challenge to coffee breeding programme in this region.That is why currently TaCRI is not only involving HdTs; natural hybrids between C. arabica and C. canephora which have genes responsible for rust resistance (Diniz et al., 2012), but catimors also confirmed to be resistant to local races existing in Tanzania.It is therefore important that while studying host resistance, parallel studies of pathogen variability in relation to disease resistance should also be made.

Conclusion
Coffee leaf rust disease (Hemileia vastatrix) and coffee berry disease (Colletotrichum kahawae) are in Tanzania the most important disease in Arabica coffee (Coffea arabica).They cause annually considerable economic damage due to crop losses, as most of Arabica coffee is still produced on susceptible cultivars.There is an urgent need for improving the levels of resistance to CLR and CBD in Arabica coffee, by exploiting resistant genes from Catimor genotypes like Ctr086, Ctr088 and Ctr127 in breeding programmes.The Catimor genotypes crossed to known hybrids with good agronomic attributes may assist in recommending the best commercial coffee genotypes with resistance to CLR and CBD.In the light of breakdown of resistance due to emerging of new physiologic disease strains, studies of pathogen variability in relation to disease resistance should also be made.
van der Vossen for commenting on a draft of this paper.The Government of Tanzania through Tanzania Commission for Science and Technology (COSTECH) for the funding support of the study.

Figure 2 .
Figure 2. Reaction of rust isolates collected from coffee regions on differentials to detect available races

Table 1 .
Sites for inoculum collection of Hemileia vastatrix and Colletotrichum kahawae Coffea spp.differential hosts for physiological races of Hemileia vastatrix were used.These clones were originally obtained by vegetative propagation from the original collection of coffee leaf rust disease centre at Oeiras Portugal (CIFC) (Table2).The 18 clones of Coffea spp.involves: Coffea arabica, Coffea canephora, Coffea excels, Coffea racemosa and Coffea congensis.This could assist in distinguishing reaction of the races of the CLR pathogen Hemileia vastatrix which is so far found in coffee growing regions in Tanzania.Another form of coffee rust Hemileia coffeicola is restricted to Central and Western Africa especially the higher and cooler coffee regions.

Table 2 .
Clones of coffee leaf rust differential hosts for Hemileia vastatrix

Table 3 .
Reaction of coffee rust differentials to isolates of Hemileia vastatrix in specific areas

Table 5 .
Disease Intensity Reaction (DIR) of the four C. kahawae strains on coffee genotypes